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 (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.
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