Improving phytase enzyme activity in a recombinant phyA mutant phytase from Aspergillus niger N25 by error-prone PCR

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Journal Article
Applied Biochemistry and Biotechnology, 2012, 166 (3), pp. 549 - 562
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The mutant acid phytase (phyAm) 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-NPep-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 phyAep 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 km value than that of PP-NPm-8 (P<0.05). In addition, the overall catalytic efficiency (kcat/km) of the mutant one was 84% higher (P<0.05) than that of PP-NPm-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. © Springer Science+Business Media, LLC 2011.
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