| Chitin is aβ-1,4-linked,insoluble linear polymer of N-acetylglucosamine (GlcNAc)and is the second most abundant organic compound on our planet following cellulose, biosynthesize 10 billion per year. And it's distributed in animal, microbe. As the exclusive alk polysaccharide of the nature, there are some special characteristics of chitin. It is became the focal point of the exploratory development for its particular function.Chitinase(EC3.2.1.14) is a glycosyl hydrolase that catalyzes the hydrolytic degradation of chitin. It has good prospects in territories as follow, for higher plants is a part of defense mechanism against fungal pathogens, cure disease of human inducing by fungal, deep processing of chitin, and so on. So it's imperative to produce chitinase in large-scale. In this study a recombinant strain of Pichia pastoris with a phenotype of Mut+ was used to produce alk rice chitinase, which was secreted into the culture. The aim of the present work was to enhance the expression level of chitinase expressed by the recombinant Pichia pastoris, and analyze the biochemistry character.In shake-flask, the conditions of the induction phase were investigated. The optimal medium was BMMY. The concentration of (NH4)2SO4 was 0.5%, oleic acid was 0.05%,YNB was 0.67%,yeast extract was 1.5%,1.0% methanol mixed-feed with 0.1% glycerol. The optimal pH condition was 6.0. Add methanol to final concentration of 1.0% every 12 hours to maintain induction without centrifuging after the growth phase. The highest productivity of chitinase was obtained when inducing for 108 hours. And it was propitious to enhance production by enhanced the stability of methanol concentration. But the oxygen vector, surface active agent, lower the induction temperature were not propitious to production. The above data could be used to direct the fed-batch fermentation.By 7 L fermentor, different methanol feeding modes were evaluated. For the strong ability to utilize methanol, the methanol feed rate could be directed by the dissolve oxygen vibration. Continue feed methanol through peristaltic pump, the highest production level of recombinant chitinase obtained earlier nearly 24 hours than the shake-flask, and high to 462.41 mg/L, was 2.7 folds than shake-flask. And the chitinase activity was 77.61 U/mL.In order to further enhance the recombinant chitinase expression level, the glycerol-methanol mixed-feed mode and the feeding mode of two carbon sources alternatively were evaluated. The feed of glycerol during glycerol-methanol mixed feed phase, improved the growth environment for cell, and the biomass became more. The improved activity of cell's physiology, prolong the induction phase, to spur the methanol induced more recombinant chitinase, the expression level was high to 495.33 mg/L and the chitinase activity was 88.31 U/mL. When feeding of two carbon sources alternatively, the blance of AOX1 was broken. And the prolong of saturation time of AOX1 activity, staved the decline rate of heterologous protein production rate, and at the 120 hours the highest level was 516.45 mg/L,99.3 U/mL.The fast purification of fermentation broth was conducted with (NH4)2SO4 and Sephadex G-100, and got pure chitinase. The pure chitinase was thermostable, keep higher activity in acid environment, and the profile showed the double summit in wide pH limit. In alk environment the activity became lower, proved the recombinant chitnase was alk. The pathogen(Rhizopus stolonifer, Botrytis cinerea) could be inhibited by the enzyme in a certain extent.
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