Study On The Thermostability Of 1,3-1,4-?-glucanase From Bacillus Species

1,3-1,4-?-glucanase is an important industrial enzyme, which could hydrolyze high molecular weight ?-glucans into oligosaccharides by strictly cutting 1,4-?-glycosidic bonds. In the brewing industry, the addition of ?-glucanases into Congress mashing can increase the yield of extract and the filtration rate of wort, thus enhancing the non-biological stability of the finished beer. In the animal feed industry, ?-glucanases can eliminate the effect of ‘anti-nutritional factor', increase the digestibility of cereal-based diets and reduce sanitary problems. However, the wild-type ?-glucanases show low thermostability and catalytic properties, which are soon inactivated in the malting, mashing and animal-feed pelleting processes. Therefore, the thermostability and catalytic properties of mesophilic ?-glucanases need to be improved to meet the industrial application standard. In this thesis, a highly thermostable ?-glucanase was successfully constructed by rational/semi-rational strategies including lysine-based mutagenesis, disulfide bonds engineering and spatial compartmentalization of mutational hotspots. The high thermostable ?-glucanase was heterogously expressed in Bacillus subtilis WB600 and applied in Congress mashing processes. The main conclusions were as follows:(1) The role of lysine residues in ?-glucanase thermostability was investigated. The ?-glucanase thermostability was moderately enhanced by chemical modification in surface lysine residues by nitrous acid. The modified enzyme showed a 2.5oC increase in T50 value and a 76.8% rise in half-life values at 60 oC compared to the wild-type enzyme. Based on the modification results, the lysine residues in ?-glucanase from B. tequilensis were mutated into serine residues by comparison of the protein total energy values, hydrogen bonds number and enzyme specific activities. The thermostability of mutants K20 S, K117 S and K165 S were superior to the wild-type enzyme. After combinational mutagenesis, the triple mutant K20S/K117S/K165 S displayed a 15 oC increase in optimal temperature and a 14 oC rise in T50 value. Its half-life values at 50 oC and 60 oC were also largely extended. More structured residues were observed and more hydrogen bonds were formed in the mutant, which might be the reason for the enhancement of thermostability.(2) The thermostability positive disulfide bonds in ?-glucanase were screened through a three-step method based on the analysis of spatial configuration of secondary structures, catalytic center and local/overall protein flexibility. The introduction of two disulfide bonds N31C-T187 C and P102C-N125 C was predicted to be able to reduce the flexibility of overall protein and the engineered regions without disturbing the enzyme catalytic properties. The Tm values of the mutant N31C-T187 C and P102C-N125 C were 1.4oC and 2.3oC higher than that of the wild-type enzyme. After combinational mutagenesis, the N31C-T187C/P102C-N125 C mutant showed a 4.1oC rise in Tm value and a 48.3% increase in half-life value at 60 oC compared to the wild-type enzyme. The catalytic property of the double mutant was unaffected. The introduction of two disulfide bonds and the newly formed hydrogen bonds led to the rigidification of both overall protein structure and the engineered regions, which should be responsible for the enhanced thermostability of the mutant enzyme.(3) The thermostability crucial regions/residue sites in homologous ?-glucanases were predicted through a novel spatial compartmentalization of mutational hotspots method which combined alignment of homologous protein sequences, spatial compartmentalization and molecular dynamics simulation. The calcium binding region was predicted to be the crucial region for thermostability of bacterial ?-glucanases. The engineering of six non-conserved residues in a mesophilic ?-glucanase showed that residue sites No.40, No.43, No.46 and No.205 were important to ?-glucanase thermostability. Optimization of these four residues by iterative saturation mutagenesis significantly enhanced the thermostability of a mesophilic ?-glucanase. The optimal temperature, T50 value and Tm value of the E46P/S43E/H205P/S40 E mutant were 20 oC, 14.5oC and 13.8oC higher than those of the wild-type enzyme while its half-life values at 60 oC and 70 oC were 3.86 times and 7.13 times as high as those of the wild-type enzyme. More negative charge was found at the calcium binding region, which could strength the binding between protein and calcium ion. More structured residues and hydrogen bonds were also observed in the mutant, which could lead to better thermostability.(4) The thermostability positive sites in ?-glucanases were combinational mutated and heterogously expressed in B. subtilis WB600. The optimal temperature, T50 value and Tm value of mutant rB-BglTO were 70 oC, 81.7oC and 56.2oC, which were 25 oC, 19.7oC and 15.9oC higher than the wild-type enzyme. The half-life values of rB-BglTO at 60 oC and 70 oC were 153.2 min and 99.6 min, which were 4.71 times and 9.05 times as high as those of the wild-type enzyme. Besides, the optimal pH of r B-BglTO was reduced from pH6.5 to pH6.0 and its stability under acidic conditions was also improved. Moreover, its specific activity and kcat value were 72.4% and 37.5% higher than those of the wild-type enzyme. In flask fermentation, the highest activity of ?-glucanase from recombinant B. subtilis reached 4840.4 U·m L-1 after fermentation condition optimization, which was 3.31-fold and 2.03-fold higher than recombinant E. coli harbored wild-type enzyme and rE-BglTO. The addition of rB-BglTO in Congress mashing process decreased the wort filtration time and viscosity by 29.7% and 12.3%, respectively, which were superior to two commercial ?-glucanases.