Skip to main content
SpringerLink
Log in

High yield expression of novel glutaminase free l-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N

  • Original Paper
  • Published:
Bioprocess and Biosystems Engineering Aims and scope Submit manuscript

Abstract

Gene encoding glutaminase-free l-asparaginase II (ans B2) from Pectobacterium carotovorum MTCC 1428 was cloned into pHT43, transformed in Bacillus subtilis WB800N and optimised the expression levels of recombinant enzyme. A three-fold higher enzyme production was observed with an efficient transformant as compared to native strain. Enzyme localization studies revealed that >90 % of recombinant enzyme is secreted extracellularly, a little fraction is attached to the membrane (>6 %) and localised intracellularly (3 %). The expression of recombinant l-asparaginase II was confirmed by SDS-PAGE, IMAC (Immobilised metal ion affinity chromatography) purification followed by Western blotting. Process parameter optimization with OFAT (one factor at a time) revealed that rpm (120), temperature (37 °C), Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration (1 mM) and time of induction (0.8 OD600nm) plays a vital role where a maximum of 55 IU/ml was achieved. Further, consecutive induction by IPTG improved the enzyme production up to 105 IU/ml with a specific activity of 101 IU/mg of protein. Molecular modelling analysis depicted that amino acids, GLY60, GLY119 and ALA252 in the active site are responsible for the glutaminase free l-asparaginase II activity. This is the first report on enhanced expression of recombinant glutaminase-free l-asparaginase II by intermediate addition of IPTG.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Rytting ME (2012) Role of l-asparaginase in acute lymphoblastic leukemia: focus on adult patients. Blood Lymphat Cancer Targets Ther 2012(2):117–124

    Article  Google Scholar 

  2. Pedreschi F, Mariotti S, Granby K, Risum J (2011) Acrylamide reduction in potato chips by using commercial asparaginase in combination with conventional blanching. LWT Food Sci Technol 44(6):1473–1476

  3. Verma N, Kumar K, Kaur G, Anand SE (2007) E coli K-12 asparaginase-based asparagine biosensor for leukemia. Artif Cell Blood Substit Immobil Biotechnol 35:449–456

    Article  CAS  Google Scholar 

  4. Borek and Jaskólski (2001) Sequence analysis of enzymes with asparaginase activity. Acta Biochim Pol 48:893–902

    Google Scholar 

  5. Kozak M, Jurga J (2002) A comparison between the crystal and solution structures of Escherichia coli asparaginase II. Acta Biochim Pol 49:509–513

    CAS  Google Scholar 

  6. Jain S, Naithani R, Kapoor G, Nath T (2009) l-Asparaginase induced severe hypertriglyceridemia in acute lymphoblastic leukemia with 11q23 abnormality. Leuk Res 33:e194

    Article  CAS  Google Scholar 

  7. Reinert RB, Oberle LM, Wek SA, Bunpo P, Wang XP, Mileva I, Goodwin LO, Aldrich CJ, Durden DL, McNurlan MA, Wek RC, Anthony TG (2006) Role of glutamine depletion in directing tissue-specific nutrient stress responses to l-asparaginase. J Biol Chem 281:31222–31233

    Article  CAS  Google Scholar 

  8. Kumar S, Pakshirajan K, Venkata Dasu V (2009) Development of medium for enhanced production of glutaminase free l-asparaginase from Pectobacterium carotovorum MTCC 1428. Appl Microb Biotechnol 84:477–486

    Article  CAS  Google Scholar 

  9. Kumar S, Pakshirajan K, Venkata Dasu V (2010) Localization and production of novel l-asparaginase from Pectobacterium carotovorum MTCC 1428. Process Biochem 45:223–229

    Article  CAS  Google Scholar 

  10. Harwood C, Wipat A (1996) Sequencing and functional analysis of the genome of Bacillus subtilis strain 168. FEBS Lett 389(1):84

    Article  CAS  Google Scholar 

  11. Xie J, Zhao Y, Zhang H, Liu Z, Lu Z (2013) Improving methyl parathion hydrolase to enhance its chlorpyrifos hydrolyzing efficiency. Lett Appl Microbiol 58:53–59

    Article  Google Scholar 

  12. Jung J, Yu KO, Ramzi AB, Choe SH, Kim SW, Han SO (2012) Improvement of surfactin production in Bacillus subtilis using synthetic wastewater by over expression of specific extracellular signaling peptides, comX and phrC. Biotechnol Bioeng 109:2349–2356

    Article  CAS  Google Scholar 

  13. Oh C, De Zoysa M, Kang DH, Lee Y, Whang I, Nikapitiya C, Heo SJ, Yoon KT, Affan A, Lee J (2011) Isolation, purification, and enzymatic characterization of extracellular chitosanase from marine bacterium Bacillus subtilis CH2. J Microbiol Biotechnol 21:1021–1025

    Article  CAS  Google Scholar 

  14. Wu SC, Yeung JC, Duan Y, Ye R, Szarka SJ, Habibi HR, Wong SL (2002) Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound protease on antibody fragment production. Appl Environ Microbiol 68(7):3261–3269

    Article  CAS  Google Scholar 

  15. Luo Z, Gao Q, Li X, Bao J (2014) Cloning of LicB from Clostridium thermocellum and its efficient secretive expression of thermostable β-1, 3-1, 4-glucanase. Appl Biochem Biotechnol

  16. Nguyen Thao Thi, Quyen Thi Dinh, Le Hoang Thanh (2013) Cloning and enhancing production of a detergent and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800. Microb Cell Fact 12:79

    Article  Google Scholar 

  17. Vojcic L, Despotovic D, Martinez R, Maurer KHH, Schwaneberg U (2012) An efficient transformation method for Bacillus subtilis DB104. Appl Microbiol Biotechnol 94:487–493

    Article  CAS  Google Scholar 

  18. Mashburn LT, Wriston JC Jr (1964) Tumor inhibitory effect of l-asparaginase from Escherichia coli. Arch Biochem Biophys 105:450–452

    Article  CAS  Google Scholar 

  19. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  20. Kaufmann SH, Ewing CM, Shaper JH (1987) The erasable western blot. Anal Biochem 161:89–95

    Article  CAS  Google Scholar 

  21. Sambrook J, Russell DW (2001) Protein interaction technologies, protocol #3: detection of protein-protein interactions using the GST fusion protein pull-down technique. In: Chapter 18: Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Plainview

  22. Triantafillou D, Georgatsos J, Kyriakidis D (1988) Purification and properties of a membrane-bound l-asparaginase of Tetrahymena Pyriformis. Mol Cell Biochem 81(1):43–51

    Article  CAS  Google Scholar 

  23. Geckil H, Ates B, Gencer S, Uckun M, Yilmaz I (2005) Membrane permeabilization of gram-negative bacteria with a potassium phosphate/hexane aqueous phase system for the release of l-asparaginase: an enzyme used in cancer therapy. Process Biochem 40(2):573579

    Article  Google Scholar 

  24. Sun D, Setlow P (1991) Cloning, nucleotide sequence, and expression of the Bacillus subtilis ans operon, which codes for l-asparaginase and l-aspartase. J Bacteriol 173(12):3831–3845

    CAS  Google Scholar 

  25. Hamoen Leendert W, Venema Gerard, Kuipers Oscar P (2003) Controlling competence in Bacillus subtilis: shared use of regulators. Microbiology 149:9–17

    Article  CAS  Google Scholar 

  26. Dubnau D (1991) Genetic competence in Bacillus subtilis. Microbiol Rev 55:395–424

    CAS  Google Scholar 

  27. Phan T, Nguyen H, Schumann W (2006) Novel plasmid-based expression vectors for intra and extracellular production of recombinant proteins in Bacillus subtilis. Protein Expr Purif 46:189195

    Article  Google Scholar 

  28. Kumar S, Venkata Dasu V, Pakshirajan K (2011) Purification and characterization of glutaminase-free l-asparaginase from Pectobacterium carotovorum MTCC 1428. Bioresour Technol 102:2077–2082

    Article  CAS  Google Scholar 

  29. Durban M, Silbersack J, Schweder T, Schauer F, Bornscheuer U (2006) High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis. Appl Microbiol Biotechnol 74(3):634639

    Google Scholar 

  30. Mahajan R, Saran S, Kameswaran K, Kumar V, Saxena R (2012) Efficient production of l-asparaginase from Bacillus licheniformis with low-glutaminase activity: Optimization, scale up and acrylamide degradation studies. Bioresour Technol 125:1116

    Article  Google Scholar 

  31. Prakasham R, Hymavathi M, Rao C, Arepalli S, Rao J, Kennady P, Nasaruddin K et al (2010) Evaluation of antineoplastic activity of extracellular asparaginase produced by isolated Bacillus circulans. Appl Biochem Biotechnol 160(1):72–80

    Article  CAS  Google Scholar 

  32. Singh S, Preez J, Pillay B, Prior B (2000) The production of hemi-cellulases by Thermomyces lanuginosus strain SSBP: influence of agitation and dissolved oxygen tension. Appl Microbiol Biotechnol 54(5):698–704

    Article  CAS  Google Scholar 

  33. Chen T, Liu W, Fu J, Zhang B, Tang Y (2013) Engineering Bacillus subtilis for acetoin production from glucose and xylose mixtures. J Biotechnol 168:499505

    Google Scholar 

  34. Kenari S, Alemzadeh I, Maghsodi V (2011) Production of l-asparaginase from Escherichia coli ATCC 11303: optimization by response surface methodology. Food Bio Prod Process 89:315321

    Google Scholar 

  35. Rastogi G, Muppidi G, Gurram R, Adhikari A, Bischoff K, Hughes S, Apel W et al (2009) Isolation and characterization of cellulose-degrading bacteria from the deep subsurface of the Homestake gold mine, Lead, South Dakota, USA. J Ind Microbiol Biotechnol 36(4):585–598

    Article  CAS  Google Scholar 

  36. Rastogi G, Bhalla A, Adhikari A, Bischoff K, Hughes S, Christopher L, Sani R (2010) Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains. Bioresour Technol 101(22):8798–8806

    Article  CAS  Google Scholar 

  37. Deka D, Das S, Sahoo N, Das D, Jawed M, Goyal D, Goyal A (2013) Enhanced cellulase production from Bacillus subtilis by Optimizing physical parameters for bio ethanol production. ISRN Biotechnol 2013:111

    Article  Google Scholar 

  38. Jo KI, Lee YJ, Kim BK, Lee BH, Chung CH, Nam SW, Kim SK et al (2008) Pilot-scale production of carboxy methyl cellulase from rice hull by Bacillus amyloliquefaciens DL-3. Biotechnol Bioprocess Eng 13(2):182188

    Article  Google Scholar 

  39. Luan C, Zhang H, Song D, Xie Y, Feng J, Wang Y (2013) Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology. Appl Microbiol Biotechnol 98(8):36513658

    Google Scholar 

  40. Nandana V, Singh S, Singh A, Dubey V (2014) Procerain B, a cysteine protease from Calotropis procera, requires N-terminus pro-region for activity: cDNA cloning and expression with pro-sequence. Protein Expr Purif 103:1622

    Article  Google Scholar 

  41. Glick BR (1995) Metabolic load and heterologous gene expression. Biotechnol Adv 13:247–261

    Article  CAS  Google Scholar 

  42. Norsyahida A, Rahmah N, Ahmad R (2009) Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli. Lett Appl Microbiol 49:544–550

    Article  CAS  Google Scholar 

  43. Meena B, Anburajan L, Dheenan P, Begum M, Vinith kumar N, Dharani G, Kirubagaran R (2015) Novel glutaminase free l-asparaginase from Nocardiopsis alba NIOT-VKMA08: production, optimization, functional and molecular characterization. Bioprocess Biosyst Eng 38(2):373–388

  44. Jia M, Xu M, He B, Rao Z (2013) Cloning, expression, and characterization of l-asparaginase from a newly isolated Bacillus subtilis B11–06. J Agric Food Chem 61(39):94289434

    Article  Google Scholar 

  45. Bentley WE, Mirjalili N, Andersen DC, Davis RH, Kompala DS (1990) Plasmid–encoded protein: the principal factor in the “metabolic burden” associated with recombinant bacteria. Biotechnol Bioeng 35:668–681

    Article  CAS  Google Scholar 

  46. Lecina M, Sarro E, Casablancas A, Godia F, Cairo J (2013) IPTG limitation avoids metabolic burden and acetic acid accumulation in induced fed-batch cultures of Escherichia coli M15 under glucose limiting conditions. Biochem Eng J 70:7883

    Article  Google Scholar 

  47. Fernandez-Castane A, Caminal G, Lopez-Santin J (2012) Direct measurements of IPTG enable analysis of the induction behaviour of E. coli in high cell density cultures. Microb Cell Fact 11:58

    Article  CAS  Google Scholar 

  48. Smith H, Wiersma K, Bron S, Venema G (1983) Transformation in Bacillus subtilis: purification and partial characterization of a membrane-bound DNA-binding protein. J Bacteriol 156:101–108

    CAS  Google Scholar 

  49. Ilk N, Schumi CT, Bohle B, Egelseer E, Sleytr U (2011) Expression of an endotoxin-free S-layer/allergen fusion protein in gram-positive Bacillus subtilis 1012 for the potential application as vaccines for immunotherapy of atopic allergy. Microb Cell Fact 10:6

    Article  CAS  Google Scholar 

  50. Shatsky M, Nussinov R, Wolfson H J (2004) A method for simultaneous alignment of multiple protein structures. Protein Struct Funct Bioinform 56(1):143–156

  51. Miller M, Rao JK, Wlodawer A, Gribskov MR (2001) A left-handed crossover involved in amidohydrolase catalysis Crystal structure of Erwinia chrysanthemi l-asparaginase with bound l-aspartate. FEBS Lett 328:275–279

    Article  Google Scholar 

  52. Aghaiypour K, Wlodawer A, Lubkowski J (2001) Structural basis for the activity and substrate specificity of Erwinia chrysanthemi l-asparaginase. Biochemistry 40:5655–5664

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Authors acknowledge the Department of Biotechnology, New Delhi for the financial support in the form of project (BT/PR6653/PID/6/710/2012) and are grateful to the Department of Biotechnology, Indian Institute of Technology Guwahati and CSIR-Indian Institute of Chemical Technology, Hyderabad for providing facilities for research work.

Author information

Authors and Affiliations

  1. Biochemical Engineering Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology (IIT) Guwahati, Guwahati, Assam, 781039, India

    Sushma Chityala & Veeranki Venkata Dasu

  2. Bioengineering and Environmental Sciences, Indian Institute of Chemical Technology, Hyderabad, 500007, India

    Jamal Ahmad & Reddy Shetty Prakasham

Authors
  1. Sushma Chityala
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Veeranki Venkata Dasu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jamal Ahmad
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Reddy Shetty Prakasham
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Veeranki Venkata Dasu.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chityala, S., Venkata Dasu, V., Ahmad, J. et al. High yield expression of novel glutaminase free l-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N. Bioprocess Biosyst Eng 38, 2271–2284 (2015). https://doi.org/10.1007/s00449-015-1464-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00449-015-1464-x

Keywords

Search

Navigation