Isolation And Molecular Basis Of The Chi-elastase, Bombyx Mori Chitinase With Strong Hydrolytic Activity Of Elastase

Chitin is the main component of fungi cell wall, septum and insect exoskeleton. Chitinase plays a very important role in the life-form containing chitin. Insect ecdysis and separation of fungal mother and daughter cell need chitinase for partially degradating chitin. Chitinase can be the target of potential novel bio-pesticides and antifungal agents. Chitinase inhibitor can prevent insect larve and pupa ecdysis and fungal cell separation through inhibiting chitinase activity. The relative research of chitinase inhibitor is mainly carried out in Japan. So it is significant that exploration novel chitinase inhibitor with independent intellectual property in china.Chitinase inhibitor-producing strain was screened out from the microorganism that isolated from soil samples with silkworm chitinase as target. Then the study including identification of chitinase inhibitor-producing strain, optimization of fermentation, purification of chitinase inhibitor and cloning gene of chitinase inhibitor were carried out and preliminary hypothesis of the inhibition mechanism was proposed.1. Screen and identification of chitinase inhibitor-producing strainA simple detection method of chitinase inhibitor: transparent zone assay was set up, for preliminary screen chitinase inhibitor-producing strain. Five strains were screened out from 1350 strains that isolated from 200 soil samples with the method. Chitinase inhibitor producing strain 1237~# was further confirmed by iron potassium cyanide method and 3,5- Dinitrosalicylic acid method.The inhibition spectrum of 1237~# strain chitinase inhitior on several enzymes was detected. The results show that 1237~# strain chitinase inhitior only inhibits silkworm chitinase, has little effect on Serratia marcescens chitinase, Streptomyces griseus chitinase, P. aeruginosa chitinase, phytase, cellulase and a-amylase.2. Identification of chitinase inhibitor-producing strainChitinase inhibitor- producing strain 1237~# was characterized. Results show that the strain is gram-negative aerobic rod bacteria(0.5-0.8μm×1.5-2μm), without spore, with f lagellum and mobility. The strain could grow on PDA solid culture medium at 37℃ with blue colony. It is catalase positive and oxidase positive. It could fluidify glutin. Pyocyanine test is negative. The optimum growing temperature is 37 ℃, cannot grow at 4 ℃ and can grow at 41 ℃.