Mutation research on Q23L and Q23LG272E in phytase derivated from Aspergillus fumigatus.
- Author:
Wei-Na GU
1
;
Pei-Long YANG
;
Ya-Ru WANG
;
Hui-Ying LUO
;
Kun MENG
;
Ning-Feng WU
;
Bin YAO
;
Yun-Liu FAN
Author Information
1. Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
- Publication Type:Journal Article
- MeSH:
6-Phytase;
chemistry;
genetics;
metabolism;
Amino Acid Substitution;
Aspergillus fumigatus;
enzymology;
genetics;
Biocatalysis;
Electrophoresis, Polyacrylamide Gel;
Fungal Proteins;
chemistry;
genetics;
metabolism;
Hydrogen-Ion Concentration;
Models, Molecular;
Mutagenesis, Site-Directed;
Mutant Proteins;
genetics;
metabolism;
Mutation;
Pichia;
genetics;
Polymerase Chain Reaction;
Protein Conformation;
Protein Engineering;
methods;
Recombinant Proteins;
metabolism;
Structure-Activity Relationship;
Substrate Specificity
- From:
Chinese Journal of Biotechnology
2007;23(2):273-277
- CountryChina
- Language:Chinese
-
Abstract:
Aspergillus fumigatus wild-type phytase has many favorable properties, such as a good thermorstability and a broad pH optimum. However, the specific activity of the enzyme is relative low. A. fumigatus Q23L phytase resulted in a remarkable increase in specific activity around pH4.5 - 7.0, but the pH stability of Q23L was lower than A. fumigatus wild-type phytase. To increase the pH stability of Q23L, the mutant Q23LG272E was constructed by site-directed mutagenesis with PCR. The gene of A. fumigatus wild-type phytase and the mutant genes encoding the Q23LG272E and the Q23L were correctly expressed in Pichia pastoris GS115. Enzymes were purified and their enzymatic properties were determined. The results revealed that the specific activity of the Q23L improved remarkably, which increased from 51 u/mg of the wild type to 109 u/mg at pH5.5. Meanwhile, the pH stability of Q23L, decreased evidently, especially from pH3.0 to pH4.0.The pH stability of Q23LG272E in pH3.0 - 4.5 and pH6.5 - 7.0 has been improved compared with Q23L. The specific activity of Q23LG272E basically maintained at the level of Q23L. Analysis of 3-D structure and sequence similarity were used to reveal the presumable factors influencing the enzymatic properties of Q23LG272E, and discussion for the relationship between structure and function of phytase was given.
