Breeding Of High-Yield β-1,3-1,4-Glucanase Producing Strain By Mutagenesis Approaches And Directed Evolution Of β-1,3-1,4-Glucanase

β-1,3-1,4-glucanase (EC.3.2.1.73) is an important industrial enzyme, which can effectively hydrolyzes the β-glucans in the barley seed of gramineae plant through reacting synergistically with other β-glucanases. In this way, the filtration rate in the saccharification of beer and the feed utilization rate of animal are improved. Currently, β-1,3-1,4-glucanase used in industry are mainly from microorganisms, particularly Bacillus. However, screened and identified β-1,3-1,4-glucanase from Bacillus are always of low enzyme activity or low thermostability, which to some extent limited its industrial application.Thus, the breeding of high yield β-1,3-1,4-glucanase producing strain through mutation of original strain, or improving the properties of the enzyme through genetic engineering or protein engineering has great theoretical and practical value.This paper mainly focus on the mutation breeding of Bacillus altitudinis YC-9 which produces thermostable β-1,3-1,4-glucanase, β-1,3-1,4-glucanase gene cloning and heterologous expression, and directed evolution of β-1,3-1,4-glucanase, to lay a foundation for industrial production and application of the enzyme.1. The mutation breeding of Bacillus altitudinis YC-9 by physical and chemical mutagenesis approachesFirstly, the NTG and LiCl mutagenesis were carried on Bacillus altitudinis YC-9, and the optimal concentration of them was respectively determined as 0.08mg/ml and 0.08g/ml. After the first round of mutagenesis and screening, a strain named N-1-7 whose enzyme activity increased 26.8% was obtained. After a second round of NTG mutagenesis on N-1-7, a high-yield strain with 134.4% increase of enzyme activity was acquired and named N-2-2. At last, N+ beam implantation was conducted on N-2-2, and a strain whose enzyme activity improved 26.2% was screened out and named 10-30s-3.2. The heterologous expression of β-1,3-1,4-glucanase gene from Bacillus altitudinis YC-9 in E. coli and the study of recombinant enzyme propertiesThe β-1,3-1,4-glucanase gene was connected to the expression vector pET-28a, and the recombinant vector was transformed into E. coli BL21 (DE3). Finally, the enzyme was successfully expressed and the intracellular activity reached to 79.2U/ml with 2% inoculation amount and induced at 30℃ for 6h.The β-1,3-1,4-glucanase was purified to electrophoretically homogeneity by combination of purification steps including ammonium sulfate fraction precipitation, DEAE-sephacel ion-exchange chromatography and Sephadex G-100 gel chromatography. The molecular weight of the enzyme was 27KDa consistent with theoretical size. The optimum reaction temperature and pH of the enzyme was 60℃ and 7.0, respectively. The enzyme appeared to be stable in the range of ≤60℃ and pH 5.0-9.0, respectively.3.Directed evolution of the β-1,3-1,4-glucanase gene from Bacillus altitudinis YC-9The high-throughput screening methods were determined and then the Mg+ and Mn2+ concentration in Error-prone PCR were further studied. Two appropriate concentration combination of Mg2+ and Mn2+ for Error-prone PCR reaction were respectively 4mM and 0.45mM, as well as 6mM and 0.3mM. After one round of Error-prone PCR, two mutated enzyme Glu-167 and Glu-1544 were screened, and the activity of them went up 42.4% and 75.9%, respectively.The sequence analysis of the mutated enzyme revealed that there were 3 amino acids changed in mutant Glul67, including K142N, Q203L and N214D, and there was just 1 amino acids changed in mutant Glul544, namely K142N. The three dimensional structure predicted by SWISS-MODLE of the mutated enzyme Glu-167 showed that T31P was at the bottom of the substrate binding pocket, while N41S was in the first loop which connects the two β-fold-layers, and I135L was in the conserved region EIDIE of the β-folding-stocks of the second β-fold-layer, next to the a-helix and the site 213W interacting with the subsrate. The three amino acids substitution made effect on the catalytic activity of the enzyme, through influencing the forming of the secondary keys and changing the structure of the active center, and the sum effects of all the factors made the activity of mutated enzyme increased at last。...