Preparation,enzymatic Properties And Structure Characterization Of Thermotolerant ?-glucosidase From Tridchoderma Viride

?-glucosidase is one of the key enzyme of cellulose hydrolysis.However,the development of cellulose enzymolysis technology was seriously restricted due to the low content and degradation activity of the ?-glucosidase.In this study,Trichoderma viride 3.139 was research object,optimization of fermentation contions were investigated.Under the best condition,Electrophoretic pure ?-glucosidase was obtained.The enzyme properties and structure identification of ?-glucosidase were characterized,bgl gene in vitro amplification,sequence and active site analysis were also investigated.The main contents and resluts of this dissertation were as follows:1.The culture medium and optimum fermentation conditions were optimized using means of single-factor orthogonal and response surface analysis test.The optimum medium were determined,which were as follows: lactose of 3%,ammonium chloride of 0.1%,MgSO₄·7H₂O of 0.1%.The optimum conditions were as follows: inoculum amount of 5%,rotational rate of 160 r/min,and medium volume of 100 mL/250 mL.Under this condition,the ?-glucosidase activity was 1.953 U/mL,the activity was increased to 144% comparing with original activity of ?-glucosidase.2.Crude enzyme was isolated and purified by centrifugal ultrafiltration,Sephadex G-200 gel chromatography.Electrophoretic pure ?-glucosidase with one electrophoresis band was obtained using polyacrylamide gel electroresis?PAGE?,the enzymatic properties were characterized as well.The results showed that the optimum reaction temperature was 80 ?.The ?-glucosidase displayed a strong stability under acidic and alkaline conditions and its optimum pH value was 6.16.Some metalions including Fe³⁺?Mg²⁺?K⁺ inhibited its activity,which Fe³⁺ inhibited enzyme activity significantly.However,Fe²⁺?Mn²⁺ increased the enzyme activity,which Mn²⁺ increased enzyme activity significantly.Some organic solvent including methanol and acetone increased the enzyme activity,which methanol increased enzyme activity significantly.However,the ethyl acetate had obvious inhibition effect.3.Trichoderma viride genomic DNA was extracted as a template,the 2 278 bp bgl gene was obtained after the PCR amplification.The sequence was analyzed,the results showed the molecular weight of the ?-glucosidase was about 78.214 3 kDa,containing 744 amino acid residues,splice site at 19 and 20 amino acid,which was also a secretory protein.Hydrophilic amino acids and hydrophobic amino acids were uniform distribution.It was a transmembrane protein with 5 times across the membrane.The secondary structure was 60.08% of randon coil,17.07% of ?-sheet,22.85% of ?-helix.The active site of ?-glucosidase were characterized using the online software of PDBsumGenerate and SWISS-MODEL Workspace.The three-dimensional structure homology models of the ?-glucosidase were developed.4.The molecular mass of the ?-glucosidase was 57.628 17 kDa using the Mass spectrometry system of 5 800MALDI-TOF/TOF.The N-end 22 kinds of amino acids were determined using the LC-MSMS.The C-terminal sequence was characterized using LC – MSMS.The result showed there was no useful information on the mass spectrogram.The secondary structure was determined using the CD.The result showed the protein secondary structure of ?-glucosidase was randon coil.