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Research Project: Zero Waste Agricultural Processing

Location: Bioproducts Research

Title: Metabolic engineering of bacillus subtilis with an endopolygalacturonase gene isolated from pectobacterium carotovorum, a plant pathogenic bacterial strain

Author
item RAFIQUE, NAGINA - University Of Poonch Rawalakot
item BASHIR, SIQA - University Of Poonch Rawalakot
item KHAN, MUHAMMAD ZUBAIR - University Of Poonch Rawalakot
item HAYAT, IMRAN - University Of Poonch Rawalakot
item Orts, William - Bill
item Wong, Dominic

Submitted to: PLoS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/20/2021
Publication Date: 12/22/2021
Citation: Rafique, N., Bashir, S., Khan, M., Hayat, I., Orts, W.J., Wong, D. 2021. Metabolic engineering of bacillus subtilis with an endopolygalacturonase gene isolated from pectobacterium carotovorum, a plant pathogenic bacterial strain. PLoS ONE. http://doi.org/10.1371/journal.pone.0256562.
DOI: https://doi.org/10.1371/journal.pone.0256562

Interpretive Summary: Pectobacterium carotovorum, a gram-negative species, produces pectinolytic enzymes that hydrolyse pectin-polysaccharides within plant cells. It is a very economically important plant pathogen in terms of postharvest losses and causes decay in stored fruits and vegetables. The virulence factors of Pectobacterium are grouped under pectinases, which include pectate lyase (Pel), pectin lyases (Pnl), and polyglacturonase (or PGase). Endo-polygalacturonase (endo-PGase) enzymes have been extensively studied for random hydrolysis of a-1, 4 glycosidic bond in the linear chain of pectin, and have wide applications in food, feed, paper, fruit juice and textile industries. Many endo-PGases are recombinantly expressed in E. coli and in Pichia pastoris. Gram positive Bacillus strains are remarkable alternatives for gene expression as they have high secretion capacity and direct export of proteins into the culture medium. The present report describes for the first time the expression of an endo-PG gene from Pectobacterium carotovorum into Bacillus subtilis. The recombinant enzyme has been characterized by performing different molecular and biochemical analyses.

Technical Abstract: Pectinolytic enzymes 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. For enhanced expression analysis, competent cells of Bacillus subtilis (WB800N) were prepared at stationary phase using high salt medium and screen for enzyme activity at various temperatures and pH ranges. Optimal activity was found at pH 5.0 and a temperature of 40°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 35 minutes. and Ca++, EDTA and Zn++ were found strongly inhibitory of the enzyme activity. Chromatographic analysis of enzymatic hydrolysate of pectin substrates using HPLC and TLC revealed that tri and tetra- galacturonates were the end products. The study led to the conclusion that the endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis and the Bacillus expression system might be safe for commercial enzyme production as compared to yeast and fungi.