Thermostability Modification Of β-mannanase And Its Efficient Expression

β-mannanase（EC 3.2.1.78）can cut theβ-1,4 glycosidic bond in mannan and produce 2-10 oligomanosaccharide,which has been applied in food,oil exploitation,pulp,medical treatment,feed addition and other industries.It can not only create industrial benefits,and the mannose-oligosaccharide produced is dietary fiber,which is also great for human health.However,the industrial application conditions ofβ-mannanase are relatively strict,such as feed granulation,oil gumbreaking,coffee extraction and other fields often need greater heat resistance and high enzyme activity.Meanwhile,the heat resistance performance of the main commercialβ-mannanase is not good enough.Therefore,more and more researchers pay attention to the development ofβ-mannanase strain with greater heat resistance.In this study,the mannanase gene Anman of Aspergillus niger CBS513.88（A.niger CBS513.88）was modified for thermostability and optimized for fermentation.The main research contents are as follows:（1）High expression of Anman gene in Pichia pastoris X33.The recombinant plasmid p PICZ-Anman was constructed and transferred into host strain P.pastoris X33 to obtain recombinant strain P.pastoris X33-Anman.The recombinant strains were induced with methanol in the flask for 72 h,and their enzymatic properties were determined.The enzyme activity and protein concentration of the recombinant strains were 159.8 U·m L^-1,the protein concentration was 0.17 mg·m L^-1,the protein molecular weight was about 47 k Da,and the optimal temperature and p H were 72.5°C and 3.0,respectively.It is stable below 60°C and p H 3.0-7.0.（2）High-density fermentation of Anman recombinant strain.The recombinant strain P.pastoris X33-Anman was fermented in a 5 L fermenter with high density.After 108 h of fermentation,the enzyme activity of the supernatant reached 1166 U·m L^-1 and the protein concentration reached 1.27 mg·m L^-1,7.3 folds and 7.5 folds of the shaking flask fermentation level,respectively.It was found that the highest enzyme activity of mannanase powder obtained by spray drying supernatant was 10.6 folds that of commercial mannanase,and the temperature stability and p H stability were almost consistent.（3）Flexible modification of Anman thermostability.Six flexible sites were screened from Anman by B-factor and Gibbs unfolding free energy change,and then six single-point mutants were designed by combining the codon preference ofβ-turns.Five of the mutants were characterized and found to be thermally stable with no significant decrease in enzymatic activity.The combination of the five single-site mutations obtained Mut5,whose thermostability was increased from 46.2%to 82.4%,and the half-life was increased 8.8-folds and the T_m value was increased by 2°C.Moreover,the mutations did not affect the kinetic properties of Mut5.Molecular dynamics simulations and constraint network analysis revealed the enhanced stability of Mut5 compared to Anman in both the global and local conformations,and revealed the reasons for the enhanced thermostability of Mut5 at the molecular level with the addition of hydrogen bonds,salt bridges and hydrophobic interactions.（4）Fermentation optimization.The induction temperature was optimized at the shake flask level using P.pastoris X33-Mut5 as the starting strain,and it was found that the induction temperature of 25°C increased the enzyme activity 0.3-folds compared to 28°C.Furthermore,the induced carbon source was optimized at the fermenter level,and it was found that the enzyme activity of supernatant induced by two carbon sources reached 2465U·m L^-1,which was 14.8 folds and 2.1 folds of that by shaker fermentation and single carbon fermentation,respectively.