| The current demand for energy is increasing and the fossil fuel is on the decrease,energy shortages in most countries could become an important issue,so it is need to find an available resources to replace fossil fuels.Cellulose is formed of ?-1-4 glycosidic bond of polymer.Cellulose is the largest and widely distributed on the earth.It is the most cheap renewable resources.Lignocellulose is converted to into biological energy and chemical products which has important significance to the sustainable development of mankind.It is also play an important role to deal with the problem of environment.In many cases,cellulose must be converted into glucose can be used.The main method of converting cellulose into glucose monomers with enzymatic or acid hydrolysis method.Compared with acid hydrolysis,enzymatic hydrolysis has the advantages of mild conditions and more product.But the enzyme is easy deactivation and inhibited by the end product.So far,the cost of cellulose is still large.In industrial production,the cellulase by end product inhibition will greatly reduce the activity of the cellulose enzyme.Many researchers put forward their own solutions,someone with mixed culture,adding yeast make glucose convert into ethanol,reduce the concentration of glucose,thereby reducing glucose inhibit cellulose enzyme;some people think that improve the various enzymes activity and stability,and find effective method is to improve of bacteria culture medium.Because different components of culture medium have different influence to the enzyme production,optimize the culture medium,make it toward the optimal ratio of enzyme production,improve the ability of cellulose enzyme collaborative lignocellulose degradation;some scholar s want to use genetic modification ways to engineered cellulase to keep it reduce the influence of enzyme inhibition of cellulase research mainly focused on Trichoderma reesei,penicillium decumbens,spines spore aspergillus fungus,to Rhizopus stolonifer production cellulase reports are rare.How to reduce the inhibition of end product to improve the effect of cellulose enzyme activity,make it can be widely used in production is an open question.Rhizopus stolonifer was found on the decaying leaves of China HuangShan,now be kept in our laboratory.Analysis the reasons of glucose and sorbitol different inhibition effect on ?-glucoside enzyme and directional change P-glucoside enzyme structure,observe whether reduced glucose of P-glucoside enzyme inhibition.We puritied cellulose form Rhizopus stolonifer fermentation liquor using ammonium sulfate?Sephadex G-100?DEAE-SepharoseFF anion exchange column chromatography and CM-SepharoseFF cation exchange chromatography column,which contain endoglucanase?exoglucosidase and P-glucosidase.The SDS-PAGE analysis of the purified enzyme preparation shows one band byprotein staining.We researched the enzymology properties of three enzymes(optimum temperature,the optimum pH and Km),the results show that endoglucanase reaction optimum temperature of 50?,the optimum pH 5,Km is 5.0 mg.mL-1?exoglucosidase reaction optimum temperature of 55?,the optimum pH 5,Km is 7.2mg.mL-1??-glucoside enzyme optimum temperature of 55 ?,the optimum pH 5,Km is 6.49 mg.mL-1Cellulose P-glucosidase was purified from Rhizopus stolonifer.The inhibition of it from glucose,mannose,sorbitol and mannitol were studied.While the sorbitol and mannitol have no effect on the catalytic activity of ?-glucosidase.Structure disparity of these sugars and polyols was located in the aldehyde group.Molegro Virtual Docker(MVD)was used to simulate the docking model of glucose,sorbitol and cellobiose with the ?-glucosidase.It was found that glucose and cellobiose have two competing binding sites(Asp244 and Asn242)in the substrates binding region,and a competitive relationship was found in these two molecules when lie in the active region.The aldehyde group of glucose played a leading role in the process of combination with active sites.However,sorbital and cellobiose displayed noncompetitive relationship in the catalytic center and binding region of ?-glucosidase.It indicated that the aldehyde group played a key role in the product inhibition of?-glucosidase by glucose.Virtual Docker(MVD)was used to simulate the docking model of glucose,sorbitol and cellobiose with the ?-glucosidase.We decided to increase active center of enzyme,the glucose can more easily eliminate the catalytic center,reduce the inhibition of glucose in the catalytic center for P-glucosidase.In order to optimize the structure of the P-glucosidase,we design primer for the two mutants(E173G,Q186G,A355G,L357G).Expression of mutant genes and original gene into BL21 got three mutations bacteria(including the original bacteria).We puritied ?-glucoside form recombinant bacteria and bacteria respectively,using ammonium sulfate.Sephadex G-100?HisTrapTMFF?DEAE-SepharoseFF anion exchange column chromatography.The SDS-PAGE analysis of the purified enzyme preparation shows one band by protein staining.We researched theproperties of three enzymes(optimum temperature,the optimum pH and Km),and observe whether reduced glucose inhibition of BG1 and BG2.In this paper,we had purified of cellulose enzyme from Rhizopus stolonifer,using HPLC measuring the end product of cellulase hydrolysis filter paper.Research the influence of all kinds of sugar and alcohol on cellulose enzyme,found that glucose has a strong inhibitory effect on ?-glucoside enzyme,sorbitol has no effect on ?-glucoside,structure disparity of these sugars and polyols was located in the aldehyde group.Virtual Docker(MVD)was used to simulate the docking model of glucose,sorbitol and cellobiose with the ?-glucosidase,found aldehyde group of glucose played a leading role in the process of combination with active sites.On the basis,we decided to optimized the structure of?-glucoside enzyme,directed transformation,purified the recombinant protein and researched enzymology 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