同源表达的木霉Cel7B催化模块的异质性。

Torny Eriksson, Ingeborg Stals, Anna Collén, Folke Tjerneld, Marc Claeyssens, Henrik Stålbrand, Harry Brumer
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引用次数: 31

摘要

通过菌株QM9414的转化,同源表达了jecorina(原reesei木霉)Cel7B的催化模块。采用酶解、高效色谱、质谱和定点诱变等方法分析纯化Cel7B制剂的翻译后修饰。在野生型酶中发现的五个潜在位点中,只有Asn56和Asn182被发现是n糖基化的。GlcNAc(2)Man(5)被确定为主要的n -聚糖,尽管少量的GlcNAc(2)Man(7)和携带甘露磷酸二酯键的聚糖也被检测到。中性和带电荷的糖基结构在两个糖基化位点上的重分配是观察到的蛋白质微观异质性的主要原因。然而,Asn259的部分脱酰胺和部分占据的o糖基化位点增加了酶制备的复杂性。
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Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module.

The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations.

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