-Fructofuranosidase and –D-Fructosyltransferase from New

Research Article120573-Fructofuranosidase and 120573 ndashD-Fructosyltransferasefrom New Aspergillus carbonarius PC-4 Strain Isolatedfrom Canned Peach Syrup Effect of Carbon and NitrogenSources on Enzyme Production

Gustavo Carvalho do Nascimento1 Ryhaacutera Dias Batista2

Claudia Cristina Auler do Amaral Santos3 Ezequiel Marcelino da Silva1

Fabr-cio Coutinho de Paula2 Danylo Bezerra Mendes 3

Deyla Paula de Oliveira4 and Alex Fernando de Almeida 5

1Bioprocess Engineering and Biotechnology Federal University of Tocantins Gurupi Tocantins Brazil2Graduate Program on Food Science and Technology Federal University of Tocantins Palmas Tocantins Brazil3Graduate Program on Biotechnology and Biodiversity of AmazonianndashBionorte Federal University of Tocantins Tocantins Brazil4Superintendencia de Desenvolvimento Cientıfico e Tecnologico Fundacao de Amparo a Pesquisa do Estado do Tocantins (FAPT)Tocantins Brazil

5Habite Biotechnology-Based Companies Incubator Federal University of Tocantins Gurupi Tocantins Brazil

Correspondence should be addressed to Alex Fernando de Almeida alexfernandouftedubr

Received 29 March 2018 Revised 16 November 2018 Accepted 2 December 2018 Published 8 January 2019

Academic Editor Maurizio Petruccioli

Copyright copy 2019 Gustavo Carvalho do Nascimento et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

120573-fructofuranosidase (invertase) and 120573-D-fructosyltransferase (FTase) are enzymes used in industrial processes to hydrolyzesucrose aiming to produce inverted sugar syrup or fructooligosaccharides In this work a black Aspergillus sp PC-4 was selectedamong six filamentous fungi isolated from canned peach syrup which were initially screened for invertase production Cultivationswith pure carbon sources showed that invertase and FTase were produced from glucose and sucrose but high levels were alsoobtained from raffinose and inulin Pineapple crown was the best complex carbon source for invertase (671 UmL after 3 days ofcultivation) and FTase production (1460 UmL after 5 days of cultivation) Yeast extract and ammonium chloride nitrogen sourcesprovided higher production of invertase (680UmL and 630UmL respectively)whereas ammoniumnitrate and soybean proteinwere the best nitrogen sources for FTase production (2400 UmL and 2490 UmL respectively) Fermentation parameters forinvertase using yeast extract were 119884119875119878 = 53685 Ug and 119875P = 149 Ugh FTase production showed values of 119884119875119878 = 262793 Ugand 119875119875 = 44 Uh using soybean protein The screening for best culture conditions showed an increase of invertase productionvalues by 510-fold after 96 h cultivation compared to initial experiments (fungi bioprospection) while FTase production increasedby 1460-fold (4440UmL) after 168 h cultivationA carbonariusPC-4 is a newpromising strain for invertase andFTase productionfrom low cost carbon sources whose synthesized enzymes are suitable for the production of inverted sugar fructose syrups andfructooligosaccharides

1 Introduction

Invertases (1 2-120573-D-fructofuranosidase fructohydrolase EC32126) are members of the GH32 family of glycoside hydro-lases that catalyze the hydrolysis of 120572-14-glycosidic bonds

from nonreducing fructofuranoside terminal residues of 120573-fructofuranosides (sucrose) to give an equimolar mixtureof monosaccharide D-glucose and D-fructose called invertsugar [1ndash3] Aqueous solutions of both sucrose and glucoseare slightly dextrorotatory ie causing rotation of plane

Hindawie Scientific World JournalVolume 2019 Article ID 6956202 13 pageshttpsdoiorg10115520196956202

2 The Scientific World Journal

polarized light to right while the solution of fructose isstrongly levorotatory ie causing rotation of plane polar-ized light to left [4] Invertases are widely used in foodand beverage industries One of important applications ofinvertase is the production of noncrystallizable sugar syrup(invert sugar syrup) from sucrose Invert sugar is used as ahumectant in the manufacture of soft candies and fondants[5] Invertase can be also used when sucrose containingsubstrates are subjected to fermentation viz production ofalcoholic beverages lactic acid and glycerol Furthermoreinvertases are also used for the manufacture of artificialhoney plasticizing agents and fructooligosaccharides [5]

Fructosyltransferase (FTase EC 2419) hydrolyzes su-crose and transfer fructosyl group to a sucrose moleculeacceptor to generate fructooligosaccharides along with glu-cose and fructose which have a wide range of applicationsin food and pharmaceutical industries [6 7] Fructooligosac-charides can be obtained synthetically from agave fructansby acid-catalyzed hydrolysis although the formation offructooligosaccharides via enzymatic methods is preferreddue to high substrate specificity and selectivity of enzymescompared to chemical routes [8] Fructooligosaccharidesfrom sucrose are considered as new alternative sweetenerswith functional properties also called soluble fibres withseveral desirable characteristics such as low calories non-cariogenicity and safety for diabetics Fructooligosaccharidesare also known as prebiotics since they stimulate the growthof probiotic organisms [1] However fructosyltransferase andfructofuranosidase production is rather confusing differingfrom one source to another from one microorganism toanother and even from one strain to another [9] In additioninvertases exhibit transfructosylating activity at high sucroseconcentrations in order to release glucose and fructose fromsucrose Microbial invertases may catalyze the synthesis ofshort-chain fructooligosaccharides such as 1-kestose nystoseand fructofuranosyl nystose in which one to three fructosylmoieties are linked to the sucrose by different glycosidicbonds depending on the enzyme source [4 5]

Invertases can be found by wide range of organismsincluding plants animal bacteria yeasts and filamentousfungi Among filamentous fungi Aspergillus genus has beenextensively used to produce invertase under submerged andsolid-state cultivations and its mechanisms of enzyme pro-ductionhave also been investigatedAspergillus niger canpro-duce invertase in the presence of 120573-fructofuranoside sugarsdemonstrating that the synthesis of invertase is inducible [10]This strain produces invertase with only hydrolytic activityproducing equimolar amounts of fructose and glucose Onthe other hand the FTase produced by this strain showsexclusively fructosyl transfer reaction producing glucose 1-kestose nystose and fructofuranosyl nystose [9] Aspergillusflavus produced high yields of invertase under optimizedconditions in submerged cultures [11] Guimaraes et al [12]observed that Aspergillus ochraceus produced high levelsof a thermostable extracellular invertase when cultured inKhanna medium supplemented with sugarcane bagasse ascarbon source

Enzyme production requires long time of laboratorialexperiment to find the ideal conditions and cost-effectiveness

for microbial cultivation Submerged and solid-state cultiva-tions are commonly employed to enzyme production usingalternative substrates as an attempt to reduce bottlenecksinvolved in the initial steps of production eg high costs ofproductionusing high value-added substratesTheutilizationof renewable resources such as agroindustrial residues ascheap and readily available substrates for enzyme productionhas been a subject of intense research as an alternative forcost-effective enzyme production and waste management[2] Agroindustrial residues are generated in large amountsevery year and their usage for bioprocesses is particularlyinteresting due to their renewability low-cost and suitablecharacteristics which allow the production of different value-added metabolites [13] Giraldo et al [14] used agroindustrialbyproducts eg sugarcane bagasse oat meal cassava flourcorn cob soy bran and wheat bran for invertase productionunder submerged cultivation Their experiments demon-strated that agroindustrial residues provided the productionof higher levels of extracellular invertase by Paecilomycesvariotii Guimaraes et al [12] observed that highest levels ofextracellular invertase activity were obtained when Khannamedium was supplemented by sugar cane bagasse as carbonsource and the extracellular invertase activity comprised98 total enzyme activity (intracellular + extracellular) fromcultivation Oyedeji et al [2] reported that agroindustrialresidues are suitable substrates for microbial cultivationand consequent production of molecule of biotechnologicalinterest such as enzymes and other value-added chemicals

The aims of this work were to select an invertase-producing Aspergillus spp strain with high fructosyltrans-ferase activity from canned peach syrups under submergedcultivations Nutritional parameters such as pure and com-plex carbon sources and nitrogen sources were evaluatedto obtain high yield of enzymes Agroindustrial residueswere also evaluated as alternative substrates for enzymeproduction

2 Material and Methods

21 Microorganisms Filamentous fungi were isolated froma jar surface containing canned peach syrup Canned peachsyrup was obtained from local market Samples of cannedpeach syrup were maintained at 30∘C for five days Coloniesgrown on the surface of syrup were transferred to Petriplates containing potato dextrose agar (PDA) and incubatedat 30∘C for five days Isolated colonies were inoculated atthe center of the Petri dishes to ensure the purity of fungistrains Filamentous fungi preservation was conducted insterile distilled water [15] Dishes (approximately 5mm)containing small portion of culture medium mycelium andspores were aseptically transferred into 6mL sterile antibioticflasks which were added to 4mL sterile distilled water andsealed with rubber stopper Castellani flasks were kept at 4∘Cand the viability of strains was verified periodically every sixmonths

Fungi were isolated as monocultures on two media maltextract agar and potato dextrose agar Fungi were identifiedto genus level according to morphological structures [16ndash18]

The Scientific World Journal 3

22 Inoculum Preparation Dishes containing mycelium andspores were aseptically inoculated in PDA plates and incu-bated at 30∘C for five days Filamentous fungi colonies weretransferred to PDA slants and incubated at 30∘C for fivedays The spore suspension was carried out using sterileNaCl 085 (wv) Culture media for screening of enzymeproduction were inoculated with 1mL of 106 sporesmLsuspension

23 Culture Conditions for Enzyme Production Filamentousfungi cultures were performed in Erlenmeyer flasks (125mL)containing 20mL of Vogel medium solution [19] A stockmedium 50-fold concentrated was prepared containing (gL)sodium citratelowast5H2O 150 KH2PO4 250 MgSO4lowast7H2O10 CaCl2lowast2H2O 5 trace elements solution 5mL stocksolution (gL) citric acidlowastH2O 50 ZnSO4lowast7H2O 50Fe(NH4)2(SO4)2lowast6H2O 10 CuSO4lowast5H2O 25 MnSO4lowastH2O 005 H3BO3 005 Na2MoO4lowast2H2O 005 biotin solu-tion (01mgmL) 5mL Chloroform (02mL) was addedas a preservative The solutions were maintained at 4∘CMedium preparation consisted of 50-fold dilution of stockmedium supplemented with carbon sources (1 wv) Yeastextract (02 wv) was used as nitrogen source The finalpH was adjusted to 60 Flasks containing culture mediawere sterilized in autoclave at 121∘C for 15min inoculatedwith spore suspension previously prepared and incubated inorbital shaker for 72 andor 120 hours at 28∘C 180 rpm

24 Crude Extract Preparation Biomass was separated fromfermentation broth by filtration (cellulose acetate membrane045120583m) and dried at 105∘C until constant weight Cell freebroth (crude extract) was used for enzyme activity assays

25 Bioprospecting of Filamentous Fungi Filamentous fungiwere screened for invertase production using Vogelrsquos mediumsupplemented with sucrose as sole carbon source (10 wv)and 02 (wv) yeast extract as nitrogen source The cultureswere carried out in Erlenmeyer flasks (125ml) containing20mLmedium and pHwas adjusted to 60The cultures wereincubated at 180 rpm and 28∘C for 120 hours All experimentswere performed in triplicate

26 Molecular Identification of Aspergillus sp PC-4 TotalDNA from filamentous fungal strains was extracted fromPDA growth medium using IllustraTM Nucleon PhytoPureplant and fungal DNA extraction kits (GE Healthcare Amer-sham England) DNA was quantified using NanoDrop 2000spectrophotometer (Thermo Scientific Uniscience Brazil)and then diluted to a concentration of 50 ng for polymerasechain reaction (PCR) using the primers ITS1 (51015840-TCCGTA-GGTGAACCTGCGG-31015840) and ITS4 (51015840-TCCTCCGCTTAT-TGATATGC-31015840) [20] Amplifications were performed onMastercycler nexus (Eppendorf) thermocycler using theGoTaq DNA Polymerase kit (Promega Madison-USA) withfinal reaction volume of 25120583LThereafter PCRproducts werechecked by 1 (w v) agarose gel electrophoresis [21] andvisualized under ultraviolet light on LPIX-EX photodocu-mentator (Loccus Biotechnology Sao Paulo Brazil) The 1 Kb

DNA Ladder (Promega Canada USA) was used as molecularweight marker The amplified products were purified withExonuclease I (ExoSap-IT) and Alkaline Phosphatase solu-tion (USB Corporation Austin-USA) followed by sequencingusing the same PCR primers on ABI 3500 xl automaticsequencer (Life Technologies) according to the Dideoxy orchain termination method [22] using the BigDye Terminatorv 31 sequencing kit (Life Technologies Foster city USA)Thisstage was carried out at the Laboratory of Polar Microbiologyand Tropical Connections of Federal University of MinasGerais (UFMG) and Laboratory of Cellular and Molec-ular Parasitology (LCPM) of Oswaldo Cruz Foundation-FiocruzRene Rachou Institute

27 Phylogenetic Analysis The sequence data were obtainedfrom NCBI Genbank database available under Aspergilluscarbonarius with accession no AJ876878 and were used forcomparisons with the 599 bp from our isolate Sequenceswith high query coverage and homology were selected forphylogenetic analysis using the neighbor-joining method[23] The sequences were aligned using software Clustal Wanalysis [24] and the phylogenetic analyses were performedusingMEGA 60 for neighbor-joining and bootstrap analysisSequence from this study was indicated in the tree bycollection code while GenBank sequences were indicated byaccession numbers

28 Effect of Pure and Complex Carbon Sources Pure carbonsources (fructose glucose raffinose cellulose cellobiose sor-bitol inulin sucrose maltose lactose and xylose) were usedas sole carbon sources Complex carbon sources (pineapplecrown sweet potato flour sugarcane bagasse corn cobcassava peel soybean peel soybean bran corn straw bacabapeel sorghum bran and wheat bran) were used as solecarbon sources which were previously washed with distilledwater dried until constant weight and sieved (18 mesh)The cultures were carried out in Erlenmeyer flasks (125ml)containing 20mL medium supplemented with 10 (wv)pure or complex carbon sources 02 (wv) yeast extract asnitrogen source and pH was adjusted to 60 The cultureswere incubated at 180 rpm and 28∘C for 72 h for invertaseproduction and 120 h for FTase production Cultures werecarried out in triplicate

29 Effect of Nitrogen Sources Nitrogen sources (ammo-nium nitrate ammonium chloride ammonium sulfate yeastextract peptone soybeanprotein and corn steep liquor)wereused as sole inorganic or organic sources The cultures wereprepared in Erlenmeyer flasks (125ml) containing 20mL ofmedium supplemented with 02 (wv) inorganic or organicsource individually 10 (wv) pineapple peel as carbonsource with pH adjusted to 60 The cultures were incubatedat 180 rpm and 28∘C for 72 h for invertase production and120 h for FTase production

210 Time-Course of Enzyme Production Time-course ofinvertase production was carried out in Erlenmeyer flasks(125mL) containing 20mL of Vogelrsquos medium supplemented


with 1 pineapple peel 02 yeast extract and pH 60The cultures were incubated at 180 rpm and 28∘C for 120 hSamples were withdrawing each 12 h of cultivation

Time-course of FTase production was carried out inErlenmeyer flasks (125mL) containing 20mL of Vogelrsquosmedium supplemented with 1 pineapple peel 02 ammo-nium chloride and pH adjusted to 60 The cultures wereincubated at 180 rpm 28∘C for 240 hours Samples wereperiodically analyzed at intervals of 12 h cultivation

211 Biomass Characterization Total organic carbon was car-ried out by Walkley-Black method [25] Total carbohydratewas determined by differences amongmoisture gray proteinand lipid [26] Total nitrogen was determined by micro-Kjeldahl procedures [27]

212 Enzyme Assays Invertase activity was assayed bymeasuring the amount of reducing sugars from sucroseEight hundred microliter of sucrose (2 wv) diluted inMcIlvaine buffer solution pH 50 were dispensed into testtube and kept at 50∘C for 5min Then 200120583L of crudeextract were added to this reaction medium The reactionwas stopped in different time intervals (0 5 and 10min)by taking 200120583L reaction medium and dispensed intoanother test tube containing 200120583L of 35-dinitrosalicylicacid Reducing sugars were estimated colorimetrically with35-dinitrosalicylic acid [28] using fructose as standardsOne unit of enzymatic activity is defined as the amount ofenzyme that releases 1120583mol of reducing sugars perminute permilliliter

FTase activity was determined by incubating 100 120583Lculture filtrate with 400 120583L sucrose (20 wv in 01Msodium acetate buffer pH 50) at 50∘C for 1 h The reac-tion was stopped by boiling the mixture at 100∘C for10min FTase activity was estimated by adding 10120583L ofappropriately diluted reaction to 1ml test reagent (glu-cose oxidasendashperoxidase GOPOD kit Sigma) The glucosereleased was measured at 510 nm One unit of FTase wasdefined as the amount of enzyme required to produce 1 120583molof glucose per minute under assayed conditions

213 Fermentation Parameters Enzyme yield (Y unit ofenzymeg of carbon source) is

119884(119875119878) =119875119891 minus 1198750

1198780(1)

where 119875119891 is total enzyme produced after fermentation 1198750 istotal enzyme in initial fermentation 1198780 is substrate concen-tration in initial fermentation

Productivity assay (P unit of enzymetime of cultivation)is

119875119875 =119864119905119905

(2)

where 119864119905 is total enzyme produced after fermentation t istime of the fermentation process

214 Statistical Analysis The results were expressed as meanplusmn standard error of the mean and subjected to analysis ofvariance (ANOVA) followed by Tukey test

3 Results and Discussion

Initially filamentous fungi were isolated from canned peachsyrup and cultivated in submerged cultures containingsucrose as sole carbon source (Table 1) Among six isolatesidentified as Aspergillus sp and Penicillin sp the PC-4 strainpresented the highest invertase production (247 UmL)Fermentation parameters also demonstrated that Aspergillussp PC-4 can be a promising invertase producer whoseproduction yield from sucrose was 22230 Ug and theproductivity was 037 Uh The highest biomass productionwas observed for PC-6 (602 gL) followed by Aspergillus spPC-1 PC-2 PC-4 and PC-5 (572 gL 556 gL 554 gL and537 gL respectively) Aspergillus genus has been highlightedas an invertase producer under both submerged and solid-state cultivations [29] Veana et al [3] related invertase pro-duction by xerophilic A niger under submerged cultivationamong eight strains evaluated only A niger GH1 produced86UmL invertase after 48 h cultivation In the present studyPC-5 and PC-1 strains produced respectively 196 and 186UmL invertase followed by PC-6 PC-2 (130 UmL 123UmL respectively)

Penicillium sp PC-3 showed invertase production of 106UmL and production yield of 9520 Ug and productivityof 016 Uh Penicillium genus was also reported in theliterature as invertase producer and its enzyme has potentialfor the production of inverted sugar and fructooligosaccha-rides Poonawalla et al [30] related that maximum invertaseproduction by Penicillium chrysogenum was intracellularand maximum extracellular invertase was reached after cellautolysis On the other hand Penicillium purpurogenumPenicillium pinophilum and Penicillium citrinum producedextracellular invertase under submerged conditions fromsucrose as carbon source [3] Invertase from Penicilliumexpansum was also produced under submerged conditionsusing cells immobilized in synthetic fibers and polyurethanefoam with fructooligosaccharides production being simul-taneously evaluated along enzyme production with fruc-tooligosaccharides production of 1203 and 1048 gL respec-tively and high invertase activity (2301 UmL and 3242UmL respectively) [31]

For the next step of this work Aspergillus sp PC-4 wasselected to parametric optimization of culture medium for120573-fructofuranosidase and 120573 ndashD-fructosyltransferase produc-tion under submerged conditions Although low invertaseproduction values have been obtained from other screenedfungi it was clear that these fungal strains are potentialenzyme producers for food industry and their cultivationparameters demand future investigation

31 Molecular Identification of Aspergillus sp PC-4 Mo-lecular identification revealed that the isolated strainsshowed 100 sequence similarity with Aspergillus carbonar-ius (GeneBank accession no AJ876878) (Figure 1) There-fore the isolated strain was referred to in this work as


Table1Filamentous

fung

istrains

morph

ologiesisolatedfro

mcann

edpeachsyrups

andinvertasep

rodu

ction

Filamentous

fung

iColon

ymorph

olog

yInvertasea

ctivity

Yps

PpBiom

ass

(Um

l)lowast(gL)

Aspergillus

spP

C-1

Colon

iesa

recompactyellowmycelium

whitewith

denselayer

ofblackconidiaheadsCon

idialh

eads

are

glob

osedark

brow

nCon

idiaareg

lobo

sedarkbrow

n18

6b16317

027

572

Aspergillus

spP

C-2

Colon

iesa

regranularw

hitemycelium

with

yello

w-green

conidiah

eadsC

onidialh

eads

arer

adiateC

onidiaare

glob

osepalegreen

123c

11090

018

556

Penicilliu

mspP

C-3

Colon

iesa

recompactw

hitemycelium

with

denselayer

ofgreenconidiaCon

idiaareh

yalin

eand

inlong

chain

glob

osegreenish

106c

9520

016

267

Aspergillus

spP

C-4

Colon

iesa


with

denselayer


idialh

eads

areg

lobo

se

dark

brow

nCon

idiaareg

lobo

sedarkbrow

n247

a22230

037

554

Aspergillus

spP

C-5

Colon

iesa

reflo

ccosewhite-yellowmyceliumw

ithbrow

nconidiah

eadsC

onidialh

eads

arer

adiateC

onidia

ares

spheric

alpaleb

rown

196b

1518

2025

537

Aspergillus

spP

C-6

Colon

iesa

recompactw

hite-yellowmycelium

with

denselayer

ofblackconidiah

eadsC

onidialh

eads

are

glob

osedark

brow

nCon

idiaareg

lobo

sedarkbrow

n13

0c9728

016

602

Cultu

recond

ition

sCu

ltivatio

nwe

recarriedou

tat28∘C

180r

pmfor7

2ho

urslowastMeanvalues

with

thes

amelette

rdono

tstatisticallydifferfrom

each

otherb

ytheANOVA

Tukeytest(p

=005)


Aspergillus_tubingensis_NR_131293_189

89

95

52

100

100

99

99

Aspergillus_tubingensis_GQ379912_1Aspergillus_niger_MH562047_1Aspergillus_niger_NR_111348_1

Aspergillus_heteromorphus_MF379647_1Aspergillus_heteromorphus_NR_106306_1

Aspergillus_ellipticus_NR_103605_1

Aspergillus_brasiliensis_NR_111414_1Aspergillus_brasiliensis_MH858557_1

Aspergillus_carbonarius_AJ876878_1Aspergillus_carbonarius_PC-4

Aspergillus_ellipticus_MH858677_1

Figure 1 The neighborjoining tree inferred from the partial sequence of the ITS region dataset using Mega 60 The bootstrap was 2000replications to assess the reliable level to the nods of the tree

0123456789

10

Pure carbon sources (1 wv)InvertaseFtase

Enzy

me a

ctiv

ity (U

mL)

Glu

cose

Fruc

tose

Xylo

se

Sucr

ose

Lact

ose

Cell

obio

se

Mal

tose

Raffi

nose

Inul

in

Cel

lulo

se

Gly

cero

l

Sorb

itol

Figure 2 Pure carbons sources on invertase and120573-D-fructosyltransferaseproduction by blackAspergillus sp PC-4 strain Culture conditionscultivation was carried out at 28∘C 180 rpm for 120 hours

Aspergillus carbonarius PC-4 A carbonarius strains havebeen extensively studied for ochratoxin A production infruits and beverages [32ndash34] Regarding enzyme productionA carbonarius have been studied for cell plant polysac-charides degradation [35] polygalacturonase and pectinaseproduction [36ndash38] and also 120573-Xylosidase [39] among otherbiotechnological studies To our knowledge in this study Acarbonarius PC-4 strain was first time reported to produceinvertase and fructosyltransferase under cost-effective fer-mentation media

32 Effect of Pure Carbon Sources on Invertase and FTaseProduction In order to determine the regulatory mechanismof invertase and FTase synthesis by A carbonarius PC-4several carbohydrates besides glycerol and sorbitol wereused as sole carbon sources in the cultures (Figure 2) Inthese experiments mono- di- and poly-carbohydrate andalcohol were evaluated as energy and inducing carbon sourceto distinguish the production mechanism and secretion ofenzymes Inducible enzymes are produced in the presence ofa potential substrate which stimulates the enzyme productionand secretion whereas the constitutive enzymes can be

produced in the absence of inducing substrate and thiscarbon source can be used as adjunct to increase the enzymeproduction

The highest invertase production was observed fromsucrose (265 UmL) whereas highest FTase production wasobtained from glucose (865 UmL) Glucose and raffinosewere intermediary sources for invertase production (185UmL and 180 UmL respectively) Sucrose and raffinosewere also intermediary sources to FTase production (660UmL and 307 UmL respectively) The lowest enzymeproduction values were observed using fructose lactosecellobiose maltose inulin cellulose glycerol and sorbitol

Fermentation parameters showed highest invertase yieldof 2650 Ug and productivity of 073 Uh from sucrosefollowed by glucose (119884119875119878 = 1850 Ug 119875119875 = 051 Uh) andraffinose (119884119875119878 = 1800 Ug 119875119875 = 050 Uh) RegardingFTase production the highest fermentation parameter valueswere observed from glucose (119884119875119878 = 8650 Ug 119875119875 = 240Uh) followed by sucrose (119884119875119878 = 6600 Ug 119875119875 = 183Uh) (Table 2) Biomass produced by filamentous fungifrom pure carbon source ranged from 381 gL to 672 gLwith sucrose and glucose resulting in 631 gL and 641 gL


Table 2 Fermentation parameters for invertase and FTase production by A carbonarius PC-4 on pure carbon sources

Pure Carbon SourcesFermentation Parameters

Invertase Fructosyltransferase BiomassYps Pp Yps Pp (gL)

Glucose 4775 008 9840 016 561Frutose 993 002 2999 005 527Xylose 1091 002 3924 007 602Sucrose 1747 003 3199 005 679Lactose 1327 002 1236 002 630Cellobiose 1280 002 4598 008 621Maltose 1911 003 8710 015 627Raffinose 10051 016 14570 024 451Inulin 7597 013 14334 024 668Cellulose 2380 004 984 002 951Glycerol 2737 004 6583 011 364Sorbitol 1459 002 4598 008 656Culture conditions Cultivation were carried out at 28∘C 180 rpm 72 hours

biomass respectively Raffinose was observed as pure carbonsources for invertase production using Paecilomyces variotii[14] Aspergillus nidulans and Aspergillus fumigatus [40]Rezende and Felix [40] related that raffinose as well asother oligosaccharides may be hydrolyzed by 120572-galactosidaseor invertase (120573-fructofuranosidase) which cleaves the 120572-12linkage of sucrose moiety producing melibiose and fructoseVainstein and Peberdy [41] observed that invertase activitieswere found inA nidulans cultures using sucrose and raffinoseas the best inducers and raffinose seemed to be the bestcarbon source for invertase secretion

Our results suggest that A carbonarius PC-4 producesa constitutive enzyme using a wide variety of carbohydratesand glycerol alcohol Vainstein and Peberdy [41] related thatthe A nidulans produced a constitutive level of invertase ina wide variety of carbohydrates with 3 glucose resulting inthe lowest activity while 1 raffinose and sucrose providedhighest activities In this study maltose melibiose glyceroland trehalose (1 wv) also induced invertase productionat similar values Rubio and Navarro [10] evaluated theregulation of invertase production by Aspergillus niger strainisolated from rotten lemons The highest invertase produc-tions were observed from inulin raffinose turanose andsucroseThe authors suggest that inducing sugars of invertasesynthesis exhibit the 120573 linkage and fructose located at theend of molecule as a common feature of their structure The120573 linkage does not cause induction of enzyme productionas shown with cellobiose and gentiobiose whereas the 120572linkage has not effect at all as demonstrated with maltoseandmelibiose On the other hand glucose and fructose failedto induce the invertase synthesis a fact that suggests thesecarbohydrates are not involved in the mechanism of invertasesynthesis induction for A niger strain

Glucose has extensively been related as repressor ofinvertase production by many filamentous fungi [14 4243] Glucose supplementation (10 wv) was the secondcarbon source which induced more efficiently the invertaseproduction by A carbonarius PC-4 (Figure 2 and Table 2)

Alegre et al [44] observed an intracellular invertase level 5-fold higher from 1 glucose than the absence of this carbonsource in cultivations of Aspergillus caespitosus Howeverall glucose concentrations were tested with exception of10 and 20 glucose (wv) the extracellular activity washigher than the intracellular one Giraldo et al [14] reportedthe effect of glucose or fructose addition to the culturemedia containing soy bran as main carbon source over intra-and extracellular invertase production by P variotii undersubmerged fermentation

33 Effect of Complex Carbon Sources on Invertase and FTaseProduction Themajor bottleneck in enzyme production andits industrial application is the relative high production costand low enzyme yields The use of agroindustrial residuesas raw materials decreases the impact on environment sincethe reuse of waste materials can reduce their accumulation inthe environment with additional economic value due to theirbiotechnological application [45] Besides agroindustrialresidues can be used as carbon sources by several microor-ganisms to produce high-value added chemicals such as car-bohydrases including invertases and fructosyltransferasesRecent studies have focused on the use of agroindustrialresidues for industrial enzyme production since it is one ofthe possible ways to reduce the production costs significantly[46]

In this study eleven agroindustrial residues were usedas carbon sources for invertase and FTase production byA carbonarius PC-4 (Figure 3) The invertase and FTaseproduction presented significance (plt005) when pineapplecrown was used as carbons sources The highest invertaseactivity (671 UmL) was observed after 3 days of cultivationand highest FTase activity was observed after 5 days ofcultivation (1460 UmL) representing 145-fold higher thanvalues obtained from other tested carbon sources

Fermentation parameters showed the highest values forinvertase production (119884119901119904 = 58697 Ug of substrate and119875119901 =


0123456789

10

Complex carbon sources (1 wv)72 hours120 hours

A

BC

G

D D

GE

F F G

a

b bcc

d

g

de e

g

f

gInve

rtas

e act

ivity

(Um

L)

pine

appl

e cro

wn

swee

t pot

ato

flour

suga

rcan

e bag

asse

corn

cob

cass

ava p

ee1

soyb

ean

peel

soyb

ean

bran

corn

stra

w

baca

ba p

eel

sorg

hum

bra

n

whe

at b

ran

(a)

05

101520253035


a

e

b

g

cd c

g

c

f f

d

AB

CCD

HF

G

D D

IF

Ftas

e act

ivity

(Um

L)

pine

appl

e cro

wn

swee

t pot

ato

flour

suga

rcan

e bag

asse

corn

cob

cass

ava p

ee1

soyb

ean

peel

soyb

ean

bran

corn

stra

w

baca

ba p

eel

sorg

hum

bra

n

whe

at b

ran

(b)

Figure 3 Complex carbon sources on invertase (a) and 120573-D-fructosyltransferase (b) by black Aspergillus sp PC-4 Culture conditionscomplex carbon sources (10 wv) yeast extract (02 wv) 28∘C 180 rpm Mean values with the same letter do not statistically differfrom each other by the ANOVA Tukey test (p = 005)

098 Uh) and FTase (119884119901119904 = 145900 Ug of substrate and 119875119901= 194 Uh) after 3 and 5 days of cultivations respectivelyusing pineapple crown as carbon source (Table 3) Oyedejiet al [2] observed invertase production of 2146 UmL after120 h incubation using pineapple peel as substrate Theseauthors stated that the increased invertase production mightbe due to utilization of pineapple peel by A niger IBK1 forgrowth and hence enzyme production However decreasingvalues of enzyme production could be due to exhaustion ofglucose a primary metabolite obtained in the degradationof substrate which is required for growth or increase ofmetabolic intermediates secreted into fermentation medium

In this study pineapple crownwas chemical characterizedpresenting 286 total carbon 18 total nitrogen with aCN ratio of 157 77 humidity 60 ashes 67 totallipids t115 total protein and 681 total carbohydratesPineapple wastes were also used to produce other carbohy-drate hydrolases such as 120573-glucosidase [46] and cellulases[47] Pineapple peel is a byproduct resulted from canningprocessing of pineapple which produces about 35 fruitwaste and lead to serious environmental pollutionThis wastecontains considerable amounts of soluble sugars such assucrose whichmakes it suitable as substrate for microbial fer-mentations [2 48]Therefore the pineapple peel compositionis an attractive alternative for fungal growth due to its lowcost and availability of carbon and nitrogen sources besideminerals Agroindustrial residues such as pineapple peel aretherefore suitable substrates for microbial cultivation andproduction of molecules with biotechnological importancesuch as enzymes and other value-added products [49] Onthe other hand pineapple crown is firstly related in this studyas a potential carbon source substrate for enzyme productionand to obtain value-added products Pineapple crown usedfor invertase and FTase production presented centesimal

composition as follows () moisture 770 plusmn 075 lipids 670plusmn 132 proteins 1148 plusmn 022 carbohydrates 6810 plusmn 103ashes 605 plusmn 007 carbon 2865 plusmn 018 and nitrogen 184 plusmn003

Intermediary values of invertase production were foundwith soybean peel and corn straw (439UmL and 424UmLrespectively) after 3 days of cultivation and sweet potato flour(435 UmL) after 4 days of cultivation Sugarcane bagassecassava peel and bacaba peel can be considered substrateswith potential use for invertase production after optimizationprocess of culture conditions since these substrates inducedthe enzyme production ranging from 25 to 322 UmLwhereas wheat bran soybean bran sorghum bran and corncob were weak inducers for invertase production by black Acarbonarius PC-4 On the other hand intermediary values ofFTase production were observed from cassava peel soybeanpeel corn straw sugarcane bagasse sweet potato and wheatbran (640 to 1165 UmL) and low production was observedfrom corn cob soybean bran bacaba peel and sorghumbran The fermentation parameters using sweet potato floursugarcane bagasse corn cob and wheat bran can indicatethat these substrates would be potential carbon sources forinvertase and FTase production under optimized conditionsor used as mixed substrates in fermentation processesGuimaraes et al [12] stated that many enzymes of industrialsignificance are regulated by carbon source composition andthe use of agroindustrial residues for extracellular invertaseproduction by Aspergillus ochraceus using sugarcane bagasseand corn cob was effective in promoting the production ofextracellular invertase substantially lowering the productioncosts Giraldo et al [14] have reported the promising uti-lization of liquid medium supplemented with agroindustrialwastes as carbon source especially soy bran or wheat branresulting in higher extracellular activity of invertase produced


Table3Ferm

entatio

nparametersfor

invertasea

nd120573-D

-fructosyltransfe

raseprod

uctio

nby

blackAspergillus

spP

C-4on

complex

carbon

sources

Com

ponent

Ferm

entatio

nparameters

72ho

urs

120ho

urs

Invertase

FTase

Biom

ass

Invertase

FTase

Biom

ass

Yps

PpYp

sPp

Yps

PpYp

sPp

Com

plex

carbon

sources(

10w

v)

Pineapplec

rown

58697

098

133843

223

670

57416

096

145900

194

788

Sweetp

otatoflo

ur35280

059

3314

3055

524

40221

067

73035

121

576

Sugarcaneb

agasse

3813

8064

1240

01

210

910

1079

2018

7310

012

199

3Corncob

26324

044

nd

nd

904

1870

3031

72390

120

908

Cassava

peel

1319

8022

40272

142

678

3310

006

24832

041

634

Soybeanpeel

2612

004

1041

002

967

2715

005

10276

006

1057

Soybeanbran

27352

046

nd

nd

561

31226

052

47618

079

521

Bacaba

peel

840

001

582

027

980

2151

004

nd

nd

999

Cornstr

aw2268

004

2476

004

1098

1386

002

4062

006

1163

Sorghu

mbran

2291

3038

28202

050

571

3791

006

25240

042

648

Wheatbram

40575

068

5177

1086

647

17885

030

67015

111

678

Nitr

ogen

sources(

02

wv)

Ammon

ium

nitrate

34082

094

108247

300

772

2316

7064

240

786

400

576

Ammon

ium

choride

49325

137

209681

582

790

5292

814

72114

51

352

600

Ammoinium

sulfate

51457

143

135937

377

824

41471

116

200

623

334

620

Yeastextract

53685

149

117666

327

833

39428

109

179910

300

640

Pepton

e42617

118

132282

367

853

39874

111

184956

310

592

Soybeanprotein

3296

8091

135120

375

848

25636

071

26279

3438

850

Cornste

epliq

uor

28717

098

111773

310

770

24058

067

184704

308

562

Cultu

recond

ition

sCu

ltivatio

nwe

recarriedou

tat28∘Cfor180

rpm


0123456789

10

Inve

rtas

e act

ivity

(Um

L)

Nitrogen sources (02 wv)72 hours120 hours

aab

FG

de

ef fg

ABA

C

DE

FA

mm

oniu

m n

itrat

e

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

(a)


a a aab abc

bc

d

AABBC

CDEF DE DE

Am

mon

ium

nitr

ate

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

05

101520253035

FTas

e act

ivity

(Um

L)

(b)

Figure 4 Organic and inorganic nitrogen sources on invertase (a) and 120573-D-fructosyltransferase (b) production by A carbonarius PC-4Culture conditions pineapple crown (10 wv) nitrogen sources (02 wv) 28∘C 180 rpm Mean values with the same letter do notstatistically differ from each other by the ANOVA Tukey test (p = 005)

by Paecilomyces variotii which was 7 and 46 times higherfrom soy bran than those estimated for sucrose and glucoserespectively

34 Effect of Nitrogen Sources on Invertase and FTase Pro-duction Nitrogen sources have an important role in fer-mentation processes in which the microbial growth andenzyme secretion are directly affected by nitrogen com-position Organic and inorganic nitrogen sources play animportant role in the synthesis of enzymes since inorganicnitrogen sources can be used quickly and organic sources cansupply many cell growth factors and amino acids needed forcell metabolism and enzyme production [50] The invertaseand FTase production by A carbonarius PC-4 using differ-ent nitrogen sources such as ammonium ions (ammoniumnitrate ammonium chloride and ammonium sulfate) yeastextract peptone soybean protein and corn steep liquor afterthree and five days of cultivation can be observed in Figure 4Highest invertase activities were observed from ammoniumchloride and yeast extract (640 UmL and 613 UmLrespectively) which did not present differences in analysisof variance (pgt005) followed by ammonium sulfate andpeptone (590 UmL and 550 UmL respectively) after threedays of cultivation Ammonium nitrate soybean protein andcorn steep liquor induced the lowest invertase productionAfter five days of cultivation the invertase activity decreasedfrom all evaluated nitrogen sources indicating a microbialgrowth suppression caused by exhaustion of nutrient in theculture medium

Fermentation parameters for invertase productionshowed highest yield and productivity from yeast extract(119884119875119878 = 53685 Ug 119875P = 149 Uh) followed by ammoniumsulfate (119884119875119878 = 51457 Ug 119875119875 = 137 Uh) after three daysof cultivation (Table 3) The maximum biomass production

was observed after three days of cultivation using organic orinorganic nitrogen sources (770 ndash853 gL) After five daysof cultivation biomass production decreased though FTaseproduction was observed suggesting an extracellular enzymeproduction which was secreted to the medium (Figure 3)Giraldo et al [14] also observed the influence of organic andinorganic nitrogen sources on enzyme production Maximalproduction of extracellular invertase was obtained whenpeptonewas added to the culturemedium while peptone andammonium phosphate were both the best nitrogen sourcesfor the intracellular form Vandakova et al [6] observedsimilar results using yeast extract as nitrogen source Theseauthors related that the advantage of using yeast extractcompared to inorganic nitrogen substrates possibly is basedon supplementation of some essential growth factors for themicroorganism

FTase productionwas not statistically significant (pgt005)for enzyme activity using inorganic and organic nitrogensources although the highest activity was observed withsoybean protein and ammonium nitrate (2490 UmL and2400 UmL respectively) after five days of cultivationFermentation parameters for FTase production under theseevaluated conditions were 119884119875119878 = 262793 Ug and 119875119875 = 440Uh for soybean protein and 119884119875119878 = 240786 Ug and 119875119875 =400 Uh for ammonium nitrate Ammonium chloride wasalso a promising nitrogen source for enzyme productionfrom which were obtained high invertase production value(2140 UmL) and significant fermentation parameters ofyield and productivity (119884119875119878 = 211451 Ug and 119875119875 = 312Uh respectively)The lowest FTase productionwas observedusing yeast extract (1780 UmL 119884119875119878 = 179910 Ug and119875119875 = 300 Uh respectively) FTase production after threedays of cultivation showed similar activities among cornsteep liquor peptone soybean protein ammonium sulfate


12 120967260483624Time-course of cultivation (hours)

0

2

4

6

8

10

12

14

Inve

rtas

e act

ivity

(Um

L)

(a)

06

121824303642485460

24 240216192168144120967248

FTas

e act

ivity

(Um

l)

Time-course of cultivation (hours)

(b)

0500

10001500200025003000350040004500

24 48 72 96 120 144 168 192 216 240

Yiel

d (U

g o

f sub

stra

te)

Time-course (hours)

Invertase(c)

0

1

2

3

4

5

6

7

24 48 72 96 120 144 168 192 216 240

Prod

uctiv

ity (U

gh

)

Time-course (hours)

Invertase(d)

Figure 5 Time-course and fermentation parameters of invertase and 120573-D-fructosyltransferase (FTase) produced by 119860 carbonarius PC-4Legend (a) invertase production (b) FTase production (c) yield of production for invertase and FTase (d) productivity for invertase andFTase Culture conditions invertase productionwas carried out using pineapple crown (10wv) yeast extract (02wv) FTase productionwas carried out using pineapple crown (10 wv) and soybean protein (02 wv) cultures were carried out at 28∘C pH 60 and 180 rpm

yeast extract and ammonium chloride ranging from 1120to 1520 UmL except for ammonium nitrate from whichwas observed to have lower FTase activity of 780 UmLUnder these conditions productivity after three days ofcultivations showed higher enzyme activity values comparedto those obtained after five days indicating which the cultureparameters should be optimized to improve the enzymeproduction

35 Time-Course of Invertase and FTase Production Time-course of invertase and FTase production by A carbonariusPC-4 were carried out using the culture conditions estab-lished previously using the carbon and nitrogen sourcesselected (Figure 5) Invertase production was carried outusing pineapple crown (10 wv) as carbon source andammonium chloride (02 wv) as nitrogen source whileFTase production was carried out using pineapple crown(10 wv) as carbon source and soybean protein (02 wv)as nitrogen source Both cultivations were carried out at180 rpm and 28∘C during 120 h and 240 h respectively Max-imum invertase production was observed among 60 h and96 h of cultivation (1100 and 1252 UmL respectively) with

the highest production after 96 h (1252 UmL) (Figure 4(a))Under these culture conditions fermentation parametersshowed maximum yield after 72 h with 107390 Ug andproductivity of 630 Uh after 24 h (Figures 4(c) and 4(d))For FTase the maximum production was observed after168 h (4440 UmL) (Figure 4(b)) Fermentation parame-ters showed also maximum yield and productivity (119884119875119878= 598271 Ug and 119875119875 = 1000 Uh respectively) after168 h

4 Concluding Remarks

The screening of filamentous fungi from canned peach syruppresented potential Aspergillus spp for invertase productionunder submerged fermentation Among six potential isolatesblack Aspergillus sp PC-4 strain was selected to invertase andFTase production This strain was identified as Aspergilluscarbonarius PC-4 To our knowledge it is the first time thatA carbonarius strain is reported as an invertase and FTaseproducer in the literature Cultivation using pure carbonsources showed that this strain produces invertase in a con-stitutive way and FTase was inducible Agroindustrial wastes


were used as complex carbon sources and the pineapplecrown was a promising substrate for invertase and FTaseproduction Furthermore pineapple crown is firstly related inthis study as carbon source for enzyme production Nitrogensources showed that organic and inorganic sources can beused to both enzymes production though the ammoniumchloride and yeast extract were the best nitrogen sourcesfor invertase production (sim66 UmL) after three days ofcultivation while soybean protein and ammonium nitratewere the best sources for FTase production (sim24 UmL) afterfive days of cultivation Time-course of enzyme productionshowed an increase of 510-fold in the invertase productioncompared to initial experiments (screening of fungi) andFTase production increased 30-fold after 196 h of cultivationcompared to complex carbon sources (1460 UmL) Thisstudy showed that A carbonarius PC-4 is a promising fungalstrain for invertase and FTase production considering cost-benefit conditions and the enzymes produced are suitable forfood and nutraceuticals industries in order to obtain invertedsugar fructose syrups and fructooligosaccharides

Data Availability

The datasets analysed during the current study are notpublicly available due to continuing of the doctoral thesis butare available from the corresponding author on reasonablerequest

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

Alex Fernando de Almeida thanks the Federal Universityof Tocantins by Productivity grant (Edital 312015) andCoordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior Brasil (CAPES) for the scholarship awarded to RyharaDias Batista

References

[1] T Chand Bhalla Bansuli N Thakur Savitri and N ThakurldquoInvertase of Saccharomyces cerevisiae SAA-612 Produc-tion characterization and application in synthesis of fructo-oligosaccharidesrdquo LWT- Food Science and Technology vol 77pp 178ndash185 2017

[2] O Oyedeji M K Bakare I O Adewale P O Olutiola andO O Omoboye ldquoOptimized production and characterizationof thermostable invertase from Aspergillus niger IBK1 usingpineapple peel as alternate substraterdquo Biocatalysis and Agricul-tural Biotechnology vol 9 pp 218ndash223 2017

[3] F Veana C N Aguilar and R R Herrera ldquoKinetic studies ofinvertase production by xerophilic Aspergillus and Penicilliumstrains under submerged culturerdquo Micologia Aplcada Interna-cional vol 23 no 2 pp 37ndash45 2011

[4] H Nadeem M H Rashid M H Siddique et al ldquoMicrobialinvertases A review on kinetics thermodynamics physio-chemical propertiesrdquo Process Biochemistry vol 50 no 8 pp1202ndash1210 2015

[5] E Akardere B Ozer E B Celem and S Onal ldquoThree-phasepartitioning of invertase from Bakerrsquos yeastrdquo Separation andPurification Technology vol 72 no 3 pp 335ndash339 2010

[6] M Vandakova Z Platkova M Antosova V Bales and MPolakovic ldquoOptimization of cultivation conditions for pro-duction of fructosyltransferase by Aureobasidium pullulansrdquoChemical Papers vol 58 no 1 pp 15ndash22 2004

[7] M A Ganaie U S Gupta and N Kango ldquoScreening of biocat-alysts for transformation of sucrose to fructooligosaccharidesrdquoJournal of Molecular Catalysis B Enzymatic vol 97 pp 12ndash172013

[8] D C Khandekar T Palai A Agarwal and P K BhattacharyaldquoKinetics of sucrose conversion to fructo-oligosaccharidesusing enzyme (invertase) under free conditionrdquo Bioprocess andBiosystems Engineering vol 37 no 12 pp 2529ndash2537 2014

[9] L LrsquoHocine Z Wang B Jiang and S Xu ldquoPurification andpartial characterization of fructosyltransferase and invertasefrom Aspergillus niger AS0023rdquo Journal of Biotechnology vol81 no 1 pp 73ndash84 2000

[10] M C Rubio and A R Navarro ldquoRegulation of invertase syn-thesis in Aspergillus nigerrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 601ndash606 2006

[11] C Uma D Gomathi CMuthulakshmi and V K Gopalakrish-nan ldquoProduction purification and characterizationof invertaseby Aspergillus flavus using fruit peel waste as substraterdquoAdvances in Biological Research vol 4 no 1 pp 31ndash36 2010

[12] L H S Guimaraes H F Terenzi M D L T D M Polizeli andJ A Jorge ldquoProduction and characterization of a thermostableextracellular 120573-d-fructofuranosidase produced by Aspergillusochraceus with agroindustrial residues as carbon sourcesrdquoEnzyme andMicrobial Technology vol 42 no 1 pp 52ndash57 2007

[13] D De Freitas Borghi R Guirardello and L C Filho ldquoStoragelogistics of fruits and vegetables Effect of temperaturerdquoChem-ical Engineering Transactions vol 17 pp 951ndash956 2009

[14] M A Giraldo T M da Silva F Salvato H F Terenzi J AJorge and L H S Guimaraes ldquoThermostable invertases fromPaecylomyces variotii produced under submerged and solid-state fermentation using agroindustrial residuesrdquoWorld Journalof Microbiology and Biotechnology vol 28 no 2 pp 463ndash4722012

[15] A Castellani ldquoMaintenace and cultivation of the commonpathogenic fungi of man in sterile distilled waterrdquo Journal ofTropical Medicine Hygine vol 70 pp 181ndash184 1967

[16] C R Benjamin K B Raper and D I Fennell The GenusAspergillus vol 58 Robert Erieger Publishing CompanyHuntimgton New York NY USA 1977

[17] K A Seifert ldquoCompendium of Soil Fungi - by KH DomschW Gams amp T-H Andersonrdquo European Journal of Soil Science2009

[18] J I Pitt The Genus Penicillium Academic Press London UK1979

[19] H J Vogel ldquoA convenient growth medium for Neurospora(Medium N)rdquo Microbial Genetics Bulletin vol 13 pp 42-431956

[20] M A Innis D H Gelfand J J Snlnsky and T J White PCRprotocols a guide to methods and applications Academic PressNew York NY USA 1990


[21] J Sambrook P Maccallum and D RusselMolecular cloning alaboratory manual Cold Spring Harbor Laboratory Press USA

[22] F Sanger S Nicklen and A R Coulson ldquoDNA sequencingwith chain-terminating inhibitorsrdquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 74 no12 pp 5463ndash5467 1977

[23] Q-Z Lou M Lu and J-H Sun ldquoYeast Diversity Associatedwith Invasive Dendroctonus valens Killing Pinus tabuliformisin China Using Culturing and Molecular Methodsrdquo MicrobialEcology vol 68 no 2 pp 397ndash415 2014

[24] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weightmatrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[25] A Walkley ldquoA critical examination of a rapid method fordetermining organic carbon in soilsmdasheffect of variations indigestion conditions and of inorganic soil constituentsrdquo SoilScience vol 63 no 4 pp 251ndash264 1947

[26] Association of Official Analytical Chemists (AOAC) OfficialMethods of Analysis S Williams Ed Association of OfficialAnalytical Chemists 15th edition 2005

[27] C A Lang ldquoSimple Microdetermination of Kjeldahl Nitrogenin Biological MaterialsrdquoAnalytical Chemistry vol 30 no 10 pp1692ndash1694 1958

[28] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959

[29] S J Romero-Gomez C Augur and G Viniegra-GonzalezldquoInvertase production by Aspergillus niger in submerged andsolid-state fermentationrdquo Biotechnology Letters vol 22 no 15pp 1255ndash1258 2000

[30] F M Poonawalla K L Patel and M R Iyengar ldquoInvertaseproduction by Penicillium chrysogenum and other fungi insubmerged fermentationrdquo Journal of Applied Microbiology vol13 no 5 pp 749ndash754 1965

[31] S I Mussatto M B Prata L R Rodrigues and J ATeixeira ldquoProduction of fructooligosaccharides and 120573-fructofuranosidase by batch and repeated batch fermentationwith immobilized cells of Penicillium expansumrdquo EuropeanFood Research and Technology vol 235 no 1 pp 13ndash22 2012

[32] C Jiang J Shi Y Cheng and Y Liu ldquoEffect of Aspergilluscarbonarius amounts on winemaking and ochratoxin A con-taminationrdquo Food Control vol 40 no 1 pp 85ndash92 2014

[33] I K Lappa S Mparampouti B Lanza and E Z PanagouldquoControl of Aspergillus carbonarius in grape berries by Lac-tobacillus plantarum A phenotypic and gene transcriptionstudyrdquo International Journal of Food Microbiology vol 275 pp56ndash65 2018

[34] L Freire T M Guerreiro E T Carames et al ldquoInfluence ofmaturation stages in different varieties of wine grapes (Vitisvinifera) on the production of ochratoxin a and its modifiedforms by Aspergillus carbonarius and Aspergillus nigerrdquo Journalof Agricultural and Food Chemistry vol 66 no 33 pp 8824ndash8831 2018

[35] R P de Vries and J Visser ldquoAspergillus enzymes involved indegradation of plant cell wall polysaccharidesrdquo MicrobiologyandMolecular Biology Reviews vol 65 no 4 pp 497ndash522 2001

[36] N Anjana Devi and A G Appu Rao ldquoFractionation purifi-cation and preliminary characterization of polygalacturonasesproduced by Aspergillus carbonariusrdquo Enzyme and MicrobialTechnology vol 18 no 1 pp 59ndash65 1996

[37] E Nakkeeran S Umesh-Kumar and R Subramanian ldquoAs-pergillus carbonarius polygalacturonases purified by integratedmembrane process and affinity precipitation for apple juiceproductionrdquo Bioresource Technology vol 102 no 3 pp 3293ndash3297 2011

[38] S A Singh M Ramakrishna and A G Appu Rao ldquoOptimisa-tion of downstream processing parameters for the recovery ofpectinase from the fermented bran of Aspergillus carbonariusrdquoProcess Biochemistry vol 35 no 3-4 pp 411ndash417 1999

[39] T Kiss and L Kiss ldquoPurification and characterization of anextracellular 120573-D-xylosidase from Aspergillus carbonariusrdquoWorld Journal of Microbiology and Biotechnology vol 16 no 5pp 465ndash470 2000

[40] S T de Rezende and C R Felix ldquoRaffinose-hydrolyzing activityofAspergillus fumigatusrdquo Biotechnology Letters vol 19 no 3 pp217ndash220 1997

[41] M H Vainstein and J F Peberdy ldquoRegulation of invertase inAspergillus nidulans Effect of different carbon sourcesrdquo Journalof General Microbiology vol 137 no 2 pp 315ndash321 1991

[42] L H S Guimaraes A F Somera H F TerenziM D L T DMPolizeli and J A Jorge ldquoProduction of 120573-fructofuranosidasesby Aspergillus niveus using agroindustrial residues as carbonsources Characterization of an intracellular enzyme accumu-lated in the presence of glucoserdquo Process Biochemistry vol 44no 2 pp 237ndash241 2009

[43] J N Alves J A Jorge and L H Guimaraes ldquoProduction ofInvertases by Anamorphic (Aspergillus nidulans) and Teleo-morphic (Emericela nidulans) Fungi under Submerged Fer-mentation Using Rye Flour as Carbon Sourcerdquo Advances inMicrobiology vol 03 no 05 pp 421ndash429 2013

[44] A C P Alegre M D L T D M Polizeli H F Terenzi JA Jorge and L H S Guimaraes ldquoProduction of thermostableinvertases by Aspergillus caespitosus under submerged or solidstate fermentation using agroindustrial residues as carbonsourcerdquoBrazilian Journal ofMicrobiology vol 40 no 3 pp 612ndash622 2009

[45] L R D S Moreira G V Ferreira S S T Santos A P SRibeiro F G Siqueira and E X F Filho ldquoThe hydrolysis ofagro-industrial residues by holocellulose-degrading enzymesrdquoBrazilian Journal of Microbiology vol 43 no 2 pp 498ndash5052012

[46] J M de Almeida V A de Lima P C G de Lima and AKnob ldquoEffective and Low-Cost Saccharification of PineapplePeel by Trichoderma viride Crude Extract with Enhanced 120573-Glucosidase Activityrdquo Bioenergy Research vol 9 no 3 pp 701ndash710 2016

[47] P Saravanan R Muthuvelayudham and T Viruthagiri ldquoEn-hanced Production of Cellulase from Pineapple Waste byResponse SurfaceMethodologyrdquo Journal of Engineering (UnitedStates) vol 2013 2013

[48] A M Siti Roha S Zainal A Noriham and K Z NadzirahldquoDetermination of sugar content in pineapple waste varietyN36rdquo International Food Research Journal vol 20 no 4 pp1941ndash1943 2013

[49] E Rosales S Rodrıguez Couto and M A Sanroman ldquoIn-creased laccase production by Trametes hirsuta grown ongroundorange peelingsrdquoEnzyme andMicrobial Technology vol40 no 5 pp 1286ndash1290 2007

[50] T Tan M Zhang J Xu and J Zhang ldquoOptimization ofculture conditions and properties of lipase from Penicilliumcamembertii Thom PG-3rdquo Process Biochemistry vol 39 no 11pp 1495ndash1502 2004

Hindawiwwwhindawicom

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polarized light to right while the solution of fructose isstrongly levorotatory ie causing rotation of plane polar-ized light to left [4] Invertases are widely used in foodand beverage industries One of important applications ofinvertase is the production of noncrystallizable sugar syrup(invert sugar syrup) from sucrose Invert sugar is used as ahumectant in the manufacture of soft candies and fondants[5] Invertase can be also used when sucrose containingsubstrates are subjected to fermentation viz production ofalcoholic beverages lactic acid and glycerol Furthermoreinvertases are also used for the manufacture of artificialhoney plasticizing agents and fructooligosaccharides [5]

Fructosyltransferase (FTase EC 2419) hydrolyzes su-crose and transfer fructosyl group to a sucrose moleculeacceptor to generate fructooligosaccharides along with glu-cose and fructose which have a wide range of applicationsin food and pharmaceutical industries [6 7] Fructooligosac-charides can be obtained synthetically from agave fructansby acid-catalyzed hydrolysis although the formation offructooligosaccharides via enzymatic methods is preferreddue to high substrate specificity and selectivity of enzymescompared to chemical routes [8] Fructooligosaccharidesfrom sucrose are considered as new alternative sweetenerswith functional properties also called soluble fibres withseveral desirable characteristics such as low calories non-cariogenicity and safety for diabetics Fructooligosaccharidesare also known as prebiotics since they stimulate the growthof probiotic organisms [1] However fructosyltransferase andfructofuranosidase production is rather confusing differingfrom one source to another from one microorganism toanother and even from one strain to another [9] In additioninvertases exhibit transfructosylating activity at high sucroseconcentrations in order to release glucose and fructose fromsucrose Microbial invertases may catalyze the synthesis ofshort-chain fructooligosaccharides such as 1-kestose nystoseand fructofuranosyl nystose in which one to three fructosylmoieties are linked to the sucrose by different glycosidicbonds depending on the enzyme source [4 5]

Invertases can be found by wide range of organismsincluding plants animal bacteria yeasts and filamentousfungi Among filamentous fungi Aspergillus genus has beenextensively used to produce invertase under submerged andsolid-state cultivations and its mechanisms of enzyme pro-ductionhave also been investigatedAspergillus niger canpro-duce invertase in the presence of 120573-fructofuranoside sugarsdemonstrating that the synthesis of invertase is inducible [10]This strain produces invertase with only hydrolytic activityproducing equimolar amounts of fructose and glucose Onthe other hand the FTase produced by this strain showsexclusively fructosyl transfer reaction producing glucose 1-kestose nystose and fructofuranosyl nystose [9] Aspergillusflavus produced high yields of invertase under optimizedconditions in submerged cultures [11] Guimaraes et al [12]observed that Aspergillus ochraceus produced high levelsof a thermostable extracellular invertase when cultured inKhanna medium supplemented with sugarcane bagasse ascarbon source

Enzyme production requires long time of laboratorialexperiment to find the ideal conditions and cost-effectiveness

for microbial cultivation Submerged and solid-state cultiva-tions are commonly employed to enzyme production usingalternative substrates as an attempt to reduce bottlenecksinvolved in the initial steps of production eg high costs ofproductionusing high value-added substratesTheutilizationof renewable resources such as agroindustrial residues ascheap and readily available substrates for enzyme productionhas been a subject of intense research as an alternative forcost-effective enzyme production and waste management[2] Agroindustrial residues are generated in large amountsevery year and their usage for bioprocesses is particularlyinteresting due to their renewability low-cost and suitablecharacteristics which allow the production of different value-added metabolites [13] Giraldo et al [14] used agroindustrialbyproducts eg sugarcane bagasse oat meal cassava flourcorn cob soy bran and wheat bran for invertase productionunder submerged cultivation Their experiments demon-strated that agroindustrial residues provided the productionof higher levels of extracellular invertase by Paecilomycesvariotii Guimaraes et al [12] observed that highest levels ofextracellular invertase activity were obtained when Khannamedium was supplemented by sugar cane bagasse as carbonsource and the extracellular invertase activity comprised98 total enzyme activity (intracellular + extracellular) fromcultivation Oyedeji et al [2] reported that agroindustrialresidues are suitable substrates for microbial cultivationand consequent production of molecule of biotechnologicalinterest such as enzymes and other value-added chemicals

The aims of this work were to select an invertase-producing Aspergillus spp strain with high fructosyltrans-ferase activity from canned peach syrups under submergedcultivations Nutritional parameters such as pure and com-plex carbon sources and nitrogen sources were evaluatedto obtain high yield of enzymes Agroindustrial residueswere also evaluated as alternative substrates for enzymeproduction

2 Material and Methods

21 Microorganisms Filamentous fungi were isolated froma jar surface containing canned peach syrup Canned peachsyrup was obtained from local market Samples of cannedpeach syrup were maintained at 30∘C for five days Coloniesgrown on the surface of syrup were transferred to Petriplates containing potato dextrose agar (PDA) and incubatedat 30∘C for five days Isolated colonies were inoculated atthe center of the Petri dishes to ensure the purity of fungistrains Filamentous fungi preservation was conducted insterile distilled water [15] Dishes (approximately 5mm)containing small portion of culture medium mycelium andspores were aseptically transferred into 6mL sterile antibioticflasks which were added to 4mL sterile distilled water andsealed with rubber stopper Castellani flasks were kept at 4∘Cand the viability of strains was verified periodically every sixmonths

Fungi were isolated as monocultures on two media maltextract agar and potato dextrose agar Fungi were identifiedto genus level according to morphological structures [16ndash18]



















119884(119875119878) =119875119891 minus 1198750

1198780(1)



119875119875 =119864119905119905

(2)









Table1Filamentous

fung

istrains

morph

ologiesisolatedfro

mcann

edpeachsyrups

andinvertasep

rodu

ction

Filamentous

fung

iColon

ymorph

olog

yInvertasea

ctivity

Yps

PpBiom

ass

(Um

l)lowast(gL)

Aspergillus

spP

C-1

Colon

iesa


whitewith

denselayer


idialh

eads

are

glob

osedark

brow

nCon

idiaareg

lobo

sedarkbrow

n18

6b16317

027

572

Aspergillus

spP

C-2

Colon

iesa

regranularw

hitemycelium

with

yello

w-green

conidiah

eadsC

onidialh

eads

arer

adiateC

onidiaare

glob

osepalegreen

123c

11090

018

556

Penicilliu

mspP

C-3

Colon

iesa

recompactw

hitemycelium

with

denselayer

ofgreenconidiaCon

idiaareh

yalin

eand

inlong

chain

glob

osegreenish

106c

9520

016

267

Aspergillus

spP

C-4

Colon

iesa


with

denselayer


idialh

eads

areg

lobo

se

dark

brow

nCon

idiaareg

lobo

sedarkbrow

n247

a22230

037

554

Aspergillus

spP

C-5

Colon

iesa

reflo


ithbrow

nconidiah

eadsC

onidialh

eads

arer

adiateC

onidia

ares

spheric

alpaleb

rown

196b

1518

2025

537

Aspergillus

spP

C-6

Colon

iesa

recompactw

hite-yellowmycelium

with

denselayer

ofblackconidiah

eadsC

onidialh

eads

are

glob

osedark

brow

nCon

idiaareg

lobo

sedarkbrow

n13

0c9728

016

602

Cultu

recond

ition

sCu

ltivatio

nwe

recarriedou

tat28∘C

180r

pmfor7

2ho

urslowastMeanvalues

with

thes

amelette

rdono


each

otherb

ytheANOVA

Tukeytest(p

=005)



89

95

52

100

100

99

99








0123456789

10


Enzy

me a

ctiv

ity (U

mL)

Glu

cose

Fruc

tose

Xylo

se

Sucr

ose

Lact

ose

Cell

obio

se

Mal

tose

Raffi

nose

Inul

in

Cel

lulo

se

Gly

cero

l

Sorb

itol




















0123456789

10


A

BC

G

D D

GE

F F G

a

b bcc

d

g

de e

g

f

gInve

rtas

e act

ivity

(Um

L)

pine

appl

e cro

wn

swee

t pot

ato

flour

suga

rcan

e bag

asse

corn

cob

cass

ava p

ee1

soyb

ean

peel

soyb

ean

bran

corn

stra

w

baca

ba p

eel

sorg

hum

bra

n

whe

at b

ran

(a)

05

101520253035


a

e

b

g

cd c

g

c

f f

d

AB

CCD

HF

G

D D

IF

Ftas

e act

ivity

(Um

L)

pine

appl

e cro

wn

swee

t pot

ato

flour

suga

rcan

e bag

asse

corn

cob

cass

ava p

ee1

soyb

ean

peel

soyb

ean

bran

corn

stra

w

baca

ba p

eel

sorg

hum

bra

n

whe

at b

ran

(b)







Table3Ferm

entatio

nparametersfor

invertasea

nd120573-D

-fructosyltransfe

raseprod

uctio

nby

blackAspergillus

spP

C-4on

complex

carbon

sources

Com

ponent

Ferm

entatio

nparameters

72ho

urs

120ho

urs

Invertase

FTase

Biom

ass

Invertase

FTase

Biom

ass

Yps

PpYp

sPp

Yps

PpYp

sPp

Com

plex

carbon

sources(

10w

v)

Pineapplec

rown

58697

098

133843

223

670

57416

096

145900

194

788

Sweetp

otatoflo

ur35280

059

3314

3055

524

40221

067

73035

121

576

Sugarcaneb

agasse

3813

8064

1240

01

210

910

1079

2018

7310

012

199

3Corncob

26324

044

nd

nd

904

1870

3031

72390

120

908

Cassava

peel

1319

8022

40272

142

678

3310

006

24832

041

634

Soybeanpeel

2612

004

1041

002

967

2715

005

10276

006

1057

Soybeanbran

27352

046

nd

nd

561

31226

052

47618

079

521

Bacaba

peel

840

001

582

027

980

2151

004

nd

nd

999

Cornstr

aw2268

004

2476

004

1098

1386

002

4062

006

1163

Sorghu

mbran

2291

3038

28202

050

571

3791

006

25240

042

648

Wheatbram

40575

068

5177

1086

647

17885

030

67015

111

678

Nitr

ogen

sources(

02

wv)

Ammon

ium

nitrate

34082

094

108247

300

772

2316

7064

240

786

400

576

Ammon

ium

choride

49325

137

209681

582

790

5292

814

72114

51

352

600

Ammoinium

sulfate

51457

143

135937

377

824

41471

116

200

623

334

620

Yeastextract

53685

149

117666

327

833

39428

109

179910

300

640

Pepton

e42617

118

132282

367

853

39874

111

184956

310

592

Soybeanprotein

3296

8091

135120

375

848

25636

071

26279

3438

850

Cornste

epliq

uor

28717

098

111773

310

770

24058

067

184704

308

562

Cultu

recond

ition

sCu

ltivatio

nwe

recarriedou

tat28∘Cfor180

rpm


0123456789

10

Inve

rtas

e act

ivity

(Um

L)


aab

FG

de

ef fg

ABA

C

DE

FA

mm

oniu

m n

itrat

e

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

(a)


a a aab abc

bc

d

AABBC

CDEF DE DE

Am

mon

ium

nitr

ate

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

05

101520253035

FTas

e act

ivity

(Um

L)

(b)









0

2

4

6

8

10

12

14

Inve

rtas

e act

ivity

(Um

L)

(a)

06

121824303642485460

24 240216192168144120967248

FTas

e act

ivity

(Um

l)


(b)

0500

10001500200025003000350040004500

24 48 72 96 120 144 168 192 216 240

Yiel

d (U

g o

f sub

stra

te)

Time-course (hours)

Invertase(c)

0

1

2

3

4

5

6

7

24 48 72 96 120 144 168 192 216 240

Prod

uctiv

ity (U

gh

)

Time-course (hours)

Invertase(d)









Data Availability




Acknowledgments


References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




















119884(119875119878) =119875119891 minus 1198750

1198780(1)



119875119875 =119864119905119905

(2)









Table1Filamentous

fung

istrains

morph

ologiesisolatedfro

mcann

edpeachsyrups

andinvertasep

rodu

ction

Filamentous

fung

iColon

ymorph

olog

yInvertasea

ctivity

Yps

PpBiom

ass

(Um

l)lowast(gL)

Aspergillus

spP

C-1

Colon

iesa


whitewith

denselayer


idialh

eads

are

glob

osedark

brow

nCon

idiaareg

lobo

sedarkbrow

n18

6b16317

027

572

Aspergillus

spP

C-2

Colon

iesa

regranularw

hitemycelium

with

yello

w-green

conidiah

eadsC

onidialh

eads

arer

adiateC

onidiaare

glob

osepalegreen

123c

11090

018

556

Penicilliu

mspP

C-3

Colon

iesa

recompactw

hitemycelium

with

denselayer

ofgreenconidiaCon

idiaareh

yalin

eand

inlong

chain

glob

osegreenish

106c

9520

016

267

Aspergillus

spP

C-4

Colon

iesa


with

denselayer


idialh

eads

areg

lobo

se

dark

brow

nCon

idiaareg

lobo

sedarkbrow

n247

a22230

037

554

Aspergillus

spP

C-5

Colon

iesa

reflo


ithbrow

nconidiah

eadsC

onidialh

eads

arer

adiateC

onidia

ares

spheric

alpaleb

rown

196b

1518

2025

537

Aspergillus

spP

C-6

Colon

iesa

recompactw

hite-yellowmycelium

with

denselayer

ofblackconidiah

eadsC

onidialh

eads

are

glob

osedark

brow

nCon

idiaareg

lobo

sedarkbrow

n13

0c9728

016

602

Cultu

recond

ition

sCu

ltivatio

nwe

recarriedou

tat28∘C

180r

pmfor7

2ho

urslowastMeanvalues

with

thes

amelette

rdono


each

otherb

ytheANOVA

Tukeytest(p

=005)



89

95

52

100

100

99

99








0123456789

10


Enzy

me a

ctiv

ity (U

mL)

Glu

cose

Fruc

tose

Xylo

se

Sucr

ose

Lact

ose

Cell

obio

se

Mal

tose

Raffi

nose

Inul

in

Cel

lulo

se

Gly

cero

l

Sorb

itol




















0123456789

10


A

BC

G

D D

GE

F F G

a

b bcc

d

g

de e

g

f

gInve

rtas

e act

ivity

(Um

L)

pine

appl

e cro

wn

swee

t pot

ato

flour

suga

rcan

e bag

asse

corn

cob

cass

ava p

ee1

soyb

ean

peel

soyb

ean

bran

corn

stra

w

baca

ba p

eel

sorg

hum

bra

n

whe

at b

ran

(a)

05

101520253035


a

e

b

g

cd c

g

c

f f

d

AB

CCD

HF

G

D D

IF

Ftas

e act

ivity

(Um

L)

pine

appl

e cro

wn

swee

t pot

ato

flour

suga

rcan

e bag

asse

corn

cob

cass

ava p

ee1

soyb

ean

peel

soyb

ean

bran

corn

stra

w

baca

ba p

eel

sorg

hum

bra

n

whe

at b

ran

(b)







Table3Ferm

entatio

nparametersfor

invertasea

nd120573-D

-fructosyltransfe

raseprod

uctio

nby

blackAspergillus

spP

C-4on

complex

carbon

sources

Com

ponent

Ferm

entatio

nparameters

72ho

urs

120ho

urs

Invertase

FTase

Biom

ass

Invertase

FTase

Biom

ass

Yps

PpYp

sPp

Yps

PpYp

sPp

Com

plex

carbon

sources(

10w

v)

Pineapplec

rown

58697

098

133843

223

670

57416

096

145900

194

788

Sweetp

otatoflo

ur35280

059

3314

3055

524

40221

067

73035

121

576

Sugarcaneb

agasse

3813

8064

1240

01

210

910

1079

2018

7310

012

199

3Corncob

26324

044

nd

nd

904

1870

3031

72390

120

908

Cassava

peel

1319

8022

40272

142

678

3310

006

24832

041

634

Soybeanpeel

2612

004

1041

002

967

2715

005

10276

006

1057

Soybeanbran

27352

046

nd

nd

561

31226

052

47618

079

521

Bacaba

peel

840

001

582

027

980

2151

004

nd

nd

999

Cornstr

aw2268

004

2476

004

1098

1386

002

4062

006

1163

Sorghu

mbran

2291

3038

28202

050

571

3791

006

25240

042

648

Wheatbram

40575

068

5177

1086

647

17885

030

67015

111

678

Nitr

ogen

sources(

02

wv)

Ammon

ium

nitrate

34082

094

108247

300

772

2316

7064

240

786

400

576

Ammon

ium

choride

49325

137

209681

582

790

5292

814

72114

51

352

600

Ammoinium

sulfate

51457

143

135937

377

824

41471

116

200

623

334

620

Yeastextract

53685

149

117666

327

833

39428

109

179910

300

640

Pepton

e42617

118

132282

367

853

39874

111

184956

310

592

Soybeanprotein

3296

8091

135120

375

848

25636

071

26279

3438

850

Cornste

epliq

uor

28717

098

111773

310

770

24058

067

184704

308

562

Cultu

recond

ition

sCu

ltivatio

nwe

recarriedou

tat28∘Cfor180

rpm


0123456789

10

Inve

rtas

e act

ivity

(Um

L)


aab

FG

de

ef fg

ABA

C

DE

FA

mm

oniu

m n

itrat

e

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

(a)


a a aab abc

bc

d

AABBC

CDEF DE DE

Am

mon

ium

nitr

ate

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

05

101520253035

FTas

e act

ivity

(Um

L)

(b)









0

2

4

6

8

10

12

14

Inve

rtas

e act

ivity

(Um

L)

(a)

06

121824303642485460

24 240216192168144120967248

FTas

e act

ivity

(Um

l)


(b)

0500

10001500200025003000350040004500

24 48 72 96 120 144 168 192 216 240

Yiel

d (U

g o

f sub

stra

te)

Time-course (hours)

Invertase(c)

0

1

2

3

4

5

6

7

24 48 72 96 120 144 168 192 216 240

Prod

uctiv

ity (U

gh

)

Time-course (hours)

Invertase(d)









Data Availability




Acknowledgments


References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









119884(119875119878) =119875119891 minus 1198750

1198780(1)



119875119875 =119864119905119905

(2)









Table1Filamentous

fung

istrains

morph

ologiesisolatedfro

mcann

edpeachsyrups

andinvertasep

rodu

ction

Filamentous

fung

iColon

ymorph

olog

yInvertasea

ctivity

Yps

PpBiom

ass

(Um

l)lowast(gL)

Aspergillus

spP

C-1

Colon

iesa


whitewith

denselayer


idialh

eads

are

glob

osedark

brow

nCon

idiaareg

lobo

sedarkbrow

n18

6b16317

027

572

Aspergillus

spP

C-2

Colon

iesa

regranularw

hitemycelium

with

yello

w-green

conidiah

eadsC

onidialh

eads

arer

adiateC

onidiaare

glob

osepalegreen

123c

11090

018

556

Penicilliu

mspP

C-3

Colon

iesa

recompactw

hitemycelium

with

denselayer

ofgreenconidiaCon

idiaareh

yalin

eand

inlong

chain

glob

osegreenish

106c

9520

016

267

Aspergillus

spP

C-4

Colon

iesa


with

denselayer


idialh

eads

areg

lobo

se

dark

brow

nCon

idiaareg

lobo

sedarkbrow

n247

a22230

037

554

Aspergillus

spP

C-5

Colon

iesa

reflo


ithbrow

nconidiah

eadsC

onidialh

eads

arer

adiateC

onidia

ares

spheric

alpaleb

rown

196b

1518

2025

537

Aspergillus

spP

C-6

Colon

iesa

recompactw

hite-yellowmycelium

with

denselayer

ofblackconidiah

eadsC

onidialh

eads

are

glob

osedark

brow

nCon

idiaareg

lobo

sedarkbrow

n13

0c9728

016

602

Cultu

recond

ition

sCu

ltivatio

nwe

recarriedou

tat28∘C

180r

pmfor7

2ho

urslowastMeanvalues

with

thes

amelette

rdono


each

otherb

ytheANOVA

Tukeytest(p

=005)



89

95

52

100

100

99

99








0123456789

10


Enzy

me a

ctiv

ity (U

mL)

Glu

cose

Fruc

tose

Xylo

se

Sucr

ose

Lact

ose

Cell

obio

se

Mal

tose

Raffi

nose

Inul

in

Cel

lulo

se

Gly

cero

l

Sorb

itol




















0123456789

10


A

BC

G

D D

GE

F F G

a

b bcc

d

g

de e

g

f

gInve

rtas

e act

ivity

(Um

L)

pine

appl

e cro

wn

swee

t pot

ato

flour

suga

rcan

e bag

asse

corn

cob

cass

ava p

ee1

soyb

ean

peel

soyb

ean

bran

corn

stra

w

baca

ba p

eel

sorg

hum

bra

n

whe

at b

ran

(a)

05

101520253035


a

e

b

g

cd c

g

c

f f

d

AB

CCD

HF

G

D D

IF

Ftas

e act

ivity

(Um

L)

pine

appl

e cro

wn

swee

t pot

ato

flour

suga

rcan

e bag

asse

corn

cob

cass

ava p

ee1

soyb

ean

peel

soyb

ean

bran

corn

stra

w

baca

ba p

eel

sorg

hum

bra

n

whe

at b

ran

(b)







Table3Ferm

entatio

nparametersfor

invertasea

nd120573-D

-fructosyltransfe

raseprod

uctio

nby

blackAspergillus

spP

C-4on

complex

carbon

sources

Com

ponent

Ferm

entatio

nparameters

72ho

urs

120ho

urs

Invertase

FTase

Biom

ass

Invertase

FTase

Biom

ass

Yps

PpYp

sPp

Yps

PpYp

sPp

Com

plex

carbon

sources(

10w

v)

Pineapplec

rown

58697

098

133843

223

670

57416

096

145900

194

788

Sweetp

otatoflo

ur35280

059

3314

3055

524

40221

067

73035

121

576

Sugarcaneb

agasse

3813

8064

1240

01

210

910

1079

2018

7310

012

199

3Corncob

26324

044

nd

nd

904

1870

3031

72390

120

908

Cassava

peel

1319

8022

40272

142

678

3310

006

24832

041

634

Soybeanpeel

2612

004

1041

002

967

2715

005

10276

006

1057

Soybeanbran

27352

046

nd

nd

561

31226

052

47618

079

521

Bacaba

peel

840

001

582

027

980

2151

004

nd

nd

999

Cornstr

aw2268

004

2476

004

1098

1386

002

4062

006

1163

Sorghu

mbran

2291

3038

28202

050

571

3791

006

25240

042

648

Wheatbram

40575

068

5177

1086

647

17885

030

67015

111

678

Nitr

ogen

sources(

02

wv)

Ammon

ium

nitrate

34082

094

108247

300

772

2316

7064

240

786

400

576

Ammon

ium

choride

49325

137

209681

582

790

5292

814

72114

51

352

600

Ammoinium

sulfate

51457

143

135937

377

824

41471

116

200

623

334

620

Yeastextract

53685

149

117666

327

833

39428

109

179910

300

640

Pepton

e42617

118

132282

367

853

39874

111

184956

310

592

Soybeanprotein

3296

8091

135120

375

848

25636

071

26279

3438

850

Cornste

epliq

uor

28717

098

111773

310

770

24058

067

184704

308

562

Cultu

recond

ition

sCu

ltivatio

nwe

recarriedou

tat28∘Cfor180

rpm


0123456789

10

Inve

rtas

e act

ivity

(Um

L)


aab

FG

de

ef fg

ABA

C

DE

FA

mm

oniu

m n

itrat

e

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

(a)


a a aab abc

bc

d

AABBC

CDEF DE DE

Am

mon

ium

nitr

ate

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

05

101520253035

FTas

e act

ivity

(Um

L)

(b)









0

2

4

6

8

10

12

14

Inve

rtas

e act

ivity

(Um

L)

(a)

06

121824303642485460

24 240216192168144120967248

FTas

e act

ivity

(Um

l)


(b)

0500

10001500200025003000350040004500

24 48 72 96 120 144 168 192 216 240

Yiel

d (U

g o

f sub

stra

te)

Time-course (hours)

Invertase(c)

0

1

2

3

4

5

6

7

24 48 72 96 120 144 168 192 216 240

Prod

uctiv

ity (U

gh

)

Time-course (hours)

Invertase(d)









Data Availability




Acknowledgments


References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Table1Filamentous

fung

istrains

morph

ologiesisolatedfro

mcann

edpeachsyrups

andinvertasep

rodu

ction

Filamentous

fung

iColon

ymorph

olog

yInvertasea

ctivity

Yps

PpBiom

ass

(Um

l)lowast(gL)

Aspergillus

spP

C-1

Colon

iesa


whitewith

denselayer


idialh

eads

are

glob

osedark

brow

nCon

idiaareg

lobo

sedarkbrow

n18

6b16317

027

572

Aspergillus

spP

C-2

Colon

iesa

regranularw

hitemycelium

with

yello

w-green

conidiah

eadsC

onidialh

eads

arer

adiateC

onidiaare

glob

osepalegreen

123c

11090

018

556

Penicilliu

mspP

C-3

Colon

iesa

recompactw

hitemycelium

with

denselayer

ofgreenconidiaCon

idiaareh

yalin

eand

inlong

chain

glob

osegreenish

106c

9520

016

267

Aspergillus

spP

C-4

Colon

iesa


with

denselayer


idialh

eads

areg

lobo

se

dark

brow

nCon

idiaareg

lobo

sedarkbrow

n247

a22230

037

554

Aspergillus

spP

C-5

Colon

iesa

reflo


ithbrow

nconidiah

eadsC

onidialh

eads

arer

adiateC

onidia

ares

spheric

alpaleb

rown

196b

1518

2025

537

Aspergillus

spP

C-6

Colon

iesa

recompactw

hite-yellowmycelium

with

denselayer

ofblackconidiah

eadsC

onidialh

eads

are

glob

osedark

brow

nCon

idiaareg

lobo

sedarkbrow

n13

0c9728

016

602

Cultu

recond

ition

sCu

ltivatio

nwe

recarriedou

tat28∘C

180r

pmfor7

2ho

urslowastMeanvalues

with

thes

amelette

rdono


each

otherb

ytheANOVA

Tukeytest(p

=005)



89

95

52

100

100

99

99








0123456789

10


Enzy

me a

ctiv

ity (U

mL)

Glu

cose

Fruc

tose

Xylo

se

Sucr

ose

Lact

ose

Cell

obio

se

Mal

tose

Raffi

nose

Inul

in

Cel

lulo

se

Gly

cero

l

Sorb

itol




















0123456789

10


A

BC

G

D D

GE

F F G

a

b bcc

d

g

de e

g

f

gInve

rtas

e act

ivity

(Um

L)

pine

appl

e cro

wn

swee

t pot

ato

flour

suga

rcan

e bag

asse

corn

cob

cass

ava p

ee1

soyb

ean

peel

soyb

ean

bran

corn

stra

w

baca

ba p

eel

sorg

hum

bra

n

whe

at b

ran

(a)

05

101520253035


a

e

b

g

cd c

g

c

f f

d

AB

CCD

HF

G

D D

IF

Ftas

e act

ivity

(Um

L)

pine

appl

e cro

wn

swee

t pot

ato

flour

suga

rcan

e bag

asse

corn

cob

cass

ava p

ee1

soyb

ean

peel

soyb

ean

bran

corn

stra

w

baca

ba p

eel

sorg

hum

bra

n

whe

at b

ran

(b)







Table3Ferm

entatio

nparametersfor

invertasea

nd120573-D

-fructosyltransfe

raseprod

uctio

nby

blackAspergillus

spP

C-4on

complex

carbon

sources

Com

ponent

Ferm

entatio

nparameters

72ho

urs

120ho

urs

Invertase

FTase

Biom

ass

Invertase

FTase

Biom

ass

Yps

PpYp

sPp

Yps

PpYp

sPp

Com

plex

carbon

sources(

10w

v)

Pineapplec

rown

58697

098

133843

223

670

57416

096

145900

194

788

Sweetp

otatoflo

ur35280

059

3314

3055

524

40221

067

73035

121

576

Sugarcaneb

agasse

3813

8064

1240

01

210

910

1079

2018

7310

012

199

3Corncob

26324

044

nd

nd

904

1870

3031

72390

120

908

Cassava

peel

1319

8022

40272

142

678

3310

006

24832

041

634

Soybeanpeel

2612

004

1041

002

967

2715

005

10276

006

1057

Soybeanbran

27352

046

nd

nd

561

31226

052

47618

079

521

Bacaba

peel

840

001

582

027

980

2151

004

nd

nd

999

Cornstr

aw2268

004

2476

004

1098

1386

002

4062

006

1163

Sorghu

mbran

2291

3038

28202

050

571

3791

006

25240

042

648

Wheatbram

40575

068

5177

1086

647

17885

030

67015

111

678

Nitr

ogen

sources(

02

wv)

Ammon

ium

nitrate

34082

094

108247

300

772

2316

7064

240

786

400

576

Ammon

ium

choride

49325

137

209681

582

790

5292

814

72114

51

352

600

Ammoinium

sulfate

51457

143

135937

377

824

41471

116

200

623

334

620

Yeastextract

53685

149

117666

327

833

39428

109

179910

300

640

Pepton

e42617

118

132282

367

853

39874

111

184956

310

592

Soybeanprotein

3296

8091

135120

375

848

25636

071

26279

3438

850

Cornste

epliq

uor

28717

098

111773

310

770

24058

067

184704

308

562

Cultu

recond

ition

sCu

ltivatio

nwe

recarriedou

tat28∘Cfor180

rpm


0123456789

10

Inve

rtas

e act

ivity

(Um

L)


aab

FG

de

ef fg

ABA

C

DE

FA

mm

oniu

m n

itrat

e

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

(a)


a a aab abc

bc

d

AABBC

CDEF DE DE

Am

mon

ium

nitr

ate

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

05

101520253035

FTas

e act

ivity

(Um

L)

(b)









0

2

4

6

8

10

12

14

Inve

rtas

e act

ivity

(Um

L)

(a)

06

121824303642485460

24 240216192168144120967248

FTas

e act

ivity

(Um

l)


(b)

0500

10001500200025003000350040004500

24 48 72 96 120 144 168 192 216 240

Yiel

d (U

g o

f sub

stra

te)

Time-course (hours)

Invertase(c)

0

1

2

3

4

5

6

7

24 48 72 96 120 144 168 192 216 240

Prod

uctiv

ity (U

gh

)

Time-course (hours)

Invertase(d)









Data Availability




Acknowledgments


References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




89

95

52

100

100

99

99








0123456789

10


Enzy

me a

ctiv

ity (U

mL)

Glu

cose

Fruc

tose

Xylo

se

Sucr

ose

Lact

ose

Cell

obio

se

Mal

tose

Raffi

nose

Inul

in

Cel

lulo

se

Gly

cero

l

Sorb

itol




















0123456789

10


A

BC

G

D D

GE

F F G

a

b bcc

d

g

de e

g

f

gInve

rtas

e act

ivity

(Um

L)

pine

appl

e cro

wn

swee

t pot

ato

flour

suga

rcan

e bag

asse

corn

cob

cass

ava p

ee1

soyb

ean

peel

soyb

ean

bran

corn

stra

w

baca

ba p

eel

sorg

hum

bra

n

whe

at b

ran

(a)

05

101520253035


a

e

b

g

cd c

g

c

f f

d

AB

CCD

HF

G

D D

IF

Ftas

e act

ivity

(Um

L)

pine

appl

e cro

wn

swee

t pot

ato

flour

suga

rcan

e bag

asse

corn

cob

cass

ava p

ee1

soyb

ean

peel

soyb

ean

bran

corn

stra

w

baca

ba p

eel

sorg

hum

bra

n

whe

at b

ran

(b)







Table3Ferm

entatio

nparametersfor

invertasea

nd120573-D

-fructosyltransfe

raseprod

uctio

nby

blackAspergillus

spP

C-4on

complex

carbon

sources

Com

ponent

Ferm

entatio

nparameters

72ho

urs

120ho

urs

Invertase

FTase

Biom

ass

Invertase

FTase

Biom

ass

Yps

PpYp

sPp

Yps

PpYp

sPp

Com

plex

carbon

sources(

10w

v)

Pineapplec

rown

58697

098

133843

223

670

57416

096

145900

194

788

Sweetp

otatoflo

ur35280

059

3314

3055

524

40221

067

73035

121

576

Sugarcaneb

agasse

3813

8064

1240

01

210

910

1079

2018

7310

012

199

3Corncob

26324

044

nd

nd

904

1870

3031

72390

120

908

Cassava

peel

1319

8022

40272

142

678

3310

006

24832

041

634

Soybeanpeel

2612

004

1041

002

967

2715

005

10276

006

1057

Soybeanbran

27352

046

nd

nd

561

31226

052

47618

079

521

Bacaba

peel

840

001

582

027

980

2151

004

nd

nd

999

Cornstr

aw2268

004

2476

004

1098

1386

002

4062

006

1163

Sorghu

mbran

2291

3038

28202

050

571

3791

006

25240

042

648

Wheatbram

40575

068

5177

1086

647

17885

030

67015

111

678

Nitr

ogen

sources(

02

wv)

Ammon

ium

nitrate

34082

094

108247

300

772

2316

7064

240

786

400

576

Ammon

ium

choride

49325

137

209681

582

790

5292

814

72114

51

352

600

Ammoinium

sulfate

51457

143

135937

377

824

41471

116

200

623

334

620

Yeastextract

53685

149

117666

327

833

39428

109

179910

300

640

Pepton

e42617

118

132282

367

853

39874

111

184956

310

592

Soybeanprotein

3296

8091

135120

375

848

25636

071

26279

3438

850

Cornste

epliq

uor

28717

098

111773

310

770

24058

067

184704

308

562

Cultu

recond

ition

sCu

ltivatio

nwe

recarriedou

tat28∘Cfor180

rpm


0123456789

10

Inve

rtas

e act

ivity

(Um

L)


aab

FG

de

ef fg

ABA

C

DE

FA

mm

oniu

m n

itrat

e

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

(a)


a a aab abc

bc

d

AABBC

CDEF DE DE

Am

mon

ium

nitr

ate

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

05

101520253035

FTas

e act

ivity

(Um

L)

(b)









0

2

4

6

8

10

12

14

Inve

rtas

e act

ivity

(Um

L)

(a)

06

121824303642485460

24 240216192168144120967248

FTas

e act

ivity

(Um

l)


(b)

0500

10001500200025003000350040004500

24 48 72 96 120 144 168 192 216 240

Yiel

d (U

g o

f sub

stra

te)

Time-course (hours)

Invertase(c)

0

1

2

3

4

5

6

7

24 48 72 96 120 144 168 192 216 240

Prod

uctiv

ity (U

gh

)

Time-course (hours)

Invertase(d)









Data Availability




Acknowledgments


References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018















0123456789

10


A

BC

G

D D

GE

F F G

a

b bcc

d

g

de e

g

f

gInve

rtas

e act

ivity

(Um

L)

pine

appl

e cro

wn

swee

t pot

ato

flour

suga

rcan

e bag

asse

corn

cob

cass

ava p

ee1

soyb

ean

peel

soyb

ean

bran

corn

stra

w

baca

ba p

eel

sorg

hum

bra

n

whe

at b

ran

(a)

05

101520253035


a

e

b

g

cd c

g

c

f f

d

AB

CCD

HF

G

D D

IF

Ftas

e act

ivity

(Um

L)

pine

appl

e cro

wn

swee

t pot

ato

flour

suga

rcan

e bag

asse

corn

cob

cass

ava p

ee1

soyb

ean

peel

soyb

ean

bran

corn

stra

w

baca

ba p

eel

sorg

hum

bra

n

whe

at b

ran

(b)







Table3Ferm

entatio

nparametersfor

invertasea

nd120573-D

-fructosyltransfe

raseprod

uctio

nby

blackAspergillus

spP

C-4on

complex

carbon

sources

Com

ponent

Ferm

entatio

nparameters

72ho

urs

120ho

urs

Invertase

FTase

Biom

ass

Invertase

FTase

Biom

ass

Yps

PpYp

sPp

Yps

PpYp

sPp

Com

plex

carbon

sources(

10w

v)

Pineapplec

rown

58697

098

133843

223

670

57416

096

145900

194

788

Sweetp

otatoflo

ur35280

059

3314

3055

524

40221

067

73035

121

576

Sugarcaneb

agasse

3813

8064

1240

01

210

910

1079

2018

7310

012

199

3Corncob

26324

044

nd

nd

904

1870

3031

72390

120

908

Cassava

peel

1319

8022

40272

142

678

3310

006

24832

041

634

Soybeanpeel

2612

004

1041

002

967

2715

005

10276

006

1057

Soybeanbran

27352

046

nd

nd

561

31226

052

47618

079

521

Bacaba

peel

840

001

582

027

980

2151

004

nd

nd

999

Cornstr

aw2268

004

2476

004

1098

1386

002

4062

006

1163

Sorghu

mbran

2291

3038

28202

050

571

3791

006

25240

042

648

Wheatbram

40575

068

5177

1086

647

17885

030

67015

111

678

Nitr

ogen

sources(

02

wv)

Ammon

ium

nitrate

34082

094

108247

300

772

2316

7064

240

786

400

576

Ammon

ium

choride

49325

137

209681

582

790

5292

814

72114

51

352

600

Ammoinium

sulfate

51457

143

135937

377

824

41471

116

200

623

334

620

Yeastextract

53685

149

117666

327

833

39428

109

179910

300

640

Pepton

e42617

118

132282

367

853

39874

111

184956

310

592

Soybeanprotein

3296

8091

135120

375

848

25636

071

26279

3438

850

Cornste

epliq

uor

28717

098

111773

310

770

24058

067

184704

308

562

Cultu

recond

ition

sCu

ltivatio

nwe

recarriedou

tat28∘Cfor180

rpm


0123456789

10

Inve

rtas

e act

ivity

(Um

L)


aab

FG

de

ef fg

ABA

C

DE

FA

mm

oniu

m n

itrat

e

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

(a)


a a aab abc

bc

d

AABBC

CDEF DE DE

Am

mon

ium

nitr

ate

Am

mon

ium

chlo

ride

Am

mon

ium

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te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

05

101520253035

FTas

e act

ivity

(Um

L)

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0

2

4

6

8

10

12

14

Inve

rtas

e act

ivity

(Um

L)

(a)

06

121824303642485460

24 240216192168144120967248

FTas

e act

ivity

(Um

l)


(b)

0500

10001500200025003000350040004500

24 48 72 96 120 144 168 192 216 240

Yiel

d (U

g o

f sub

stra

te)

Time-course (hours)

Invertase(c)

0

1

2

3

4

5

6

7

24 48 72 96 120 144 168 192 216 240

Prod

uctiv

ity (U

gh

)

Time-course (hours)

Invertase(d)









Data Availability




Acknowledgments


References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



0123456789

10


A

BC

G

D D

GE

F F G

a

b bcc

d

g

de e

g

f

gInve

rtas

e act

ivity

(Um

L)

pine

appl

e cro

wn

swee

t pot

ato

flour

suga

rcan

e bag

asse

corn

cob

cass

ava p

ee1

soyb

ean

peel

soyb

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bran

corn

stra

w

baca

ba p

eel

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bra

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whe

at b

ran

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05

101520253035


a

e

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CCD

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ivity

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Table3Ferm

entatio

nparametersfor

invertasea

nd120573-D

-fructosyltransfe

raseprod

uctio

nby

blackAspergillus

spP

C-4on

complex

carbon

sources

Com

ponent

Ferm

entatio

nparameters

72ho

urs

120ho

urs

Invertase

FTase

Biom

ass

Invertase

FTase

Biom

ass

Yps

PpYp

sPp

Yps

PpYp

sPp

Com

plex

carbon

sources(

10w

v)

Pineapplec

rown

58697

098

133843

223

670

57416

096

145900

194

788

Sweetp

otatoflo

ur35280

059

3314

3055

524

40221

067

73035

121

576

Sugarcaneb

agasse

3813

8064

1240

01

210

910

1079

2018

7310

012

199

3Corncob

26324

044

nd

nd

904

1870

3031

72390

120

908

Cassava

peel

1319

8022

40272

142

678

3310

006

24832

041

634

Soybeanpeel

2612

004

1041

002

967

2715

005

10276

006

1057

Soybeanbran

27352

046

nd

nd

561

31226

052

47618

079

521

Bacaba

peel

840

001

582

027

980

2151

004

nd

nd

999

Cornstr

aw2268

004

2476

004

1098

1386

002

4062

006

1163

Sorghu

mbran

2291

3038

28202

050

571

3791

006

25240

042

648

Wheatbram

40575

068

5177

1086

647

17885

030

67015

111

678

Nitr

ogen

sources(

02

wv)

Ammon

ium

nitrate

34082

094

108247

300

772

2316

7064

240

786

400

576

Ammon

ium

choride

49325

137

209681

582

790

5292

814

72114

51

352

600

Ammoinium

sulfate

51457

143

135937

377

824

41471

116

200

623

334

620

Yeastextract

53685

149

117666

327

833

39428

109

179910

300

640

Pepton

e42617

118

132282

367

853

39874

111

184956

310

592

Soybeanprotein

3296

8091

135120

375

848

25636

071

26279

3438

850

Cornste

epliq

uor

28717

098

111773

310

770

24058

067

184704

308

562

Cultu

recond

ition

sCu

ltivatio

nwe

recarriedou

tat28∘Cfor180

rpm


0123456789

10

Inve

rtas

e act

ivity

(Um

L)


aab

FG

de

ef fg

ABA

C

DE

FA

mm

oniu

m n

itrat

e

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

(a)


a a aab abc

bc

d

AABBC

CDEF DE DE

Am

mon

ium

nitr

ate

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

05

101520253035

FTas

e act

ivity

(Um

L)

(b)









0

2

4

6

8

10

12

14

Inve

rtas

e act

ivity

(Um

L)

(a)

06

121824303642485460

24 240216192168144120967248

FTas

e act

ivity

(Um

l)


(b)

0500

10001500200025003000350040004500

24 48 72 96 120 144 168 192 216 240

Yiel

d (U

g o

f sub

stra

te)

Time-course (hours)

Invertase(c)

0

1

2

3

4

5

6

7

24 48 72 96 120 144 168 192 216 240

Prod

uctiv

ity (U

gh

)

Time-course (hours)

Invertase(d)









Data Availability




Acknowledgments


References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Table3Ferm

entatio

nparametersfor

invertasea

nd120573-D

-fructosyltransfe

raseprod

uctio

nby

blackAspergillus

spP

C-4on

complex

carbon

sources

Com

ponent

Ferm

entatio

nparameters

72ho

urs

120ho

urs

Invertase

FTase

Biom

ass

Invertase

FTase

Biom

ass

Yps

PpYp

sPp

Yps

PpYp

sPp

Com

plex

carbon

sources(

10w

v)

Pineapplec

rown

58697

098

133843

223

670

57416

096

145900

194

788

Sweetp

otatoflo

ur35280

059

3314

3055

524

40221

067

73035

121

576

Sugarcaneb

agasse

3813

8064

1240

01

210

910

1079

2018

7310

012

199

3Corncob

26324

044

nd

nd

904

1870

3031

72390

120

908

Cassava

peel

1319

8022

40272

142

678

3310

006

24832

041

634

Soybeanpeel

2612

004

1041

002

967

2715

005

10276

006

1057

Soybeanbran

27352

046

nd

nd

561

31226

052

47618

079

521

Bacaba

peel

840

001

582

027

980

2151

004

nd

nd

999

Cornstr

aw2268

004

2476

004

1098

1386

002

4062

006

1163

Sorghu

mbran

2291

3038

28202

050

571

3791

006

25240

042

648

Wheatbram

40575

068

5177

1086

647

17885

030

67015

111

678

Nitr

ogen

sources(

02

wv)

Ammon

ium

nitrate

34082

094

108247

300

772

2316

7064

240

786

400

576

Ammon

ium

choride

49325

137

209681

582

790

5292

814

72114

51

352

600

Ammoinium

sulfate

51457

143

135937

377

824

41471

116

200

623

334

620

Yeastextract

53685

149

117666

327

833

39428

109

179910

300

640

Pepton

e42617

118

132282

367

853

39874

111

184956

310

592

Soybeanprotein

3296

8091

135120

375

848

25636

071

26279

3438

850

Cornste

epliq

uor

28717

098

111773

310

770

24058

067

184704

308

562

Cultu

recond

ition

sCu

ltivatio

nwe

recarriedou

tat28∘Cfor180

rpm


0123456789

10

Inve

rtas

e act

ivity

(Um

L)


aab

FG

de

ef fg

ABA

C

DE

FA

mm

oniu

m n

itrat

e

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

(a)


a a aab abc

bc

d

AABBC

CDEF DE DE

Am

mon

ium

nitr

ate

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

05

101520253035

FTas

e act

ivity

(Um

L)

(b)









0

2

4

6

8

10

12

14

Inve

rtas

e act

ivity

(Um

L)

(a)

06

121824303642485460

24 240216192168144120967248

FTas

e act

ivity

(Um

l)


(b)

0500

10001500200025003000350040004500

24 48 72 96 120 144 168 192 216 240

Yiel

d (U

g o

f sub

stra

te)

Time-course (hours)

Invertase(c)

0

1

2

3

4

5

6

7

24 48 72 96 120 144 168 192 216 240

Prod

uctiv

ity (U

gh

)

Time-course (hours)

Invertase(d)









Data Availability




Acknowledgments


References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



0123456789

10

Inve

rtas

e act

ivity

(Um

L)


aab

FG

de

ef fg

ABA

C

DE

FA

mm

oniu

m n

itrat

e

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

(a)


a a aab abc

bc

d

AABBC

CDEF DE DE

Am

mon

ium

nitr

ate

Am

mon

ium

chlo

ride

Am

mon

ium

sulfa

te

Yeas

t ext

ract

Pept

one

Soyb

ean

prot

ein

Cor

n ste

ep li

quor

05

101520253035

FTas

e act

ivity

(Um

L)

(b)









0

2

4

6

8

10

12

14

Inve

rtas

e act

ivity

(Um

L)

(a)

06

121824303642485460

24 240216192168144120967248

FTas

e act

ivity

(Um

l)


(b)

0500

10001500200025003000350040004500

24 48 72 96 120 144 168 192 216 240

Yiel

d (U

g o

f sub

stra

te)

Time-course (hours)

Invertase(c)

0

1

2

3

4

5

6

7

24 48 72 96 120 144 168 192 216 240

Prod

uctiv

ity (U

gh

)

Time-course (hours)

Invertase(d)









Data Availability




Acknowledgments


References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




0

2

4

6

8

10

12

14

Inve

rtas

e act

ivity

(Um

L)

(a)

06

121824303642485460

24 240216192168144120967248

FTas

e act

ivity

(Um

l)


(b)

0500

10001500200025003000350040004500

24 48 72 96 120 144 168 192 216 240

Yiel

d (U

g o

f sub

stra

te)

Time-course (hours)

Invertase(c)

0

1

2

3

4

5

6

7

24 48 72 96 120 144 168 192 216 240

Prod

uctiv

ity (U

gh

)

Time-course (hours)

Invertase(d)









Data Availability




Acknowledgments


References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Data Availability




Acknowledgments


References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Documents

-Fructofuranosidase and –D-Fructosyltransferase from New