内酯酶产生菌的筛选及其催化拆分手性内酯的性能

Screening of Lactonase-Producing Strains and Their Performance in Catalytic Resolution of Chiral Lactones

以γ-丁内酯系列化合物作为唯一碳源,通过富集培养从土壤中分离得到180株产内酯酶的菌株.经反复筛选,从中挑选出两株内酯水解活性和选择性均较高的菌株.经鉴定,这两株真菌皆为镰孢霉菌属,分别命名为Fusarium moniliforme ECU2001和Fusarium proliferatum ECU2002.对这两株菌的产酶特性进行了研究,发现ECU2001的内酯酶属于胞内酶,而ECU2002的内酯酶同时存在于胞内和胞外.选择戊二醛交联的方法对这两株菌进行了细胞固定化,结果表明,ECU2002固定化细胞的活力、选择性和稳定性都优于ECU2001.ECU2002固定化细胞反应的适宜温度和pH分别为50℃和7.0-7.5.考察了ECU2002固定化细胞的底物专一性,发现底物为α-羟基-γ-丁内酯时,固定化细胞的活性最高,其催化速率是γ-丁内酯的54.3倍.利用ECU2002固定化细胞催化α-羟基-γ-丁内酯的对映选择性水解,固定化细胞重复使用10批后活力仍保持为初始活力的92.8%,水解产物经内酯化后得到（R）-α-羟基-γ-丁内酯的光学纯度为92.8%-96.1%ee.

Fusarium moniliforme strain ECU2001 and Fusarium proliferatum strain ECU2002, capable of catalyzing the enantioselective hydrolysis of chiral lactones, were newly identified from 180 strains isolated from soil samples through two steps of screening. The lactonase produced by ECU2001 was an intracellular enzyme, whereas that by ECU2002 existed in both inside and outside of the mycelia. Simple immobilization of fungal mycelia of ECU2001 and ECU2002 by glutaraldehyde cross-linking led to stable and easy-tohandle biocatalysts for hydrolytic resolution of chiral lactones. The activity and selectivity of immobilized ECU2002 cells were better than those of ECU2001. The catalytic performance of immobilized ECU2002 cells was investigated, giving temperature and pH optima at 50 ℃ and pH 7.0-7.5, respectively. The substrate specificity of immobilized ECU2002 cells was also examined using several useful lactones, among which α-hydroxy-γ-butyrolactone was the best substrate, with 54.3-fold higher lactonase activity as compared to γ-butyrolactone. The immobilized cells of ECU2002 were used for repeated resolution of α-hydroxy-γ-butyrolactone at a substrate concentration as high as 1 mol/L, affording the hydrolytic product with 92.896-96.1% ee. The immobilized ECU2002 cells were very stable, retaining 92.8 % of initial activity after 10 cycles of repeated reaction.