Extraction And Purification Of Chitin Deacetylase From Rhodococcus Sp.

Chitin deacetylase (CDA, E.C.3.2.1.41) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-D-glucosamine in chitin, convering it to chitosan. Chitin deacetylase producting chitosan from chitin don't pollute the environment, and product the specific chitosan, and so on. The research of CDA aroused much interesting. We researched the CDA from Rhodococcus sp, optimized the fermentation medium and purified the CDA in the paper.The strain Rhodococcus sp. was selected from the soil, which was preserved in our laboratory. In order to enhance the level of CDA activity, the culture medium and the fermentation conditions of CDA production by Rhodococcus sp. were optimized. The optimal culture medium for enzyme production was as follows (g/L): corn pulp 12.5, sucrose 12.5, sodium acetate 2.0, KH2PO4 1.0, ammonium chloride 1.0, peptone 0.5. The optimal fermentation conditions for CDA production was as follows: seed age 20h, initial pH 7.5, temperature 30℃, rotation speed of rocking bed 180r/min, volume in shake flask 30mL/250mL, the quantity of inoculation 4mL/30mL. The enzyme activity was 5119.10U/mL with the optimal culture medium and optimal fermentation conditions, with enzyme activity enhanced by 1.38 times.We found that the CDA activity in fermentation lost severely by the experimental results. The CDA activity lost about half after 24h. So it was important to find the best storage condition. By investigating enzyme storage methods and its influence factors, we determined the optimal storage conditions: collected the cell after centrifuge the fermentation liquid, made the dry cell and stored in -20℃. After two mounths, enzyme activity loss was only 0.98%. The problem of enzyme inactivation was solved basically, which provided the foundation for the further analysis of the chitin deacetylase.Because the CDA from strain Y104 was the intracellular enzyme, the enzyme should release the cell outside firstly by cell disruption. Then, compared several commonly methods of cell disruption, the optimal method to extract CDA was as follows: fermenting liquor was washed three times, and the thallus was suspended with pH8.0 Tris-Hcl buffer. The solution was repeated freezed and then treated 6 min by glass homogenizer. Finally the supernatant was obtained by centrifuge. The recovery of lactoperoxidase activity was 40%.Separation purification of the chitin deacetylase after cell disruption was by ammonium sulfate precipitation, bag filter desalting, Sephadex G-25 gel filtration chromatography, Sephadex G-75 gel filtration chromatography, the activity recovery and purification factor of the fibrinolytic enzyme were 33.94% and 4.44; the pure enzyme was single strap verification by SDS-PAGE identification and the relative molecular weight was 58.4 kDa.