Enzymatic Properties Of β-glucosidase And Amplification Of Its Gene In Vitro From Trichoderma Viride

β-glucosidase, also known as β-D-glucoside hydrolase glucose,plays a key rolein cellulose enzymatic hydrolysis.Its activity level directly impacts the efficiency ofthe cellulose hydrolysis,but its low content and low vitality is a bottleneck ofcellulose enzymatic hydrolysis and severely restrict its application. Trichodermaviride is one of the groups secreted the highest cellulase activity. This study focuseson the optimization of β-glucosidase fermentation conditions, fermentation kinetics,enzymatic properties,bgl gene in vitro amplification and recipient bacteriumscreening.The main contents of this dissertation are as follows.1.By single factor test, the carbon source,nitrogen source and initial pH in thefermentation process were studied and optimized them by orthogonal test,the resultsshowed:the best carbon source is5%microcrystalline cellulose;best nitrogen sourceis0.15%sodium nitrite;optimal initial pH is4.0.The activity of β-glucosidase couldreached2.879U/mL under the optimized culture conditions, which is1.175times thanthe initial fermentation conditions.2.The kinetic models of Trichoderma viride for cell concentration,β-glucosidasesynthesis and substrate consumption were studied.The parameters were obtained withnonlinear curve fit by Origin.The fitting curves of the experimental data wereobtained in fitting formula.3.The enzymatic properties of Trichoderma viride was studied,the resultsshowed that:the optimum temperature is70℃, the optimum pH is4.6; optimumsubstrate concentration is0.05mol/L; optimum reaction time is35min; methanol hasa positive role for enzyme activity, ethanol and ethyl acetate inhibited enzymeactivity.4.Taking Trichoderma viride genomic DNA as a template,the PCR amplificationgot the2506bp bgl gene.The nucleotide and amino acid sequence were analyzed,theresults showed that:the gene encoding protein is alkaline and secretory protein,themolecular weight is about80.55kDa, containing726amino acid residues,splice site at20amino acid, the top20amino acid is signal peptide region.5.The defective acidic proteinase producing strains were screened after compound mutation of UV and diethy sulfate(DES).The acidic protease activity was8.27%of original strain.The optimum mutation parameters were as follows:1.5%DES, and quivering for30min at30℃. Genetic stability test showed that its acidicprotease relative activity was stabilized. This could be the first step for theheterologous expression of bgl gene in Aspergillus niger.