从甲基营养型酵母多形汉逊酵母中克隆麦芽糖酶基因。

Cloning of maltase gene from a methylotrophic yeast, Hansenula polymorpha.

作者信息

Liiv L, Pärn P, Alamäe T

机构信息

Institute of Molecular and Cell Biology, Riia 23, University of Tartu, 51010, Tartu, Estonia.

出版信息

Gene. 2001 Mar 7;265(1-2):77-85. doi: 10.1016/s0378-1119(01)00359-6.

DOI:10.1016/s0378-1119(01)00359-6

PMID:11255010

Abstract

The Hansenula polymorpha maltase structural gene (HPMAL1) was isolated from a genomic library by hybridization of the library clones with maltase-specific gene probe. An open reading frame of 1695 nt encoding a 564 amino-acid protein with calculated molecular weight of 65.3 kD was characterized in the genomic DNA insert of the plasmid p51. The protein sequence deduced from the HPMAL1 exhibited 58 and 47% identity with maltases from Candida albicans and Saccharomyces carlsbergesis encoded by CAMAL2 and MAL62, respectively, and 44% identity with oligo-alpha-1,6-glucosidase from Bacillus cereus. The recombinant Hansenula polymorpha maltase produced in Escherichia coli hydrolyzed p-nitrophenyl-alpha-D-glucopyranoside (PNPG), sucrose, maltose and alpha-methylglucoside and did not act on melibiose, cellobiose, trehalose and o-nitrophenyl-beta-D-galactopyranoside (ONPG). The affinity of the recombinant enzyme for its substrates increased in the order maltose <alpha-methylglucoside <sucrose <PNPG. Southern analysis revealed presence of a single maltase gene in H. polymorpha. This is the first report on the maltase gene sequence from a methylotrophic yeast.

摘要

通过用麦芽糖酶特异性基因探针与文库克隆进行杂交，从多形汉逊酵母基因组文库中分离出麦芽糖酶结构基因（HPMAL1）。在质粒p51的基因组DNA插入片段中鉴定出一个1695 nt的开放阅读框，其编码一个564个氨基酸的蛋白质，计算分子量为65.3 kD。从HPMAL1推导的蛋白质序列与分别由CAMAL2和MAL62编码的白色念珠菌和卡尔斯伯酵母麦芽糖酶的同一性分别为58%和47%，与蜡样芽孢杆菌的寡聚-α-1,6-葡萄糖苷酶的同一性为44%。在大肠杆菌中产生的重组多形汉逊酵母麦芽糖酶可水解对硝基苯基-α-D-吡喃葡萄糖苷（PNPG）、蔗糖、麦芽糖和α-甲基葡萄糖苷，而对蜜二糖、纤维二糖、海藻糖和邻硝基苯基-β-D-吡喃半乳糖苷（ONPG）不起作用。重组酶对其底物的亲和力按麦芽糖＜α-甲基葡萄糖苷＜蔗糖＜PNPG的顺序增加。Southern分析显示多形汉逊酵母中存在单个麦芽糖酶基因。这是关于甲基营养型酵母麦芽糖酶基因序列的首次报道。