Discovering Novel Glycosidases Based On Genomics Analysis And Their Applications

With increasing attention to environmental protection and sustainable development,it is crucial to develop efficient,mild,and eco-friendly manufacturing processes to replace traditional chemical processes with high pollution,high consumption,and low efficiency.Enzyme catalysis exhibits the advantages of high efficiency,high specificity,mild reaction conditions,environmental friendliness,and low cost.It has played an essential role in the preparation of rare natural compounds.However,the reported enzymes cannot meet industrial production requirements effectively in enzyme activity,stability,sugar tolerance,and function.In this study,novel glycosidase mining was performed based on the genomic analysis of Talaromyces stollii CLY-6,and the enzymatic properties of novel glycosidases were investigated.Then,the applications of new glycosidases in diosgenin,icaritin,and baohuoside I production were explored.The main results in the present study are included as follows:(1).In this study,a microorganism was screened and identified as Talaromyces stollii CLY-6,which could efficiently convert steroidal saponins and epimedin C into diosgenin and icaritin,respectively.Furthermore,genome sequencing was performed,and the whole-genome sequence data of Talaromyces stollii CLY-6 was obtained(Accession number:ASM1406522vl),filling the gap of the existing database.Notably,we constructed a carbohydrate-active enzyme library by genomic analysis to support subsequent protein identification.(2).Through stepwise protein purification,we obtained four novel enzymes:Rhase-TS(removing the terminal rhamnose of steroidal saponins),Gluase-TS removing the glucose of steroidal saponins),Rhase Ⅰ(removing the inner rhamnose of epimedin C),and Rhase Ⅱ(removing the outer rhamnose of epimedin C).Protein fingerprinting was performed using the carbohydrateactive enzyme library as the database,and the Rhase-TS,Gluase-TS,Rhase-Ⅰ,and Rhase-Ⅱ were identified as protein 10806,3425,10410,and 7880,respectively.(3).The protein 10806,3425,10410,and 7880 were over-expressed using Pichia pastoris.Preliminary substrate test showed that proteins 10806,3425,10410,and 7880 are the critical glycoside hydrolase Rhase-TS(α-Lrhamnosidase,accession number:MT779018),Gluase-TS(β-D-glucosidase,accession number:MT779019),Rhase-I(α-L-rhamnosidase,accession number:MT779021),and Rhase-Ⅱ(α-L-rhamnosidase,accession number:MT779022).The studies of substrate catalytic pathway showed that Rhase-TS could hydrolyze the terminal rhamnosidic bond of steroidal saponins,GluaseTS could gradually hydrolyze the glucosidic bond steroidal saponins,Rhase-I could gradually hydrolyze the outer and inner rhamnosidic bonds of epimedin C,and Rhase-Ⅱ could specifically hydrolyze the outer rhamnosidic bond of epimedin C.These results provided theoretical guidance for the future development of efficient enzymatic catalysis processes for diosgenin,icaritin,and baohuoside I.Besides,compared with previously reported glycosidases,both Rhase-TS and Gluase-TS have the highest hydrolysis activity against steroidal saponins,while Rhase-I has the highest hydrolysis activity of the inner rhamnosidic bond of epimedin C.Substrate profile studies show that Rhase-TS is a specific steroidal saponin α-L-1,2-rhamnosidase;Gluase-TS could also hydrolyze the glucosidic bond of epimedin C;Rhase-I and Rhase-II showed a broad spectrum that could hydrolyze rutin,neohesperidin,and naringin.(4).Enzymatic properties of Rhase-TS,Gluase-TS,Rhase-I,and Rhase-II were further investigated.Results showed that Rhase-TS and Gluase-TS have high thermal stability and pH stability,and low-concentration of organic reagents can activate Rhase-TS to a certain extent.Notably,Rhase-TS has outstanding rhamnose tolerance(Ki:0.5 M),indicating potential application in the industry.Compared with Rhase-TS,Gluase-TS needs to be further improved in terms of enzyme activity and sugar tolerance.Rhase-I and RhaseII also have better thermal stability,but they are more stable in acidic environments.Kinetic studies showed that Rhase-I and Rhase-II have good catalytic efficiency against flavonoid glycosides,indicating the excellent application prospect.Besides,through computational analysis,the potential catalytic residues of each enzyme and the substrate-binding mechanism were predicted for understanding the enzymatic catalysis at the molecular level,laying the groundwork for improving the performance of enzymes through protein engineering in the future.(5).Enzymatic productions of diosgenin,icaritin,and baohuoside I using the new glycosidases discovered in this study were explored.The one-pot and two-step enzymatic methods for the preparation of diosgenin were established.Under the optimized conditions,10 g E-DZW could be converted into 1.86 g diosgenin by the one-pot reaction using Rhase-TS and cellulase Cel-TL4 within 24 h(yield:98.5%).The two-step enzymatic method can achieve the same production level as the one-pot method and realize the separation of rhamnose.In short,the high-efficiency enzymatic production of diosgenin has significant potential in controlling pollution and increase economic efficiency.We also established a two-step enzymatic process using Rhase-I/Bglsk for icaritin(yield:98.5%)production from epimedin C with the highest effective productivity of icaritin(3.72×102 g/L/h/g Rhase-I)ever reported.Finally,we explored a two-step enzymatic catalysis for the production of baohuoside I by Rhase-II and Bglsk,and 15 mg of epimedin C could be converted to 9.05 mg of baohuoside I within 80 minutes(yield:96.5%),laying the foundation for the future industrial production of baohuoside I by enzymatic conversion of Chaohuidine C.In summary,four novel glycoside hydrolases were mined from Talaromyces stollii CLY-6 by microbial screening,microbial genome analysis,and protein purification.The discovered enzymes played essential roles in hydrolyzing steroidal saponins and epimedin C into diosgenin,icaritin,and baohuoside I.The amino acid sequences of these enzymes were further identified by mass spectrometry analysis,followed by heterologous expression in Pichia pastoris,and the functions of the enzymes were verified.In addition,detailed enzymatic characterization of each novel glycosidase was carried out to assess their potential application.Finally,an enzyme-catalyzed process for the efficient production of diosgenin,icaritin,and baohuoside I was developed,laying a foundation for future industrial applications.