Discovery Of Novel ?-L-Rhamnosidases From Bacteroides Thetaiotaomicron VPI-5482 And Structural Analysis

The biocatalysis that use enzyme as catalyst has many advantages and is widely used in various industrial fields.?-L-Rhamnosidase?E.C.3.2.1.40?,a kind of glycoside hydrolase,can effectively hydrolysis natural glycosides and has potential biocatalytic application in biotechnology.At present,fewer than twenty?-L-rhamnosidase genes belonging to glycoside hydrolase family78?GH78?have been reported.Enzymatic properties and catalytic mechanism of?-L-rhamnosidase need to be further studied.Human gut bacteria Bacteroides thetaiotaomicron can utilize wide variety of glycan and glycoside and its many glycoside hydrolase systems have been elucidated in detail.However,there were no reports about?-L-rhamnosidase of Bacteroides thetaiotaomicron VPI-5482.By searching CAZy database,we learned that genome of Bacteroides thetaiotaomicron VPI-5482 contains six predicated?-L-rhamnosidase genes,in this paper three?-L-rhamnosidase genes were selected for the study.The research adopts genome of Bacteroides thetaiotaomicron VPI-5482 as PCR template amplifying the target gene BtRha78A,BtRha78D and BtRha78E successfully.The length of nucleotide sequence were 2190 bp,3459 bp and2646 bp respectively,encoding amino acid for 729,1152 and 881correspondingly.The gene ligated to vector pET-28a and heterologously over-expressed in Escherichia coli BL21?DE3?,then purifying the objective protein adopt Ni-NTA agarose affinity chromatography.Further,the recombinant proteins were characterized systematically.The study of enzymatic properties indicated that three recombinant?-L-rhamnosidases could efficiently hydrolyze p-nitrophenyl?-L-rhamnopyranoside?pNPR?.The optimum pH of?-L-rhamnosidase BtRha78A,BtRha78D and BtRha78E were 6.5,6.0 and 7.0,respectively.The optimum temperature of BtRha78A was 60 ^oC,while BtRha78D and BtRha78E were 50 ^oC.The V_maxax of BtRha78A,BtRha78D and BtRha78E on pNPR were 48.7?mol min^-11 mg^-1,6.77?mol min^-11 mg^-11 and 3.21?mol min^-11 mg^-1;k_cat/K_Mwere 130000 M^-1s^-1,105600 M^-1s^-11 and 12500 M^-1s^-1,respectively.The experimental results of organic solvents on enzymatic activities were shown that BtRha78A could be tolerant of a low concentration of alcohols,it remained catalytic activity of78%-85%at the concentration of 1%.BtRha78D and BtRha78E could tolerate various organic solvents at low concentration?1%?.Most organic solvents significantly lowered catalytic activity at the concentration of 10%.However,BtRha78D and BtRha78E were well tolerant of dimethyl sulfoxide?DMSO?,and remained catalytic activities of 42%and 53%at the concentration of 10%,respectively.In addition,the stability experiment of BtRha78A has shown that it exhibited a good pH stability and relatively high thermostability.Based on sequence alignment and structure analysis,utilizing the alanine scanning mutation to determine the functional residues of BtRha78A.The site-directed mutants of the key residues of BtRha78A were constructed by whole plasmid PCR.The enzymatic activities of mutants were reduced significantly.So we came to the conclusion that the catalytic general acid of BtRha78A is Asp335 and catalytic general base is Glu595.Conserved residues Asp330,Arg334,Trp339,Asp342,Tyr383,Trp440 and His620 were important functional residues of BtRha78A.The research on conserved general acid motif?Asp330-Asp342?which contain catalytic general acid and many functional residues indicated that it is crucial for enzymatic catalysis.The research revealed that three?-L-rhamnosidases of Bacteroides thetaiotaomicron VPI-5482 with good enzymatic properties,which is benefical to the application of biocatalysis in industrial production.The analysis of the structure and the determination of key residues lay the foundation for the expounding of catalytic mechanism and designing of the molecular structure.