油脂酵母斯达氏油脂酵母中苹果酸酶基因的克隆与特性分析。
Molecular cloning and characterization of a malic enzyme gene from the oleaginous yeast Lipomyces starkeyi.
机构信息
Dalian Institute of Chemical Physics, CAS, Dalian 116023, People's Republic of China.
出版信息
Mol Biotechnol. 2010 Jun;45(2):121-8. doi: 10.1007/s12033-010-9255-8.
The malic enzyme-encoding cDNA (GQ372891) from the oleaginous yeast Lipomyces starkeyi AS 2.1560 was isolated, which has an 1719-bp open reading frame flanked by a 290-bp 5' untranslated sequence and a 92-bp 3' untranslated sequence. The proposed gene, LsME1, encoded a protein with 572 amino acid residues. The protein presented 58% sequence identity with the malic enzymes from Yarrowia lipolytica CLIB122 and Aspergillus fumigatus Af293. The LsME1 gene was cloned into the vector pMAL-p4x to express a fusion protein (MBP-LsME1) in Escherichia coli TB1. The fusion protein was purified and then cleaved by Factor Xa to give the recombinant LsME1. This purified enzyme took either NAD(+) or NADP(+) as the coenzyme but preferred NAD(+). The K (m) values for malic acid, NAD(+) and NADP(+) were 0.85 +/- 0.05 mM, 0.34 +/- 0.08 mM, and 7.4 +/- 0.32 mM, respectively, at pH 7.3.
从产油酵母 Lipomyces starkeyi AS 2.1560 中分离到苹果酸酶编码 cDNA（GQ372891），其开放阅读框为 1719bp，侧翼为 290bp 的 5'非翻译序列和 92bp 的 3'非翻译序列。该基因被命名为 LsME1，编码一个含有 572 个氨基酸残基的蛋白质。该蛋白质与 Yarrowia lipolytica CLIB122 和 Aspergillus fumigatus Af293 的苹果酸酶具有 58%的序列同一性。将 LsME1 基因克隆到载体 pMAL-p4x 中，在大肠杆菌 TB1 中表达融合蛋白（MBP-LsME1）。纯化融合蛋白后，用 Factor Xa 切割得到重组 LsME1。该纯化酶既可以 NAD(+)也可以 NADP(+)作为辅酶，但更偏好 NAD(+)。在 pH 7.3 时，苹果酸、NAD(+)和 NADP(+)的 K (m) 值分别为 0.85 +/- 0.05 mM、0.34 +/- 0.08 mM 和 7.4 +/- 0.32 mM。
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