A spectrophotometric method for high-throughput screening of α-l-rhamnosidase activity on rutin coupled with a β-d-glucosidase assay.

Abstract

α-l-Rhamnosidase may biotransform rutin into isoquercetin with better bioavailability and bioactivity. To date, the high-throughput screening for the activity of α-l-rhamnosidases on rutin could not be achieved. Herein, based on the spectral differences between rutin and its aglycone quercetin in alkaline pH 10.0, we have developed a novel and simple spectrophotometric method for high-throughput screening of α-l-rhamnosidase activity on rutin by combining with a highly active β-d-glucosidase. Quercetin showed the maximum absorbance at 320nm in alkaline pH 10.0, and could be considered as the characteristic peak of quercetin because rutin had low absorption at 320nm. Meanwhile, rutin exhibited the maximum absorption at 400nm and quercetin showed low absorption at 400nm in pH 10.0. With this novel spectrophotometric method, the relative abilities of nine different α-l-rhamnosidases on rutin had been evaluated by monitoring the absorption values of the reaction mixture in alkaline pH 10.0 at 320nm and 400nm, and the trend in the activity on rutin was consistent with that obtained by HPLC. Moreover, the library from site-directed saturation mutagenesis at the residue Val338 in the α-l-rhamnosidase BtRha78A from Bacteroides thetaiotaomicron was constructed for high-throughput screening by this novel spectrophotometric method, and the mutant V338S with improved activity on rutin was obtained. The conversion rate of the mutant V338S on rutin increased by 21.7% and 16.8% than wild type when using whole cells and purified enzymes, respectively. Our findings demonstrated that this novel spectrophotometric method coupled with the β-d-glucosidase assay might be applied for high-throughput screening of different α-l-rhamnosidases and a great number of mutants from semi-rational design and directed evolution for α-l-rhamnosidase.