Cloning, Expression And Characterization Of Alcohol Dehydrogenase From Lactobacillus Kefir

Alcohol dehydrogenases （E.C.1.1.1.1, also known as keto-reductase） belong to the class of oxidoreductases. They play an important role in metabolic pathway by catalyzing alcohol metabolism in organism. Among the class of oxidoreductases, alcohol dehydrogenases represent an important group due to their ability to stereospecifically reduce prochiral carbonyl compounds. Alcohol dehydrogenases （ADH） can be used in high-efficient synthesis of optically active alcohols （chiral alcohol）, which are key building blocks for the medical and pharmaceutical industry. Finding and developing novel alcohol dehydrogenase applicable in the production of chiral drug or other field is very popular scientific research. In this work, a novel gene named LK-adh encoding alcohol dehydrogenase was cloned from Lactobacillus kefir DSM20587. An engineering strain expressing the recombinant LK-ADH was constructed and the conditions for its expression and enzymology were investigated. The cofactor preference and enantioselectivity of this novel alcohol dehydrogenase in asymmetric reduction were also determined. Finally, a preliminary research was carried out to study the transcription and expression of LK-ADH in Lactobacillus kefir. Details are as follows:A fragment which consists of an open reading frame （ORF） of 1,044bp, coding for 347 amino acids, was cloned from the genomic DNA of Lactobacillus kefir DSM20587 with the help of bioinformatics and TAIL-PCR. The ORF was analyzed and named as LK-adh by a Blast similarity search in GenBank database. The highest homology was found less than 78% at protein level. This novel gene was deposited into GenBank with the accession number of EU877965 and the corresponding protein sequence could be found under the accession number of ACF95832.LK-adh was then subcloned into the plasmid pET-28a（+） followed by transforming into E.coli BL21（DE3） to obtain the engineering strain E.coli BL21（DE3）/pYG461. The recombinant LK-ADH could be efficiently expressed by IPTG induction. The conditions for enzyme activity such as incubation temperature, IPTG concentration, rotation speed of shaker and so on were studied.The optimum conditions for incubation of engineering strain were as follows:temperature 27℃,initial pH value of medium 6.0, rotation speed of shaker 150rpm; the optimum conditions for the induction of recombinant LK-ADH were:optical density of engineering strain （OD600） 0.8, 0.2mmol/L IPTG,induction time 6 h. Under these conditions, the recombinant enzyme activity was increased from 7.2×10^-2U/ml to 2.17×10^-1U/ml.The recombinant LK-ADH was purified to apparent homogeneity in SDS-PAGE and the purification factor was 4.5 fold while the specific activity of the purified enzyme was 6.7 U/mg. LK-ADH showed high enantioselectivity in the reduction of acetophenone to （S）-phenylethanol, the enantiomeric excess （ee） value was calculated as 99.4%. Coenzyme dependent assay indicated that NADH was preferably used as a cofactor. Therefore, LK-ADH was identified to be a NADH-dependent （S）-specific alcohol dehydrogenase.The zymological properties of recombinant enzyme were characterized. The optimum pH in oxidative and reductive reaction is 5.6 and 5.0 respectively and the optimum reaction temperature is 35℃. The enzyme activity had no obvious loss after stored at 20³5℃for 1h . However, only 70% activity was remained if stored at -20℃for 4 weeks. The Km values of the LK-ADH in reductive and oxidative reaction were 55.37mmol/L and 88.60mmol/L respectively. The enzyme activity could be activated by Zn²⁺ and Mg²⁺, but inhibited by Cu²⁺, 2-mercaptoethanol and DTT. LK-ADH showed relatively high activity with all the tested aldehydes as substrates but low activity in oxidation of alcohols. Ketones with bulky side chains, such as acetophenone and its derivative were also found to be effectively transformed to their corresponding alcohols. .LK-adh was identified to be continuously expressed in L.kefir cell by RT-PCR analysis. The transcription level at 24h was 130% as compared with that at 12h, however, 38%⁶3% lower than those by a previously reported alcohol dehydrogenase gene LK-adh（R） in L.kefir cell at 12h and 24h. These results showed that at least two alcohol dehydrogenase genes with different enantioselectivity are existed in the genome of Lactobacillus kefir. Transformation of acetophenone by L.kefir cell also showed that both （R） and （S）-phenylethanol were acquired in the products.