来自棘孢曲霉的一种内切阿拉伯聚糖酶的功能克隆及其在米曲霉和烟草中的异源表达。

Functional cloning of an endo-arabinanase from Aspergillus aculeatus and its heterologous expression in A. or oryzae and tobacco.

作者信息

Skjøt M, Kauppinen S, Kofod L V, Fuglsang C, Pauly M, Dalbøge H, Andersen L N

机构信息

Biotechnology Group, Danish Institute of Agricultural Sciences, Copenhagen.

出版信息

Mol Genet Genomics. 2001 Jul;265(5):913-21. doi: 10.1007/s004380100489.

DOI:10.1007/s004380100489

PMID:11523809

Abstract

Functional cloning in yeast has been used to isolate full-length cDNAs encoding an endo-alpha-1,5-L-arabinanase from the filamentous fungus Aspergillus aculeatus. Screening of a cDNA library constructed in a yeast expression vector for transformants that hydrolysed AZCL-arabinan identified 44 Saccharomyces cerevisiae clones all harbouring the same arabinanase-encoding cDNA. The cloned cDNA was expressed in A. oryzae and the recombinant enzyme was purified and characterized. The mode of action of the enzyme was studied by analysis of the digestion pattern towards debranched arabinan. The digestion profile obtained strongly suggests that the enzyme is an endo-arabinanase. In addition, the feasibility using Nicotiana tabacum as an alternative host for arabinanase expression was investigated.

摘要

酵母中的功能克隆已被用于从丝状真菌棘孢曲霉中分离编码内切α-1,5-L-阿拉伯聚糖酶的全长cDNA。通过筛选构建在酵母表达载体中的cDNA文库，寻找能够水解AZCL-阿拉伯聚糖的转化体，鉴定出44个酿酒酵母克隆，它们都含有相同的阿拉伯聚糖酶编码cDNA。将克隆的cDNA在米曲霉中表达，并对重组酶进行纯化和表征。通过分析该酶对去支链阿拉伯聚糖的消化模式来研究其作用方式。所获得的消化图谱有力地表明该酶是一种内切阿拉伯聚糖酶。此外，还研究了使用烟草作为阿拉伯聚糖酶表达替代宿主的可行性。