??Heterologous Expression And Characterization Of Two Endoglucanases From Sorangium Cellulosum So0157-2 ??The Research For Biotransfomation Method Of MPCA

Part one:Heterologous expression and characterization of two endoglucanases from Sorangium cellulosum So0157-2Sorangium cellulosum is the only myxobacteria which degrades crystalline cellulose and can use it as the sole carbon source.S.cellulosum So0157-2 has rich cellulases,however,there are few reports on its cellulose degradation,so the features and functions of a single enzyme component are worth studying.The previous work in our lab revealed the cellulose degradation process of S.cellulosum relied highly on the contact between the cells and the substrate filter paper,which makes it become a tough work to separate the single enzyme component.Moreover,this limits the research of the biochemical characteristic of the enzyme and cellulose-degrading mechanism of S.cellulosum.Firstly,we analyzed the motif character of the cellulases from S.cellulosum So0157-2 and So ce56.Neither organisms appears to encode the dockerin and cohesin domains that are key components of cellulosomes.Besides,few of their cellulases have CBMs.Furthermore,they have only one exoglucanase and various englucanases.According to the previous research of our lab,S.cellulosum showed different approaches from individual cellulases and cellulosomes to degrade cellulose,so we concluded that S.cellulosum might have its unique strategy in cellulose degradation.One englucanase from GH8 was selected and named ScCel8A in accordance with the genome sequencing of S.cellulosum So0157-2.ScCel8A was cloned into pET-22b to construct pET-22b-ScCel8A,and the recombinant plasmid was expressed in heterogenous host E.coli.The recombinant protein was purified,named r-ScCel8A.The optimal pH of r-ScCel8A was 6.0.The optimal temperature was 45?.After the preincubation in the buffer with pH7 to pH10 for 18 h in 4?,80%of the activity was retained,with a strong alkali tolerance.The r-ScCel8A could not have function under high temperature.After 5min in 60?,20%of the activity was retained.No activity was detected at 60? for 1h.Most of the metal ions detected,such as Na+?K+?Ca2+?Mg2+?Li+(in the concentration of 1 mM)could slightly enhance the activity.In the concentration of 10 mM,all of the metal ions could inhibit the activity of r-ScCel8A,especially,Fe3+?Mn2+ made the enzyme loss activity.CTAB could strongly inhibit the activity of r-ScCel8A.The recombinant enzyme showed the activity on lichenan?barley?CMC as substrates.The Km was 3.2mg/ml,Vmax was 96.697?molmin-1mg-1,Kcat was 73.74 s-1 and Kcat/Km was 23.67mLmg-1 s-1while barley was used as substrate.The main enzymatic products from barley were cellobiose,cellotriose,cellotetraose,which showed the enzyme was typical englucanase.One englucanase that contains a CelD-N domain from GH9 was selected and named ScCel9A in accordance with the genome sequencing of S.cellulosum So0157-2.ScCel9A was cloned into pBAD g? A to construct pBAD g? A-ScCel9A,and the recombinant plasmid was expressed in heterogenous host E.coli.The recombinant protein was purified,named r-ScCel9A.The enzyme showed the greatest activity on xyloglucan,and also showed activity on CMC?barley?lichenan.The Km was 1.38mg/ml,Vmax was 5.813?molmin-1mg-1 when xyloglucan was used as substrate.The optimal pH of r-ScCel9A was 4.0.After the preincubation in the buffer with pH7.5 to pH12(except pH8.5)for 17 h in 4? 60%of the activity was retained,but it is strange that only 10%of the activity was retained in Tris-HCl buffer.Generally speaking,the enzyme had a strong alkali tolerance.The optimal temperature was 25?,and r-ScCel9A exhibited 20%activity at 0 ?.r-ScCel9A was a cold-active cellulose with low thermostability.After 1h under different temperatures,less than 35?,80%of the activity was retained,but more than 35?,there was a sudden drop in the activity.So the r-ScCel9A could not tolerate high temperature.Most of the metal ions could inhibit the activity of r-ScCel9A in the concentration of 10 mM.DTT and imidazole had a little influence on the activity,but different concentrations of SDS could make the enzyme loss activity.Furthermore,we got a truncated protein without CelD-N domain,named r-ScCel9A-CD.The deletion of the entire CelD-N module reduced its solubility and promoted the complete loss of enzymatic activity.The enzyme showd no activity on CMC.Part two:The research for biotransfomation method of MPCAThe molecular formula of 5-Methylpyrazine-2-carboxylic acid is C6H6N2O2,and it is a kind of important medicinal intermediate,mainly used for synthesizing the second generation sulphonylureas hypoglycemic agent,new generation long effect blood-lipid lowering drug and the medicine used to efficiently treat bacillary phthisis.At present,there are chemical synthesis,electrochemical oxidation synthesis and biotransfomation method.We can synthesize MPCA using dimethylpyrazine as a substract.Chemical synthesis can pollute our environment and produce by-products,electrochemical oxidation synthesis needs much energy,but biotransfomation method can produce MPCA by one step and protect environment,so it is the best method to synthesize MPCA.Biological synthesis of MPCA is not yet mature,and most of DMP is produced as spice in our country.There are almost no cases to synthesize medicinal intermediate using biotransfomation method.So this thesis did a preliminary research on biological synthesis.Pseudomonas putida ATCC33015 can grow on toluene,p-xylene as sole carbon and energy source.Moreover,the microorganism can take advantage of methyl groups on aromatic heterocycles.According to its characteristics,we did some research on the methods of adding p-xylene,selected the way that adding p-xylene to absorbent cotton in 10ml centrifuge tube whose lid had two holes.p-Xylene was offered by volatilization.When we added 10mM,50mM DMP to the medium,microorganisms could grow normally.So we could add 50mM to medium to increase conversions in the future.In the LB medium,MPCA was very easy to produce carboxylate because it is acidic compound,so we needed to add acid to adjust pH to 1 or 2 when extraction.It was found that the substract conversion efficiency was highest and no intermediates were produced while using 1271 medium.In addition,we made standard curve of MPCA so that we could do quantitative analysis.The quantitative analysis of conversion was made by HPLC and the results were in keeping with TLC,but conversion efficiency needed to be increased.Disappointed,it was difficult to control p-xylene addition manner and concentration,that made unable to continue to produce MPCA,so we considered to transform DMP in E.coli.The plasmid pWWO of Pseudomonas putida ATCC33015 has genes that are related to degradation of DMP.xylM and xylA encode xylene monooxygenase(XMO)which can oxidize toluene to benzyl alcohol,xylB encodes benzyl alcohol dehydrogenase(BADH)which can oxidize benzyl alcohol to benzaldehyde,xylC encodes benzaldehyde dehydrogenase(BZDH)which can oxidize benzaldehyde to benzoic acid.According to other literatures,Xylene monooxygenase(XMO),encoded by the plasmid pWWO of Pseudomonas putida ATCC33015,is a key enzyme system in the degradation of toluene and xylenes and oxidizes toluene to benzoic acid.To research the influence of different enzymes on DMP transformation,xylC,xylM,xylA,xylB genes were cloned into pET-28a to construct pET-28a-xylMA?pET-28a-xylMAB?pET-28a-xylCMA?pET-28a-xylCMAB,and the recombinant plasmids were expressed in heterogenous host E.coli.We added DMP to the medium and took samples to detect whether or not producing MPCA at different times.The results indicated that only intermediate was detected in pET-28a-xylMA fermentation system;few MPCAs could be detected and main product was intermediate in pET-28a-xylMAB fermentation system;we detected some MPCAs,but intermediate was main product in pET-28a-xylCMA fermentation system;MPCA was main product in pET-28a-xylCMAB fermentation system.In conclusion,the system of the recombinant plasmid pET-28a-xyICMAB is the best method to transform DMP to MPCA.But we need to optimize conditions to increase the rate of transformation for industrial applications in the future.