Screening Of High-producing Chitinase Strains And Purification And Authentication Of CK₁-3

Chitin is one of the most abundant organics in the world. Every year, the syntheticamount of chitin can reach more than ten billion, the stockpile of chitin comes second after cellulose, it's an inexhaustible renewable natural resources in the world.Chitinase specifically can catalyse the reaction of hydrolyzing chitin into N-acetylglucosamine(GlcNac), Water-soluble chitin and(GlcNac)n . Study on screening chitinase producing bacateria from microbe have evident science value, and also has great practical significance in cleaner production and wide use of natural chitin, such as biological control, environmental protection, food and medical exploitation.This experiment have extensive screened with the high-producing chitinase srtains, and have selected out eighty-nine chitinase-producing strains which have been used as candidate stains. We also Isolated and depurated the chitinase of a high-producing chitinase strain which named CK₁-3.CK₁-3 is a saccharomycetes by primary identification.We have optimize the fermentation conditions's of CK₁-3, and have been surethat the optimal compositions is: colloidal chitin 8.0, peptone 5, yeast extract 5, K₂HPO₄ 0.7, KH₂PO₄ 0.3, MgSO₄ï¹'7H₂O 0.5, FeSO₄·7H₂O 0.01, ZnSO₄ 0.01, PH 6.8; The optimal cultivation conditions for CK₁-3 is: Original pH is 6.8, inoculum concentration is 1.4%(w/v), 250mL conical flask contain 50mL fermentation liquor, 30 oC incubating temperature and shaking cultivation at 180r/min for 120h. The maximal chitinase activity of fermented liquid can reach 2.51U/mL.We have established the Isolated and depurated condition of strain CK₁-3's fermentation liquor. Refrigerated centrifuge the fermentation liquor, sedimente the supernate by 85% ice ethanol, centrifuge, dissolve in a small amount of water, then we have got the rough chitinase. After purified by Sephadex G-100, we have got the macromolecular chitinase immediately. The purification fold is 6.61, and yield coefficient is 37.2%. SDS-PAGE cataphoresis display as electrophoretically pure, and it's molecular weight is 46000Da.We have studied the character of the chitinase which secreted by strain CK₁-3. The result is as follows: the optimal temperature for hydrolysis of chitinase is 50 oC, the optimal pH is 6.0 respectively. The chitinase activity is stable in the pH range of 6.0-7.5. The enzyme activity is slightly activated by Mg²⁺,Fe²⁺, whereas Zn²⁺,Fe³⁺,Cu²⁺,Mn²⁺ can inhibit the enzyme activity.