Gene Cloning, Expression And Molecular Evolution Of Glycosidase From Thermophilic Fungi

Cellulose is the most abundant carbohydrate available on earth, it can be converted into glucose by cellulases. It has great practical significance to solve the energy crisis, food shortage and environmental pollution. Traditionally, the chemical method to break down cellulose uncontrollable, costly and environmental pollution, but the biological degradation of cellulose effectively overcome these problems. Cellulase is a general term for multi-component enzyme that can hydrolyze cellulose byβ-D-glucoside bond. The completely degradation of cellulose requires synergy of three components: endoglucanase, exo-glucanase andβ-glucosidase, theβ-glucosidase is the bottleneck of cellulose hydrolysis. Increased mount or enhencedβ-glucosidase activity, will effectively improve the efficiency of cellulose hydrolysis. In recent years, many experts and scholars continue to improve the performance of activity and access to new recombinant cellulase by means of molecular biology.Glucoamylase is an exo-acting enzyme that removes the glucose unites from the non-reducing ends of amylose, amylopectin and glycogen by hydrolyzingα-1, 4 linkages in a consecutive manner, producingβ-D-glucose as the sole product. Glucoamylase was widely used in dextrose production, baking industry, brewing of low calorie beer and whole grain hydrolysis for the alcohol industry. In recent years glucoamylases from thermophilic fungi, which are expected to be thermostable, have aroused increasing attention among researchers. The glucoamylase used in the market mainly from room temperature microorganisms. Due to high production costs, poor thermal stability, low activity and yield, the application of glucoamylase is restricted in agriculture and industrial applications. Thus, the research and development of thermorphilic glucoamylase have great importance.T.lanuginosus and C.thermophilum are widely distributed soil-inhibiting fungi of considerable interest producers of thermostable enzymes, which have great value in research and application. In this study, aβ-glucosidase gene was isolated firstly from C.thermophilum CT2 by RT-PCR and RACE-PCR. The GenBank accession number was EF648280, sequence analysis showed that the full nucleotide sequence of the gene was 3213 bp, contained a 2604bp open reading frame, encoding 867 amino acids. The amount of enzyme protein was 1.644 U/mg after yeast expression. The recombinant protein was purified by ammonium sulfate precipitation, DEAE-Sepharose Fast Flow anion exchange. The SDS-PAGE showed that the recombinant protein was about 119 kDa, slightly larger than the deduced molecular weight, which may be related to the glycosylation. The optimal temperature and optimal pH were 60℃and 5.0 separately, the enzyme was relatively stable, after incubated at 70℃for 10 min still remained 29.7 % activity.In this study, a glucoamylase gene from T.lanuginosus was also cloned (GenBank accession number is EF545003) and highly expressed in P.pastoris, the enzyme activity was 11.6 U/mL. The recombinant protein was purified by ammonium sulfate precipitation, DEAE-Sepharose Fast Flow anion exchange. SDS-PAGE showed that the recombinant protein was about 67 kDa, the optimal temperature and the optimum pH were 60℃and 5.0, separately. The enzyme was stable in the range pH 4.0-9.0.The site-directed mutagenesis was acted toβ-glucosidase from C.thermophilum CT2. Three non-conservative amino acid mutation K275R, N276S, and 279D were expressed in p.pastoris, three mutants activities were 81.3 %, 63 % and 65 % respectively compared to GS-BGL, but the optimum temperature of three mutants were increased 5℃; mutants N276S and G279D optimum pH changed from 5 into 4 and mutant K275R optimum pH remained at 5. The performance of the three mutants were different in thermal stability, after incubated at 60℃for 60 min, G279D still have 80.6 % of the activity, N276S have 72.1 % of the activity, the activity of K275R was 59.5 %, while the original enzyme activity remaining 69.55 %. The conservative amino acid Asp287(nucleophile) mutated to Gly(non-nucleophile), After expressed in p.pastoris, found the mutant lost the glycoside hydrolase activity, but not yet detected glycoside synthase activity.Since site-directed mutagenesis is mainly directed against a small number of amino acid residues, the high-level protein structure remained unchanged. The structure ofβ-glucosidase in this study enzyme was not reported so far, in order to improve theβ-glucosidase activity and stability, the directed evolution was used to alert theβ-glucosidase gene. In this study, the mutant library was constructed by error-prone PCR, fluorescence screening and small amount ferment were used to screen mutants with higher activity. A mutant BE857 with 0.74-fold increased enzyme activity was acquired. The sequencing showed the mutant have 6 amino acids changed. The optimum temperature of BE857 was 65℃, 5℃higher than the starting enzyme, the optimum pH kept invariant; the stability also increased, after treatment at 60℃for 1 h, the mutant still remaining 75.58 % activity, while the remaining activity of wild enzyme was 69.55 %.