Screening And Identifying Of A Fucoidan-degrading Marine Bacterial Strain, Optimization Of Enzyme Production Condition, And Characterization Of The Fucoidanase

In this contest, a marine bacterium strain named HDJ3 was isolated from sea cucumber cultivation instrument of Weihai Homey Group that using fucoidan from A. Molpadioides as inducing substrate. The strain could produce fucoidanase and was gram-negative. Phylogenetic analysis of its 16S ribosomal DNA sequence indicated that it was a member of the family Favobacteriaceae.Flavobacteriaceae sp. HDJ3 which produces fucoidanase was isolated for researching. We choose DNS method which is a classic method for test reducing sugar to do quantitative measurement.The optimal fucoidanase fermentation conditions of strain Flavobacteriaceae sp. HDJ3 were conformed by single-factor experiments and response surface method. The optimum culture medium has been obtained with sea cucumber fucoidan 0.28%, yeast 0.09%, MgSO4 0.1%, NaCl 2.09%, CaCl₂ 0.02%, FeSO4 0.002%, K₂HPO₄ 0.01%. After cultivated in 200mL medium of 500mL shake flask, initial pH is 7.5, 25℃, 150rpm for 36h, the activity reached the highest value of 21.67U/mL which is 3.43 times higher than that under the original conditions.HDJ3 can produce fucoidanase which is an intracellular enzyme. This fucoidanase is unstable and more difficult to be separated. Here we just groped the separation conditions. Fucoidanase producing is incubated at 25℃, pH 7.5 for 36 hours. The fucoidanase recovered from intracellular supertanant with a procedure of ultrasonic disruption, ultrafiltration, Q-sepharose Fast Flow ion exchange chromatography. Results of enzymatic property study showed that the the optimum pH and temperature for enzyme activity are 7.5 and 30℃, respectively. The fucoidanase is stable in low temperature condition. Its activity remains 80.56% after 24 hours under 4℃. But is unstable at high temperature, the activity remains only 47.44% after 24 hours under 20℃. The enzyme has a high activity when the Na+ concentration reaches 3%. Ba²⁺,Ca²⁺ could enhance the enzyme activity, whereas K⁺,Cd²⁺,Ni²⁺,Zn²⁺,Mn²⁺,Hg²⁺and Cu²⁺ inhibit the activity. Fcoidanase can easily degrade fucoidan to get high active fucoidan-oligosaccharides. This research will be a base of utilization and structure analysis of fucoidan.