Improvement Of Chitinase Activity And Its Immobilization

Chitinase, which can hydrolyze the β-1,4 glucosidic bond of chitin or its derivative and generate N-acetylglucosamine, is one of glycosyl hydrolase. After the chitinase inordinately hydrolyze chitin, there will product some chitooligosaccharides which have special biological activity, such as the activity of antibacterial, antifungal, anticancer and regulation of body’s immune ability. And chitinase can destroy the chitin in the intestinal of plant-parasitic nematode and the cell wall of pathogenic fungus. Therefore, chitinase has potential in the field of medicine, environmental protection and agroforestry.In this study, the random mutagenesis library of gene pachi from our previous work was constructed with error-prone PCR. We used 1% chitosan as substrate, DNS as indicator and screened the mutants with the bacteriophage T7 lysis-based method for 96 deep-well plates high-throughput screening system. We obtained one mutant PachiN35 D with great activity from 12 000 mutants, and amino acid sequence analysis between wild-type and the mutant revealed a glutamic acid substitution of the glutamine at position 35. After the characterization of Pachi and Pachi N35 D, the optimal temperature of Pachi N35 D is 50?C, which has decreased 10?C compared to 60?C of wild-type. The wild-type Pachi retained 76% activity after the incubation at 60?C for 1 h, while PachiN35 D has lost all activity after the incubation at 55?C for 1 h. The Km of Pachi and PachiN35 D is 36.1 mg/m L and 13.3 mg/m L respectively; the kcat/Km of Pachi and PachiN35 D is 0.37 mL/mg/min and 1.14 m L/mg/min respectively; the specific activity of Pachi and PachiN35 D is 22.3 U/mg and 49.9 U/mg respectively. The enzyme kinetic parameters showed that the mutant chitinase exhibited a 63% increase in substrate affinity, a 2.1-fold enhancement in catalytic efficiency and a 1.2-fold increase in specific activity. Interestingly, the mutant PachiN35 D showed a LC50 value of 309.6±1.1 μg/ml, more than 20% increase over WT in mortality rate.We used nano-silica as vector, glutaraldehyde as cross-linking agent to immobilize chitinase Pachi N35 D with the method of covalent cross-linking. According to the analysis of FTIR and SEM, there are some special peak of chitinase in the immobilized chitinase. The immobilization rate id 98%, retained specific activity rate is 70%. The optimal temperature of Im Pachi N35 D has increased to 55?C, and it retained 56% activity after the incubation at 55?C for 1 h. The Km,kcat,kcat/Km value of PachiN35 D and Im PachiN35 D showed very less difference, the specific activity of Im PachiN35 D was 70% of PachiN35 D. The enzyme kinetic parameters showed that the immobilized chitinase has almost the same substrate affinity, catalytic efficiency and specific activity. These results provide useful information for the study of structure-function relationship of Pachi and lay a foundation for its potential applications in agro-biotechnology.