Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli.
Nitrilases constitute an important class of biocatalysts for chiral synthesis. This work was undertaken with the aim to optimize nitrilase production in a host that is well-studied for protein production. Process parameters were optimized for high cell density fermentation, in batch and fed-batch modes, of Escherichia coli BL21 (DE3) expressing Pseudomonas fluorescens nitrilase with a T7 promoter based expression system. Effects of different substrates, temperature and isopropyl β-D-1-thiogalactopyranoside (IPTG) induction on nitrilase production were studied. Super optimal broth containing glycerol but without an inducer gave best results in batch mode with 32 °C as the optimal temperature. Use of IPTG led to insoluble protein and lower enzyme activity. Optimized fed-batch strategy resulted in significant improvement in specific activity as well as volumetric productivity of the enzyme. On a volumetric basis, the activity improved 40-fold compared to the unoptimized batch process.
Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India; DBT-Pan IIT Center for Bioenergy, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India.
Sujata Vijay
SV
Sohoni
Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India.
Dhanaraj
D
Nelapati
Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India.
Sneha
S
Sathe
Hi Tech Biosciences India Ltd., C-2, 102/103, Saudamini Complex, Right Bhusari Colony, Paud Road, Kothrud, Pune 411038, India.
Vaishali
V
Javadekar-Subhedar
Hi Tech Biosciences India Ltd., C-2, 102/103, Saudamini Complex, Right Bhusari Colony, Paud Road, Kothrud, Pune 411038, India.
Raghavendra P
RP
Gaikaiwari
Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India; DBT-Pan IIT Center for Bioenergy, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India; Wadhwani Research Center for Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India. Electronic address: wangikar@iitb.ac.in.
Pramod P
PP
Wangikar
D000619:Aminohydrolases / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000096:biosynthesis; D019149:Bioreactors; D002244:Carbon / Q000737:chemistry; D003470:Culture Media / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005285:Fermentation; D005947:Glucose / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry*; D007218:Industrial Microbiology; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis*
D001426:Bacterial Proteins; D003470:Culture Media; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D002244:Carbon; D000619:Aminohydrolases; C021867:nitrilase; D005947:Glucose; D005990:Glycerol
E. coli BL21 (DE3); High cell density fermentation; Nitrilase