Heterologous Expression,Fermentation Optimization And Immobilization Of α-glucanase From Chaetomium Globosum

α-glucanase,also called dextranase（EC 3.2.1.11）usually,is one of glycoside hydrolase that can degrade high-molecular glucan specifically.α-1,6 glycosidic bond of the substrate could be cleavaged randomly by it to produce oligosaccharides and glucose.Therefore,it is applied to sugar,medicine,food and chemical industries widely,and has better potentials for applicaton.In this paper,α-glucanase（NCBI number:MH 122516.1）derived from Chaetomium globosum was the object of study.Heterologous expression,optimization of fermentation conditions,separation and purification,and immobilization ofα-glucanase were carried out.The codon adaptation index（CAI）was increased from 0.56 to 0.91,and the gene sequence identity before and after optimization was 73.38%afterα-glucanase gene optimization.The engineered plasmid p PIC9k-DEX was constructed by genetic engineering technology.After linearization with endonuclease Sac I,it was integrated into chromosome of Pichia Pastoris by electric shock successfully.The recombinant strain with Mut⁺phenotype was obtained.G418-YPD resistant plates with different concentrations were prepared to screen out recombinant strains with high copy numbers.A recombinant strain with an initialα-glucanase production of 2.69 U·m L^-1 was screened through methanol-induced fermentation,and the production reached 6.16 U·m L^-1 after high-copy screening.The optimal fermentation conditions of the recombinant strain at the shake flask level:the recombinant strain was grown in medium BMGY at 30℃,210 r·min^-1 for 26 h,then replaced them in medium BMMY with initial p H of 9 and sorbitol of 5 mg·m L^-1 at 24℃,230 r·min^-1,2.5%methanol was added every 24 hours.The best enzyme activity was 47.92 U·m L^-1,which was 17.80 times higher than the initial.The preliminary optimization results of 5 L fermentor:When the wet weight of biomass was 120 g·L^-1,the highest expression level of recombinantα-glucanase was 28.19 U·m L^-1,which is about 50%of the fermentation level in shake flasks;When the wet weight of biomass was 260 g·L^-1,the highest expression level of recombinantα-glucanase was 66.22 U·m L^-1,which is 38%higher than that of shake flask fermentation.The recombinantα-glucanase was purified by Hitrap Q HP and Hitrap HP SP chromatography step-by-step and validated to up to the electrophoresis purity by SDS-PAGE.The molecular weight of the recombinant protein was about 65 k Da.The purification factor reached 40.83,with the recovery of 24.15%.And the specific activity reached 277.61 U·mg^-1.Subsequently,the dynamic characteristics of the purified enzyme were investigated.The optimal reaction temperature and p H are 60℃and 5.5,respectively.The enzymatic activity keeps stable within 20℃～50℃and p H 4.5～8.5.The presence of 10 mmol·L^-1 Fe²⁺activates the enzyme whereas 0.1 mmol·L^-1～10 mmol·L^-1 Cu²⁺inhibits the enzyme.Affinity study indicates that dextran T2000 was the optimal substrate of the enzyme.α-glucanase was immobilized by crossbonding oxidized graphene.The immobilization conditions were optimized:GO was activated for 3 h,and under the acion of 1%genipin for 8h.The recovery rate of enzyme activity reached 88.16±2.47%.The optimal reaction temperature and p H were 55℃and 5.5.The half-life of the immobilized enzyme at 50℃was300 min.After reused for 4 times,the catalytic efficiency of the substrate remained above 60%.After being placed in a refrigerator at 4℃for 80 d,the enzyme activity could be above 65%.