Kluyveromyces Lactis ARTP Mutagenesis Breeding For High Ketoreductase Production And Its Catalytic Properties

The Kluyveromyces lactis culture medium was optimized by single factor,and the fermentation process of acetophenone catalyzed by Kluyveromyces lactis was further optimized by orthogonal experiment.The optimum conditions for Kluyveromyces lactis to catalyze the acetophenone catalysis were obtained.Kluyveromyces lactis can transform the acetophenone up to 27.28%under the conditions of the substrate concentration 20g/L at a time,the sucrose 22.0g/L,the yeast extract 10.0g/L,KH₂PO₄3.0g/L,Mg SO₄·7H₂O 1g/L at 28?,200 rpm for 36 h.The mutant strain KL5 with 2.72 U/m L ketorecutase activity was obtained by the screening of Kluyveromyces lactis using atomspheric room temperature plasma(ARTP)through the selection maker of the resistance to 20g/L acetophenone and high ketoreductase activity.The growth characteristic and the biotransformation ability to acetophenone for the mutant strain KL5 were further investigated.The results showed that the sucrose and the yeast extract were respectively the best carbon and nitrogen sources and the mutant strain KL5 can transform the acetophenone up to 91.8%under the conditions of the substrate concentration 20g/L at a time,the sucrose 22.0g/L,the yeast extract 10.0g/L,KH₂PO₄3.0g/L,Mg SO₄·7H₂O 1g/L at 28?,200rpm for 36 h.The mutant strain KL5 is used to catalyze the intermediates of typical chiral drugs to evaluate the enzymatic activity of keto-reductase,for three kinds of cyclopropyl ketone like:Cyclopropyl methyl ketone,acetyl cyclopentane and acetyl cyclohexane their conversion rates were 78.9%,50%,and 30.1%;The conversion ratios of the 1-,4-naphthalenone and 1-naphthyl ketone of the different heterocyclic groups were 16.7%and 10.3%;The conversion rates of phenyl methyl ketone,2-Chloroacetophenone and4-Chloroacetophenone were 91.7%,96.8%and 98.2%;The conversion rate of CPMK was 90.6%.