Discovery Of Novel Bacterial ?-L-Rhamnosidases And Application For Biotransformation

?-L-rhamnosidase(E.C.3.2.1.40)catalyzes the hydrolysis of terminal ?-L-rhamnose from the flavonoid glycosides to improve their bioavailability and biological properties.?-L-rhamnosidase has broad application prospects.At present,only nineteen bacterial ?-L-rhamnosidases have been reported,and their reports on biocatalytic applications mainly focus on fungi.Therefore,the discovery of novel bacterial ?-L-rhamnosidases and the biotransformation of natural flavonoid glycosides are of great significance.By genome mining,three novel ?-L-rhamnosidases from Pedobacter heparinus have been discovered.The ?-L-rhamnosidase genes PhRha78 D,PhRha78E and PhRha78 G were amplified from the genome of Pedobacter heparinus and were ligated into the expression vector pET-28 a.The recombinant plasmids were transformed into E.coli BL21(DE3).Interestingly,PhRha78 D,PhRha78E and PhRha78 G were over-expressed as the catalytically active inclusion bodies(CatIBs)in E.coli determined by SDS-PAGE and enzymatic activity assays.PhRha78 D was less active on p-nitrophenyl ?-Lrhamnopyranoside(pN PR),so the enzymatic properties of only PhRha78 E and PhRha78 G have been studied.PhRha78 E and PhRha78 G displayed the highest activity at pH 6.5 and 7.0,respectively,and the optimum temperatures were very different remarkably,60 oC and 40 oC,respectively.The recombinant enzyme had a KM value of 0.55 mM and a kcat value of 0.18 s-1 for PhRha78 E,a calculated KM of 0.4 mM and kcat value of 0.12 s-1 for PhRha78 G.PhRha78E displayed the T50 at 51°C and retained more than 59% activity after incubation 30 min at 40-50 °C,while PhRha78 G showed a T50 at 36 °C,remaining more than 50% activity between 20 °C and 36 °C for 30 min;They could tolerate various organic solvents at low concentration(1%).For discovery of novel enzymes using the strategy genome mining,there are disadvantages such as unpredictable catalytic activity and uncontrollableexpression,so we introduced the new strategy for discovery of novel enzymes by high-throughput sequencing for metagenome coupled with virtual screening for catalytic key motifs.Three target candidate enzyme genes HFM-RhaA,HFM-RhaB and HFM-RhaC were obtained from human fecal metagenome.By seamless cloning technique,these genes were cloned into the vector pET-28 a after codon optimization and whole-gene synthesis.The recombinant gene products were expressed in E.coli,with higher expression in the supernatant confirmed by SDS-PAGE,then,they were purified by Ni-NTA agarose affinity chromatography and characterized.The values of optimum pH of HFMRhaA,HFM-RhaB and HFM-RhaC were 6.0,6.0,and 5.7,respectively.With regard to temperature,they presented highest activity at 40 °C,60 °C,40 °C.HFM-RhaA and HFM-RhaC had lower KM values of 1.59 mM and 0.053 mM,higher V max values of 57.6 and 37.6 ?mol min-1 mg-1 and kcat values of 92.8 s-1and 62.9 s-1,respectively.The T50 of HFM-RhaA and HFM-RhaC were 35 °C and 28 °C,respectively.HFM-RhaA showed very well tolerances to 5% or even 10% of the alcoholic organic solvents,but only 5% for HFM-RhaC.With a certain understanding of the enzymatic properties,we initially explored the biotransformation of rutin and naringin by HFM-Rha.HFM-RhaA might hydrolyze rutin and naringin efficiently,but higher for the latter.30 ?g/mL of HFM-RhaA catalyzed the reaction of naringin into prunin for 60 min with conversion rate of 88.6%,while 120 ?g/mL of HFM-RhaA hydrolysed rutin which was a molar conversion of 80.8%.HFM-RhaB at 200 ?g/mL hydrolysed naringin for 120 minutes with a corresponding conversion yield of 58.8%,but showed little catalytic activity on rutin.HFM-RhaC had good catalytic ability on rutin and naringin,with conversion of 66.1% and 49.2%,respectively,when HFM-RhaC at 180 ?g/mL hydrolyzed rutin and naringin for 120 min.This study provided a novel enzyme discovery strategy to obtain high probability of activity,acquired novel bacterial ?-L-rhamnosidases with inde-pendent intellectual property rights,carried out biotransformation of flavonoids with bacterial ?-L-rhamnosidase,and will guide the biotransformation of natural flavonoids by enzymatic biocatalysis or using whole cells as the biocatalysts.