Metabolic engineering of Bacillus subtilis with an endopolygalacturonase gene isolated from Pectobacterium. carotovorum; a plant pathogenic bacterial strain.

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science

Bacillus subtilis/*growth & development ; Metabolic Engineering/*methods ; Pectobacterium carotovorum/*enzymology ; Polygalacturonase/*metabolism; Bacillus subtilis/genetics ; Bacterial Proteins/genetics ; Bacterial Proteins/metabolism ; Cloning, Molecular ; Hexuronic Acids/metabolism ; Pectins/metabolism ; Pectobacterium carotovorum/genetics ; Polygalacturonase/genetics ; Potassium Chloride/metabolism ; Promoter Regions, Genetic

Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector 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 safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.
Competing Interests: The authors have declared that no competing interests exist.

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0 (Bacterial Proteins)
0 (Hexuronic Acids)
4JK6RN80GF (galacturonic acid)
660YQ98I10 (Potassium Chloride)
89NA02M4RX (Pectins)
EC 3.2.1.15 (Polygalacturonase)
VV3XD4CL04 (polygalacturonic acid)

Date Created: 20211222 Date Completed: 20220110 Latest Revision: 20240819

20250114

PMC8694468

10.1371/journal.pone.0256562

34936645