Study On The Thermal Protection And Immobilization Technology Of Cellulase

As one of the widely used enzymes,cellulase composed of endo-β-1,4-glucanase(EG),cellobiohydrolase(CBH),and β-glucosidase(β-G),which plays an important role in many industries,such as food,paper making,textile and animal feed.However,the free cellulase is vulnerable to external environment which would lead to the loss of enzyme activity,and it is also difficult for recycling,which causes the high cost that limits the large-scale industrial application.These current problems can be solved by the protective effects of enzymes.Cellulose is one of the most abundant renewable resources on the earth,but it has not been effectively used,and even its waste has caused damage to the environment.Cellulase can degrade cellulose to produce glucose,and further fermentation steps can produce biofuel ethanol,which can effectively alleviate the energy crisis.The hydrophilic macromonomer PEG-MA was synthesized on the basis of methoxypolyethylene glycol.Then the copolymer PEG-g-PDMAEMA with a polymerization degree of 46 was synthesized by 2-(dimethylamino)ethyl methacrylate(DMAEMA)and PEG-MA.4 mg/mL of the positively charged copolymer was combined with 0.5 mg/mL of the cellulase of negatively charged amino acids in the active regions by electrostatic interaction under neutral condition.The active regions of enzyme were covered by the copolymer,thus shielding the unfavorable factors such as external environment.With the addition of 20 mg/mL negatively charged polyacrylic acid at 25 oC,causing the more power electrostatic interaction with PEG-g-PDMAEMA,then the cellulase was desorbed,resulting in the recovery of enzyme activity almost absoultly.The construction of the two step artificial chaperone PEG-g-PDMAEMA/PAAc system has a thermal protection effect on cellulase based on the "on/off" regulation of enzyme activity.When PEG-g-PDMAEMA/cellulase complex was cultivated at 90 oC for 20 min,58% of the relative enzyme activity was restored.The soluble-insoluble UCST copolymer PMAAc was synthesized by two monomers of methacrylamide(MAA)and acrylic acid(AAc).The amino groups of PMAAc was activated by 40 μg/mL of glutaraldehyde at 50 oC for 5 h,then production was covalently immobilized cellulase with the UCST of 19 oC.The immobilization conditions were optimized,when 4 mg/mL of cellulase was immobilized in the buffer solution(pH 5.0)at 35 oC for 4 h,the enzyme loading amount was reached 46.6 mg/g.The immobilized cellulase widened the range of pH and temperature,and the stability was significantly improved.As the content of β-glucosidase is very low in the compound cellulase,however the β-G is the last key step in the synergistic action of the hydrolysis of each enzyme,so β-G is added to the cellulase(1:2.5,w/w)as a supplementary enzyme to be co-immobilized.The hydrolysis reaction was up to a balance after 24 h,and the glucose yield of PMAAc-cellu&β-G was nearly 3 times of that of PMAAc-cellulase.The glucose yield of the PMAAc-cellu&β-G was also remained about 61% of its original productivity after eight cycles.The β-glucosidase was coprecipitated with copper acetate monohydrate and 4-aminobenzoic acid under the protection of polymer PVP,and the β-G@Cu(PABA)was based on the co-synthesized metal organic frameworks.The optimum concentrations of copper acetate monohydrate and 4-aminobenzoic acid were 50 mM and 12.5 mM,and the enzyme concentration was 2 mg/mL,the mixture was cultivated for 8 h in sodium acetate-acetate acid buffer solution(pH 7.0),then the enzyme loading amount and the relative activity were achieved the best value of 162.95 mg/g and 81.89% respectively.The three-dimensional channel structure of MOFs provides a rigid shielding environment for β-glucosidase,which significantly improved its stability and the tolerance of organic solvent.The hydrolysis equilibrium of CMC by β-G@Cu(PABA)with PMAAc-cellulase was reached after 12 h,The glucose yield of the two kinds of immobilized enzyme was nearly double compared with that of PMAAc-cellulase,and also over 70% of its original productivity was remained after eight cycles.