Abstract

Endoglucanases are key elements in several industrial applications, such as cellulosic biomass hydrolysis, cellulose fiber modification for the production paper and composite materials, and in nanocellulose production. In all of these applications, the desired function of the endoglucanase is to create nicks in the amorphous regions of the cellulose. However, endoglucanase can be diverted from its activity on the fibers by other substrates—soluble oligosaccharides. This issue was addressed in the current study using enzyme engineering and an enzyme evolution approach. To this end, a hypothetical endoglucanase from a thermostable bacterium Spirochaeta thermophila was for the first time cloned and characterized. The wild-type enzyme was used as a starting point for mutagenesis and molecular evolution toward a preference for the higher molecular weight substrates. The best of the evolved enzymes was more active than the wild-type enzyme toward high molecular weight substrate at temperatures below 45 °C (3-fold more active at 30 °C) and showed little or no activity with low molecular weight substrates. These findings can be instrumental in bioeconomy sectors, such as second-generation biofuels and biomaterials from lignocellulosic biomass.

Key points

• A new thermostable endoglucanase was characterized.

• The substrate specificity of this endoglucanase was changed by means of genetic engineering.

• A mutant with a preference for long molecular weight substrate was obtained and proposed to be beneficial for cellulose fiber modification.

中文翻译：

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嗜热螺旋藻及其底物偏好改变的突变体的新型热稳定内切葡聚糖酶

摘要

内切葡聚糖酶是几种工业应用中的关键元素，例如纤维素生物质水解，生产纸和复合材料的纤维素纤维改性以及纳米纤维素生产。在所有这些应用中，内切葡聚糖酶的所需功能是在纤维素的无定形区域中产生切口。但是，内切葡聚糖酶可以通过其他底物（可溶性寡糖）改变其对纤维的活性。当前的研究使用酶工程和酶进化方法解决了这个问题。为此，从热稳定细菌嗜热螺旋藻中获得了一种假定的内切葡聚糖酶首次克隆和鉴定。野生型酶被用作诱变和分子进化的起点，倾向于偏高分子量的底物。在低于45°C的温度下，最好的进化酶比野生型酶对高分子量底物的活性更高（在30°C时，活性高3倍），而对低分子量底物则几乎没有或没有活性。这些发现可能对生物经济领域有帮助，例如第二代生物燃料和木质纤维素生物质的生物材料。

关键点

•表征了一种新的热稳定的内葡聚糖酶。

•此内切葡聚糖酶的底物特异性已通过基因工程方法改变。

•获得了一种偏爱长分子量底物的突变体，并提出对纤维素纤维改性有益。