Directed Evolution and Characterization of a Novel d-Pantonohydrolase from Fusarium moniliforme

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Abstract
D-Pantonohydrolase has attracted increasing attention as a biocatalyst for stereospecific production of d-pantoic acid. The Fusarium moniliformed-pantonohydrolase was selected for directed evolution through error-prone Polymerase Chain Reaction (PCR) combined with DNA shuffling for improved activity and pH stability using a convenient two-step high-throughput screening method based on the product formation and pH indicator. After three sequential error-prone PCRs and two rounds of DNA shuffling followed by screening, about 60 positive mutants were produced and a best mutant, Mut H-1287, with improved activity and pH stability was obtained. As compared to wild-type d-pantonohydrolase, Mut H-1287 showed a 10.5-fold higher specific activity; moreover, it could retain 85% of its original activity after incubation under low pH. Gene analysis indicated that the Mut H-1287 had D63H, K118Q, and V241I substitutions. The wild-type and evolved d-pantonohydrolase (Mut H-1287) was purified in three steps. The activities and characteristics of purified wild-type and evolved d-pantonohydrolase were also studied and compared. Keywords: d-Pantonohydrolase; Fusarium moniliforme; d-pantolactone; error-prone PCR; DNA shuffling; characterization

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