Cloning and overexpression of ketopantoic acid reductase gene from Stenotrophomonas maltophilia and its application to stereospecific production of D-pantoic acid
Cloning and overexpression of ketopantoic acid reductase gene from Stenotrophomonas maltophilia and its application to stereospecific production of D-pantoic acid
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DOI：
10.1007/s00253-011-3664-x
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发表时间：
2012-02-01
影响因子：
5
通讯作者：
Kataoka, Michihiko
中科院分区：
文献类型：
--
作者：
Si, Dayong;Urano, Nobuyuki;Kataoka, Michihiko
酮泛酸(KPA)还原酶催化酮泛酸立体定向还原为D-泛酸。根据嗜麦芽窄食单胞菌845的KPA还原酶N端氨基酸序列，克隆了嗜麦芽窄食单胞菌NBRC14161的KPA还原酶基因，并进行了序列测定。该基因包含一个777bp的开放阅读框，编码258个氨基酸残基，推导的氨基酸序列与SDR超家族蛋白高度相似。构建了含KPA还原酶全长基因的表达载体pETSmKPR，并将其导入大肠杆菌BL21(DE3)中进行高效表达。利用共表达KPA还原酶和辅因子再生酶基因的大肠杆菌转化细胞对KPA进行了生物还原。当底物浓度达到1.17M时，KPA转化率可达88%以上。
Ketopantoic acid (KPA) reductase catalyzes the stereospecific reduction of ketopantoic acid to d-pantoic acid. Based on the N-terminal amino acid sequence of KPA reductase from Stenotrophomonas maltophilia 845, the KPA reductase gene was cloned from S. maltophilia NBRC14161 and sequenced. This gene contains an open reading frame of 777 bp encoding 258 amino acid residues, and the deduced amino acid sequence showed high similarity to the SDR superfamily proteins. An expression vector, pETSmKPR, containing the full KPA reductase gene was constructed and introduced into Escherichia coli BL21 (DE3) to overexpress the enzyme. Bioreduction of KPA using E. coli transformant cells coexpressing KPA reductase together with cofactor regeneration enzyme gene was also performed. The conversion yield of KPA to d-pantoic acid reached over 88% with a substrate concentration up to 1.17 M.