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Protein Expr Purif. 2019 Oct;162:67-71. doi: 10.1016/j.pep.2019.06.002. Epub 2019 Jun 8.

Enzymatic characteristics of a recombinant protease (rPepD) from Aspergillus niger expressed in Pichia pastoris.

Author information

1
School of Life Sciences, Shaoguan University, Shaoguan, Guangdong, 512005, PR China. Electronic address: sgu_keye@sgu.edu.cn.
2
School of Life Sciences, Shaoguan University, Shaoguan, Guangdong, 512005, PR China. Electronic address: sgu_weimengyao0621@sgu.edu.cn.
3
School of Life Sciences, Shaoguan University, Shaoguan, Guangdong, 512005, PR China. Electronic address: sgu_fuyutong2329@sgu.edu.cn.
4
School of Life Sciences, Shaoguan University, Shaoguan, Guangdong, 512005, PR China. Electronic address: sgu_zhuyanmei1467@sgu.edu.cn.
5
School of Life Sciences, Shaoguan University, Shaoguan, Guangdong, 512005, PR China. Electronic address: sgu_zhanxuanlin0629@sgu.edu.cn.

Abstract

The Aspergillus niger AS3.350 protease gene (pepD) was successfully cloned and expressed in Pichia pastoris KM71. The rPepD activity was 331.5 U/ml, and the optimum temperature and pH were 45 °C and 8-9 respectively. In addition, enzyme activity was significantly inhibited by PMSF, EDTA, Mg2+, Fe2+ and Zn2+ ions, and stimulated by Ca2+ which selectively bound to the T302 and D323 residues. Mutation in either or both of the residues inhibited rPepD expression, indicating that binding to Ca2+ is necessary for PepD expression and activity. The rPepD showed a wide substrate range, and was particularly selective to those with hydrophobic amino acids. The degree of rPepD-mediated hydrolysis of soy protein isolate, corn flour and gluten meal were 8.7%, 38.1% and 33.6% respectively, which was higher than that by Alcalase, indicating that rPepD has potential applications in the food processing industry.

KEYWORDS:

Aspergillus niger; Cloning and expression; Enzymatic characteristics; Hydrolysis characteristics; Protease

PMID:
31181254
DOI:
10.1016/j.pep.2019.06.002

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