Zhang Xian, Xu Jian-He, Xu Yi, Pan Jiang
Laboratory of Biocatalysis and Bioprocessing, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
Appl Microbiol Biotechnol. 2007 Jul;75(5):1087-94. doi: 10.1007/s00253-007-0941-9. Epub 2007 May 26.
A fungus strain ECU2002, capable of enantioselectively hydrolyzing chiral lactones to optically pure hydroxy acids, was newly isolated from soil samples through two steps of screening and identified as Fusarium proliferatum (Matsushima) Nirenberg. From the crude extract of F. proliferatum ECU2002, a novel levo-lactonase was purified to homogeneity, with a purification factor of 460-folds and an overall yield of 9.7%, by ultrafiltration, acetone precipitation, and chromatographic separation through DEAE-Toyopearl, Butyl-Toyopearl, Hydroxyapatite, Toyoscreen-Super Q, and TSK-gel columns. The purified enzyme is a monomer; with a molecular mass of ca 68 kDa and a pI of 5.7 as determined by two-dimensional electrophoresis. The catalytic performance of the partially purified levo-lactonase was investigated, giving temperature and pH optima at 50 degrees C and 7.5, respectively, for gamma-butyrolactone hydrolysis. The substrate specificity of the partially purified lactonase was also examined using several useful lactones, among which alpha-hydroxy-gamma-butyrolactone was the best substrate, with 448-fold higher lactonase activity as compared to gamma-butyrolactone. The F. proliferatum lactonase preferentially hydrolyzed the levo enantiomer of butyrolactones, including beta-butyrolactone, alpha-hydroxy-gamma-butyrolactone, alpha-hydroxy-beta,beta-dimethyl-gamma-butyrolactone (pantolactone), and beta-hydroxy-gamma-butyrolactone, affording (+)-hydroxy acids in high (94.8 approximately 98.2%) enantiomeric excesses (ee) and good conversions (38.2 approximately 44.2%). A simple immobilization of the crude lactonase with glutaraldehyde cross-linking led to a stable and easy-to-handle biocatalyst for catalytic resolution of chiral lactones. The immobilized lactonase also performed quite well in repeated batch resolution of dl-pantolactone at a concentration of 35% (w/v), retaining 67% of initial activity after ten cycles of reaction (corresponding to a half life of 20 cycles) and affording the product in 94 approximately 97% ee, which can be easily enhanced to >99% ee after the d-hydroxy acid was chemically converted into l-lactone and crystallized.
从土壤样品中通过两步筛选新分离出一株能够对映选择性地将手性内酯水解为光学纯羟基酸的真菌菌株ECU2002，并鉴定为层出镰刀菌（Matsushima）Nirenberg。通过超滤、丙酮沉淀以及使用DEAE - Toyopearl、Butyl - Toyopearl、羟基磷灰石、Toyoscreen - Super Q和TSK - 凝胶柱进行色谱分离，从层出镰刀菌ECU2002的粗提物中纯化出一种新型左旋内酯酶，纯化倍数为460倍，总收率为9.7%。纯化后的酶是单体；通过二维电泳测定其分子量约为68 kDa，pI为5.7。研究了部分纯化的左旋内酯酶的催化性能，其对γ - 丁内酯水解的最适温度和pH分别为50℃和7.5。还使用几种有用的内酯检测了部分纯化的内酯酶的底物特异性，其中α - 羟基 - γ - 丁内酯是最佳底物，其内酯酶活性比γ - 丁内酯高448倍。层出镰刀菌内酯酶优先水解丁内酯的左旋对映体，包括β - 丁内酯、α - 羟基 - γ - 丁内酯、α - 羟基 - β,β - 二甲基 - γ - 丁内酯（泛内酯）和β - 羟基 - γ - 丁内酯，以高（94.8%至98.2%）对映体过量（ee）和良好的转化率（38.2%至44.2%）生成（+） - 羟基酸。用戊二醛交联简单固定粗内酯酶可得到一种稳定且易于操作的生物催化剂，用于手性内酯的催化拆分。固定化内酯酶在35%（w/v）浓度的dl - 泛内酯的重复分批拆分中也表现良好，在十个反应循环后保留67%的初始活性（相当于半衰期为20个循环），并以94%至97% ee生成产物，在将d - 羟基酸化学转化为l - 内酯并结晶后，ee值可轻松提高到>99%。
