| Chitin is a kind of high molecular polymer made of N-acetyl glucosamine（GlcNAc） and linked by (3-1,4-glycosidic linkages. It’s secondary to cellulose which is also a renewable resource. Chito-oligosaccharides, an intermediate degradtion product of chitin, is a kind of functional oligosaccharides with quality of low molecular weight, good solubility and easy to absorb for which it’s quite useful in biomedical, food, cosmetics, textiles, agriculture and other fields. However, it will cause severe environmental pollution by using the traditional technology to extract and prepare endochitinase. As a result, it is an economic and theoretical study to find a pollution-free solution to make the production of endochitinase industrialized, such as synthesizing new endochitinase gene and constructing efficient expressed engineering bacteria.In this paper, we synthesized endochitinase gene SECH which based on pchia pastoris bias codons by successive PCR. The sequences were cloned into plasmid pPIC9K to generate the recombinant expression vectors pPIC9K-SECH. They were linearized with Bpu1102I and transformed into Pichia pastoris GS115 by electroporation method to acquire transformant ZJGSU04 which had the highest endochitinase activity. The protein was analyzed by DNS, SDS-PAGE and zymogram. Finally, we used Fractional Factorial Design and Respond Surface to optimize the production of endochitinase. They are as follows:1. According to the P.pastoris bias codons and the sequences of endochitinase gene, we used 14pairs primer to synthesize 1185bp of DNA by successive PCR. Compared the synthetic sequences with the endochitinase gene which was cloned from Trichoderma viride, we found that the sequences of amino acid of both were the same, although the sequences of the gene were different, which means we synthesized the endochitinase gene based on P.pastoris bias condons successfully.2. Used restriction enzyme EcoRⅠand NotⅠto double digest the synthetic sequences SECH and plasmid pPIC9K, linked the restriction product of SECH and pPIC9K with T4DNA to acquire eukaryotic vectors pPIC9K-SECH. Transformed the vector into E.coli DH5a by CaCl2 method, and screened the single colony of Kana resistance. Analyzed the transformant by PCR, digestion and DNA sequencing, it turned out that the cloning was exactitude.3. Linearized by the restriction enzyme Bpu1102I, this constructed expression vector was transformed into the genome of Pichia pastoris GS115 by electroporation method. The transformant ZJGSU04 with the highest endochitinase activity was screened, the endochitinase activity was 23.09 U/mL which was tested by DNS. SDS-PAGE and zymogram indicated that the molecular weight of the recombinant endochitinase was about 43 kDa.4. The optimal conditions for production of endochitinase by engineered ZJGSU04 ware investigated in this study. With the endochitinase activity as the target, the concentration of several important factors, metahanol, oleic acid, Tween-80, PTM1, and pH were optimized, respectively. The optimal concentration of methanol was 0.5%, the oleic acid was 0.05%, Tween-80 was 0.3%, PTM1 was 0.8%, the optimal pH was 6.0, respectively. In this condition the activity of recombinant endochitinase could be improved greatly. Furthermore, culture conditions were optimized by the Fractional Factorial Design and Box-Behnken design. The most optimal culture medium components obtained were as follows: 1.00% yeast extract,2.00% tryptone,1.34%YNB, 100mM potassium phosphate, 0.71% methanol,0.086% oleic acid,0.31% Tween-80,0.80% PTM1 and 4.00×10-5% biotin. The activity of endochitinase under the above optimal conditions was about 30.92 U/mL, which was similar with the theoretical value and higher 44.35% than that of non-optimal endochitinase. |
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