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首页> 外文期刊>African Journal of Biotechnology >Overexpression, purification and characterization of the Aspergillus niger endoglucanase, EglA, in Pichia pastoris
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Overexpression, purification and characterization of the Aspergillus niger endoglucanase, EglA, in Pichia pastoris

机译：黑曲霉内切葡聚糖酶EglA在巴斯德毕赤酵母中的过表达，纯化和鉴定

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摘要

Cellulases are industrially important hydrolytic enzymes applicable in the bioconversion of cellulosic biomass to simple sugars. In this work, an endoglucanase from?Aspergillus niger?ATCC 10574,?EglA, was expressed in the methylotrophic yeast?Pichia pastoris?and the properties of the recombinant protein were characterized. The full length cDNA of?eglA?has been cloned into a pPICZαC expression vector and expressed extracellularly as a ~30 kDa recombinant protein in?P. pastoris?X-33. Pure?EglA?displayed optimum activity at 50°C and was stable between 30?and 55°C. The pH stability of this enzyme was shown to be in the range of pH 2.0 to 7.0 and optimum at pH 4.0.?EglA?showed the highest affinity toward β-glucan followed by carboxymethyl cellulose (CMC) with a specific activity of 63.83 and 9.47 U/mg, respectively. Very low or no detectable hydrolysis of cellobiose, laminarin, filter paper and avicel were observed. Metal ions such as Mn2+, Co2+, Zn2+, Mg2+, Ba2+, Fe2+, Ca2+?and K+?showed significant augmentation of endoglucanase activity, with manganese ions causing the highest increase in activity to about 2.7 fold when compared with the control assay, whereas Pd2+, Cu2+, SDS and EDTA showed inhibition of?EglA?activity.
机译：纤维素酶是工业上重要的水解酶，可用于将纤维素生物质生物转化为单糖。在这项工作中，来自黑曲霉ATCC 10574的EglA内切葡聚糖酶在甲基营养酵母巴斯德毕赤酵母中表达，并表征了重组蛋白的特性。已将egegAA的全长cDNA克隆到pPICZαC表达载体中，并以〜30kDa的重组蛋白在细胞外表达。 Pastoris X-33。纯的EglA 2在50℃下表现出最佳活性，在30℃和55℃之间稳定。该酶的pH稳定性显示在pH 2.0到7.0的范围内，最适在pH 4.0时。EglA2对β-葡聚糖的亲和力最高，其次是羧甲基纤维素（CMC），比活性为63.83和9.47。 U / mg。观察到纤维二糖，laminarin，滤纸和avicel的水解非常低或没有检测到。诸如Mn2 +，Co2 +，Zn2 +，Mg2 +，Ba2 +，Fe2 +，Ca2 +α和K +α等金属离子显示出内切葡聚糖酶活性的显着增强，其中锰离子引起的活性最高增加，约为对照的2.7倍，而Pd2 + ，Cu 2+，SDS和EDTA显示出对βEglAβ活性的抑制。

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