The High-expression Of β-Mannanase In Pichia Pastoris

β-Mannanase(β-1,4-D-mannan mannohydrolase, EC 3.2.1.78),which catalyzes the random cleavage of β-1,4-mannopyranosyl linkages within the main chain of glucomannan, galactomannan, galactoglucomannan and mannan, is a kind of hemicellulose. It can be produced mainly by various microorganisms,β-mannanase can be useful in several provesses in the food, feed, textile, as well as in the pulp and paper industries. In order to reduce the production cost, the feed-batch fermentation of Pichia pastoris engineering strain Pichia pastoris GS115/Ppic9K-(3-MAN was studied, and then, we used the Pichia pastoris recombinant VHb, Bip and PDI gene, and achieve co-expression.Firstly, the fermentation conditions of genetically engineered Pichia pastoris for β-Mannanase were studied in this paper. Basic conditions of the carbon source, immunization rates, temperature, pH were optimized in shake-flask. The tests in 30L fermenter was applied to study the effects of DCW, methanol concentration and DO on enzyme production. Through the orthogonal design to optimize the fermentation conditions. The results indicated that the optimum conditions were as follows:10% inoculum size,30 g/L initial glucose concentration,10%～20%DO, pH5.0 at 28℃. The dry cell weight was 135 g/L, the target protein concentration could reach to 5.04 g/L. And the β-Mannanase activity got to be 29600 U/mL, which was 24 times of that the results in the shake flask under these conditions.Secondly, in order to improve the β-Mannanase stability, the effect of different substances on β-Mannanase stability was studied through single factor experiments, and the orthogonal experiment was used to optimize the complex protective agents, and then, stability of enzyme with or without complex protective agents was measured. Results indicate that the glycine, sorbitol, glycerol, mannitol and calcium chloride can enhance the enzyme stability. The complex protective agents consists of 120 g/L glycine,25 g/L sorbitol,600 g/L glycerol and 120 g/L mannitol. The t1/2 value of enzyme with protective agents is 7.5℃ higher than that of enzyme with no protective agent. The results above suggest that the complex protective agents can effectively enhance the thermost ability of P-Mannanase.Thirdly, the aim of this study is to alleviate the dissolved oxygen(DO) limitation and improve β-mannanase production during high-cell density fermentation, the β-mannanase gene and Vitreoscilla hemoglobin gene vgb were co-expressed in Pichia pastoris under the control of AOX1 promoter. VHb gene was synthesized by optimization of codon and inserted into vector pPICZαaA, The constructed recombinant plasmid pPICZαA-VHb was transformed to recombinant P.pastoris GS115/Ppic9K-β-MAN, and the positive transformants for co-expressing VHb was screened by G418 and Zeocin. By high-density fermentation in 30 L bioreactor, the expression of β-mannanase in strain VHb+ co-expressed with VHb was compared with that of VHb" co-expressed without anything. Compared with the control strain, expression of β-mannanase in VHb+ and VHb- under oxygen limitation was improbed by 90%. Meanwhile, expression of VHb improved tolerance for alcohol. VHb+ enables shortening the fermentation period of 40 hours, which could be taken advantage of for its industrial application.Finally, primers were designed according to the PDI and Bip sequence of P.pastoris reported in GenBank, with which the target gene fragment was amplified from the genome of P.pastoris by PCR and inserted into expression vector pPICZαA. The constructed recombinant plasmid pPICZαA-PDI and pPICZαA-Bip was transformed to recombinant P.pastoris GS115/Ppic9K-β-MAN, and the positive transformants for co-expressing PDI or Bip were screened. By high-density fermentation in 30 L bioreactor, the expression of β-mannanase in strain PM115 co-expressed with PDI and BM115 co-expressed with Bip were compared with that of BM115 co-expressed without any chaperone. The results showed that the highest β-mannanase activity, the protein concentration and specific production in BM115 reached 40900 U/mL,13.22 g/L and 3150 U/mg, which yield 38%,22%,18% than those in GM115 and PM115. The high-level expression of β-mannanase could be achieved by co-expression of chaperones Bip, which meted the conditions of β-mannanase in industry.