Expression And Characterization Of Mannanase From Aspergillus Niger CBS 513.88 In Pichia Pastoris

Mannan is a linear polymer connected with each other by the beta 1,4-D-pyran mannose,and is widely existed in various plants in the nature.Mannanase can specificity degrade mannan,thus can improve the animal's gut microbes environment,promote the nutrients absorption and improve animal's immunity.Therefore mannanase has important use in animal fodder.Except for the animal fodder,mannanase can also be applied to the papermaking.The mannanase gene from Aspergillus niger CBS 513.88 has 1149bp,encoding a protein that is consisted of 383 amino acids,and the first 21 amino acids is the signal peptide sequence.Based on the important applications of mannanase,this study synthesized the mannanase gene from Aspergillus niger CBS513.88 after removal of the signal peptide sequence,and achieved an efficient expression of the gene in Pichia pastoris.The main contents are as follows:1.The synthesis of the Mannanase(Man)GeneFound the amino acid sequence of mannanase from Aspergillus niger CBS 513.88 in NCBI,then predicted the signal peptide using SignalP 4.1 Server Online.After removal of the signal peptide and adding 6×His tag in the C-terminal,the amino acid sequence was divided into four sections and added a specific sequence with each small fragment ends.Then designed corresponding primers based on Pichia codon bias by DNAWorks at Helix Systems,and synthesized these primers.On the basis of the primers,synthesized four small fragments,then stitched the four small fragments together to a whole gene to pUC19 carrier through Golden Gate cloning.2.Construction of the recombinant vectorThe mannanase gene was cloned from pUC19 carrier to pHBM905BDM carrier,constructed recombinant plasmid pHBM905BDM-MAN through digestion and connection.Then constructed two copies recombinant plasmid pHBM905BDM-MAN2C on the basis of recombinant plasmid pHBM905BDM-MAN.3.Transformed into Pichia pastoris and screening recombinantAfter linearized recombinant plasmid with restriction enzyme digestion Sal I,transformed Pichia competent cells.And after a preliminary screening by yeast colony PCR,then get fermentation supernatant for SDS-PAGE detection and werstern blotting identification screening recombinant Pichia pastoris.The single copy and two copies of the recombinant yeast were named pHBM905BDM-Man and pHBM905BDM-Man2C,respectively.4.Purification of the recombinant mannanaseThe yeast recombinants was inoculated into BMGY liquid medium,transferred to BMMY liquid medium after 28 ? 200 rpm for 48h,and added 1%methanol to induce the expression of heterologous protein,and added 1%methanol each 24h.After the induction,the yeast supernatant was collected,the first preliminary purification by ammonium sulfate precipitation and then purified by Ni-NTA affinity chromatography.5.Characterization of the mannanaseAfter determination,the mannanase's optimum temperature was 80 ? the optimum pH was 4.5.It was stable within the range of pH3.5-9.The residual activity was more than 50%after incubation in 50 ?,60 ? 70 ? for 4h.6.Substrate specificity of the mannanaseAfter enzymatic degradation at a certain temperature for konjac flour,locust bean gum,guar gum,then carried out thin layer chromatography analysis,the results indicated that the effect of hydrolysis of konjac flour was the best.