Short Communication Partitioning studies of L-glutaminase production by Bacillus cereus MTCC 1305 in different PEG–salt/dextran Priyanka Singh, Rathindra Mohan Banik ⇑ School of Biochemical Engineering, Institute of Technology, Banaras Hindu University, Varanasi, India article info Article history: Received 6 January 2012 Received in revised form 14 March 2012 Accepted 14 March 2012 Available online 21 March 2012 Keywords: Bacillus cereus MTCC 1305 L-Glutaminase PEG 4000/dextran T500 pH Temperature abstract Partitioning studies of L-glutaminase production by Bacillus cereus MTCC 1305 was carried out in different PEG–salt/PEG–dextran system. The partitioning value of L-glutaminase increased with increasing molec- ular weight of PEG from 2000–4000 kDa and decreased with higher molecular weight of 6000 kDa. Phase system of PEG 4000 (8.5%)/dextran T500 (9.5%) was selected for the extractive fermentative production of L-glutaminase on the basis maximum partition coefficient (1.31). The production of L-glutaminase was found higher in top phase of ATPS (2.09 U/ml) than control media (1.42 U/ml). Overall production of L- glutaminase (1.83 U/ml) was found lower than top phase (2.09 U/ml) in ATPS system. The growth profile with short lag phase and higher cell concentration was obtained for ATPS. The partition coefficient of L- glutaminase increased with increase of system pH and temperature and optimum production was obtained at pH 7.5 and temperature 30 °C in top phase of PEG 4000/dextran T500 system. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Aqueous two-phase systems (ATPS) provide alternative extrac- tion process to conventional method for purification of biomole- cules by preferentially partitioning product biomolecules in one phase and interfering substances into other phase. The partitioning of product in one phase during extractive fermentation broth gets improved with careful adjustment of the phase composition and other physicochemical parameters. The biocompatibility of aque- ous two phase systems provides a very low interfacial tension be- tween the phases, which results in high mass transfer and ease of scale up. Aqueous polymer/polymer or polymer/salt two-phase systems have been shown to be useful for the extractive separation of biologic materials such as cells, organelles, proteins and nucleic acids. Recently, aqueous two-phase systems has led to their use for extraction and recovery of a variety of intracellular enzymes from disrupted cell broth (Kuboi et al., 1995), affinity purification of en- zymes and proteins (Rito-Palomares, 2004; Saravanan et al., 2008), purification of interferon from mammalian cell cultures (Guan et al., 1996). Polyethylene glycol (PEG)/salt system are particularly useful because of their low cost and ease of handling for many bio- molecules like protease (Lee and Chang, 1990; Hotha and Banik, 1997), a-amylase (Kim and Yoo, 1991; Stredansky et al., 1993), b-galactosidase (Kuboi et al., 1995), chitinase (Chen and Lee, 1995), endoglucanase (Sinha et al., 2000), alkaline phosphatase (Pandey and Banik, 2011), glucose oxidase (Singh and Verma, 2010). L-Glutaminase (L-glutamine amidohydrolase E.C. 3.5.1.2), a key enzyme for conversion of L-glutamine to glutamic acid, is commer- cially used as enzyme therapy for cancer especially acute lympho- cytic leukemia (Satomura et al., 2005) and as flavor enhancing agent in food industry (Nandakumar et al., 2003). The production and purification of L-glutaminase has been reported in many mi- crobes like Micrococcus luteus (Moriguchi et al., 1994), Bacillus pasteurii (Klein et al., 2002), Escherichia coli (Guan et al., 1996), Bacillus subtilis (Satomura et al., 2005). The extractive fermentation production of microbial L-glutaminase in aqueous two phase sys- tem has been not reported till now. The present study was focused on partitioning of L-glutaminase produced from Bacillus cereus MTCC 1305 in polymeric phase sys- tem composed of PEG X (X = 2000, 4000, 6000)/salts (magnesium sulfate, sodium sulfate, sodium citrate)/polymers (dextran 40, dex- tran T500). The effect of fermentation time, pH and temperature on the extractive production of L-glutaminase was also studied. 2. Methods 2.1. Materials B. cereus MTCC 1305 was obtained from Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chan- digarh, India. Poly ethylene glycol (PEG) of various molecular weights (2000 kDa, 4000 kDa and 6000 kDa) used were purchased from Sisco Research Laboratories, Mumbai, (India). The enzyme 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.03.046 ⇑ Corresponding author. Tel.: +91 542 2312232; fax: +91 542 2368428. E-mail address: [email protected](R.M. Banik). Bioresource Technology 114 (2012) 730–734 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Transcript
Bioresource Technology 114 (2012) 730–734
Contents lists available at SciVerse ScienceDirect
Partitioning studies of L-glutaminase production by Bacillus cereus MTCC 1305 was carried out in differentPEG–salt/PEG–dextran system. The partitioning value of L-glutaminase increased with increasing molec-ular weight of PEG from 2000–4000 kDa and decreased with higher molecular weight of 6000 kDa. Phasesystem of PEG 4000 (8.5%)/dextran T500 (9.5%) was selected for the extractive fermentative production ofL-glutaminase on the basis maximum partition coefficient (1.31). The production of L-glutaminase wasfound higher in top phase of ATPS (2.09 U/ml) than control media (1.42 U/ml). Overall production of L-glutaminase (1.83 U/ml) was found lower than top phase (2.09 U/ml) in ATPS system. The growth profilewith short lag phase and higher cell concentration was obtained for ATPS. The partition coefficient of L-glutaminase increased with increase of system pH and temperature and optimum production wasobtained at pH 7.5 and temperature 30 �C in top phase of PEG 4000/dextran T500 system.
� 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Aqueous two-phase systems (ATPS) provide alternative extrac-tion process to conventional method for purification of biomole-cules by preferentially partitioning product biomolecules in onephase and interfering substances into other phase. The partitioningof product in one phase during extractive fermentation broth getsimproved with careful adjustment of the phase composition andother physicochemical parameters. The biocompatibility of aque-ous two phase systems provides a very low interfacial tension be-tween the phases, which results in high mass transfer and ease ofscale up. Aqueous polymer/polymer or polymer/salt two-phasesystems have been shown to be useful for the extractive separationof biologic materials such as cells, organelles, proteins and nucleicacids. Recently, aqueous two-phase systems has led to their use forextraction and recovery of a variety of intracellular enzymes fromdisrupted cell broth (Kuboi et al., 1995), affinity purification of en-zymes and proteins (Rito-Palomares, 2004; Saravanan et al., 2008),purification of interferon from mammalian cell cultures (Guanet al., 1996). Polyethylene glycol (PEG)/salt system are particularlyuseful because of their low cost and ease of handling for many bio-molecules like protease (Lee and Chang, 1990; Hotha and Banik,1997), a-amylase (Kim and Yoo, 1991; Stredansky et al., 1993),b-galactosidase (Kuboi et al., 1995), chitinase (Chen and Lee,1995), endoglucanase (Sinha et al., 2000), alkaline phosphatase
ll rights reserved.
: +91 542 2368428.
(Pandey and Banik, 2011), glucose oxidase (Singh and Verma,2010).
L-Glutaminase (L-glutamine amidohydrolase E.C. 3.5.1.2), a keyenzyme for conversion of L-glutamine to glutamic acid, is commer-cially used as enzyme therapy for cancer especially acute lympho-cytic leukemia (Satomura et al., 2005) and as flavor enhancingagent in food industry (Nandakumar et al., 2003). The productionand purification of L-glutaminase has been reported in many mi-crobes like Micrococcus luteus (Moriguchi et al., 1994), Bacilluspasteurii (Klein et al., 2002), Escherichia coli (Guan et al., 1996),Bacillus subtilis (Satomura et al., 2005). The extractive fermentationproduction of microbial L-glutaminase in aqueous two phase sys-tem has been not reported till now.
The present study was focused on partitioning of L-glutaminaseproduced from Bacillus cereus MTCC 1305 in polymeric phase sys-tem composed of PEG X (X = 2000, 4000, 6000)/salts (magnesiumsulfate, sodium sulfate, sodium citrate)/polymers (dextran 40, dex-tran T500). The effect of fermentation time, pH and temperature onthe extractive production of L-glutaminase was also studied.
2. Methods
2.1. Materials
B. cereus MTCC 1305 was obtained from Microbial Type CultureCollection and Gene Bank, Institute of Microbial Technology, Chan-digarh, India. Poly ethylene glycol (PEG) of various molecularweights (2000 kDa, 4000 kDa and 6000 kDa) used were purchasedfrom Sisco Research Laboratories, Mumbai, (India). The enzyme
substrate, L-glutamine, Tris–HCl buffer and polymers (dextran 40and dextran T500) were purchased from Hi Media, Bombay (India).Magnesium sulfate, sodium sulfate and sodium citrate salts werepurchased from Qualigens, Bombay (India). All other reagents wereused of analytical grade.
2.2. Microorganism and culture maintenance conditions
The culture was grown in 50 ml media (pH 7.0) containing beefextract 1.0 g/l, yeast extract 2.0 g/l, NaCl 5.0 g/l, peptone 5.0 g/l,and agar 15.0 g/l at 35 �C. The organism was subcultured everymonth and maintained at 4 ± 1 �C. The production of L-glutaminasehas been studied in 100 ml semi-synthetic medium (pH 7.5) con-taining glucose 2 g/l, Na2HPO4�2H2O 6.0 g/l, KH2PO4 3 g/l, NaCl0.5 g/l, MgSO4�7H2O 0.5 g/l, CaCl2�2H2O 0.015 g/l and peptone2.0 g/l.
2.3. Assay of L-glutaminase
The glutaminase activity was estimated by modified method(Imada et al., 1973) in which reaction mixture (pH 7.5) containing0.5 ml of crude extract of enzyme, 0.5 ml of 0.04 M L-glutaminesolution, 0.5 ml of distilled water and 0.5 ml of 0.1 M phosphatebuffer was incubated at 37 �C for 30 min. The reaction was termi-nated by the addition of 0.5 ml of 1.5 M trichloroacetic acid to0.1 ml of the reaction mixture. Reagent blank and substrate blankwas also prepared subsequently. 3.7 ml of distilled water wasadded to 0.1 ml reaction mixture and then 0.2 ml of Nessler’s re-agent was added. The absorbance was measured at 450 nm after2 min. Standard graph using NH4Cl (12 � 10�4 M) was plotted asthe standard for computation of the concentration of ammonia.One unit of glutaminase activity was defined as enzyme requiredfor deamination of 1.0 lmol of glutamine per min per ml of en-zyme solution at pH 7.5, 35 �C (Curthoys and Watford, 1995).
2.4. Estimation of cell biomass
Cell biomass in the fermentation broth was quantified by dry-cell weight analysis and by measurement of the optical densityof the broth. For dry weight determinations, the cells were recov-ered by centrifugation at 4 �C, 8000 rpm and washed twice withdistilled water. The recovered biomass was dried to constantweight in an oven at 80 �C for 24 h. Absorbance was read at600 nm using a Shimadzu UV/Vis spectrophotometer for opticaldensity.
2.5. Physicochemical parameters for aqueous two phase system
Phase diagramme for aqueous two phase system was con-structed by stepwise titration of 25% (w/v) dextran T500 with fixedamounts of 20% (w/v) PEG 4000 and diluted with water to thepoint of disappearance of turbidity. One milliliter of cell extractwas added to each phase system, mixed thoroughly in orbital sha-ker for 20–30 min and allowed to separate for 40–50 min at rest.Samples for each phase were collected by the help of disposablesyringe and analyzed for L-glutaminase activity in each system.The composition of top and bottom phases was determined gravi-metrically in term of phase volume ratios (Vt/Vb). The tie-linelength (%) is the distance between the composition of top and bot-tom phases in the phase diagram. To conduct any extractive fer-mentation, it is essential to know the exact composition, pH,temperature and phase volume ratio (Vt/Vb) at which phase separa-tion occurs. The partition coefficient (k) for L-glutaminase is deter-mined by the ratio of L-glutaminase activity in the top (Ct) andbottom phase (Cb). Percent yield of L-glutaminase in top phasewas determined from the formula as
Y% ¼ 100VtKVtK þ Vb
where Vt and Vb are the volumes of the top and bottom phase,respectively.
2.6. Extractive batch fermentation in shake culture
L-Glutaminase fermentation by B. cereus MTCC 1305 was carriedout in production media with 8.5% (w/v) PEG 4000/9.5% (w/v) dex-tran T500 and in media without aqueous two phase components.The aqueous two phase fermentation medium was prepared bymixing separately sterilized stock solutions of PEG 4000 and dex-tran T500 with sterilized culture medium under aseptic conditions.Fermentations were carried out in by adding 2% inoculum to100 ml production medium and incubating in orbital shaker at35 �C, 180 rpm for 40 h. Samples from aqueous two-phase fermen-tation were withdrawn at regular intervals and allowed to settle ingraduated tubes for 30 min. The activity of L-glutaminase was esti-mated in both phase and dry cell weight was estimated in bottomphase. Similarly, samples from medium without aqueous twophase components were withdrawn at regular interval, centrifugedand culture filtrate was measured for L-glutaminase activity. Effectof pH and temperature on glutaminase production in media of PEG4000/dextran T500 was studied by varying pH from 5 to 8.5 andtemperature 25–50 �C.
3. Results and discussion
In an extractive fermentation with aqueous two-phase systems,microbial cells are retained in the dispersed phase and enzyme istransferred to a continuous phase in order to increase the overallproductivity. In the system using PEG/salts and PEG/dextran, thetop phase is continuous and rich in PEG while the bottom phaseis rich in salt/dextran. Microbial cells were retained in the lowerphase and at the interface. Aqueous two phase systems was devel-oped by using PEG X (X = 6000, 4000, 2000) with salts (magnesiumsulfate, sodium sulfate, tri-sodium citrate) or polymer (dextranT500, dextran 40). The most suitable aqueous two-phase systemfor partitioning of L-glutaminase was selected on basis of greaterpartition coefficient (Tables 1–3). Seventy to eighty percentage ofL-glutaminase partitioned toward bottom phase in case of PEG/salts phase system. This result may be due to ionic interactions be-tween metal salts and anionic nature of L-glutaminase (Benavidesand Rito-Palomares, 2008). PEG/dextran was selected as suitablesystem for partitioning study of L-glutaminase. Sixty to ninety per-centage of L-glutaminase was partitioned towards the top phase ofPEG/dextran aqueous two-phase systems as shown in Tables 1–3.The partitioning efficiency of L-glutaminase in PEG/dextran in-creased with increase of molecular weight of PEG 2000–4000 andthen decreased to PEG 6000. The large molecular size of PEG6000 results high hydrophobicity in top phase which causes L-glu-taminase to interact with opposite dextran phase and resulting lowpartition coefficient in top phase. PEG 4000/dextran T500 was se-lected as most suitable systems for partitioning studies of L-gluta-minase on basis of maximum partition coefficient value (1.31).
Phase diagram of partitioning of L-glutaminase in PEG 4000/dextran T500 separated the heterogeneous area from homoge-neous and showed the exact composition of top and bottomphases (Fig. 1). The phase composition becomes more and moresensitive with shortening of tie-line. The plait point (P) representsthe composition of 8.5% (w/w) PEG 4000 and 9.5% (w/w) dextranT500 and tie-line near plait point favor the partitioning of L-gluta-minase into upper phase. L-Glutaminase transferred more towardtop phase due to least polarity difference between PEG and dex-tran. Volume ratio (Vt/Vb) corresponding to various tie-line length
Table 1Partitioning of L-glutaminase from Bacillus cereus using PEG 2000/salt and PEG 2000/dextran systems.
Systems %PEG % Salt Phase volume ratio (Vt/Vb) Enzyme activity (U/ml) Partition coefficient (k)
of different concentrations of dextran T500 was also estimated inorder to supplement the exact phase system compositions atthose points (Table 4). Total estimated yield of enzyme from entirephase system was approximately same to the enzyme assayedfrom the same amount of the culture filtrate used for formingthe phase system.
The effect of the phase forming polymers on cell growth and en-zyme production is necessary to complete the evaluation of thesuitability of the phase system for enzyme fermentation. It was ob-served that in shake culture, B. cereus MTCC 1305 produced 2.09 U/ml of L-glutaminase at 32 h of fermentation in the top phase of theaqueous two-phase medium (Fig. 2) with an overall production of1.83 U/ml of the enzyme (Table 5), whereas 1.42 U/ml of L-gluta-
minase was obtained at 40 h of fermentation in the medium with-out aqueous two-phase components (Fig. 3 and Table 5). Theoverall production of enzyme in aqueous two-phase medium refersto the enzyme production based on the entire volume of the phasesystem. Overall L-glutaminase production was found better inaqueous two-phase medium compared to fermentation in mediumwithout aqueous two-phase components in shake culture. Higherenzyme activity in the extractable top phase showed that L-gluta-minase with high activity could be obtained free of cells and insol-uble substrate in an aqueous two-phase system (Sinha et al., 2000).The overall volumetric productivity obtained in aqueous two-phase medium based on extractable top phase was 65.3 U/l/h (Ta-ble 5) while in medium without aqueous two-phase componentswas 35.5 U/l/h (Table 5). Cell concentration rapidly increased dur-ing the first 16 h to reach a maximum of 1.262 g/l in aqueous two-phase medium, while in the medium without aqueous two-phasecomponents, the maximum cell concentration obtained was1.244 g/l after 20 h of fermentation. It showed that aqueous two-phase medium was more supportive of both cell growth and en-zyme production compared to medium without aqueous two-phase components.
The amount of enzyme production was affected by altering pHof media (Rito-Palomares, 2004; Saravanan et al., 2008). Extractivefermentations were carried out by applying different pH (5.0–8.5)to media with phase component for 32 h and maximum activity ofL-glutaminase in top phase was obtained at pH 7.5 (Fig. 4). The par-titioning value of many enzymes has been reported to increasewith high pH (Saravanan et al., 2008). The effect of temperatureon L-glutaminase production in PEG 4000/dextran T500 systemwas studied with different temperature (20–50 �C) at constantpH 7.5 (Fig. 5). The binodial curve moved down with increase oftemperature of ATPS system and the larger two-phase region
Table 4Effect of Vt/Vb on L-glutaminase partitioning in PEG 4000/dextran T500 system.
above the binodial curve resulted in increased differences in thephase compositions. The variation in the phase compositions re-sults change in partition coefficient of enzyme (Gautam and Simon,2006; Ratanapongleka, 2010).Optimum temperature for extractive
fermentation of L-glutaminase was obtained as 30 �C and maxi-mum activity of L-glutaminase was found in top phase of PEG4000/dextran T500 system (Fig. 5).
4. Conclusions
L-Glutaminase has been widely used as flavor enhancing agentin food industry. Aqueous two phase system as powerful extractivemethod improves production of enzyme by filtering cells andinsoluble substrate in the extractable phase of the fermentationmedium. Polymeric phase system (PEG 4000/dextran T500) wasselected as suitable ATPS system for partitioning of L-glutaminase.The optimization of physicochemical parameters like phase com-position, pH and temperature of system improve extractive pro-duction of L-glutaminase.
Acknowledgements
The authors are thankful to the School of Biochemical Engineer-ing, Banaras Hindu University, Varanasi, India for providing re-search facilities to carry this work. Mrs. Priyanka singh is also
thankful to DST-INSPIRE to provide financial support in the form ofJRF-Professional.
References
Benavides, J., Rito-Palomares, M., 2008. Practical experiences from the developmentof aqueous two-phase processes for the recovery of high value biologicalproducts. J. Chem. Technol. Biot. 83, 133–142.
Chen, J.P., Lee, M.S., 1995. Enhanced production of Serratia marcescens chitinase inPEG/dextran aqueous two-phase systems. Enzyme Microb. Technol. 17, 1021–1027.
Curthoys, N.P., Watford, M., 1995. Regulation of glutaminase activity and glutaminemetabolism. Annu. Rev. Nutr. 15, 133–159.
Gautam, S., Simon, L., 2006. Partitioning of b-glucosidase from Trichoderma reesei inpoly (ethylene glycol) and potassium phosphate aqueous two-phase systems:influence of pH and temperature. Biochem. Eng. J. 30, 104–108.
Guan, Y., Lilley, T.H., Treffry, T.E., Wilkinson, P.B., 1996. Use of aqueous two-phasesystems in the purification of human interferon-1 from recombinant Escherichiacoli. Enzy. Microb. Technol. 19, 446–455.
Hotha, S., Banik, R.M., 1997. Production of alkaline protease by Bacillus thuringiensisH 14 in aqueous two-phase systems. J. Chem. Tech. Biotechnol. 69, 5–10.
Imada, A., Igarasi, S., Nakahama, K., Isono, M., 1973. Asparaginase and glutaminaseactivities of microorganisms. J. Gen. Microbiol. 76, 85–99.
Kim, S.H., Yoo, Y.J., 1991. Characteristics of an aqueous two-phase system for a-amylase production. J. Ferment. Bioeng. 71, 373.
Klein, M., Kaltwasser, H., Jahns, T., 2002. Isolation of a novel, phosphate-activatedglutaminase from Bacillus pasteurii. FEMS Microbiol. Lett. 206, 63–67.
Kuboi, R., Umakoshi, H., Komasawa, I., 1995. Extractive cultivation of Escherichia coliusing poly (ethylene glycol)/phosphate aqueous two-phase systems to produceintracellular b-galactosidase. Biotechnol. Prog. 11, 202.
Lee, Y.H., Chang, H.N., 1990. Production of alkaline protease by Bacillus lichiniformisin an aqueous two-phase system. J. Ferment. Bioeng. 2, 89–92.
Moriguchi, M., Sakai, K., Tateyama, R., Furuta, Y., Wakayama, M., 1994. Isolation andcharacterization of salt-tolerant glutaminases from marine Micrococcus luteusK-3. J. Ferment. Bioeng. 77, 621–625.
Nandakumar, R., Yoshimune, K., Wakayama, M., Moriguchi, M., 2003. Microbialglutaminase: biochemistry, molecular approaches and applications in the foodindustry. J. Mol. Catal. B: Enzyme 23, 87–100.
Pandey, S.K., Banik, R.M., 2011. Extractive fermentation for enhanced production ofalkaline phosphatase from Bacillus licheniformis MTCC 1483 using aqueous two-phase systems. Bioresour. Technol. 102 (5), 4226–4231.
Ratanapongleka, K., 2010. Recovery of biological products in aqueous two phasesystems. Int. J. Chem. Eng. Appl. 1 (2), 191–197.
Rito-Palomares, M., 2004. Practical application of aqueous two-phase partition toprocess development for the recovery of biological products. J. Chromatogr. B807, 3–11.
Saravanan, S., Raghava Rao, J., Unni Nair, B., Ramasami, T., 2008. Aqueous two-phasepoly (ethylene glycol)–poly (acrylic acid) system for protein partitioning:Influence of molecular weight, pH and temperature. Process Biochem. 43, 905–911.
Sinha, J., Dey, P.K., Panda, T., 2000. Extractive fermentation for improved productionof endoglucanase by an intergeneric fusant of Trichoderma reesei/Saccharomycescerevisiae using aqueous two-phase system. Biochem. Eng. J. 6, 163–175.
Satomura, T., Shimura, D., Asai, K., Sadaie, Y., Hirooka, K., Fujita, Y., 2005.Enhancement of glutamine utilization in Bacillus subtilis through the GlnK-GlnL two-component regulatory system. J. Bacteriol. 187, 4813–4821.
Stredansky, M., Svorc, R., Sturdik, E., Dercova, K., 1993. Repeated batch a-amylaseproduction in aqueous two-phase system with Bacillus strains. J. Biotechnol. 27,181.
Singh, J., Verma, N., 2010. Partition of glucose oxidase from Aspergillus niger inaqueous two-phase systems based on salt and polyethylene glycol. Braz. Arch.Biol. Technol. 53 (5), 1051–1056.
