Dietrich Diane, Crooks Casey
USDA Forest Products Laboratory, Madison, WI 53726, USA.
Biotechnol Lett. 2009 Aug;31(8):1223-8. doi: 10.1007/s10529-009-9983-7. Epub 2009 Apr 3.
A pyranose 2-oxidase gene from the brown-rot basidiomycete Gloeophyllum trabeum was isolated using homology-based degenerate PCR. The gene structure was determined and compared to that of several pyranose 2-oxidases cloned from white-rot fungi. The G. trabeum pyranose 2-oxidase gene consists of 16 coding exons with canonical promoter CAAT and TATA elements in the 5'UTR. The corresponding G. trabeum cDNA was cloned and contains an ORF of 1,962 base pairs encoding a 653 amino acid polypeptide with a predicted molecular weight of 72 kDa. A Hisx6 tagged recombinant G. trabeum pyranose 2-oxidase was generated and expressed heterologously in Escherichia coli yielding 15 U enzyme activity per ml of induced culture. Structural alignment and phylogenetic analysis were performed and are discussed.
利用基于同源性的简并PCR从褐腐担子菌密粘褶菌中分离出一个吡喃糖2-氧化酶基因。确定了该基因结构，并与从白腐真菌中克隆的几种吡喃糖2-氧化酶的基因结构进行了比较。密粘褶菌吡喃糖2-氧化酶基因由16个编码外显子组成，在5'UTR中有典型的启动子CAAT和TATA元件。克隆了相应的密粘褶菌cDNA，其包含一个1962个碱基对的开放阅读框，编码一个653个氨基酸的多肽，预测分子量为72 kDa。构建了一个带有Hisx6标签的重组密粘褶菌吡喃糖2-氧化酶，并在大肠杆菌中进行异源表达，每毫升诱导培养物产生15 U的酶活性。进行了结构比对和系统发育分析并加以讨论。
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