Abstract

Endocellulase is a key cellulase for cellulosic material pretreatment in the industry by hydrolyzing long cellulose chains into short chains. To investigate the endocellulase characteristics from Bacillus subtilis 1AJ3, and increase its production yield, this paper cloned an endocellulase gene denoted CEL-5A from strain 1AJ3 and expressed in E. coli BL21 (DE3). The CEL-5A gene was sequenced with a full-length of 1500 bp, encoding a totally of 500 amino acids, and containing two domains: the GH5 family catalytic domain (CD) and the CBM3 family cellulose-binding domain (CBD). Recombinant endocellulase Cel-5A with a His-tag was purified of the Ni-NTA column, and SDS-PAGE results demonstrated that Cel-5A exhibited a molecular weight of 56.4 kDa. The maximum enzyme activity of Cel-5A was observed at pH 4.5 and 50 °C. Moreover, it was active over the broad temperature region of 30–60 °C, and stable within the pH range of 4.5–10.0. In addition, Co²⁺ was able to increase enzyme activity, while the majority of metal ions demonstrated stable enzyme activity under low- concentration. The substrate specificity of Cel-5A exhibited a high specific activity on the β-1,3-1,4 glucan linkage from barley. The Michaelis–Menten constant and the maximum velocity of the recombinant Cel-5A for CMC-Na were determined as 14.87 mg/mL and 19.19 μmol/min/mg, respectively. When Cel-5A was applied to the switchgrass and coffee grounds, its color became lighter and the biomass was observed to loosen following hydrolyzation. The saccharification rate reached 12% of the total weight of switchgrass in 20 h. These properties highlight the potential application of Cel-5A as an endocellulase in the pretreatment of biomass, for example, in the coffee grounds/waste, and related industries.

中文翻译：

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枯草芽孢杆菌1AJ3的低温适应性GH5-CBM3内切纤维素酶的克隆，鉴定及其在柳枝switch和咖啡渣糖化中的应用

摘要

内切纤维素酶是工业上用于纤维素材料预处理的关键纤维素酶，它可以将长纤维素链水解为短链。为了研究枯草芽孢杆菌1AJ3的内切纤维素酶特性，并提高其产量，本文从1AJ3菌株中克隆了一种内切纤维素酶基因CEL -5A，并在大肠杆菌BL21（DE3）中表达。该CEL-5A基因的全长为1500 bp，编码总共500个氨基酸，并包含两个域：GH5家族催化域（CD）和CBM3家族纤维素结合域（CBD）。Ni-NTA柱纯化​​了带有His-tag的重组内切酶Cel-5A，SDS-PAGE结果表明Cel-5A的分子量为56.4 kDa。在pH 4.5和50°C下观察到Cel-5A的最大酶活性。而且，它在30–60°C的宽温度范围内都具有活性，并且在4.5–10.0的pH范围内稳定。另外，Co ²⁺能够增加酶的活性，而大多数金属离子在低浓度下表现出稳定的酶活性。Cel-5A的底物特异性对大麦的β-1,3-1,4葡聚糖键具有较高的比活性。测定CMC-Na的Michaelis-Menten常数和重组Cel-5A的最大速度分别为14.87 mg / mL和19.19μmol/ min / mg。当将Cel-5A应用于柳枝and和咖啡渣时，其颜色变浅，并且观察到水解后生物质松弛。在20小时内，糖化率达到了柳枝total总重量的12％。这些特性突出了Cel-5A作​​为内切酶在生物质预处理中的潜在应用，例如在咖啡渣/废料以及相关行业中。