The Screening Of Chondroitinase Producing Strain, Optimization Of Fermentation Conditions And Analysis Of Hydrolysatese

Chondroitinase (ChSase)is a lyase,which exists in microorganisms,and degrades a variety of acidic mucopolysaccharides such as sulfuricacid,dermatan sulfate,chondroitin sulfate and hyaluronic acid tooligosaccharide and unsaturated disaccharides. The enzyme has a broadapplication prospects in many aspects such as researching the function,structure and distribution of the vivo chondroitin sulfateglycosaminoglycan and the treatment of atherosclerosis, arthritis, etc.Meanwhile, the enzyme is also used for the preparation oflow-molecular-weight chondroitin sulfate. Low-molecular-weightchondroitin sulfate whose molecular is2-10kDa has a better efficiencythan large molecular weight chondroitin sulfate in prevention andtreatment of rheumatic inflammation and atherosclerosis.Relative to thecurrently widely used methords such as acid hydrolysis and ionexchange,the enzymatic production of low molecular weight chondroitinsulfate does not require advanced laboratory equipments, and will notpollute the environment.Therefore, the preparation of getting a lot ofhigh enzyme activity ChSase has important theoretical and practicalsignificance,and it’s also the purpose of this experiment. Thus thisthesis systematically studies the screening of the ChSase producingstrains, enzyme production characteristics of the strain, theidentification of the strain, separation and purification of ChSase,and enzymatic degradation products.In this experiment, we used tablet rapid screening method andrescreening method to filter out a high-yield ChSase strain No.12from the soil. Through its morphology and16SrRNA gene sequencecomparison,this strain is identified as Arthrobacter sp.And its enzymeproduction is secretory expression.In order to improve the level of enzyme production of NO.12strain,the fermentation conditions for chondroitinase production was optimizedusing one-factor-at-a-time methodology and orthogonal experiment method.The optimal composition of fermentation medium is as follows (w/v):chondroitin sulfate0.8%,yeast extract0.6%, KH2PO40.02%, MgSO47H2O0.8%,pH6. And the optimal cultivation conditions were settled as follows:1.5%seed culture was transferred to a50mL sterilized fermentation mediain250mL cotton-plugged flasks and incubated at25℃for72h withreciprocation (150r/min). When fermenting Arthrobacter sulfonivoransunder the conditions described above, the enzyme activity reached3.981U/mL,which is higher than most of the strains reported.Finally, this paper preliminarily analysis the enzymatic degradationproducts degradated by chondroitinase produced by No.12strain. Theresult of mass analysis shows that the enzyme degradation product ofchondroitin sulfate are almost all monosaccharide and disaccharides.And the product has a high purity. The molecular of the unsaturateddisaccharide in the degradation products is about458Da, and the molecularof the monosaccharide is about228Da.