Direct Evolution Of Endochitinase

Chitin is a kind of high molecular polymer made of N-acetyl glucosamine(GlcNAc) and linked byβ-1,4-glycosidic linkages. It's secondary to cellulose which is also an renewable resource. Chito-oligosaccharides, an intermediate degradtion product of chitin, is a 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. Compared with foreign countries, our study on chito-oligosaccharides started later. Endochitinase could hydrolyze chitin, but it's activity can't meet the need for industrialization. Thus, if there were some methods to improve this, it will be very significant.The research group has completed the cloning and recombinant of endochitinase gene using E. coli expression system. My subject set this endochitinase gene as starting material for Directed Evolution, by high-throughput screening method we get a strain of high enzyme activity mutant. For it, we do some research on optimizing the fermentation medium, recombinant protein purification and its enzymatic properties. The main results state as follows:Recombinat plasmid pET-28a(+)-MECHs is set as error-prone PCR template for building high-throughput screening mutants. Through a round of directed evolution, we get a strain of high enzyme activity mutant whose activity is 1.81 times as high as the original. Gene sequencing indicates that 9 bases changed:G69A, T276C, C300T, A554T, T676C, A1002G, T1168A, A1169G, G1170C. Result from this, its amino acid sequence has some changes as follow:Y185F, S226P.FFD and BBD are used for optimizing fermentation medium. The former finds out three main fators:Glucose, Na2HPO4·12H2O and KH2PO4. The later finds out an optimal fermentation medium compsition(g/L):Tryptone 14, Glucose 8.8, Na2HPO4·12H2O 16.8, NH4Cl 1, MgSO4·7H2O 0.14, Yeast extract 6, KH2PO4 2. Comparing the specific activity before and after optimization, the later is 1.67 times the number of the former.Fermentation liquor is purified into endochitinase by ammonium sulfate precipitation, DEAE-52 anion-exchange chromatography and Sephadex G-100 chromatography. The results show that, the specific activity of crude extract increases from 0.537×10-3 (U/mg) to 5.194×10-3 (U/mg). Purification fold is 9.67, and the Activity recovery reachs 8.1%. SDS-PAGE shows that protein molecular weight is 46.77 kD.Research on enzymatic properties indicate that the optimal temperature(Topt) of endochitinase is 60℃and the optimal pH is 7.4. The value of Km is 0.2514 mmol/L and Vmax is 4.5872μmol/L·min-1。Metal ions play the key role in maintaining catalytic activity. Comparing 9 different metal ions, we find that Mn2+, Mg2+ and Ba2+ has obvious activation on endochitinase, while Zn2+, Cu2+ and Co2+ has obvious inbibitional effect on it.