title: Improving phytase enzyme activity in a recombinant phyA mutant phytase from Aspergillus niger N25 by error-prone PCR.
abstract: The mutant acid phytase (phyA ( m )) gene was modified by random mutagenesis to improve enzymatic activity by using an error-prone PCR (ep-PCR) strategy. The mutated gene was linearized and inserted into plasmid vector pPIC9K and transformed by electroporation into Pichia pastoris GS115. A single transformant, PP-NP(ep)-6A, showing the strongest phytase activity from among the 5,500 transformants, was selected for detailed analyses. Southern blot analysis of the mutant yeast transformant showed that phyA ( ep ) gene was integrated into the chromosome genome through single crossover with one copy of phyA. The kinetic parameters indicated that the mutant one showed 61% higher specific activity and 53% lower k (m) value than that of PP-NP(m)-8 (P < 0.05). In addition, the overall catalytic efficiency (k (cat)/k (m)) of the mutant one was 84% higher (P < 0.05) than that of PP-NP(m)-8. Nine bases were altered in the mutant sequences, which resulted in three amino acid changes, namely, Glu156Gly, Thr236Ala, and Gln396Arg. The structural predictions indicated that the mutations generated by ep-PCR somehow reorganized or remodeled the active site, which could lead to increasing catalytic efficiency.
authors.affiliation: College of Life and Physical Science, Sichuan Agricultural University, Ya'an 625014, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liao
authors.forename: Min
authors.initials: M
authors.lastname: Zeng
authors.forename: Zhen-Fang
authors.initials: ZF
authors.lastname: Wu
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.forename: Hong-Ning
authors.initials: HN
authors.lastname: Wang
authors.forename: Qi
authors.initials: Q
authors.lastname: Wu
authors.forename: Zhi
authors.initials: Z
authors.lastname: Shan
authors.forename: Xue-Yi
authors.initials: XY
authors.lastname: Han
mesh_terms: D010832:6-Phytase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain; D018274:Electroporation; D004926:Escherichia coli; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016681:Genome, Fungal; D015698:Genomic Library; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D010832:6-Phytase