Sakai Y, Akiyama M, Kondoh H, Shibano Y, Kato N
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Japan.
Biochim Biophys Acta. 1996 Jul 31;1308(1):81-7. doi: 10.1016/0167-4781(96)00075-9.
The methylotrophic yeast, Canadida boidinii, was investigated as an expression host for secretory enzyme production. The cDNA of Rhizopus oryzae glucoamylase was placed under the C. boidinii alcohol oxidase (AODl) promoter. A transformant integrated with a single-copy expression cassette to the chromosome produced glucoamylase into the medium to a high amount when the cells were grown on methanol or methanol plus glycerol as (a) carbon source(s). The transformant C. boidinii cells were grown up to ca. 95 g dry cell weight/liter medium, and the concentration of glucoamylase in the medium reached 3.4 g/liter. This showed that the signal sequence from Rhizopus glucoamylase functioned very efficiently in C. boidinii. Next, secreted glucoamylase from C. boidinii was purified and compared with the enzyme produced in S. cerevisiae. The enzyme produced in C. boidinii was found to have higher molecular weight than that produced in S. cerevisiae, which was due to the difference of the N-linked glycosylated sugar structure of the produced proteins.
对甲基营养型酵母博伊丁假丝酵母（Canadida boidinii）作为分泌酶生产的表达宿主进行了研究。米根霉葡糖淀粉酶的cDNA置于博伊丁假丝酵母乙醇氧化酶（AOD1）启动子的控制之下。当细胞在甲醇或甲醇加甘油作为碳源的条件下生长时，整合有单拷贝表达盒至染色体的转化体能将葡糖淀粉酶大量分泌到培养基中。博伊丁假丝酵母转化体细胞生长至约95克干细胞重量/升培养基，培养基中葡糖淀粉酶的浓度达到3.4克/升。这表明来自米根霉葡糖淀粉酶的信号序列在博伊丁假丝酵母中发挥了非常有效的作用。接下来，对博伊丁假丝酵母分泌的葡糖淀粉酶进行了纯化，并与酿酒酵母中产生的酶进行了比较。发现博伊丁假丝酵母中产生的酶分子量高于酿酒酵母中产生的酶，这是由于所产生蛋白质的N-连接糖基化糖结构不同所致。
