产D-海因酶微生物的筛选及其催化特性

Screening of D-Hydantoinase-Producing Microorganism and Its Catalytic Properties

为获得高活性和立体选择性海因酶生产菌株,首先采用氨基酸关环法合成了数种5'-单取代海因衍生物及N-氨甲酰-氨基酸,建立了相关液相色谱检测方法。然后以实验室保藏的野生菌株出发,利用底物诱导的方法筛选到6株具有D-海因酶活性的野生菌,其中荧光假单胞菌产生的D-海因酶具有较好的底物谱,较高的活性和立体选择性。经优化后,其最适产酶温度为30℃,培养时间为12 h,发酵活力达到92.1 U/L。该菌催化D-海因水解的最适反应温度为60℃,pH 8.5。在50 m L体系中,利用0.25 g菌体(干重)可对映选择性水解20 mmol/L对羟基苯海因底物,反应3 h转化率达到98%。

Hydantoinases are important enzymes with industrial potential in producing D-amino acids. In this study,6 strains with hydantoinase activity were selected from 23 wildtype microbial strains using 5＇-monosubstituted hydantoin derivatives as induce substrates. Four 5＇-monosubstituted hydantoins were chemically synthesized and verified with 1H-NMR. From 6 hydantoinase-producing strains,strain Pseudomonas fluorescenswas successfully identified with the highest enantioselectivity,desired substrate specificity and high activity in the hydrolysis of 5＇-monosubstituted hydantoins andwas selected for further study. After induction with hydantoin substrates at 30 ℃ for 12 h,the D-hydantoinase activity could reach 92.1 U/L. The reaction condition was optimized to be 60 ℃ and pH 8.5,which was in favor of the spontaneous racemization of substrate. In a 50 mL whole-cell biocatalytic system,20 mmol/L D,L-p-hydroxyphenyl hydantoin was enantioselectively hydrolyzed by 0.25 g dry cells with a conversion ratioof 98% within 3 hours.