Optimization Of Alginate Lyase Fermentation And Alginate Enzymolysis For Oligosaccharides Production

As a common algal polysaccharide,alginate is widely applied in many fields.Alginate oligosaccharides,derived from alginate,have been deeply studied and extended to the aspects of drug treatment,disease control,plant growth,etc.In the present work,the shaking fermentation conditions were optimized to enhance the production of alginate lyase.Then,the fermentation process at 5 L bioreactor was studied according to the experimental results carried in shaking flasks.Moreover,the fermentation process in 5 L bioreactor was scaled up from 20 L bioreactor to 500 L bioreactor.At last,alginate enzymolysis process was optimized by alginate lyase produced by Microbulbifer sp.ALW1.Culture conditions in shaking flask were carried out by single factor experiment and response surface analysis.Under the optimal medium composition?alginate lyase 1.99 g/L,?NH₄?₂SO₄ 0.44 g/L,K₂HPO₄ 3.31 g/L,MgSO₄ 0.6 g/L,NaCl 60 g/L,FeSO₄ 0.06 g/L?,alginate lyase activity reached 180.58 U/mL,which was 7.85-fold over the activity using basal medium.Meanwhile,under the optimal culture conditions?temperature 23oC,inculation volume 6.5%,initial pH 8.0,liquid volume 50 mL?,the alginate lyase activity reached 130.05 U/mL,and enzyme activity was 17%higher than that of basal conditions.After 36 h of fermentation under both of the above optimal medium composition and optimal culture conditions,alginate lyase activity reached 183.49 U/mL,which was 7.98-fold increase over that of the initial fermentation conditions?20.44 U/mL?.Alginate lyase production parameters,including carborn concentration,temperature and pH,were respectively studied in a 5 L of bioreactor.It was showed that the best alginate concentration was 1 g/L,half or double of the concentration would make the enzyme activity decrease,and 25oC was more conductive to produce enzyme than 20oC.Additionally,when pH was controlled at 7.5,the alginate lyase producing peak time was earlier than that of no pH control.Finally,pilot scale tests were conducted based on the result of 5 L bioreactor fermentation.The maximum alginate lyase activitives harvested in 20 L,200 L and 500 L bioreactors were 57.02 U/mL,43.5 U/mL and 38.25 U/mL,respectively.Alginate enzymolysis conditions were optimized by the single factor experiment.Maximum yield of reducing sugar reached 1.82 mg/mL after 210 min of enzymolysis,which was obtained under the optimum condition of 45oC,pH 7.0,the substrate concentration of agar 7 g/L,enzyme dosage 27.5 U/mL under static state.Under the optimal conditions,an increased yield of reducing sugar was 60%over the initial condition,meanwhile the conversion rate of alginate reached 35%.The conversion rate of alginate reached 39%when the reaction systerm enlarged to1 L,and it was conformed well with the laboratory test.