Kinsella B T, Larkin A, Bolton M, Cantwell B A
Guinness Brewing Worldwide Research Centre, St. James's Gate, Dublin, Ireland.
Curr Genet. 1991 Jul;20(1-2):45-52. doi: 10.1007/BF00312764.
The molecular cloning of an alpha-glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cervisiae is reported. The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S. cerevisiae. The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing alpha-1,2, alpha-1,3, alpha-1,4 and alpha-1,6 linked, as well as aryl and alkyl, D-glucosides. On the basis of its substrate specificity profile, the cloned enzyme was classified as an alpha-glucosidase (E.C. 3.2.1.20). It has a pH optimum in the range 4.2-4.6, a temperature optimum of 58 degrees C and is readily inactivated at pasteurization temperature (60 degrees C). Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts. A rapid plate-assay, based on the utilization of a chromogenic substrate X-alpha-D-glucoside to detect the expression of the cloned alpha-glucosidase in S. cerevisiae transformants, was developed.
报道了从筑波假丝酵母（CBS 6389）基因组文库中分离出的α-葡萄糖苷酶基因在酿酒酵母中的分子克隆。克隆的基因包含在一个6.2 kb的Sau3A DNA片段中，并指导一种淀粉分解酶在重组宿主酿酒酵母的细胞外培养基中合成和分泌。发现克隆的酶具有异常广泛的底物特异性，能够水解α-1,2、α-1,3、α-1,4和α-1,6连接的以及芳基和烷基的D-葡萄糖苷。根据其底物特异性谱，克隆的酶被归类为α-葡萄糖苷酶（E.C. 3.2.1.20）。它的最适pH在4.2 - 4.6范围内，最适温度为58℃，在巴氏杀菌温度（60℃）下很容易失活。Southern印迹分析未能揭示克隆基因与从其他特征明确的淀粉分解酵母中分离出的基因组DNA之间的任何同源性。开发了一种基于利用显色底物X-α-D-葡萄糖苷检测酿酒酵母转化体中克隆的α-葡萄糖苷酶表达的快速平板测定法。
