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Bench scale synthesis of p-hydroxybenzoic acid using whole-cell nitrilase of Gordonia terrae mutant E9

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Abstract

Mutants of Gordonia terrae were generated using chemical mutagens for better activity, stability and higher substrate/product tolerance of its nitrilase enzyme. Mutant E9 showed two-time increase in activity and tolerated p-hydroxybenzonitrile (p-HBN) up to 50 mM. Response surface methodology and inducer mediation approach further enhanced the production of enzyme to 2.5-fold. The bench scale production of p-hydroxybenzoic acid (p-HBA) was carried out in a fed-batch reaction (500-mL scale) using whole-cell nitrilase of mutant E9 in 0.1 M potassium phosphate buffer (pH 8.0) at 40 °C. Total six feedings each at an interval of 45 min resulted in accumulation of 360 mM (21.6 g) of p-HBA with a purity of 99 %. The catalytic and volumetric productivity of bioprocess using mutant G. terrae was improved to 1.8 g h−1 g −1DCW and 43.2 g L−1, respectively, from 0.78 g h−1 g −1DCW and 28.8 g L−1 using resting cells of wild strain. K m and V max of purified nitrilase from mutant E9 were 55 U mg−1 and 1.8 mM for p-HBN with a higher turnover number of 36 s−1 × 10−3.

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Abbreviations

EMS:

Ethyl methane sulphonate

MMS:

Methyl methane sulphonate

ENU:

Ethyl nitroso urea

MNU:

Methyl nitroso urea

p-HBN:

p-Hydroxybenzonitrile

p-HBA:

p-Hydroxybenzoic acid

RSM:

Response surface methodology

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Acknowledgments

We acknowledge University Grants Commission (UGC) New Delhi, India, for financial support in the form of SRF to Vijay Kumar and JRF to Virender Kumar. The computational facility availed at Bioinformatics Centre, HP University Shimla is also duly acknowledged.

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Authors and Affiliations

  1. Department of Biotechnology, Himachal Pradesh University, Summer Hill Shimla, 171005, Himachal Pradesh, India

    Vijay Kumar, Virender Kumar, Neerja Thakur & Tek Chand Bhalla

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  1. Vijay Kumar
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Correspondence to Tek Chand Bhalla.

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Kumar, V., Kumar, V., Thakur, N. et al. Bench scale synthesis of p-hydroxybenzoic acid using whole-cell nitrilase of Gordonia terrae mutant E9. Bioprocess Biosyst Eng 38, 1267–1279 (2015). https://doi.org/10.1007/s00449-015-1367-x

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  • DOI: https://doi.org/10.1007/s00449-015-1367-x

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