Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme

被引：15

作者：

de Almeida, Fabiana M. ^{[2
]}

Bonini, Beatriz M. ^{[2
]}

Beton, Daniela ^{[1
]}

Jorge, Joao A. ^{[2
]}

Terenzi, Hector F. ^{[2
]}

da Silva, Aline M. ^{[1
]}

机构：

[1] Univ Sao Paulo, Inst Quim, Dept Bioquim, BR-05508000 Sao Paulo, Brazil

[2] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Pret, Dept Biol, BR-14049 Ribeirao Preto, SP, Brazil

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2009年 / 65卷 / 02期

基金：

巴西圣保罗研究基金会;

关键词：

Neutral trehalase; Trehalose; Neurospora crassa; Filamentous fungi; Heterologous expression; YEAST SCHIZOSACCHAROMYCES-POMBE; DEPENDENT PROTEIN-KINASE; SACCHAROMYCES-CEREVISIAE; MOLECULAR-CLONING; CYCLIC-AMP; INTRACELLULAR TREHALOSE; FILAMENTOUS FUNGI; PICHIA-PASTORIS; NITROGEN-SOURCE; ACID TREHALASE;

D O I：

10.1016/j.pep.2008.11.010

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of similar to 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80-150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 degrees C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 degrees C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K-M was 42 mM, and V-max was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence. (C) 2008 Elsevier Inc. All rights reserved.

引用

页码：185 / 189

页数：5

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