University Centre for Research Development, Chandigarh University Gharuan, 140 413, India.
University Centre for Research Development, Chandigarh University Gharuan, 140 413, India.
Pestic Biochem Physiol. 2018 Jul;149:73-80. doi: 10.1016/j.pestbp.2018.06.001. Epub 2018 Jun 8.
The endoglucanase belonging to glycoside hydrolase family 61 are little studied. In present study, a β-endoglucanase of ~37 kDa induced on autoclaved mycelium of Fusarium oxysporum was cloned and characterized. The molecular characterization of β-endoglucanase encoding gene revealed presence of a single intron and an open reading frame of 1044-bp which encoded a protein of 347 amino acid residues. The phylogenetic analysis of Eglu revealed its similarity to endo-β-glucanases of other Trichoderma spp. The catalytic site of β-endoglucanase contained Asp, Asn, His and Tyr residues. The cDNA encoding β-glucanase was cloned into E. coli and Pichia pastoris using pQUA-30 and pPIC9K vector system, respectively. The comparison of structure revealed that most similar structure to Eglu is Hypocrea jecorina template 5o2w.1.A of glycoside hydrolase family 61.The biochemical characterization of β-endoglucanase purified from T. saturnisporum isolate and the recombinant protein expressed in E. coli and P. pastoris was active under acidic conditions with a pH optima of 5 and temperature optima of 60 °C. The purified and expressed enzyme preparation was able to inhibit growth of F.oxysporum at 1 × 10 spores/mL which clearly revealed its significance in plant pathogen suppression.
内切葡聚糖酶属于糖苷水解酶家族 61 类，目前研究较少。本研究克隆并鉴定了一种在高压灭菌的尖孢镰刀菌菌丝体上诱导表达的~37 kDa 的β-内切葡聚糖酶。β-内切葡聚糖酶编码基因的分子特征分析表明，该基因存在一个内含子和一个 1044-bp 的开放阅读框，编码一个由 347 个氨基酸残基组成的蛋白质。Eglu 的系统发育分析表明，它与其他木霉属的内切-β-葡聚糖酶具有相似性。β-内切葡聚糖酶的催化位点含有 Asp、Asn、His 和 Tyr 残基。β-葡聚糖酶的 cDNA 分别使用 pQUA-30 和 pPIC9K 载体系统克隆到大肠杆菌和毕赤酵母中。结构比较表明，与 Eglu 结构最相似的是 Hypocrea jecorina 模板 5o2w.1.A 的糖苷水解酶家族 61。从 T. saturnisporum 分离株中纯化的β-内切葡聚糖酶和在大肠杆菌和毕赤酵母中表达的重组蛋白的生化特性研究表明，该酶在酸性条件下具有活性，最适 pH 值为 5，最适温度为 60°C。纯化和表达的酶制剂能够抑制 1×10 孢子/mL 的尖孢镰刀菌的生长，这清楚地表明了它在植物病原菌抑制中的重要性。
