Batch, Fed Batch Production and Characterization of Glutaminase Free L-Asparaginase II of Pectobacterium carotovorum MTCC 1428 in Escherichia coli

Rachna Goswami, Krishnamoorthy Hegde, Venkata Dasu Veeranki

Biochemical Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India.

DOI: 10.4236/aim.2014.410072                

Abstract

The present study describes the production optimization of recombinant L-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Escherichia coli BL21 (DE3) at batch and fed batch bioreactor level. Production of recombinant L-asparaginase II in batch and fed batch mode was found to be 1.34 and 5.38 folds higher, respectively as compared to shake flask culture. SDS-PAGE and native PAGE of the purified enzyme revealed that molecular mass of the subunits and native enzyme are ~37.5 kDa and ~150 kDa, respectively. Optimum range of pH and temperature for hydrolysis of L-asparagine were found to be 7.5 - 8.5 and 47^°C - 52^°C, respectively. The recombinant enzyme is very specific for its natural substrate, L-asparagine. The activity of recombinant L-asparaginase II is improved by mono cations and diverse effectors including Na⁺, K⁺, L-cystine, L-histidine, glutathione and 2-mercaptoethanol whereas, it is moderately inhibited by different divalent cations and thiol group blocking reagent. The kinetic parameters K_m, V_max, k_cat and K_m/K_cat of purified recombinant L-asparaginase II were determined. The purified L-asparaginase II possesses no partial glutaminase activity, which is prerequisite to reduce the possibility of side effects during the course of anti-cancer therapy.

Keywords

Batch Fermentation, Fed-Batch Fermentation, Recombinant L-Asparaginase II, Taguchi’s Method

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Kotzia, G.A. and Labrou, N.E. (2005) Cloning, Expression and Characterization of Erwinia carotovora L-Asparaginase. Journal of Biotechnology, 119, 309-323. http://dx.doi.org/10.1016/j.jbiotec.2005.04.016
[2]	Hendriksen, H.V., Kornbrust, B.A., Ostergaard, P.R. and Mary, A. (2009) Evaluating the Potential for Enzymatic Acrylamide Mitigation in a Range of Food Products Using an Asparaginase from Aspergillus oryzae. Journal of Agricultural and Food Chemistry, 57, 4168-4176. http://dx.doi.org/10.1021/jf900174q
[3]	Peterson, R.E. and Ciegler, A. (1969) L-Asparaginase Production by Erwinia aroideae. Applied Microbiology, 18, 64-67.
[4]	Verma, N., Kumar, K., Kaur, G. and Anand, S. (2007) L-Asparaginase, A Promising Chemotherapeutic Agent. Critical Reviews in Biotechnology, 27, 45-62. http://dx.doi.org/10.1080/07388550601173926
[5]	Dunlop, P.C., Roon, R.J. and Even, H.L. (1976) Utilization of D-L-Asparaginase by S. cerevisiae. Journal of Bacteriology, 125, 999-1004.
[6]	Khushoo, A., Pal, Y., Singh, B.N. and Mukherjee, K.J. (2004) Extracellular Expression and Single Step Purification of Recombinant Escherichia coli L-Asparaginase II. Protein Expression and Purification, 38, 29-36. http://dx.doi.org/10.1016/j.pep.2004.07.009
[7]	Kotzia, G.A. and Labrou, N.E. (2007) L-Asparaginase from Erwinia chrysanthemi 3937, Cloning, Expression and Characterization. Journal of Biotechnology, 127, 657-669. http://dx.doi.org/10.1016/j.jbiotec.2006.07.037
[8]	Dasu, V.V., Panda, T. and Chidambaram, M. (2003) Determination of Significant Parameters for Improved Griseofulvin Production in a Batch Bioreactor by Taguchi’s Method. Process Biochemistry, 38, 877-880. http://dx.doi.org/10.1016/S0032-9592(02)00068-7
[9]	Stowe, R.A. and Mayer, R.P. (1999) Efficient Screening of Process Variables. Industrial & Engineering Chemistry Research, 56, 36-40.
[10]	Panda, T., Babu, P.S.R., Kumari, J.A., Rao, D.S., Theodore, K. and Jagannandha, K., et al. (1997) Bioprocess Optimization Challenge. Journal of Microbiology and Biotechnology, 7, 367-72.
[11]	Muller, H.J. and Boos, J. (1998) Use of L-Asparaginase in Childhood ALL. Critical Reviews in Oncology/Hematology, 28, 97-113. http://dx.doi.org/10.1016/S1040-8428(98)00015-8
[12]	Lappa, K., Kotzia, G.A. and Labrou, N.E. (2010) Labrou Studies on the Thermal Stability of the Therapeutic Enzyme L-Asparaginase from Erwinia carotovora. Biochemistry Research Trends Series, Nova Science Publisher, New York, 323-334.
[13]	Kumar, S., Pakshirajan, K. and Dasu, V.V. (2009) Development of Medium for Enhanced Production of Glutaminase-Free L-Asparaginase from Pectobacterium carotovorum MTCC 1428. Applied Microbiology and Biotechnology, 84, 477-486. http://dx.doi.org/10.1007/s00253-009-1973-0
[14]	Squez-Alvarez, E., Lienqueo, M.E. and Pinto, J.M. (2001) Optimal Synthesis of Protein Purification Processes. Biotechnology Progress, 17, 685-696. http://dx.doi.org/10.1021/bp010031d
[15]	Zhang, Y.K., Li, T.M. and Liu, J.J. (2003) Low Temperature and Glucose Enhanced T7 RNA Polymerase-Based Plasmid Stability for Increasing Expression of Glucagon-Like Peptide-2 in Escherichia coli. Protein Expression and Purification, 29, 132-139. http://dx.doi.org/10.1016/S1046-5928(03)00002-0
[16]	Studier, F.W. (2005) Protein Production by Auto-Induction in High-Density Shaking Cultures. Protein Expression and Purification, 41, 207-234. http://dx.doi.org/10.1016/j.pep.2005.01.016
[17]	Nayak, D.P. and Vyas, V.V. (1999) Improved Stability and Expression of a Recombinant Shuttle Plasmid in Escherichia coli during Fedbatch Cultivation. World Journal of Microbiology and Biotechnology, 15, 65-71. http://dx.doi.org/10.1023/A:1008870528336
[18]	Giridhar, R. and Srivastava, A.K. (2000) Fed-Batch Sorbose Fermentation Using Pulse and Multiple Feeding Strategies for Productivity Improvement. Biotechnology and Bioprocess Engineering, 5, 340-344. http://dx.doi.org/10.1007/BF02942209
[19]	Ramalingama, S., Gautama, P., Mukherjee, K.J. and Jayaraman, G. (2007) Effects of Post-Induction Feed Strategies on Secretory Production of Recombinant Streptokinase in Escherichia coli. Biochemical Engineering Journal, 33, 34-41. http://dx.doi.org/10.1016/j.bej.2006.09.019
[20]	Zhang, H., Zheng, Y., Liu, Q., Tao, X., Zheng, W., Ma, X. and Wei, D. (2009) Development of a Fed-Batch Process for the Production of Anticancer Drug TATm-Survivin(T34A) in Escherichia coli. Biochemical Engineering Journal, 43, 163-168. http://dx.doi.org/10.1016/j.bej.2008.09.013
[21]	Miller, G.L. (1959) Use of DNS Reagent for Determination of Reducing Sugar. Analytical Chemistry, 31, 426-428. http://dx.doi.org/10.1021/ac60147a030
[22]	Kumar, S., Dasu, V. and Pakshirajan, K. (2011) Purification and Characterization of Glutaminase-Free L-Asparaginase from Pectobacterium carotovorum MTCC 1428. Bioresource Technology, 102, 2077-2082. http://dx.doi.org/10.1016/j.biortech.2010.07.114
[23]	Daverey, A. and Pakshirajan, K.K. (2010) Kinetics of Growth and Enhanced Sophorolipids Production by Candida bombicola Using a Low-Cost Fermentative Medium. Applied Biochemistry and Biotechnology, 160, 2090-2101. http://dx.doi.org/10.1007/s12010-009-8797-3
[24]	Niccolai, A., Fontani, S., Kapat, A. and Olivieri, R. (2003) Maximization of Recombinant Helicobacter pylori Neutrophil Activating Protein Production in Escherichia coli: Improvement of a Chemically Defined Medium Using Response Surface Methodology. FEMS Microbiology Letters, 221, 257-262. http://dx.doi.org/10.1016/S0378-1097(03)00184-8
[25]	Hao, D.C., Zhu, P.H., Yang, S.L. and Yang, L. (2006) Optimization of Recombinant Cytochrome P450 2C9 Protein Production in Escherichia coli DH5α by Statistically-Based Experimental Design. World Journal of Microbiology and Biotechnology, 22, 1169-1176. http://dx.doi.org/10.1007/s11274-006-9158-9
[26]	Ghane, M., Yakhchali, B. and Khodabandeh, M. (2008) Over Expression of Biologically Active Interferon Beta Using Synthetic Gene in E. coli. Journal of Sciences, 19, 203-209.
[27]	Khushoo, A., Pal, Y. and Mukherjee, K.J. (2005) Optimization of Extracellular Production of Recombinant Asparaginase in Escherichia coli in Shake-Flask and Bioreactor. Applied Microbiology and Biotechnology, 68, 189-197. http://dx.doi.org/10.1007/s00253-004-1867-0
[28]	Kweon, D.H., Han, N.S., Park, K.M. and Seo, J.H. (2001) Overproduction of Phytolacca insularis Protein in Batch and Fed-Batch Culture of Recombinant Escherichia coli. Process Biochemistry, 36, 537-542. http://dx.doi.org/10.1016/S0032-9592(00)00237-5
[29]	Gummadi, S.N. and Bhavya, B. (2011) Enhanced Degradation of Caffeine and Caffeine Demethylase Production by Pseudomonas sp. in Bioreactors under Fed-Batch Mode. Applied Microbiology and Biotechnology, 91, 1007-1017. http://dx.doi.org/10.1007/s00253-011-3319-y
[30]	Kozak, M., Jasloski, M. and Rohm, K.H. (2000) Preliminary Crystallographic Studies of 425F Mutant of Periplasmic Escherichia coli L-Asparaginase. Acta Biochimica Polonica, 47, 807-814.
[31]	Aghaiypour, K., Wlodawer, A. and Lubkowski, J. (2001) Structural Basis for the Activity and Substrate Specificity of Erwinia chrysanthemi L-Asparaginase. Biochemistry, 40, 5655-5664. http://dx.doi.org/10.1021/bi0029595
[32]	Lubkowski, J., Wlodawer, A., Ammon, H.L., Copeland, T.D. and Swain, A.L. (1994) Structural Characterization of Pseudomonas 7A Glutaminase-Asparaginase. Biochemistry, 33, 10257-10265. http://dx.doi.org/10.1021/bi00200a005
[33]	Maladkar, N.K., Singh, V.K. and Naik, S.R. (1993) Fermentative Production and Isolation of L-Asparaginase from Erwinia carotovora. Hindustan Antibiotics Bulletin, 35, 77-86.
[34]	El-Sayed, M., El-Sayed, S.T., Shousha, W.G., Shehata, A.N. and Hanafy, S.S. (2011) Purification, Characterization and Antitumor Activity of L-Asparaginase from Chicken Liver. Journal of American Science, 1, 439-449.
[35]	Cunningham, T.J., Yao, L.H. and Lucena, A. (2008) Product Inhibition of Secreted Phospholipase A2 May Explain Lysophosphatidylcholines’ Unexpected Therapeutic Properties. Journal of Inflammation, 5, 17. http://dx.doi.org/10.1186/1476-9255-5-17
[36]	Jimnez, E.S.D., Evangelina, L., Torres, J. and Soberon, G. (1964) On the Mechanism of the Effect of Ionic Strength on Crystalline Aldolase Activity. Journal of Biological Chemistry, 239, 4154-4158.
[37]	Ernst, V., Levin, D.H. and London, I.M. (1979) Inhibition of Protein Synthesis Initiation by Oxidized Glutathione: Activation of a Protein Kinase that Phosphorylates the α Subunit of Eukaryotic Initiation Factor 2. Proceedings of the National Academy of Sciences of the United States of America, 75, 4110-4114.
[38]	Manna, S., Sinha, A., Sadhukhan, R. and Chakrabarty, S.L. (1995) Purification, Characterization and Antitumor Activity of L-Asparaginase Isolated from Pseudomonas stutzeri MB-405. Current Microbiology, 30, 291-298. http://dx.doi.org/10.1007/BF00295504
[39]	Warangkar, S.C. and Khobragade, C.N. (2010) Purification, Characterization, and Effect of Thiol Compounds on Activity of the Erwinia carotovora L-Asparaginase. Enzyme Research, 2010, Article ID: 165878. http://dx.doi.org/10.4061/2010/165878