Study On The Screening, Fermentation Of Chsase-Producing Strains, And Purification And Properties Of The ChSase

Chondroitinase has a variety of applications in many ways, such as studying the glycosaminoglycans’structure, properties and functions and so on. Recent years the application of ChSase in the clinical field is extensive, curing some diseases such as the KeshinBeck disease, intervertebral disc protrasion and so on. At the same time, it also has a broad use in preparing low molecular weight chondroitin sulfate. So it is of important theoretical and practical significance for getting ChSase with high enzyme activity. This study screened a ChSase-producing strain with high activity, and researched the fermentation, purification, and properties of the ChSase systematically.The strain with high ChSase activity, numbered28, was obtained from the soil specimen by methods of preliminary and second screening. It was identified to be Sphingomonas paucimobilis according to biological characteristics and biochemical reaction tests. The ChSase produced by the strain was extracellular-enzyme.In order to improve the ChSase productivity, the fermentation conditions for ChSase production was optimized using one-factor-at-a-time methodology. The optimal composition of fermentation medium was as follows (%, w/v):yeast extract1.0, chondroitin sulfate0.5, NaC10.5, KH2PO40.03, MgSO4·7H2O0.5, the initial pH8.5. And the optimal cultivation conditions were settled as:1%seed culture was transferred to a40mL sterilized fermentation media in250mL cotton-plugged flasks and incubated at25℃for36h with reciprocation (100r/min). The ChSase activity could reach1.2U/mL when the strain was incubated under the optimal fermentation conditions.The ChSase was prepared from the fermented broth by ammonium sulfate precipitation, DEAE sepharose fast flow ion-exchange chromatogram and gel filtrition with sephadex G-100chromatogram. The purified enzyme moved as a single electrophoretic band in SDS-PAGE and the moleculer weight was about82.3kDa. Through the purification, the ChSase was purified155.62-fold over culture supernatant in46.87%yield and its specific activity was about98.04U/mg.The results of properties of the ChSase were as follows. The enzyme was proved to be inducible enzyme, and there was no ChSase activity in the media without ChS. The optimal pH and temperature for enzyme activity was6.5and40℃respectively. Most inorganic metal ions had no effect on the ChSase activity. The study on substrate specificity showed that the enzyme was relatively specificity to substrates, when active degradation occurred on chondroitin sulfate, heparin and hyaluronic avid which were similar in the structures, while chitin and chitosan were not degraded by the enzyme. The products obtained from the enzyme was analysed by mass spectrometry. The products was determined as unsaturated disaccharide, and the level of unsaturated disaccharide rised to nearly100%. The molecular of the unsaturated disaccharide was about458Da.