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

Abstract

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.

Production and biological activities of oligo pectin from early immature fruit of Durio zibethinus L.

“…The physicochemical and structural of pectin from sweet lemon peel was rich in galacturonic acid, high in molecular weight and low in degree of esterification. Also, the FT-IR results confirm the major presence of low methylated galacturonic acid rich structure in the isolated samples (Rahmani et al, 2020). The FT-IR spectrum in our study is very similar to previous studies, showing that the broad and extensive signals ranged from 3000 to 3500 cm -1 are attributed to intramolecular hydroxyl groups.…”

“…XRD spectra are particularly useful for determining the atomic structure of pectin or crystalline pectin. FT-IR can provide information about the degree of methylation of pectin by identifying characteristic absorption bands of functional groups, such as methoxyl (CH 3 O) groups (Rahmani et al, 2020). Functional attributes of pectin, including its gelling, thickening, stabilizing, and emulsifying capabilities, have been evaluated in previous studies (Huang et al, 2020).…”

Effects of single- and mixed-bacterial inoculation on the colonization and assembly of endophytic communities in plant roots

“…Compared with the Bacillus sample, most of the identified lignocellulose genes were upregulated in Enterobacter samples Ent_181 and Ent_189 ( Table 4 ). The transcriptomic analysis results verified that lignocellulose-decomposing enzymes might play an important role in bacterial colonization of plant roots, and the Enterobacter sample had higher expression levels of enzymes related to lignocellulose degradation, especially pectinases, which was consistent with the pathogenic trait of the Enterobacter strains ( Rafique et al, 2021 ). In addition to the role of enzymes, the colonization by the Bac samples in plants might be related to bacterial chemotaxis and flagellar assembly, which illustrated the complexity of the colonization process.…”

Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene Repression

“…In E. coli and S. Typhimurium, reporter plasmids were constructed using pBR322 as the parent plasmid ( 47 , 48 ). In M. smegmatis , B. thuringiensis , and B. subtilis , plasmids pMV261 ( 49 , 50 ), pRP0122 ( 28 ), and pHT43 ( 51 ) were used, respectively. Except for pHT43, the transcription of turborfp along with its 5′-UTR regulatory element was carried out under a strong constitutive promoter, J23100.…”