Gene Cloning,Expression And Directed Revolution Of Thermostable ?-1,3-1,4-glucanase

?-Glucans,which occur widely in the seed of higher gramineae as Non-structural-starch polysaccharides,can cause problems to beer brewing and feed stuff industry,which use barley or other gramineae as raw material,by making wort filtration difficult and the beer haze early,or by making feed conversation ratio decreased and the animals indigestion.By adding ?-glucanases(EC 3.2.1.73;lichenase),which can hydrolyze P-glucans,can solve the problems above in food industry efficiently.To date,?-glucanases used in food industry are mainly from microorganism.However,those from the Bacillus are always of low thermostability or low activity.Thus,screening of Bacillus strains which can secrete(3-glucanases with good properties,using genetic engineering and protein engineering to improve the enzyme properties,have great theretical and application value.The thesis mainly presents the research on screening of Bacillus strains with thermostable P-glucanase,purification and characterization of ?-glucanase,the gene cloning and expression of ?-glucanase,and also directed revolution of ?-glucanase.The aim of the research is to use genetic engineering and protein engineering to highly express or modify ?-glucanase,to lay a foundation for production and application of the enzyme in the future.The main results of this study are as follows:1.A Bacillus strain which could produce high-thermostable ?-glucanase was isolated from the spring silt and identified.in the city of Yichun,JiangXi ProvinceA Bacillus strain with thermostable ?-glucanase namely YC-9,was selected from the silt samples of Yichun spring in Jiangxi Province.The activity of ?-glucanase from this strain reached 10.7U/ml.The crude ?-glucanase remained 78.7%activity after incubated under 60? for 1h.The 16S rDNA gene sequence of the Bacillus strain YC-9 was amplified by PCR,and a 1421-bp sequence was obtained.A homology search was performed on Genbank database and a neighbor-joining phylogenetic tree was constructed.The results indicated that the 16S rDNA gene sequence of strain YC-9 revealed the highest homology(100%)with that of Bacillus aerophilus.Bacillus stratosphericus and Bacillus altitudinis.In the phylogenetic tree,strain YC-9 and the above three strains were in the same branch showing the closest kinship.As identification of the morphological,physiological,and biochemical characteristics of strain YC-9,the results showed that its colony is white,round,smooth surface;it can hydrolyze starch and use D-galactose,L-Tyrosine and citrate;its V-P test is positive and methyl red test and nitrate reduction test are negative.The charactrerizations of strain YC-9 are nearly the same with that of strain Bacillus altitudinis,except citrate utilization.Thus,by comprehensive consideration of the 16S rDNA gene,the morphological,physiological,and biochemical characteristics of strain YC-9,it was identified as Bacillus altitudinis.2.The ?-glucanase was purified from Bacillus altitudinis strain YC-9 and its characterization was studied.?-Glucanase was purified to electrophoretically homogeneity from crude culture by combination of purification steps including ammonium sulfate fraction precipitation,DEAE-sephadex ion-exchange chromatography and Sephadex G-100 gel chromatography.The specific activity of 2372.9 U/mg of the purified protein was 36.96-fold from the enzymatic extracts with a yield of 25.8%.By analysis of SDS-PAGE,the molecular weight of ?-glucanase from Bacillus altitudinis YC-9 is between 27-28kDa.The optimum temperature and pH of the enzyme was 65? and 6.0,respectively.The enzyme appeared to be stable in the range of<60? and pH 5.0-9.0,respectively.The P-glucanase from Bacillus altitudinis YC-9 has very strict substrate specificity,since it can only hydrolyze ?-glucan from barely and lichenan,which only have ?-1,3 and(3-1,4 bonds.So it was identified as endo-beta-1,3-1,4-glucanase(E.C.3.2.1.73).Kinetic study indicated that the(3-glucanase had a Km value of 3.71mg/ml,a Vmax value of 4761.9U/mg,a kcat value of 2142.68/s and a kcat/Km value of 577.54ml/s/mg on lichenan.The enzyme activity was enhanced in the presence of Co2+,Fe2+ and Ca2+,while partially inhibited by Cu2+,Mn2+ and EDTA.K+,Na+,Zn2+ and Mg2+showed no obvious effect on the activity.3.The ?-glucanase gene from was cloned and the sequence analysis was performed.The whole ORF with a length of 732bp of P-glucanase from Bacillus altitudinis YC-9 was amplified.The bioinformatics analysis of the predicted protein of ?-glucanase revealed that the protein contained 243 amino acids with a predicted molecular weight of 27.47kDa.The amino acid sequence of p-glucanase showed the highest identity(97%)to the glucanase from B.licheniformis(P27051.1).The conserved motif EIDIEF of GH16 family is in the 134?140 amino acid sites,while its catalytic active center is in E134 and E138.The predicted signal peptide sequence of the enzyme was 87bp encoding 29 amino acids as follows:MSYRVKRMLM LLVTGLFLSL STFAASASA.The amino acid sequence of ?-glucanase was submitted to http://swissmodel.expasy.org to predict the three-dimensional structure of the enzyme.The result showed that the three-dimensional structure of ?-glucanase from Bacillus altitudinis YC-9 was similar with that of hybrid enzyme H(A16-M).4.The ?-glucanase gene of Bacillus altitudinis YC-9 was highly heterologous expressed in E.coli.The ?-glucanase gene from Bacillus altitudinis YC-9 was linked to two expression vectors,pET-23 and pET-32a,respectively,to construct two recombinant expression vectors,pET-23a-Glu and pET-32a-Glu,which were transformed to E.coli BL21(DE3),respectively.The recombinant(3-glucanase was found to be actively expressed both extracellular and intracellular.The recombinant E.coli harboring pET-32a-Glu yield a higher activity of 63.3 U/ml,under the culture condition of 0.4M IPTG concentration,2%inoculation amount,with induced temperature and time of 37? and 4h,respectively.After optimization of the expression condition,including the concentration of IPTG,the inoculation amount,the induced time and temperature,the recombinant enzyme finally reached a total activity of 319.34U/ml,with extracellular activity of 102.67U/ml and intracellular activity of 216.67/ml.The optimized expression condition is of 0.4M IPTG concentration,4%inoculation amount,with induced temperature and time of 28? and 8h,respectively.The optimized activity is of 5 folds higher than the unoptimized,and about 30 folds higher than the activity of the primitive strain.The specific activity of the supernatant in the culture and the cell lysate reached 2409U/mg and 2708.8U/mg,respectively,after optimization.5.The SDS-PAGE electrophoresis,purification and characterization of the recombinant enzyme was studied.The SDS-PAGE electrophoresis showed the recombinant enzyme became the major protein in the supernatant of the cell lysate and the cell precipitation,after the recombinant strain E.coliBL21(DE3)/pET-32a-Glu being induced with IPTG.There were two molecular forms in the supernatant of the cell lysate,of which one molecular weight was about 28kDa,and another was about 45kDa.The recombinant enzyme existed in the form of inclusion body in the cell precipitation,with a molecular weight of about 45kDa.The supernatant of the recombinant strain E.coliBL21(DE3)/pET-32a-Glu was collected to purify the recombinant enzyme.SDS-PAGE showed the purified enzyme to be electrophoretically homogeneity and revealed the molecular weight of the extracellular enzyme to be about 27-28kDa.The specific activity of 5392.7 U/mg of the purified recombinant enzyme was 2.24-fold from the enzymatic extracts with a yield of 56.5%.Kinetic study indicated that the recombinant ?-glucanase had a Km value of 4mg/ml,a Vmax value of 7692U/mg,a kcat value of 3461/s and a kcat/Km value of 865.28ml/s/mg on lichenan.The optimum temperature and pH of the recombinant enzyme was 65? and 7.0,respectively.The recombinant enzyme appeared to be stable in the range of ?60? and pH 5.0-9.0.By comparing characterics of the recombinant ?-glucanase and the primary one,they were comparable.6.Using Error-PCR method to study directed revolution of ?-glucanase.The appropriate concentration of Mg2+ and Mn2+ was investigated to carry on Error-PCR reactions.A final concentration combination of 3mM and 4mM of Mg2+ and Mn2+,respectively,which can cause the number of mutated bases to be about 5.67,was used in Error-PCR reactions.Through screening mutants with high enzyme activity using high temperature pyrolysis method,a high enzyme activity mutant Glu-3060 was selected among 5000 transformants.After induced with IPTG,the activity of the mutant Glu-3060 reached 474.6U/ml,including extracellular activity of 138.2U/ml and intracellular activity of 336.4U/ml,resulting in 48.6%increment of the parent enzyme activity.Research on the characterization of the mutated enzyme showed the optimal pH of the mutated enzyme is 5.0,which is lower than the parent enzyme,but thermal stability are almost the same between them.The sequence analysis of the mutated enzyme revealed that there were 3 amino acids changed compared with the parent enzyme,including K142N,Q203L and N214D.The three dimensional structure predicted by SWISS-MODLE of the mutated enzyme Glu-3060 showed K142N was near to the catalytic residues E134 and E138,while Q203L was in the position of ?-turn of the second(3-fold-layer,and N214D was in the position of the?-folding-stocks of the second P-fold-layer,next to the a-helix and the site 213 W interacting with the subsrate.The three amino acids substitution made effect on the catalytic activity,the stability and the optimal pH of the enzyme,through changing the charge properties or the electron density,the forming of the secondary keys,the acidity of the amino acids and the side chain group.The sum effects of all the factors made the mutated enzyme activity increased,the optimal pH decreased and the thermostability maintain the same.