4.5 Article

Tailoring pullulanase PulAR from Anoxybacillus sp. AR-29 for enhanced catalytic performance by a structure-guided consensus approach

期刊

BIORESOURCES AND BIOPROCESSING
卷 9, 期 1, 页码 -

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SPRINGER HEIDELBERG
DOI: 10.1186/s40643-022-00516-4

关键词

Pullulanase; Structure-guided consensus approach; Site-directed mutagenesis; Catalytic pocket; Stability; Catalytic efficiency

资金

  1. National Natural Science Foundation of China [31801466, 22178318]

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In this study, Pullulanase was modified using a structure-guided consensus approach, resulting in the creation of a quadruple mutant PulAR-A365V/V401C/T504V/H499A. This mutant exhibited enhanced thermostability, pH stability, and catalytic efficiency, making it potentially useful in the starch industry.
Pullulanase is a well-known debranching enzyme that can specifically hydrolyze a-1,6-glycosidic linkages in starch and oligosaccharides, however, it suffers from low stability and catalytic efficiency under industrial conditions. In the present study, four residues (A365, V401, H499, and1504) lining the catalytic pocket of Anoxybacillus sp. AR-29 pullulanase (PulAR) were selected for site-directed mutagenesis (SDM) by using a structure-guided consensus approach. Five beneficial mutants (PulAR-A365V, PulAR-V401C, PuIAR-A365/V401C, PulAR-A365V/V401C/T504V, and PulAR-A365V/V401C/T504V/H499A) were created, which showed enhanced thermostability, pH stability, and catalytic efficiency. Among them, the quadruple mutant PulAR-A365V/V401C/T504V/H499A displayed 6.6- and 9.6-fold higher catalytic efficiency toward pullulan at 60 degrees C, pH 6.0 and 5.0, respectively. In addition, its thermostabilities at 60 degrees C and 65 degrees C were improved by 2.6- and 3.1-fold, respectively, compared to those of the wild-type (WT). Meanwhile, its pH stabilities at pH 4.5 and 5.0 were 1.6- and 1.8-fold higher than those of WT, respectively. In summary, the catalytic performance of PulAR was significantly enhanced by a structure-guided consensus approach. The resultant quadruple mutant PulAR-A365V/V401C/T504V/H499A demonstrated potential applications in the starch industry.

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