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果胶裂解酶是果胶酶的重要成员之一,能够通过β-消除作用,催化降解(1→4)-α-D-聚半乳糖醛酸。本研究首次鉴定出禾谷镰孢菌中的一个果胶裂解酶PelA,并在体外检测了其生化特性。PelA基因(FGSG_02386)的cDNA被克隆并进行了原核表达,同时构建了单基因敲除突变体。将重组质粒pET-28a(+)-PelA转化蛋白表达菌株大肠杆菌BL21表达目的蛋白,纯化后的蛋白具有果胶裂解酶活性。酶活性测定结果显示,该酶的活性受到外界环境因素如温度、钙离子浓度和pH条件的影响,其最适反应条件为50℃,CaCl2浓度0.5 mmol·L-1,pH 8.5。在侵染小麦胚芽鞘时PelA单基因突变体的毒力较野生型菌株并没有出现明显变化。
Pectate lyase is one of the important members of pectinase, which can catalyze the degradation of (1 → 4) -α-D-galacturonic acid through β-elimination. In this study, PelA, a pectate lyase from Fusarium graminearum, was identified for the first time and its biochemical properties were examined in vitro. The cDNA of PelA gene (FGSG_02386) was cloned and prokaryotic expressed, and a single-gene knockout mutant was constructed. The recombinant protein pET-28a (+) - PelA transformed protein expression strain E. coli BL21 expression of the target protein purified pectate lyase activity. The results of enzyme activity assay showed that the enzyme activity was affected by external environmental factors such as temperature, calcium ion concentration and pH. The optimal reaction conditions were 50 ℃, CaCl2 concentration 0.5 mmol·L-1, pH 8.5. The virulence of the PelA single-gene mutant did not change significantly compared to the wild type strain when it was infected with wheat cob sheath.