Zhou Qinzheng, Ji Peng, Zhang Jianye, Li Xue, Han Chao
Department of Mycology, Shandong Agricultural University, Taian, Shandong 271018, China.
Department of Mycology, Shandong Agricultural University, Taian, Shandong 271018, China.
J Biosci Bioeng. 2017 Sep;124(3):271-276. doi: 10.1016/j.jbiosc.2017.03.017. Epub 2017 May 19.
A novel thermostable endoglucanase (CTendo45) encoding gene was cloned from Chaetomium thermophilum and heterologously expressed in Pichia pastoris. Sequence alignment indicated that the CTendo45 enzyme belonged to glycoside hydrolase family 45. The recombinant enzyme was purified by Ni affinity chromatography, and its apparent molecular mass was estimated to be 32 kDa by SDS-PAGE. The purified enzyme displayed maximum activity at 70°C and pH 4. CTendo45 was stable at 60°C for 1 h, and residual activities of 78.9% and 65.6% were estimated after 1 h at 70°C and 80°C, respectively. Ca, Zn, Mg, Cu and Mn were found to have beneficial effects on the enzyme activity to different degrees. The specific activity of purified CTendo45 was 1.52 IU mg and the K value was 59.6 μg ml with a sodium carboxymethyl cellulose substrate. Moreover, CTendo45 exhibited high hydrolysis activity towards pectin, and the hydrolysis products were mainly galacturonic acid oligosaccharides. CTendo45 is the first reported bifunctional enzyme in glycoside hydrolase family 45 from C. thermophilum that is able to hydrolyze both cellulose and pectin. The biochemical properties of this recombinant CTendo45 make it a potentially effective glycoside hydrolase for industrial applications.
从嗜热毛壳菌中克隆出一个编码新型耐热内切葡聚糖酶（CTendo45）的基因，并在毕赤酵母中进行了异源表达。序列比对表明CTendo45酶属于糖苷水解酶家族45。重组酶通过镍亲和层析进行纯化，SDS-PAGE测定其表观分子量约为32 kDa。纯化后的酶在70°C和pH 4时表现出最大活性。CTendo45在60°C下1小时内稳定，在70°C和80°C下1小时后分别测得残留活性为78.9%和65.6%。发现Ca、Zn、Mg、Cu和Mn对酶活性有不同程度的促进作用。纯化后的CTendo45以羧甲基纤维素钠为底物时的比活性为1.52 IU mg，K值为59.6 μg ml。此外，CTendo45对果胶表现出高水解活性，水解产物主要是半乳糖醛酸寡糖。CTendo45是首次报道的来自嗜热毛壳菌的糖苷水解酶家族45中的双功能酶，能够水解纤维素和果胶。这种重组CTendo45的生化特性使其成为工业应用中一种潜在有效的糖苷水解酶。
