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α-L-Rhamnosidases with desirable activity and thermostability profiles could be used for the biocatalytic production of the flavonoid glucoside isoquercetin from natural rutin for functional food. Herein, to improve the catalytic activity of GH78 α-L-rhamnosidase BtRha78A from Bacteroides thetaiotaomicron VPI-5482, a list of residues located at the conserved general acid motif were selected for targeted mutagenesis by the sequence alignment of BtRha78A with homologous α-L-rhamnosidases. Ala-scanning mutagenesis and site-directed mutagenesis based on sequence alignment were performed, and the relative activity on rutin was evaluated. Furthermore, the reaction time curves and enzyme kinetics of better mutants were determined. The results indicate that the conversion rates of mutants V338A, V338I, S340A, and G341A were increased by 21.3%, 20.1%, 13.2%, and 1.6%, respectively, compared with the wild type when using whole-cell biotransformation. Moreover, the catalytic efficiency kcat/KM value of mutant V338A was 1.3-fold higher than that of the wild type. The best mutant, V338A, was employed for the enzymatic preparation of isoquercetin via the biotransformation of rutin at a concentration of 2 mM, and 1.80 g of isoquercetin was obtained. The identification of the best mutant V338A lays the foundation for the efficient preparation of isoquercetin via the biotransformation of rutin, which in turn provides theoretical guidance for its large-scale production. [ABSTRACT FROM AUTHOR]

Effects of different vegetation types ( Ulmus pumila , Larix gmelinii , Armeniaca vulgaris , Picea asperata and Robinia pseudoacacia ) and reclamation years (15 and 20 years) on soil bacterial community structure in reclaimed Antaibao opencast mine areas were investigated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and clone sequencing. For 20-year reclaimed soils, the significantly highest and lowest bacterial diversity were found in U. pumila and A. vulgaris stand, respectively, whereas no significant differences were found between the other three vegetation types. Under 15-year plantations, soil bacterial diversity index of P. asperata was significantly higher than that of R. pseudoacacia . Soil bacterial diversity index significantly increased in R. pseudoacacia planted soils but decreased in P. asperata treatment with the increase of reclaimed years. No significant change of soil bacterial community structure was observed in the same reclamation years based on the similarity coefficient analysis, cluster analysis and principal component analysis (PCA). Pearson correlation analysis demonstrated that bacterial diversity index was significantly positively correlated with soil pH. Nitrospira , Sphingomonas , Arthrobacter , Brachybacterium , Rhizobium as well as Mesorhizobium, which play important roles in the nitrogen cycle, degradation of polycyclic aromatic hydrocarbons and other organic matter, were identified by clone sequencing of the DGGE bands. Our results indicated that U. pumila and P. asperata were conducive to the recovery of soil bacterial diversity. The most dominant bacterial community from reclaimed mine soil would be beneficial for restoring wasteland contaminated soil and improving soil fertility.