Study Of Trichoderma Harzianum BGL Gene Expression And Enzymatic Properties From The Mangroves

?-D-glucopyranoside glucohydrolases(EC 3.2.1.21)can specifically catalyze the hydrolysis of small molecule oligosaccharides,hydrocarbyl groups and non-reducing terminal ?-glycosidic bonds of aromatic groups to form glucose.As the main rate-limiting enzyme in cellulase,?-glucosaccharase is of great significance for the efficient utilization of biomass carbon source.In addition,it has important use value in the fields of food processing,breeding,biomedicine,planting,etc.Therefore,the development of new?-glucosidase resources has far-reaching production significance.This experiment aimedto screen out the novel ?-glucosidase from the mangrove environment of Hainan and explore the application potential of the enzyme resource by studying enzymatic characteristics.The main findings are as follows:1.9 fungi strains with significant ?-glucosidase activity were successfully screened from the mangrove soil in Hainan,and one of the most active fungi H1 was selected for subsequent experiments.Under the induction of crystalline cellulose,the enzyme activity in the supernatant of fermentation broth reached 19.1 U/ml;the strain H1 was identified as Trichoderma harzianum by morphological observation and ITS sequencing.2.The primers were designed using the bgl gene of Trichoderma harzianum IOC 3844(GenBank accession number:KU201604.1)based on NCBI database.The complete bgl gene was obtained from wild strain H1 by reverse transcription PCR,then pPICZaA was used as the template,the H1 bgl gene was ligated to pPICZaA vector and integrated into Pichia pastoris X33 genome,to express and determine the optimal enzyme production cycle.The results showed that the whole length of H1 bgl fragment was 1398 bp,and the homology was highest in comparison with the database of T.harzianum IOC 3844?-glucosidase,reaching the similarity of 96.07%,till no sequence of 100%agreement rate was found.The optimum fermentation cycle was 6d and the highest activity was 3.09 U/ml respectively.3.The H1bgl protein was successfully purified by anion exchange chromatography,and the purity and concentration of the protein were detected by SDS-PAGE and BCA respectively.Finally,the activity of H1 bgl ?-glucosidase and the dynamic parameters of enzyme reaction at different temperatures and pH were determined using pNPG as substrate.The results showed that after purification the target protein concentration of the solution was 0.106 mg/ml,and the specific activity of the enzyme protein was 14 U/mg.No obvious non-target bands were detected by SDS-PAGE,and the molecular weight of target band was about 80 kD.The optimum reaction pH was 7.0,the temperature was 55 C,the Michaelis constant Km=1.47 mM,and the specificity constant Kcat/K13.56(/s/mM).In summary,the test successfully found a novel ?-glucosidase resource from the mangrove Trichoderma harzianum,which has excellent alkaline environmental adaptability,but its catalytic efficiency is relatively weak and further improvements are needed.