Eriksson Torny, Stals Ingeborg, Collén Anna, Tjerneld Folke, Claeyssens Marc, Stålbrand Henrik, Brumer Harry
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, Sweden.
Eur J Biochem. 2004 Apr;271(7):1266-76. doi: 10.1111/j.1432-1033.2004.04031.x.
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.
通过转化菌株QM9414同源表达了嗜热栖热放线菌（以前的里氏木霉）Cel7B的催化模块。通过酶切、高效液相色谱、质谱和定点诱变分析了纯化的Cel7B制剂中的翻译后修饰。在野生型酶中发现的五个潜在位点中，仅发现Asn56和Asn182发生了N-糖基化。尽管也检测到少量的GlcNAc(2)Man(7)和带有甘露糖磷酸二酯键的聚糖，但GlcNAc(2)Man(5)被鉴定为主要的N-聚糖。中性和带电荷聚糖结构在两个糖基化位点上的分布主要解释了观察到的蛋白质微异质性。然而，Asn259的部分脱酰胺作用和一个部分占据的O-糖基化位点导致酶制剂中出现进一步的复杂性。
