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Studies on efficient production of a novel l-asparaginase by a newly isolated Pseudomonas resinovorans IGS-131 and its heterologous expression in Escherichia coli

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Abstract

In the current study, the production of novel glutaminase free l-asparaginase from a new microbial source (Pseudomonas resinovorans IGS-131) is reported. Optimization of l-asparaginase production using conventional and statistical optimization techniques resulted in an enzyme yield of 37.63 IU/mL, which was 3.45-fold higher than the initial enzyme activity (i.e., 10.91 IU/mL). l-Asparaginase production from P. resinovorans IGS-131 was successfully carried out at the bioreactor level and investigations on the effect of agitation rates showed a maximum asparaginase yield of 38.88 IU/mL after 24 h fermentation at 400 rpm. The l-asparaginase gene from this source, showing 78% identity with a reported sequence in GenBank, was expressed in Escherichia coli rosetta DE3. The molecular weight of the recombinant protein was determined as 35.6 kDa. Downstream processing of recombinant l-asparaginase resulted in a purified protein concentration of 62.53 mg/L, which showed good free radical scavenging activity of 62%. The current findings provide promising results for a process of l-asparaginase production from P. resinovorans IGS-131. Furthermore, the recombinant production of this enzyme could help in avoiding the complexity of down streaming processes associated with the purification of this enzyme from wild-type organisms.

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References

  • Abdel-fattah YR, Olama ZA (2002) l-Asparaginase production by Pseudomonas aeruginosa in solid-state culture: evaluation and optimization of culture conditions using factorial designs. Process Biochem 38:115–122

    Article  CAS  Google Scholar 

  • Bansal S, Gnaneswari D, Mishra P, Kundu B (2010) Structural stability and functional analysis of l-asparaginase from Pyrococcus furiosus. Biochemistry 75:375–381

    CAS  PubMed  Google Scholar 

  • Bari MN, Alam MZ, Muyibi SA et al (2009) Improvement of production of citric acid from oil palm empty fruit bunches: optimization of media by statistical experimental designs. Bioresour Technol 100:3113–3120

    Article  CAS  Google Scholar 

  • Baskar G, Renganathan S (2010) Optimization of l-asparaginase production by Aspergillus terreus MTCC 1782 using response surface methodology and artificial neural network-linked genetic algorithm. Asia Pac J Chem Eng 7:212–220

    Article  Google Scholar 

  • Brumano LP, da Silva FVS, Costa-Silva TA et al (2019) Development of l-asparaginase biobetters: current research status and review of the desirable quality profiles. Front Bioeng Biotechnol. https://doi.org/10.3389/fbioe.2018.00212

    Article  PubMed  PubMed Central  Google Scholar 

  • Ciesarová Z, Kiss E, Boegl P (2006) Impact of l-asparaginase on acrylamide content in potato products. J Food Nutr Res 45:141–146

    Google Scholar 

  • Doriya K, Kumar DS (2018) Optimization of solid substrate mixture and process parameters for the production of l-asparaginase and scale-up using tray bioreactor. Biocatal Agric Biotechnol 13:244–250

    Article  Google Scholar 

  • El-Bessoumy AA, Sarhan M, Mansour J (2004) Production, isolation, and purification of l-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation. J Biochem Mol Biol 37:387–393

    CAS  PubMed  Google Scholar 

  • El-Hadi A, El-Refai H, Shafei M et al (2016) Statistical optimization of l-asparaginase production by using Fusarium solani. Egypt Pharm J 16:16–23

    Article  Google Scholar 

  • Erva RR, Goswami AN, Suman P et al (2017) Optimization of l-asparaginase production from novel Enterobacter sp., by submerged fermentation using response surface methodology. Prep Biochem Biotechnol 47:219–228

    Article  CAS  Google Scholar 

  • Farag AM, Hassan SW, Beltagy EA, El-Shenawy MA (2015) Optimization of production of anti-tumor l-asparaginase by free and immobilized marine Aspergillus terreus. Egypt J Aquat Res 41:295–302

    Article  Google Scholar 

  • Ghosh S, Murthy S, Govindasamy S, Chandrasekaran M (2013) Optimization of l-asparaginase production by Serratia marcescens (NCIM 2919) under solid-state fermentation using coconut oil cake. Sustain Chem Process 1:8. https://doi.org/10.1186/2043-7129-1-9

    Article  CAS  Google Scholar 

  • Gulati R, Saxena RK, Gupta R (1997) A rapid plate assay for screening l-asparaginase producing micro-organisms. Lett Appl Microbiol 24:23–26

    Article  CAS  Google Scholar 

  • Haaland PD (1989) Statistical problem solving. In: Haaland PD (ed) Experimental design in biotechnology. Marcel Dekker Inc, New York, pp 1–18

    Google Scholar 

  • Imada A, Igarasi S, Nakahama K, Isono M (1973) Asparaginase and glutaminase activities of micro-organisms. J Gen Microbiol 76:85–99

    Article  CAS  Google Scholar 

  • Irshad M, Zafaryab M, Singh M, Rizvi MMA (2012) Comparative analysis of the antioxidant activity of Cassia fistula extracts. Int J Med Chem 2012:1–6

    Google Scholar 

  • Iyyappan J, Gurunathan B (2017) Statistical optimization of l-asparaginase production by E. coli K-12 MTCC 1302. Int J Ind Eng 1:178–182

    Google Scholar 

  • Kenari SLD, Alemzadeh I, Maghsodi V (2011) Production of l-asparaginase from Escherichia coli ATCC 11303: optimization by response surface methodology. Food Bioprod Process 89:315–321

    Article  CAS  Google Scholar 

  • Kishore V, Nishita KP, Manonmani HK (2015) Cloning, expression, and characterization of l-asparaginase from Pseudomonas fluorescens for large scale production in E. coli BL21. 3 Biotech 5:975–981

    Article  Google Scholar 

  • Kwak K-O, Jung S-J, Chung S-Y et al (2006) Optimization of culture conditions for CO2 fixation by a chemoautotrophic microorganism, strain YN-1 using factorial design. Biochem Eng J 31:1–7

    Article  CAS  Google Scholar 

  • Lubkowski J, Palm GJ, Gilliland GL et al (1996) Crystal structure and amino acid sequence of Wolinella succinogenesl-asparaginase. Eur J Biochem 241:201–207

    Article  CAS  Google Scholar 

  • Mahajan RV, Mihooliya KN, Saran S, Saxena RK (2014) l-asparaginase from Bacillus sp. RKS-20: process optimization and application in the inhibition of acrylamide formation in fried foods. J Proteins Proteom 5:125–132

    Google Scholar 

  • Mahajan RV, Saran S, Kameswaran K et al (2012) Efficient production of l-asparaginase from Bacillus licheniformis with low-glutaminase activity: optimization, scale-up, and acrylamide degradation studies. Bioresour Technol 125:11–16

    Article  CAS  Google Scholar 

  • McCredie KB, Ho DH, Freireich EJ (1973) l-Asparaginase for the treatment of cancer. CA Cancer J Clin 23:220–227

    Article  CAS  Google Scholar 

  • Meena B, Anburajan L, Sathish T et al (2015) l-Asparaginase from Streptomyces griseus NIOT-VKMA29: optimization of process variables using factorial designs and molecular characterization of l-asparaginase gene. Sci Rep 5:1–12. https://doi.org/10.1038/srep12404

    Article  Google Scholar 

  • Meena B, Anburajan L, Vinithkumar NV et al (2016) Molecular expression of l-asparaginase gene from Nocardiopsis alba NIOT-VKMA08 in Escherichia coli: a prospective recombinant enzyme for leukaemia chemotherapy. Gene 590:220–226

    Article  CAS  Google Scholar 

  • Mihooliya KN, Nandal J, Swami L et al (2017) A new pH indicator dye-based method for rapid and efficient screening of l-asparaginase producing microorganisms. Enzyme Microb Technol 107:72–81

    Article  CAS  Google Scholar 

  • Moharam ME, Gamal-eldeen AM, El-sayed ST (2010) Production, immobilization, and anti-tumor activity of L- asparaginase of Bacillus sp. R36. J Am Sci 6:157–165

    Google Scholar 

  • Ohnuma T, Holland JF, Freeman A, Sinks LF (1970) Biochemical and pharmacological studies with asparaginase in man. Cancer Res 30:2297–2305

    CAS  PubMed  Google Scholar 

  • Rani SA, Sundaram L, Vasantha P (2012) A study on in vitro antioxidant and anticancer activity of l-asparaginase. J Pharm Res 5:1463–1466

    Google Scholar 

  • Shekhar TC, Anju G (2014) Antioxidant activity by DPPH radical scavenging method of Ageratum conyzoides Linn. leaves. Am J Ethnomed 1:244–249

    Google Scholar 

  • Soniyamby AR, Lalitha S, Praveesh BV, Priyadarshini V (2011) Isolation, production, and anti-tumor activity of l-asparaginase of Penicillium sp. Indian J Microbiol Res 2:38–42

    Google Scholar 

  • van Amsterdam FTM, Roveri A, Maiorino M et al (1992) Lacidipine: a dihydropyridine calcium antagonist with antioxidant activity. Free Radic Biol Med 12:183–187

    Article  Google Scholar 

  • Wakil SM, Adelegan AA (2015) Screening, production and optimization of l-asparaginase from soil bacteria isolated in Ibadan, south-western Nigeria. J Basic Appl Sci 11:39–51

    Article  CAS  Google Scholar 

  • Zuo S, Zhang T, Jiang B, Mu W (2015) Recent research progress on microbial l-asparaginase s. Appl Microbiol Biotechnol 99:1069–1079

    Article  CAS  Google Scholar 

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Acknowledgements

The authors Kanti N. Mihooliya and Alka Kumari are thankful to the Council of Scientific and Industrial Research (CSIR), Jitender Nandal is thankful to the Department of Biotechnology (DBT), and Himanshu Verma is thankful to the University Grant Commission (UGC) for financial support. Special thanks to Mr. Jaideep Mehta and Mr. Dinesh for helping in bioreactor studies.

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Authors and Affiliations

  1. Biochemical Engineering Research and Process Development Centre, CSIR-Institute of Microbial Technology, Chandigarh, 160 036, India

    Kanti N. Mihooliya, Jitender Nandal, Sidhanta Nanda, Himanshu Verma & Debendra K. Sahoo

  2. Microbial Type Culture Collection, CSIR-Institute of Microbial Technology, Chandigarh, India

    Alka Kumari

Authors
  1. Kanti N. Mihooliya
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  2. Jitender Nandal
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  3. Alka Kumari
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  4. Sidhanta Nanda
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  5. Himanshu Verma
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  6. Debendra K. Sahoo
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Contributions

DKS conceived and designed the experiments. KNM carried out the experiments. JN helped in fermenter studies of l-asparaginase production. AK and SN performed the antioxidant or free radical scavenging activity of l-asparaginase. HV and AK helped in recombinant studies and purification. DKS and KNM analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Debendra K. Sahoo.

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The authors have declared that they have no conflict of interests related to this work.

Additional information

The nucleotide sequence of the l-asparaginase gene obtained in this study was submitted in NCBI GenBank and received an accession number MK799854.

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Mihooliya, K.N., Nandal, J., Kumari, A. et al. Studies on efficient production of a novel l-asparaginase by a newly isolated Pseudomonas resinovorans IGS-131 and its heterologous expression in Escherichia coli. 3 Biotech 10, 148 (2020). https://doi.org/10.1007/s13205-020-2135-4

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  • DOI: https://doi.org/10.1007/s13205-020-2135-4

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