Generation of High Substrate-Tolerant Mutant BS-7 of Rhodococcus rhodochrous PA-34 for Transformation of Acrylonitrile to Acrylamide

Sheetal; Monika; Vishal Ahuja; Savitri; Tek Chand Bhalla

doi:10.36948/ijfmr.2025.v07i05.58511

Generation of High Substrate-Tolerant Mutant BS-7 of Rhodococcus rhodochrous PA-34 for Transformation of Acrylonitrile to Acrylamide

Author(s)	Mr. Sheetal, Dr. Monika, Dr. Vishal Ahuja, Dr. Savitri, Prof. Tek Chand Bhalla
Country	India
Abstract	Nitrile hydratase (NHase) is a key biocatalyst employed for the commercial production of acrylamide from acrylonitrile. However, the existing biocatalysts exhibit substrate inhibition at high concentrations of acrylonitrile exceeding 1250-1500 mM (7 to 8 % w/v). The aim of this study was to generate a mutant of R. rhodochrous PA-34 using chemical mutagenesis, producing intracellular NHase, which can tolerate an acrylonitrile concentration of more than 8 % w/v. A stable mutant BS-7 was successfully generated using MNNG and screened at 1874 mM (10 % w/v) concentration of acrylonitrile. The nutritional and physiological parameters for the production of NHase by the mutant BS-7 of R. rhodochrous PA-34 were optimized using a one-variable-at-a-time (OVAT) strategy. The complete conversion of the high acrylonitrile concentration (10 % w/v) was analyzed using HPLC. Medium M7 (glucose 1 % w/v, yeast extract 0.1 % w/v, peptone 0.3 % w/v, K₂HPO₄ 0.05 %, MgSO₄∙7H₂O 0.05 % w/v, urea 7.5 % w/v and CoCl₂∙7H₂O 0.002 % w/v), pH 7.0, 30 °C incubation temperature, propionitrile as inducer (0.2 % v/v) and 36 h of incubation time were optimized for the maximum production of NHase from mutant BS-7. The yield of NHase was increased 4.7-fold after optimizing culture conditions strategically using the OVAT approach. The complete conversion of 1874 mM (10 % w/v) acrylonitrile was achieved in 30 min, confirmed using the HPLC system. In this study, an NHase from mutant BS-7 of R. rhodochrous PA-34 with high substrate tolerance and efficient biotransformation of acrylonitrile to acrylamide is reported.
Keywords	Chemical mutagenesis, N-methyl-N-nitro-N-nitrosoguanidine (MNNG), Rhodococcus rhodochrous PA-34, Nitrile hydratase (NHase), High substrate tolerance
Field	Biology > Genetics / Molecular
Published In	Volume 7, Issue 5, September-October 2025
Published On	2025-10-22
DOI	https://doi.org/10.36948/ijfmr.2025.v07i05.58511

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Generation of High Substrate-Tolerant Mutant BS-7 of Rhodococcus rhodochrous PA-34 for Transformation of Acrylonitrile to Acrylamide

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