Cloning, Expression, and Characterization of a Novel (S)-Specific Alcohol Dehydrogenase from Lactobacillus kefir
Cloning, Expression, and Characterization of a Novel (S)-Specific Alcohol Dehydrogenase from Lactobacillus kefir
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DOI：
10.1007/s12010-008-8442-6
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发表时间：
2010-01-01
影响因子：
3
通讯作者：
Zhu, Baoquan
中科院分区：
文献类型：
--
作者：
Chen, Qilei;Hu, Youjia;Zhu, Baoquan
采用热不对称交错聚合酶链反应（thermal asymmetric interlaced polymerase chain reaction，HEAT-PCR）技术，从开菲尔乳杆菌（Lactobacillus kefir DSM 20587）基因组DNA中克隆了一个新的（S）特异性依赖于NADH的乙醇脱氢酶（LK-ADH）基因。测定了（S）-LK-ADH基因（adhS）的核苷酸序列，其开放阅读框为1,044 bp，编码347个氨基酸，分子量为37.065 kDa。经BLAST同源性检索，（S）-LK-ADH的氨基酸序列与几种含锌的中链乙醇脱氢酶有一定的同源性。该基因已在GenBank中登录，登录号为EU 877965。将adhS基因亚克隆到原核表达载体pET-28 a（+）中，在大肠杆菌中成功表达了重组（S）-LK-ADH。coliBL 21（DE 3）中，通过异丙基-β-D-1-硫代半乳糖苷诱导表达。纯化后的酶对苯乙酮还原为（S）-苯乙醇具有较高的对映选择性，ee值为99.4%。对重组（S）-LK-ADH的底物特异性和辅因子选择性进行了检测。
A gene encoding a novel (S)-specific NADH-dependent alcohol dehydrogenase (LK-ADH) was isolated from the genomic DNA of Lactobacillus kefir DSM 20587 by thermal asymmetric interlaced-polymerase chain reaction. The nucleotide sequence of (S)-LK-ADH gene (adhS) was determined, which consists of an open reading frame of 1,044 bp, coding for 347 amino acids with a molecular mass of 37.065 kDa. After a BLAST similarity search in GenBank database, the amino acid sequence of (S)-LK-ADH showed some homologies to several zinc containing medium-chain alcohol dehydrogenases. This novel gene was deposited into GenBank with the accession number of EU877965. adhS gene was subcloned into plasmid pET-28a(+), and recombinant (S)-LK-ADH was successfully expressed in E. coli BL21(DE3) by isopropyl-beta-D-1-thiogalactopyranoside induction. Purified enzyme showed a high enantioselectivity in the reduction of acetophenone to (S)-phenylethanol with an ee value of 99.4%. The substrate specificity and cofactor preference of recombinant (S)-LK-ADH were also tested.