Gene Cloning And Characterization Of A Higly Thermal Stable Glucanase From Aspergillus Niger And Its High Level Expression In Pichia Pastois

As an important industrial enzyme,β-glucanase can hydrolyzeβ-glucan in cereals such as barley and is widely used in beer brewing and feed industries.In the beer industry,β-glucan in wort increases the viscosity of the wort and affects the filtration rate.β-glucanase can decomposeβ-glucan in wort,reducing the occurrence of beer precipitation and improving the quality of beer.In the feed industry,the addition ofβ-glucanase to wheat diets can effectively reduce the intestinal viscosity of animals and increase the absorption of nutrients by animals.In order to find thermostableβ-glucanase encoding genes,putativeβ-glucanase encoding cDNA genes were cloned from Aspergillus niger CICIM F0510,the recombinant enzymes were expressed and characterized.The main results were as follows:（1）A cDNA gene eglA6 which encodingβ-glucanase was cloned and expressed in Pichia pastoris,the recombinant enzyme designated as AnEglA6 showed high thermal stability.（2）The codon number of eglA6 was optimized to artificially synthesize a gene.The codon-optimized gene eglA6b was expressed in Pichia pastoris.The average activity of recombinant yeast transformants with eglA6b was 1.8 times higher than those with eglA6.The molecular weight of AnEglA6 was approximately 52 k Da.AnEglA6 showed low specific activity of1645.7 U·mg^-1 when the substrate was polysaccharides containing onlyβ-1,4-glucoside,such as CMC-Na,the K_m and V_max were 43.1 mg·m L^-1 and 2.6 mmol·min^-1·mg^-1,respectively.AnEglA6 showed high activity of 12450.6 U·mg^-1 when the substrate was barley glucan,the K_m and V_max were 19.1 mg·m L^-1 and 5.9 mmol·min^-1·mg^-1,respectively.The optimum temperature and pH of AnEglA6 were 75℃and 4.0.The half-lives of AnEglA6 in 75℃and70℃were 1.9 h and 4.6 h,respectively.Co²⁺and Mn²⁺could activate AnEglA6,and Fe³⁺had a significant inhibitory effect on the activity of the enzyme.（3）The auxotrophic mutant strain HB133（ura3^-）and HB133（ura3^-/trp1^-）were constructed by two-step homologous recombination method.The expression cassette pPIC9K-eglA6b-TRP1 and pPIC9K-eglA6b-URA3 were constructed and transformed into HB133（ura3^-/trp1^-）,which obtained a highly expressed strain Pichia pastoris GS115/pPIC9K-eglA6b HB133 S6.（4）The fermentation conditions of recombinant Pichia pastoris GS115/pPIC9K-eglA6b HB133 S6 were optimized at shake flask level and 5 L fermentor level.In shake flask,when the initial pH was 6.0,the amount of methanol added was 1.5%,the enzyme activity of glucanase reached a maximum of 3603.9 U·mL^-1.In the 5 L fermentor,when the pH was 4.5,the OD₆₀₀ concentration was 360,and methanol was added at a rate of 10 m L·h·^-1·L^-1,the glucanase activity of the recombinant strain reached a maximum of 33031.8 U·mL^-1,which was9.17 times that in shake flask.