Recovery of choline oxidase activity by in vitro recombination of individual segments

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• 作者：Birgit Heinze ; Nina Hoven ; Timothy O¡¯Connell ; Karl-Heinz Maurer ; Sebastian Bartsch and Uwe T. Bornscheuer
• 关键词：Choline oxidase ; In vitro recombination ; Chimeragenesis
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2008
• 出版时间：November, 2008
• 年：2008
• 卷：81
• 期：2
• 页码：275-282
• 全文大小：279.7 KB

文摘

Initial attempts to express a choline oxidase from Arthrobacter pascens (APChO-syn) in Escherichia coli starting from a synthetic gene only led to inactive protein. However, activity was regained by the systematic exchange of individual segments of the gene with segments from a choline oxidase-encoding gene from Arthrobacter globiformis yielding a functional chimeric enzyme. Next, a sequence alignment of the exchanged segment with other choline oxidases revealed a mutation in the APChO-syn, showing that residue 200 was a threonine instead of an asparagine, which is, thus, crucial for confering enzyme activity and, hence, provides an explanation for the initial lack of activity. The active recombinant APChO-syn-T200N variant was biochemically characterized showing an optimum at pH 8.0 and at 37¡ãC. Furthermore, the substrate specificity was examined using N,N-dimethylethanolamine, N-methylethanolamine and 3,3-dimethyl-1-butanol.