Codon Optimizaion, Expression And Characterization Of A ?-1,4-mannanase From Caldibacillus Cellulovorans

?-Mannanases come from various organisms, such as plants, animals and microorganism. Microorganisms are the ideal source of ?-mannanases due to their high enzyme productivity and easy cultivation. Most ?-mannanases studied are derived from bacteria. However, research of heat-resistant mannase still catches the attention of scientists. Until now, the applieation is limited because of the short supply and high price. And that the use of enzymatic properties can't be industrialized is an important reason too. Undoubtedly, developing the heat and high efficiency with utilization efficient expression system of the mannanase exogenous expression can product more ?-Mannanases with low cost and large scale for more people to use, which is one of the effective methods to solve the problem.The research that applies gene engineering, protein engineering technology and fermentation engineering obtains the mannanase producing engineering bacteria with independent intellectual property rights and provides a theoretical basis and conditions for the industrial application of mannanase in food and feed areas. The research ineludes the following items: in the basis of not changing the thermostable mannanase ManAd3 amino acid sequence, mannanase gene ManAHr gene was designed and synthesized(Accession No. KJ806637), according to the known amino acid sequence of ManAd3 thermostable mannanase and referring to Pasteur Pichia pastoris codon usage preference. During the progress, the codon preferenced best is used first and then the next codon to eliminate the two level structure of mRNA is stable. The connection of synthetic mannanase gene ManAHr gene engineering and pPIC9 k constructed the recombinant yeast expression vector pPIC9k-ManAHr and transformed into Pichia pastoris GS115 by recombinant plasmid. The recombinant strain fermentation product was identified by SDS-PAGE and the sweet dew of xylanase ManAHr content reached electrophoresis purity level with about 30 kDa molecular weight. The optimization of fermentation conditions of fermentation can find the optimum fermentation conditions for recombinant protein expression to further improve the mannanase production.The experimental results show that based on the result of detection result of recombinant mannanaseen zymatic properties enzyme, the optimum reaction temperature of ManAHr is 75°C, the optimum pH is 6.0, specific activity is up to 3200 IU/mg, and under the temperature of 75°C, 30 min can be maintained above 90% relative enzyme activity or more. The mannanase, with high expression of ManAHr and relative enzyme activity in acidic environment, which can still maintain high thermal stability and significantly, can be widely used in the industrial field of brewing, food, feed, textile and medicine etc..