Gene Mining,characterization And Molecular Modification Of Rhamnosidases With Reverse Hydrolysis Activity

α-L-rhamnosidases（EC 3.2.1.40）are glycoside hydrolases that can cleave terminal α-Lrhamnose from a variety of natural rhamnosides and rhamnosyl-glycans,and under certain reaction conditions,a few microbial α-L-rhamnosidases can catalyze the synthesis of rhamnosyl compounds by reverse hydrolysis activity with L-rhamnose as donor.At present studies on αL-rhamnosidases in the world mainly focus on the discovery of the novel enzymes,the utilization of hydrolysis activity and the molecular modification for making it a suitable catalyst for industry.However,there are a few reports about α-L-rhamnosidases with reverse hydrolysis activity for α-L-rhamnosidase-catalyzed synthesis of rhamnosides and rhamnosyl compounds,and most of those reports are about the fungi-derived enzymes.In this work,the discovery of novel α-L-rhamnosidases with reverse hydrolysis activity and the directed molecular evolution of the enzyme to improve the synthesis yield of reverse hydrolysis reaction were carried out,and the main results are as follows:1.Gene mining of novel α-L-rhamnosidases with reverse hydrolysis activityBased on sequence analysis and gene mining,18 putative α-L-rhamnosidases from intestinal microbes were selected for cloning from intestinal microbe genome,of which five genes were successfully cloned and expressed in E.coli.These five putative enzymes were two GH78 α-L-rhamnosidases,BC132（GenBank No.ALJ59132.1.83 kDa）from B.cellulosilyticus WH2 and BD192（GenBank No.ALA76192.1,149 kDa）from B.dorei isolate HS2_L2B045bBD192,and three GH106 α-L-rhamnosidases,BV915（GenBank No.ABR37915.1,104 kDa）and BV348（GenBank No.ABR40348.1.121 kDa）from B.vulgatus ATCC 8482,and BT141（GenBank No.ALJ42141.1,124 kDa）from B.thetaiotaomicron strain 7330.The results of substrate specificities in hydrolysis showed that all the five α-Lrhamnosidases could hydrolyze the artificial substrate pNP-α-L-Rha to varying degrees,but only BT141 and BV348 were able to hydrolyze α-1,2 rhamnosidic linkage of naringin.Using L-rhamnose as donor,11 receptor molecules including mannitol,mannose,fructose.glucose.galactose,sorbitol,sucrose,xylose,xylitol,and two clinical antitumor drugs cytarabine（Ara C）and 5-fluoro-2’-deoxyuridine（FUDR）were used to screen the reverse hydrolysis activity of the enzymes.The results of TLC and mass spectrometry analysis revealed that BD192,BV915.BT141 and BV348 had reverse hydrolysis activities.BD192 could catalyze the reverse hydrolysis reaction using L-rhamnose and mannitol to form rhamnosyl-mannitol.and all four enzymes,BD192,BV915,BT141 and BV348,could catalyze the synthesis of rhamnosylgalactose,rhamnosyl-sorbitol and rhamnosyl-sucrose using L-rhamnose as donor and galactose,sorbitol and sucrose as acceptor,respectively.2.Molecular modification of α-L-rhamnosidase AN995Our previous work found that an α-L-rhamnosidase AN995（GenBank No.CAK38995.1,103 kDa）from Aspergillus niger 3.09 had reverse hydrolysis activity and could catalyze the rhamnosylation of anticancer drugs including Ara C and FUDR.However,the yield of those two rhamnosyl-anticancer drugs was much lower.So,here we carried out directed molecular evolution of AN995 to improve the synthesis yield of reverse hydrolysis reaction.The ligand molecule of Ara C was docked into the enzyme model to select the amino acid residues that form direct or indirect hydrogen bonds with Ara C.Using site-directed mutagenesis,seven mutants R388A,R482A,W488A,H544A,P611A,R764A and S781A of AN995 were constructed for reducing the steric hindrance,and the reverse hydrolysis activity of mutant P611A was improved significantly.The synthesis yield of Rha-Ara C by reverse hydrolysis reaction using L-rhamnose and Ara C was 1.6-fold higher than that of the wild type enzyme（18.2 mM）.Further site-saturation mutagenesis was performed on the Pro611 site,and the synthesis yield of the mutant P611A was the highest among all 19 mutants.The affinity and maximum reaction rate（Km value of 0.2560 mM and Vmax of 1.013 nmol/min）of the mutant P611A for pNPRha were significantly improved compared with those（Km value of 24.27 mM and Vmax of 0.55 nmol/min）of the wild-type AN995,and the effects of temperature and p H on enzyme activities were almost unchanged.And then,the effects of initial substrate concentrations and reaction time were separately studied.Under optimal conditions,the maximum yield of Rha-Ara C reached 105 mM,which was approximately improved 5.8-fold compared with that of the AN995 WT（1 8.2 mM）.Protein simulation and molecular dynamics analysis showed that substitution of the amino acid residue at position 611 from Pro to Ala could reduce the steric hindrance and increase flexibility of loop（607PTAPPEEPGDAR618）,which were conducive to substrates entry or product separation,thereby improving the reverse hydrolysis activity.In this work,four novel bacterial α-L-rhamnosidases that could catalyze the synthesis of rhamnoside compounds by reverse hydrolysis activity with L-rhamnose as donor were obtained by gene mining,and it was discovered for the first time that the GH106 α-L-rhamnosidases derived from intestinal bacteria had the reverse hydrolysis activity.The mutant enzyme P611A obtained by the directed molecular evolution of a fungal α-L-rhamnosidase AN995 showed highest synthesis yield of Rha-Ara C through the reverse hydrolysis reaction,which provides a new tool enzyme for rhamnosyl-modification of antitumor drugs.