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bioRxiv · 10.1101/2021.08.17.456673

Metabolic engineering of Bacillus subtilis with an Endopolygalacturonase gene Isolated from Pectobacterium carotovorum; a Plant Pathogenic bacterial strain.

Rafique, N.·Bashir, S.·Khan, M. Z.·Hayat, I.·Orts, W.·Wong, D. W. S.
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Abstract

Pectinolytic enzymes (pectinases) produced by microbes are highly important for their biotechnological use in processing of vegetables and fruits beverages and use in pulp and paper industry. A pectinase, namely endo-polygalacturonase (endo-PGase), encoding gene isolated from Pectobacterium carotovorum, a plant pathogenic strain of bacteria was successfully cloned into a secretion vector pHT43 having {sigma}A-dependent promoter P grac. For enhanced expression analysis, competent cells of Bacillus subtilis (WB800N) were prepared at stationary phase using high salt medium. The recombinant B. subtilis competent cells, harbouring the engineered pHT43 with the endo-PGase gene were cultured in 2X-yeast extract tryptone medium. The recombinant endo-PGase enzyme was secreted directly into the medium after 72 hours of the first IPTG induction. The recombinant endo-PGase was screened for its activity at various temperatures and pH ranges. Optimal activity was found at pH 5.0 and a temperature of 40{degrees}C with a stability ranging from pH 5.0-9.0. For detection of metal ion effect, recombinant enzyme was incubated with 1mM concentration of; Ca++, Mg++, Zn++, EDTA, K++ for 45 minutes. Resultantly, Ca++, EDTA and Zn++ strongly inhibited the enzyme activity. The chromatographic analysis of enzymatic hydrolysate of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed that tri and tetra-galacturonates were the end products of hydrolysis. The study led to the conclusion that endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safe for commercial enzyme production as compared to yeast and fungi to escape endotoxins.

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Rafique, N., Bashir, S., Khan, M. Z., Hayat, I., Orts, W., Wong, D. W. S.. 2021-08-17. Metabolic engineering of Bacillus subtilis with an Endopolygalacturonase gene Isolated from Pectobacterium carotovorum; a Plant Pathogenic bacterial strain.. https://doi.org/10.1101/2021.08.17.456673

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