A Aman, S Ul Qader, S BAno, S Iqbal, A Azhar

ISPUB.COM The Internet Journal of MicrobiologyVolume 7 Number 1

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Production of Commercially Important Glucansucrase froma Newly Isolated Strain of Leuconostoc mesenteroides AA1A Aman, S Ul Qader, S BAno, S Iqbal, A Azhar

Citation

A Aman, S Ul Qader, S BAno, S Iqbal, A Azhar. Production of Commercially Important Glucansucrase from a NewlyIsolated Strain of Leuconostoc mesenteroides AA1. The Internet Journal of Microbiology. 2008 Volume 7 Number 1.

Abstract

Glucansucrase is an industrially important extracellular enzyme produced by Leuconostoc mesenteroides. This glucansucrase iswidely used for glucan production. Both the product and the enzyme have received increased attention because of their widerange of applications. Due to this, isolation of new strains of L.mesenteroides for glucansucrase production is of great interest. In this work eleven different strains of Leuconostocmesenteroides were screened and isolated from locally available fruits and vegetables. A new glucansucrase producing strain ofLeuconostoc mesenteroides AA1 have been isolated from Brassica oleracea var Capitata L. and was selected on the basis ofhigh enzyme productivity. Various experimental conditions and aspects regarding glucansucrase production were studied. Thisnewly isolated strain optimally produces maximum glucansucrase enzyme after 8 hours of incubation in a new mediumdesigned for enzyme production. Maximum glucansucrase was produced at a sucrose concentration of 2.5% when incubated at25°C and pH 7.5. Under these conditions 53.0DSU/ml/hr units were produced. This new strain Leuconostoc mesenteroidesAA1 could be used for the commercial production of glucansucrase and can also facilitate glucan production on large scale.

INTRODUCTION

Glucansucrase [2.4.1.5] commonly known as dextransucraseis a glucosyltransferase, that is responsible for glucan(dextran) synthesis from sucrose and it catalyzes the transferof glucosyl residue from sucrose to the growing glucanpolymer, liberating fructose as a byproduct (Sidebotham,1974).

Glucansucrase and glucan have applications in variousindustries like pharmaceutical, food, cosmetic, agricultural,as well as in photography and mining. Specific molecularweight fractions of glucans are used in medicine,flocculation, stabilization, lyophilization, protective colloidsin blood-expanders and cosmetic ingredients formulation(Kim & Day, 1994; Leather et al. 1995; Shamala & Prasad,1995; Sutherland et al. 1996). Crosslinked glucans are usedas gel permeation matrices in research and industries forseparation purposes of various products (Alsop, 1983).

Several species of genera Leuconostoc, Lactobacillus andStreptococcus have been found to synthesize glucansucrase.Its expression is constitutive in Streptococcus strains (Ciardiet al. 1997), while it is inducible in Leuconostoc strains(Chellapandian et al. 1998).

Attempts have been made to improve production of both

glucan and glucansucrase by various species of Leuconostocmesenteroides and for this different media compositions andfermentation conditions have been used (Kobayashi et al.1986; Lawford et al. 1979; Lopez & Monsan, 1980).

Strains of Leuconostoc mesenteroides require high sucroseconcentration, temperature range from 25-30°C and aninitial fermentation pH of 7.0 for maximum enzymeproduction (Tsuchiya et al. 1952; Santos et al. 2000).Moreover, the rate of the cell death follows an exponentialdecay-law for different values of pH. The optimum pH forcell survival has been found to be pH 5.0, whereas cell deathoccurs rapidly at extreme pH values (Kim et al. 2000). Thepresence of manganese, magnesium and calcium salts in themedia affects the enzyme activity and increases the glucanyield (Qader et al. 2001). Sugar beet molasses and wheatbran have been used as a carbon source for enzymeproduction (Behravan et al. 2003).

The objectives of this study were to discover and identify themost potential glucansucrase producing bacterium fromlocally isolated strains. Glucansucrase and glucan fromLeuconostoc mesenteroides have gained worldwide researchinterest for their diverse properties and potential industrialuses. In this article we have reviewed the factors

Production of Commercially Important Glucansucrase from a Newly Isolated Strain of Leuconostocmesenteroides AA1

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contributing to the optimization of conditions for optimalglucansucrase production using a locally isolated strain of L.mesenteroides AA1.

MATERIALS AND METHODS

Isolation and screening of strain: Bacterial culture wasisolated from Brassica oleracea var Capitata L. (Cabbage)purchased from local market using enrichment mediatechnique. Initially the culture was cultivated on MRSmedium (BioM Laboratories, USA). The isolates werescreened for dextran producing strains by inoculating inmedium containing: (g l–1): Sucrose, 50.0; Tryptone, 10.0;Yeast extract, 1.0; K2HPO4, 2.5; the pH was adjusted at 7.0

and autoclaved at 121ºC for 15 minutes. After autoclaving0.005 % sodium azide was added aseptically. Bacterial strainwas selected showing highly viscous slimy growth onsucrose agar plate.

Strain identification: Leuconostoc mesenteroides AA1 wasselected for optimization and characterization due to highenzyme activity and dextran producing characteristic. Thebacterial strain AA1 was isolated and identified by the usualmethods (Holt, 1994) and was maintained on sucrose brothmedium at 4C.

Culture media and growth conditions: For fermentationpurpose, the organism was grown at 25C in a mediumcontaining (g l–1): Sucrose, 25.0; Bacto-peptone, 5.0; yeastextract, 5.0; K2HPO4, 15.0; MnSO4.H2O, 0.01; NaCl, 0.01;

MgSO4.7H2O, 0.01; CaCl2.2H2O, 0.1. The pH of the medium

was adjusted to 7.5 before sterilization at 121ºC for 15minutes.

Enzyme isolation: Sterile sucrose broth (10 ml) wasinoculated by a loopful culture of L. mesenteroides AA1 andincubated at 25C for 24 hours. This 10 ml of 24 hours oldculture was then transferred into 90 ml of same brothmedium and incubated at 25C for 8 hours. After incubation,the cell free supernatant containing the enzyme was obtainedby centrifugation at 35000xg for 15 min at 0C and wasstored at 20C.

Enzyme assay: Glucansucrase activity was determined bymeasuring the reducing sugar (Kobayashi & Matsuda, 1974).Units of glucansucrase activity are represented in DSU.“One unit of enzyme activity was defined as the enzymequantity that converts 1.0 milligram of sucrose into fructoseand glucan in 1.0 hour at 35 C using citrate phosphatebuffer pH 5.00” (Lopez & Monsan, 1980).

Protein assay: Total protein of the cell-free supernatant wasdetermined by the Lowry’s method using bovine serumalbumin as a standard (Lowry et al. 1951).

Effect of time, substrate concentration, temperature, and pHon glucansucrase production:

Different conditions like time course, pH, substrate andtemperature was optimized for maximum glucansucraseproduction in fermentation medium. For this purpose,culture medium was incubated for different time intervals(0-48 hours) and for the determination of glucansucraseproductivity; total protein, glucansucrase activity, final pHof fermented culture broth and wet cell mass weredetermined.

After time course, sucrose concentration for the optimumenzyme production was varied from 0.5% to 3.5% in theculture media. For optimum temperature fermentation brothswere incubated at various temperatures ranging from 15C to45C with an increment of 5C. Where as for pH optima, pHof the culture broths was adjusted from 4.5-9.5 by theincrement of 0.5 before autoclaving.

Measurements of all the experiments were determined inthree independent experiments at least, each performed withthree replications.

RESULTS AND DISCUSSION

In this study some of the physicochemical parametersaffecting bacterial growth were controlled primarily by thechanges of environment of the cultural medium such asincubation temperature, pH of medium and addition of sugarto the medium composition. These factors not only influencethe cellular growth but also the enzyme production.

TAXONOMIC ASSESSMENT OF THE ISOLATEDSTRAIN

Eleven bacterial isolates identified as Leuconostocmesenteroides were isolated from the locally availablefermented fruits and vegetables (Table 1). The isolates werescreened for glucansucrase productivity. The taxonomicassessment of the isolated strain AA1 confirmed thecharacteristic properties of Leuconostoc mesenteroides andwas selected for further studies. Among various strainsisolated, bacterial strain AA1 was selected showing highlyviscous slimy growth on 5% sucrose agar plate (Fig. 1)whereas slimy, shiny transparent colonies were observed on2% sucrose agar plate (Fig. 2). The characteristics of theisolated strain are summarized in table 2. A strain of


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Leuconostoc mesenteroides AA1 was confirmed accordingto Bergey’s Manual of Systematic Bacteriology (Holt,1994).

Figure 1

Table 1: strains isolated from various sources and theirenzyme activities

Figure 2

Table 2: Morphological and Physiological Characteristics ofthe Isolated Strain of AA1

Figure 3

Figure 1: Characteristic Slime of Glucan Produced on 5 %Sucrose Agar Plate by AA1

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Figure 2: Slimy, Shiny Transparent Colonies on 2 % SucroseAgar Plate by AA1

SELECTION OF MEDIUM

After isolation of L. mesenteroides AA1, the medium wasselected for maximum glucansucrase productivity. Table-3shows the compositions of four different media used. Theexperimental culture medium-4 was compared withpreviously reported medium-1 (Lopez & Monsan, 1980),medium-2 (Tsuchiya, 1952) and medium-3 (Qader et al.2001). Among four different media prepared, medium-4


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supported maximum extracellular glucansucrase production.When this experimental medium-4 was compared with theother reported media it was observed that this mediumsupported maximal enzyme production. This medium is notpreviously reported and was designed to achieve a greaterinduction of glucansucrase from L. mesenteroides AA1(Table 4). In this medium 53.0 DSU/ml/hr activity with aspecific activity of 20.3 DSU/mg was recorded which is 5.4,4.6, and 1.6 times higher than medium-1, medium-2 andmedium-3, respectively. This medium was supplementedwith varying concentration of mineral ions along with thebasic nutrient constituents, which had a positive effect onenzyme productivity. Previously the basic constituents,concentration of sucrose, yeast extract, and K2HPO4 were

chosen as critical variables to improve enzyme productionby different strains of L. mesenteroides (Chellapandian et al.1998; Kitaoka & Robyt, 1998; Rodrigues et al. 2003).

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Table 3: Media Compositions for Maximum GlucansucraseProduction by AA1

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Table 4: Selection of Medium for Maximum GlucansucraseProduction

OPTIMIZATION OF GLUCANSUCRASEPRODUCTION

Physicochemical parameters that influence the cellulargrowth and enzyme production were studied including timecourse for enzyme production, medium composition,substrate concentration, temperature and pH.

EFFECT OF TIME COURSE ON BACTERIALGROWTH AND ENZYME PRODUCTION

In the first step the time course for maximum enzymeproduction was studied. Growth time of the bacteria infermentation broth always plays an important role and it wasobserved that the cell growth of L. mesenteroides AA1 andenzyme production is a function of time. The relationshipbetween cell growth and enzyme production duringfermentation was observed for L. mesenteroides AA1 and itwas recorded that the biomass increased within 2 hours andreached a maximum at 8 hours of incubation with a wet cellmass of 0.85g/dl. As the wet cell mass increased with time(Fig. 3) maximum enzyme production was observed at thelast stage of the exponential phase (53 DSU/ml/hr) and theearly stationary phase of the cell growth (8 hour). Thestationary phase remained up to 18 hour and was followedby a decline phase. Maximum enzyme production at 8 hoursof fermentation is a unique property of this strain. Earlier ithas been reported that L. mesenteroides PCSIR-3 showsmaximum glucansucrase production in 18 hours while L.mesenteroides NRRL B-512F in 12 hours (Qader et al.2001).

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Figure 3: Time course of glucansucrase production and cellgrowth in fermentation medium

EFFECT OF SUBSTRATE CONCENTRATION

Among many factors that influence that influence theformation of bacterial enzyme, presence of particularsubstrate is known to be the most important factor. L.mesenteroides AA1 was grown in the medium containingsucrose as a carbon source to determine its effect oninduction of extracellular glucansucrase. When sucrose


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concentration was varied from 0.5% to 3.5% duringcultivation maximum yield was achieved at 2.5% (Fig. 4).2.5% of substrate concentration greatly influenced maximumglucansucrase production. During cultivation, the bacterialcells multiply and use sucrose as a carbon source. It has beenreported that approximately 85% of sucrose is consumedduring glucansucrase production and feeding of sucrose tothe culture, increases the yield of glucansucrase up to certainlimit. Further increase of sucrose concentration in medium isnot utilized by the organism as a carbon source but isconsequently accumulated in the medium (Lopez & Monsan,1980; Neely & Nott, 1962). It has also been reported that theproduction of glucansucrase gradually declines due to theformation of glucan in the medium that could increase theviscosity of the culture medium and this viscous materialretards cells duplication and enzyme production (Lopretti etal. 1999; Michelena et al. 2003. Same was noticed for L.mesenteroides AA1 and it was observed that above thisconcentration the cells were not easily separated due to theincrease in the viscosity of the cultivation medium. Sucroseconcentration was varied for production of glucansucrasefrom L. mesenteroides NRRL B512 (f) and showed that 4%sucrose in culture medium was ideal (Santos et al. 2000), Ithas also been reported that 2% sucrose is required formaximum glucansucrase production in the cultivation media(Qader et al. 2001; Lopretti et al. 1999; Michelena et al.2003).

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Figure 4: Effect of substrate concentration on glucansucraseproduction

EFFECT OF TEMPERATURE

Temperature may affect the metabolic pattern, nutritional

requirements, and composition of bacterial cells. In addition,temperature also affects the rates of all cellular reactions.When L. mesenteroides AA1 was cultivated at varioustemperatures maximum enzyme production was observed at25C and the results showed that the strain is temperaturesensitive and enzyme is thermo labile at higher temperatures(Fig. 5). After 25C a mark decline in glucansucraseproduction was observed i.e. at 30C, 55% less enzymeproduction was observed and at 45C only 4 DSU/ml/hractivity was examined. It was also observed that L.mesenteroides AA1 is not capable of producingglucansucrase at temperature below 15C.

Different temperature maxima for glucansucrase productionhave been reported previously (Santos et al. 2000; Itaya &Yamatoto, 1975; Lopez-Mungia et al. 1998; Monsan et al.1997).

Figure 9

Figure 5: Effect of temperature on glucansucrase production

EFFECT OF PH

Enzyme production was also analyzed at different pH valuesand the optimum glucansucrase production by L.mesenteroides AA1 was achieved when the pH of thecultivation medium was adjusted to 7.5 (Fig. 6).Glucansucrase production was found maximum at pH 7.5(53.0 DSU/ml/hr) while 31.14 and 20 DSU/ml/hr weredetected at pH 7.0 and 8.0, respectively. At slight acidic andalkaline pH values, enzyme production was retarded and L.mesenteroides AA1 failed to produce extracellularglucansucrase at pH 5.0 and 9.0. At these extreme pHconditions morphological and physiological changes occurin the cell that renders enzyme production. Earlier variouspH maxima have been reported for the fermentation mediafor glucansucrase production (Tsuchiya et al. 1952; Loprettiet al. 1999; Lopez-Mungia et al. 1998; Dols et al. 1998). The


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results show that the production of the enzyme depends,inter alia, on pH.

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Figure 6: Effect of pH on glucansucrase production

CONCLUSION

The experimental data of culture conditions optimization forLeuconostoc mesenteroides AA1 is presented in this work.The newly isolated strain Leuconostoc mesenteroides AA1showed higher extracellular glucansucrase production. Inconclusion it seems highly probable that this extracellularglucansucrase is significant from an industrial perspective. Itis therefore suggested that Leuconostoc mesenteroides AA1can be used for the elaboration of the glucansucrase on alarge scale, which can allow the commercial production ofglucan in a cell free bioreactor.

References

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Author Information

Afsheen Aman, PhDPharmaceutical Research Center PCSIR Laboratories Complex Karachi-75280, Pakistan

Shah Ali Ul Qader, PhDInstitute of Sustainable Halophytes Utilization University of Karachi Karachi-75270, Pakistan.

Saeeda BAno, M.Sc.Pharmaceutical Research Center PCSIR Laboratories Complex Karachi-75280, Pakistan

Samina Iqbal, M.Sc.Pharmaceutical Research Center PCSIR Laboratories Complex Karachi-75280, Pakistan

Abid Azhar, PhDInstitute of Biotechnology & Genetic Engineering, University of Karachi Karachi-75270, Pakistan.

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A Aman, S Ul Qader, S BAno, S Iqbal, A Azhar