Skip to main content
Log in

Significantly enhanced production of recombinant nitrilase by optimization of culture conditions and glycerol feeding

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript


The production of a recombinant nitrilase expressed in Escherichia coli JM109/pNLE was optimized in the present work. Various culture conditions and process parameters, including medium composition, inducer, induction condition, pH and temperature, were systematically examined. The results showed that nitrilase production in E. coli JM109/pNLE was greatly affected by the pH condition and the temperature in batch culture, and the highest nitrilase production was obtained when the fermentation was carried out at 37°C, initial pH 7.0 without control and E. coli was induced with 0.2 mM isopropyl-β-d-thiogalactoside at 4.0 h. Furthermore, enzyme production could be significantly enhanced by adopting the glycerol feeding strategy with lower flow rate. The enzyme expression was also authenticated by sodium dodecyl phosphate polyacrylamide gel electrophoresis analysis. Finally, under the optimized conditions for fed-batch culture, cell growth, specific activity and nitrilase production of the recombinant E. coli were increased by 9.0-, 5.5-, and 50-fold, respectively.

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

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others


  • Babu KR, Seaminathan S, Marten S, Khanna N, Rinas U (2000) Production of interferon-α in high cell density cultures of recombinant Escherichia coli and its single step purification from refolded inclusion body proteins. Appl Microbiol Biotechnol 53:655–660

    Article  CAS  Google Scholar 

  • Banerjee A, Kaul P, Banerjee UC (2006) Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis. Appl Microbiol Biotechnol 72:77–87

    Article  CAS  Google Scholar 

  • Bok SH, Demain AL (1977) An improved colorimetric assay for polyols. Anal Biochem 81:21–27

    Article  Google Scholar 

  • Brady D, Beeton A, Zeevaart J, Kgaje C, Rantwijk F, Sheldon RA (2004) Characterisation of nitrilase and nitrile hydratase biocatalytic systems. Appl Microbiol Biotechnol 64:76–85

    Article  CAS  Google Scholar 

  • Choi JH, Lee SY (2004) Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 64:625–635

    Article  CAS  Google Scholar 

  • Choi JH, Jeong KJ, Kim SC, Lee SY (2000) Efficient secretory production of alkaline phosphatase by high cell density culture of recombinant Escherichia coli using the Bacillus sp. endoxylanase signal sequence. Appl Microbiol Biotechnol 53:640–645

    Article  CAS  Google Scholar 

  • Choi JH, Keum KC, Lee SY (2006) Production of recombinant proteins by high cell density culture of Escherichia coli. Chem Eng Sci 61:876–885

    Article  CAS  Google Scholar 

  • Jeong KJ, Lee SY (1999) High-level production of human leptin by fed-batch cultivation of recombinant Escherichia coli and its purification. Appl Environ Microbiol 65:3027–3032

    CAS  Google Scholar 

  • Kato Y, Asano Y (2003) High-level expression of a novel FMN-dependent heme-containing lyase, phenylacetaldoxime dehydratase of Bacillus sp. strain OxB-1, in heterologous hosts. Protein Expr Purif 28:131–139

    Article  CAS  Google Scholar 

  • Kleist S, Miksch G, Hitzmann B, Arndt M, Friehs K, Flaschel E (2003) Optimization of the extracellular production of a bacterial phytase with Escherichia coli by using different fed-batch fermentation strategies. Appl Environ Microbiol 61:456–462

    CAS  Google Scholar 

  • Korz DJ, Rinas U, Hellmuth K, Sanders EA, Deckwer WD (1995) Simple fed-batch technique for high cell density cultivation of Escherichia coli. J Biotechnol 39:59–65

    Article  CAS  Google Scholar 

  • Lammeli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  Google Scholar 

  • Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538

    CAS  Google Scholar 

  • Mare L, Cimander C, Elfwing A, Hagander P (2007) Feeding strategies for E. coli fermentations demanding an enriched environment. Bioprocess Biosyst Eng 30:13–25

    Article  CAS  Google Scholar 

  • Martinkova L, Mylerova V (2003) Synthetic applications of nitrile-converting enzymes. Curr Org Chem 7:1279–1295

    Article  CAS  Google Scholar 

  • Naik SC, Kaul P, Barse B, Banerjee A, Banerjee UC (2008) Studies on the production of enantioselective nitrilase in a stirred tank bioreactor by Pseudomonas putida MTCC 5110. Bioresour Technol 99:26–31

    Article  CAS  Google Scholar 

  • Norsyahida A, Rahmah N, Ahmad RMY (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 

  • Qui J, Swartz JR, Georgiou G (1998) Expression of active human tissue-type plasminogen activator in Escherichia coli. Appl Environ Microbiol 64:4891–4896

    Google Scholar 

  • Riesenberg D, Guthke R (1999) High-cell-density cultivation of microorganisms. Appl Microbiol Biotechnol 51:422–430

    Article  CAS  Google Scholar 

  • Shin CS, Hong MS, Bae CS, Lee J (1997) Enhanced production of human mini-proinsulin in fed-batch cultures at high cell density of Escherichia coli BL21(DE3)[pET-3aT2M2]. Biotechnol Prog 13:249–257

    Article  CAS  Google Scholar 

  • Singh R, Sharmac R, Tewarib N, Geetanjalid RDS (2006) Nitrilase and its application as a ‘green’ catalyst. Chem Biodivers 3:1279–1287

    Article  CAS  Google Scholar 

  • Sletta H, Nedal A, Aune TEV, Hellebust H, Hakvag S, Aune R, Ellingsen TE, Valla S, Brautaset T (2004) Broad-host-range plasmid pJB658 can be used for industrial-level production of a secreted hosttoxic single-chain antibody fragment in Eshcerichia coli. Appl Environ Microbiol 70:7033–7039

    Article  CAS  Google Scholar 

  • Wong HH, Kim YC, Lee SY, Chang HN (1998) Effect of post-inducing nutrient feeding strategies on the production of bioadhesive protein in Escherichia coli. Biotechnol Bioeng 60:271–276

    Article  CAS  Google Scholar 

  • Wu PH, Nair GR, Chu IM, Wu WT (2008) High cell density cultivation of Escherichia coli with surface anchored transglucosidase for use as whole-cell biocatalyst for α-arbutin synthesis. J Ind Microbiol Biotechnol 35:95–101

    Article  CAS  Google Scholar 

  • Yim SC, Jeong KJ, Chang HN, Lee SY (2001) High level secretory production of human G-CSF by fed-batch culture of recombinant Escherichia coli. Bioprocess Biosyst Eng 24:249–254

    Article  CAS  Google Scholar 

  • Yuan H, Yang X, Hua ZC (2004) Optimization of expression of an annexin ν-hirudin chimeric protein in Escherichia coli. Microbiol Res 159:147–156

    Article  CAS  Google Scholar 

  • Zhang ZJ, Xu JH, He YC, Ouyang LM, Liu YY, Imanaka T (2010) Efficient production of (R)-(−)-mandelic acid with highly substrate/product tolerant and enantioselective nitrilase of recombinant Alcaligenes sp. Process Biochem 45:887–891

    Article  CAS  Google Scholar 

Download references


This work was financially supported by the National Natural Science Foundation of China (Nos. 20672037, 20773038, and 20902023), Ministry of Science and Technology, PR China (Nos. 2009ZX09501-016 and 2009CB724706), China National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204), and Shanghai Municipal Fund for Natural Science (No. 07ZR14030).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Jian-He Xu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, JF., Zhang, ZJ., Li, AT. et al. Significantly enhanced production of recombinant nitrilase by optimization of culture conditions and glycerol feeding. Appl Microbiol Biotechnol 89, 665–672 (2011).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: