A novel protease-resistant α-galactosidase with high hydrolytic activity from Gibberella sp F75: gene cloning, expression, and enzymatic characterization

被引：67

作者：

Cao, Yanan ^{[1
]}

Wang, Yaru ^{[1
]}

Meng, Kun ^{[1
]}

Bai, Yingguo ^{[1
]}

Shi, Pengjun ^{[1
]}

Luo, Huiying ^{[1
]}

Yang, Peilong ^{[1
]}

Zhou, Zhigang ^{[1
]}

Zhang, Zhifang ^{[2
]}

Yao, Bin ^{[1
]}

机构：

[1] Chinese Acad Agr Sci, Key Lab Feed Biotechnol, Minist Agr, Feed Res Inst, Beijing 100081, Peoples R China

[2] Chinese Acad Agr Sci, Biotechnol Res Inst, Beijing 100081, Peoples R China

来源：

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY | 2009年 / 83卷 / 05期

关键词：

alpha-Galactosidase; Gibberella; Protease-resistance; High hydrolytic activity; THERMOSTABLE BETA-MANNANASE; TRICHODERMA-REESEI; FUNCTIONAL EXPRESSION; MOLECULAR-CLONING; CRYSTAL-STRUCTURE; NUTRITIVE-VALUE; SOYBEAN-MEAL; PURIFICATION; OLIGOSACCHARIDES; CLASSIFICATION;

D O I：

10.1007/s00253-009-1939-2

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 [微生物学]; 090105 [作物生产系统与生态工程];

摘要：

A novel alpha-galactosidase gene (aga-F75) from Gibberella sp. F75 was cloned and expressed in Escherichia coli. The gene codes for a protein of 744 amino acids with a 24-residue putative signal peptide and a calculated molecular mass of 82.94 kDa. The native structure of the recombinant Aga-F75 was estimated to be a trimer or tetramer. The deduced amino acid sequence showed highest identity (69%) with an alpha-galactosidase from Hypocrea jecorina (Trichoderma reesei), a member of the glycoside hydrolase family 36. Purified recombinant Aga-F75 was optimally active at 60A degrees C and pH 4.0 and was stable at pH 3.0-12.0. The enzyme exhibited broad substrate specificity and substantial resistance to neutral and alkaline proteases. The enzyme K (m) values using pNPG, melibiose, stachyose, and raffinose as substrates were 1.06, 1.75, 54.26, and 8.23 mM, respectively. Compared with the commercial alpha-galactosidase (Aga-A) from Aspergillus niger var. AETL and a protease-resistant alpha-galactosidase (Aga-F78) from Rhizopus sp. F78, Aga-F75 released 1.4- and 4.9-fold more galactose from soybean meal alone, respectively, and 292.5- and 8.6-fold more galactose from soybean meal in the presence of trypsin, respectively. The pH and thermal stability and hydrolytic activity of Aga-F75 make it potentially useful in the food and feed industries.

引用

页码：875 / 884

页数：10

共 49 条

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Cloning and characterization of Aspergillus niger genes encoding an α-galactosidase and a β-mannosidase involved in galactomannan degradation [J].