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SUMMARY
X ylan is a com plex and m ajor hetero polysaccharide constituent o f
hem icellulose, w hich is present in the form o f arabinoxylan in softw ood and grasses,
and as O-acetyl-4-O -m ethylglucurono-P-D-xylan in hardwood. T his heteropolym er
containing a backbone o f P-1,4 linked xylopyranosyl units is substituted w ith L-
arabinosyl, acetyl, feruloyl, p-coumaroyl and glucuronyl residues. Due to the
heterogeneity and com plex chem ical nature o f xylan, its hydrolysis into sim pler
constituents requires synergistic action o f w ide spectrum o f enzym es. M ost important o f
these enzym es is endoxylanase (EC 3.2.1.8) that cleaves P-1,4 linked xylose backbone,
w hile P-xylosidase (EC 3.2.1.37) hydrolyses xylo-oligom ers. In addition, a variety o f
debranching enzym es, i.e. a-arabinofuranosidase (EC 3.2.1.55), acetyl xylan esterase
(EC 3.1.1.72) and other hem icellulolytic enzym es, a-g lucuronidases (EC 3.2.1.139), a -
galactosidases (EC 3.2.1.22) and P-m annosidases (EC 3.2.1.25) are required for
effective utilization o f hem icellulosic fraction.
M any m icroorganism s, including bacteria, actinom ycetes as w ell a s fungi have
been investigated fo r their ability to produce endoxylanases that can degrade P-1,4-
xylan in random fashion, yielding a series o f linear and branched oligosaccharide
fragm ents. O f these therm ophilic/therm otolerant fungal strains are o f prim e importance
being source o f therm ostable enzym es, w hich are important for industrial applications
because o f possible econom ic benefits o f being able to carry out catalysis a t elevated
tem peratures. In recent years xylanases have received a great deal o f research attention
particularly because o f their biotechnological potential in various industrial processes
such as food, feed, biofuels and pulp & paper industries. Currently, the m ost prom ising
application o f xylanases is in the pre-bleaching o f pulps m ainly because o f a desire to
replace chlorine as a bleaching agent. Xylanases prom ote bleaching through the
hydrolysis o f relocated and re-precipitated xylan on the surface o f pulp fibre which
results in better chem ical penetration and consequently im proved lignin extractability.
This application requires that the xylanases should be active and stable at high
tem perature under alkaline conditions. Therefore search for novel m icrobial strains that
can produce h igh levels o f therm ostable alkaline active xylanases w ith an ability to
Summary
remove xylan from different kinds o f pulps is desirable. Owing to immense importance
o f alkaline active thermostable xylanases, this study was designed with the following
objectives:
• Isolation, screening and molecular characterization o f xylanase producing
thermophilic/thermotolerant fungi.
• Optimization o f xylanase production by novel fungal strain Malbranchea flava
using solid state fermentation (SSF) by one variable at a time and statistical
response surface methodology approaches.
• Study the regulation and expression o f xylanases and acetyl esterases by
Malbranchea flava.
• Purification and characterization o f xylanase isoforms and acetyl esterase from
Malbranchea flava.
• Evaluation o f the produced xylanases for bleaching o f the pulp.
6.1 Screening and molecular characterization o f therm ophilic/therm otolerant
fungi
Sixteen thermophilic/thermotolerant strains namely Acrophialophora nainiana
MTCC 6662, Aspergillus caespitosus MTCC 6326, Chaetomium thermophile MTCC
4891, Corynascus sepedonium MTCC 6490, Emericella nidulans var. lata MTCC
6327, Emericella nidulans var. nidulans MTCC 6339, Humicola fuscoatra MTCC 6329,
Humicola insolens MTCC 4617, Malbranchea flava 4889, Melanocarpus albomyces
MTCC 3922, Mucor indicus MTCC 6333, Myceliophthora sp. MTCC 6661,
Penicillium janthinellum MTCC 6564, Rhizomucor pusillus MTCC 4895, Thermoascus
aurantiacus MTCC 4890, and Thermomyces lanuginosus D2W3 isolated from
composting soils were included in the present study. These isolates were identified on
the basis o f morphological and molecular characteristics. The molecular
characterization was based on Amplified rDNA Restriction Analyses (ARDRA) o f the
18S rDNA as well as sequence analysis o f ITSI-5.8S-ITSII and D1/D2 hyper-variable
region of 26S rDNA. Thel8S rDNA amplified PCR product from different fungal
isolates ranged between 1.6-1.7 Kb in size was digested with Mbol, H infl and Rsal to
study the restriction pattern. The dendrogram constructed by combined cluster analysis
128
Summary
o f restriction pattern obtained by M bo\, ///n fl and Rsal revealed th ree d istinct clades
w ith m em bers o f m ucorales, i.e., R. pusillus and M indicus form ing an ou t group. O ther
14 fungal strains w ere resolved in tw o distinct clades. The other clade w as characterized
w ith M. Jlava show ing an early divergence, while, M y celiophthora sp. and C.
sepedonium show ing h igh sim ilarity coefficient w ere clubbed together. The
phylogenetic trees constructed on the basis gene sequences o f ITSI-5.8S-ITSII region
and D1/D2 hyper-variable region o f 26S rDN A, show ed sim ilar topology. However,
ITS gene tree w as m ore robust as m ore nodes w ithin ITS tree received higher measure
o f support from bootstrapping. M oreover, the branch lengths show ed deeper nesting and
better resolution in ITS tree as com pared to D1/D2 hyper-variable region o f 26S rDNA.
The dendrogram based on ITS sequence analysis show ed that therm otolerant fungi
belonging to A spergilli and Penicillii and therm ophilic fungi w ere clustered separately.
Therm ophilic fungal isolates, i.e., T. lanuginosns, M. Jlava and M. albomyces,
producing alkaline active xylanases show ed m uch earlier phylogenetic divergence as
com pared to other isolates included in the study. All the therm ophilic and
therm otolerant strains w ere screened for xylanase production. T he results showed that
the strains included in the study produced m ultiple xylanase. Therm ophilic fungal
isolates T. lanuginosus, M. Jlava, and M yceliophthora sp., produced 9556, 3475 and
633.7 (U /g DW substrate) xylanase, respectively. The xylanases produced by these
three isolates w ere characterized to be alkaline active.
6.2 Biobleaching o f paper pulp
T he bleaching o f D ecker pulp w ith the xylanases produced by therm ophilic and
therm otolerant fungal strains w as carried out. The treatm ent o f pulp w ith xylanase from
M. Jlava resulted in m axim al release o f chromophores (237, 254 and 280 nm). The
released chrom ophores at these wavelengths w ere also high in the pulp treated with
xylanase from 71 lanuginosus, M. albomyces and A. caespitosus, though it was
appreciably less than that observed in M. Jlava. The increase in brightness o f the
resultant hand sheets obtained after treatm ent o f pulp w ith xylanases o f M. Jlava, 71
lanuginosus, M. albomyces and A. caespitosus w as 2.04, 2.07, 1.81 and 1.63% ISO
units, respectively.
129
Summary
6.3 Optimization o f culture conditions for hemicellulase production by
M albranchea Jlava
A novel thermophilic fungal isolate Malbranchea flava which showed distinct
phylogenetic origin and found to be a good source o f xylanase. Based on its capability
to enhance brightness o f pulp by 2.04% ISO units, it was taken up for further
optimization o f culture conditions for improving the production o f hemicellulases. The
initial optimization by classical one variable at a time approach resulted in improved
levels o f hemicellulase production on solidified culture medium containing sorghum
straw (1 mm particle size) as a carbon source and casein (2%) as nitrogen source
inoculated with a spore suspension (1.2 *107/g substrate) at a moisture content o f 80%.
The optimal pH and temperature were observed to be 7.0 and 40°C, respectively. Under
these conditions the culture produced xylanase (14900 U/g DW substrate), P-xylosidase,
(9.9 U/g DW substrate), acetyl esterase (52 U/g DW substrate) and ri-L-
arabinofuranosidase (2.980 U/g DW substrate). Further optimization was carried out
using response surface methodology based on Box-Behnken design o f experiments to
study effect o f interactions between casein concentrations, inoculum age and inoculum
level as independent variables on the production o f hemicellulases. Regression analysis
showed that the R2 values computed for xylanase, P-xylosidase, acetyl esterase, and a-
L-arabinofuranosidase were 93.85, 93.79, 98 and 88.24%, respectively, showing their
fitness in predicting the effect o f casein concentration, inoculum age and inoculum level
on xylanase, p-xylosidase, acetyl esterase, and a-L-arabinofiiranosidase production
under SSF by M. flava. Furthermore significant F values for the model and insignificant
lack o f fit also showed the robustness o f the experimental design to explain the obtained
results. It was found that optimal enzyme activities, i.e., 16,078, 10.97, 68.91, 3.786
(U/g DW substrate) o f xylanase, p-xylosidase, acetyl esterase (AE) and a-L-
arabinofuranosidase, respectively, were produced on solidified culture medium. Using
combination o f one factor at a time and RSM optimization approaches, 2.26, 3.12,6.02
and 3.26 folds increase in the levels o f xylanase, P-xylosidase, acetyl esterase (AE) and
a-L-arabinofuranosidase, activities, respectively, when compared to those under
unoptimized conditions were achieved. The culture under optimal process conditions
produced, 8 functionally distinct xylanases, in addition 5 ,1 0 and 6, P-xylosidase, acetyl
130
Summary
esterase (A E) and a-L-arabinofuranosidase isoforms, respectively, as revealed by
zymogram developed against native PAGE were observed.
6.4 Two dimensional electrophoretic profiling o f secretome of Malbranchea
Jlava
T he crude enzym e extract produced under optim ized and unoptim ized culture
conditions was resolved by 2DE. Seventy tw o distinct protein spots w ere detected in the
pi range o f 3.0-5.6. T he culture under optim al conditions produced spectrum o f proteins
that show ed d istinct differences in the num ber and intensity o f protein spots when
compared to the one grown under unoptim ized conditions. Expression o f few o f the
proteins w as up-regulated under the optim ized condition w hile few others were down-
regulated. T he partial secretom e characterization using peptide m ass fingerprinting
(LC/M S/M S) identified the protein spots as xylanases (GH11) and
cellobiodehydrogenase, cellobiohydrolase constituting the com ponents o f cell wall
degrading enzym es. In addition to these a m itochondrial and cytoplasm ic glycyl-tRNA
synthase protein and tw o hypothetical proteins w ere also identified.
6.5 Induction and regulation o f xylanase and acetyl esterase production in
Malbranchea JlavaT he induction o f xylanase and acetyl esterase w as studied in presence o f various
com plex carbohydrates (oatspelt xylan, rice straw , sorghum straw, w heat straw, wheat
bran, com cob, and bagasse), m onosaccharides, disaccharides, alcohols and glycerol as
carbon sources under shake flask cultures. The zym ogram s developed from the extracts
resolved on PA G E show ed differential expression o f m ultiple xylanases in presence o f
these carbon sources. T he results show ed that the presence o f oat spelt xylan resulted in
induction o f e igh t electrophoretically distinct xylanases ou t o f w hich three o f the
xylanase isoform s (Ic, Id and Ig) w ere constitutive as these w ere expressed even in
basal m edium devoid o f any sugar/carbohydrate. However, in presence o f com cob,
bagasse, rice straw , sorghum straw and w heat bran, five xylanase isoforms were
expressed. W hereas, in presence o f arabinose, cellobiose and fructose as carbon source
only three constitutive xylanase isoform s w ere expressed. On the o ther hand xylose as a
131
Summary
carbon source supported the expression o f all five isoforms that were produced in
presence o f complex lignocellulosics (com cob, bagasse, rice straw, sorghum straw and
wheat bran). However, in presence o f glucose, glycerol, ethanol, methanol and propanol
four xylanase isoforms i.e., three constitutive and an additional inducible isoform (Ie)
was expressed. The addition o f monosaccharides, disaccharides, alcohols and glycerol
to xylan containing medium led to repression o f xylanase production. The repressive
effect o f arabinose addition however was most severe as it resulted in low xylanase
activity (12.75 units/mg protein) when compared to 85.65 (units/mg protein) observed
in presence o f xylan as sole carbon source. The zymogram showing the production
profile o f xylanase by M. flava on oat spelt xylan as carbon source revealed sequential
induction o f xylanases. The culture showed expression o f two constitutive isoforms (Ic,
Id) and an inducible isoform (Ih) after 24 h o f incubation. This was followed by
expression o f isoform (Ig) after 48 h. Next in the sequence was the expression o f
xylanase isoform (le) which was observed after 72 h o f incubation, whereas, after 96 h
o f incubation the culture expressed all eight xylanase isoforms.
The zymogram developed against extract resolved by PAGE showed that the culture
produced multiple esterases where five o f the isoforms (la, lb, Id, I f and lh) were expressed
constitutively in medium devoid o f any sugar/ complex carbohydrate. Maximal expression o f
seven esterase isoforms (la, lb, Ic, Id, Ie, I f and Ig) were produced in presence o f rice straw.
Glycerol as carbon source supported high expression o f esterase followed by fructose xylose
and glucose after 120 h o f incubation. The level o f esterase in presence o f arabinose, lactose,
cellobiose and alcohols was very low and was almost similar to that observed in basal
medium (control). However expression o f five acetyl esterase isoforms was observed in
presence o f all monosaccharides, disaccharides, alcohols and glycerol that were used in the
study. No repression in acetyl esterase activity was observed when fructose was added to the
medium. However addition o f other monosaccharides, disaccharides, alcohols or glycerol to
the xylan containing medium repressed the acetyl esterase production and the observed levels
of activity were almost similar to that observed in presence o f basal medium.
132
Summary
6.6 Purification and characterization of xylanases from Malbranchea flava
Two m ajor alkaline active xylanases (MFX I and MFX II) with molecular
masses o f 25.2 and 30 kDa and p is o f 4.5 and 3.7, respectively were purified to
homogeneity using ion exchange and hydrophobic interaction chromatography. The
purified xylanases (M FX I and MFX II) showed specific activity o f 1800 and 961.53
pmol m in '1 mg protein*1 corresponding to 21.304 and 11.39 fold purification,
respectively. Liquid chromatography/mass spectrometry (LC-M S/M S) analysis o f
tryptic digests o f MFX I and MFX II revealed similarity to endoxylanases from T.
lanuginosus and Penicillium chrysogenum belonging to GH family 11 and GH family
10, respectively. The match score was found to be 114 in case o f MFX I, however it
was 58 in case o f MFX II.
6.6.1 Temperature, pH optima and stability
The xylanases (MFX I and MFX II) were optimally active at pH 9.0 and at
70°C, exhibited a half-life o f 4 h at 60°C, while at 70°C and at pH 9.0, MFX I and II
lost 73 and 53% o f their activity, respectively, after 30 min.
6.6.2 Effect o f metal ions and other compounds
Both the xylanases were found to be resistant to inhibition by metal ions. The
enzyme activity o f MFX I was promoted by EDTA and Ca2+, whereas, DTT, (3-
mercaptoethanol, Mg2+, SDS and Fe2+, Zn2+ and Mn2+ slightly reduced the activity. On
the other hand, M FX II was moderately inhibited by Zn2+ and Fe2+while its activity was
positively modulated by other metal ions and chemicals. Complete inhibition o f both
the purified xylanases was observed in presence o f N-Bromosuccinimide (NBS)
indicating the presence o f tryptophan residues at active site o f MFX I and MFX II.
6.6.3 Substrate specificity
The endo-activity o f xylanases was tested by reducing sugar assay on a number
o f complex substrates and it was found that MFX II was more substrate specific as
compared to MFX I. The purified MFX I showed maximal activity on rye arabinoxylan
(RAX) followed by wheat arabinoxylan (WAX) and oat spelt xylan (OSX), as
compared to that on birchwood xylan (BWX) as substrate. However, MFX II showed
133
Summary
almost similar activity against different xylan types. Further MFX I showed low levels
of activity on lichenan, but MFX II was hardly active on this polysaccharide. MFX I
showed no activity against any of the pNP substrates tested, however, MFX II was
active on pNP cellobioside which is a characteristic property o f GH10 xylanases. The
purified xylanases MFX I and MFX II showed higher affinity towards larchwood xylan
(LWX) as evident from the comparatively lower Km values o f 1.2S and 3.7 mg/ml,
respectively. Kca/Km values suggested that the xylanases preferentially hydrolyzed
RAX. The results showed that 40% of the total purified MFX I adsorbed to avicel
(crystalline cellulose), however, MFX II did not show any adsorption to avicel
indicating putative absence o f carbohydrate binding module in MFX II.
6.6.4 Hydrolysis of xylan
The Thin Layer Chromatography (TLC) analysis showed that purified xylanases
(MFX I and MFX II) showed distinct mode o f action and hydrolysis product profiles
with BWX, OSX, LWX, as well as WAX and RAX. The resultant hydrolysis products
obtained with MFX I and II differed from each other. As the xylan degradation
occurred, a large amount o f intermediate products (X2, X3, X4 and X5) was obtained,
with a small amount of xylose (Xi). The hydrolysis o f BWX (which is primarily a
methylglucouronoxylan) with MFX I resulted in xylobiose (X2) as the major product
followed by xylotriose (X3) as the other main product. In addition, positional isomer
iso-Xs putatively identified aldopentauronic acid was detected which is a characteristic
property xylanase (GH11). The hydrolysis of BWX with MFX II, however, showed the
presence of X2 as the major hydrolysis product followed by xylotetraose (X4) and iso-
X4 putatively identified as aldotetrauronic acid a characteristic property o f xylanase
(GH10). Similarly the differences in the hydrolysis product profile obtained with
arabinoxylans (OSX, RAX and WAX) were observed. The hydrolysis product profile
obtained by TLC was confirmed by HPLC.
6.7 Purification and characterization of acetyl esterase from Malbranchea Jlava An acetyl esterase with a molecular mass and pi of 42 kDa and 3.0, respectively
was purified to homogeneity using hydrophobic interaction chromatography, chromato-
focusing and ion exchange chromatography. The purified esterase showed specific
134
Summary
activity o f 17.6 pm ol min*1-mg protein*1 w ith 10.11 fold purification. The purified
esterase w as optim ally active a t S0°C and a t pH 6.0 and appreciably stable a t 60°C at
pH 6.0 and 7.0 fo r 4 h . A t 70°C , ~ 80% o f activity w as still recoverable after 4 h o f
incubation. T he activity o f purified esterase w as positively m odulated by all
m onovalent, divalent m etal ions as w ell a s SDS, PM SF, DTT and m ercaptoethanol. The
purified esterase w as m ost active against pN P acetate and it could also recognise pNP
butyrate bu t show ed very low activity against pN P ferulate. T he estim ated K„, Vmax and
kcat values fo r the purified esterase w ith pN P acetate as a substrate w ere 7.42 m M , 2.78
pm ol m in 'm g p ro te in '1 and 233.52 m in '1, respectively. T he purified esterase on the
basis o f characterization w as classified as carbohydrate esterase (CE-4).
135