Cloning, expression and purification of a novel carboxypeptidase from a hypersaline lake halophilic Bacillus persicus

Document Type : Research Paper

Authors

1 M.Sc. Student of Biochemistry, Faculty of Science, University of Guilan, Rasht, Iran

2 Associate Professor in Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran

3 Scientific Member in Molecular Bank, Iranian Biological Resource Center (IBRC), Academic Center for Education, Culture and Research (ACECR), Tehran, Iran

10.22124/japb.2022.22750.1479

Abstract

Microbial proteases have a large portion of the market of industrial enzymes, because of their wide application in detergents, drug enzymes and animal feed processing. Bacillus strains potentially produce a significant quantity of proteases, due to their physiological properties. In this study, first, the gene encoding carboxypeptidase from Bacillus persicus was isolated by PCR and using primers with NdeI and XhoI restriction sites. After digestion of the PCR product, it was cloned into the appropriate site at PET28a+ vector, then recombinant plasmids transformed into E. coli BL21)DE3(­­ strain. Plasmids with positive colony PCR result were sent for sequencing for final confirmation, then, recombinant enzyme expression was optimized at different temperature and time conditions, then purified with nickel embedded agarose affinity chromatography.  IPTG concentration of 0.4mM at 32°C and incubation time of 20 hours provided the maximum amount of enzyme expression. Molecular weight of the carboxypeptidase was estimated approximately 58KDa. Considering the high amount of expression of the soluble protein and the easy optimization of its expression and purification in laboratory conditions, if the enzyme activity in the presence of standard substrates is adequate, and also if additional studies show the possibility of optimal and cost-effective production of a high amount of this enzyme in a large volume and pilot conditions, it can be hoped that this enzyme is a suitable option for industrial purposes.

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References

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Aquatic Physiology and Biotechnology
Volume 11, Issue 1
June 2023
Pages 59-75

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