Properties And Gene Analysis,Knockout And Vector Construction Of ?-glucosidase From Aspergillus Niger

?-glucosidase(?-D-Glucosidase,EC 3.2.1.21)is the rate-limiting enzyme in the process of cellulose saccharification and cellobiose degradation and is the bottleneck of the cellulose hydrolysis process.In this study,Aspergillus niger 3.316 and Aspergillus niger 3.2130 were investigated.The enzymatic properties and gene analysis of?-glucosidase produced by Aspergillus niger 3.316 and Aspergillus niger 3.2130were used to construct a heat resistance of Aspergillus niger 3.316 and a high activity of Aspergillus niger 3.2130.The?-glucosidase-producing strains were investigated in advance for this purpose,including construction of expression vectors and knockout of the?-glucosidase gene.The main findings are as follows:The main research results obtained are as follows:1.Aspergillus niger 3.2130 was fermented to obtain crude enzyme solution,and its enzymatic properties were measured.The optimum temperature and p H value of the enzyme were 55°C and 4.4,respectively.The best salicylate substrate concentration was 0.05 mol/L;0.01 mol/L Mn²⁺and Fe²⁺can increase the enzymatic activity,EDTA has the most obvious inhibitory effect on the enzyme activity;the Michaelis constant of the enzyme is 4,the affinity of this enzyme to salicin is high,salicin is suitable for the substrate.The c DNA fragment of the bgl gene was amplified by RT-PCR and its size was 2583 bp.The amino acid sequence was analyzed by software and the enzyme structure was predicted.The results are as follows:The enzyme contains 860 amino acids,of which Ala13 has the highest hydrophobicity,the maximum score is 2.211,its secondary structure has 23.02%of the alpha helix structure,16.16%of the?-sheet structure,in addition to 60.81%of the random coil structure.?-Glucosidase belongs to the family of glycoside hydrolase 3 and its protein family determines that its active site is between(?/?)₈TIM and(?/?)₆,which are Asp280 and Glu509 with carboxylic acid residues,respectively.The cleavage site of the signal peptide is at the 20 th amino acid,and the 20 th amino acid at the N-terminus is the signal peptide region.2.The gene of Aspergillus niger 3.316 bgl was amplified by RT-PCR;the Aspergillus niger glucoamylase gene Gla A strong promoter was obtained by PCR amplification;Aspergillus niger 3.2130?-glucosidase gene and its promoter and terminator.The construction of the expression vectors was finally completed.3.Two methods were used to knock out the Aspergillus niger 3.2130?-glucosidase gene.One was the CRISPR-Cas9 method,but no positive transformants were detected when the knockout vector was constructed.The vector was not constructed successfully.The reason may be due to the improper selection of the vector,resulting in low efficiency or even the connection is not possible.Another is the use of homologous recombination method to knock out the Aspergillus niger 3.2130?-glucosidase,but the probability of screening positive transformants is low,the reason may be due to the poor quality of Aspergillus protoplast preparation,the second shock conversion result in some of the protoplasts died.