Abstract

Two novel glycoside hydrolases were cloned from the genomic DNA of alklinphilic bacterium Cellulomonas bogoriensis 69B4^T and functionally expressed in Escherichia coli. The two enzymes shared less than 73% of identities with other known glycosidases and belonged to glycoside hydrolase families 5 and 9. Recombinant Cel5A exhibited optimum activity at pH 5.0 and at a temperature of 70 °C, and Cel9A showed optimum activity at pH 7.0 and at a temperature of 60 °C. The two enzymes exhibited activity at alkaline pH 11 and were stable over a wide range of pH. The maximum activities of Cel5A and Cel9A were observed in 0.5 M NaCl and 1 M KCl, respectively. In addition, these two enzymes exhibited excellent halostability with residual activities of more than 70% after pre-incubation for 6 days in 5 M NaCl or 4 M KCl. Substrate specificity analysis revealed that Cel5A and Cel9A specifically cleaved the β-1,4-glycosidic linkage in cellulose with the highest activity on carboxymethyl cellulose sodium (78.3 and 145.3 U/mg, respectively). Cel5A is an endoglucanase, whereas Cel9A exhibits endo and exo activities. As alkali-activated, thermo-tolerant, and salt-tolerant cellulases, Cel5A and Cel9A are promising candidates for further research and industrial applications.

中文翻译：

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极端嗜碱性细菌玻纤单胞菌69B4 T的两个糖苷水解酶的重组表达和表征

摘要

从嗜碱性细菌Boulomonas bogoriensis 69B4 ^T的基因组DNA中克隆了两个新型糖苷水解酶，并在大肠杆菌中进行功能表达。这两种酶与其他已知的糖苷酶的同一性少于73％，属于糖苷水解酶家族5和9。重组Cel5A在pH 5.0和70°C的温度下显示最佳活性，而Cel9A在pH 7.0和70℃时显示最佳活性。在60°C的温度下。这两种酶在碱性pH 11时表现出活性，并且在很宽的pH范围内都稳定。在0.5 M NaCl和1 M KCl中分别观察到Cel5A和Cel9A的最大活性。此外，这两种酶在5 M NaCl或4 M KCl中预培养6天后，显示出优异的光晕性，残留活性超过70％。底物特异性分析表明，Cel5A和Cel9A特异性切割了纤维素中的β-1,4-糖苷键，对羧甲基纤维素钠的活性最高（78.3和145.3 U / mg，分别）。Cel5A是一种内切葡聚糖酶，而Cel9A则具有内切和外切活性。作为碱活化，耐热和耐盐的纤维素酶，Cel5A和Cel9A是有希望的候选物，可用于进一步的研究和工业应用。