Department of Food Science and Technology, Faculty of Agriculture, University of the Poonch, Rawalakot, Azad Jammu and Kashmir, Pakistan.
Bioproducts Research Unit, Western Regional Research Centre, United States Department of Agriculture, Albany, California, United States of America.
PLoS One. 2021 Dec 22;16(12):e0256562. doi: 10.1371/journal.pone.0256562. eCollection 2021.
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.
果胶酶或果胶裂解酶由许多微生物和植物自然合成。这些酶可降解各种果胶，果胶是植物细胞壁的主要成分。从植物病原菌果胶杆菌中分离出编码内切聚半乳糖醛酸酶（endo-PGase）的果胶酶基因，并成功克隆到具有 σA 依赖性启动子的分泌载体 pHT43 中，用于枯草芽孢杆菌（WB800N）的异源表达。期望的 PCR 产物为 1209bp，编码 402 个氨基酸的开放阅读框。重组蛋白经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）证实，估计分子量为 48 kDa。含有工程化 pHT43 载体和外源内切 PG 酶基因的转化枯草芽孢杆菌感受态细胞在 2X-酵母提取物-胰蛋白胨培养基中培养，然后在不同温度和 pH 值范围内筛选酶活性。重组内切 PG 酶的最佳活性在 40°C 和 pH 5.0 时发现。为了测定金属离子的催化效应，将重组酶与 1mM 浓度的各种金属离子孵育。氯化钾增加了酶活性，而 EDTA、Zn++和 Ca++则强烈抑制了酶活性。使用 HPLC 和 TLC 对聚半乳糖醛酸（PGA）和果胶底物的酶水解产物进行色谱分析，结果表明三糖和四糖醛酸盐是重组内切 PG 酶水解的终产物。总之，首次使用 pHT43 表达载体在枯草芽孢杆菌中成功表达了来自植物病原菌的内切 PG 酶基因，并可通过 IPTG 诱导使用非常简单的培养基评估酶的产量。与酵母和真菌相比，这些发现表明枯草芽孢杆菌表达系统在商业酶生产中可能更安全，可避免内毒素。此外，重组内切 PG 酶活性产生的水解产物为食品和饮料行业的优质产品提供了有用的应用。
