Fermentation,Immobilization And Preparation Of Carrageenase From Pseudoalteromonas Carrageenovora ASY5

Carrageenase,which exists widely in nature and has many important applications,belongs to glycoside hydrolase family.Carrageenase can specifically hydrolyze carrageenan into carrageenan oligosaccharides.In order to improve enzyme production,simplify the separation process of enzyme and extend the application of carrageenase,attention has been paid to the fermentation of carrageenase,enzyme immobilization and enzyme preparation.In this study,a?-carrageenase-producing bacterium from Pseudoalteromonas carrageenovora ASY5 was isolate from mangrove soli leaf by our laboratory.At first,fermentation mediums and culture conditions in shaking flask were carried out by single factor experiment to enhance enzyme production.After optimization,the optimal medium composition and culture conditions were obtained with carrageenan 5 g/L,tryptone 5 g/L,NaCl 20 g/L,CaCl₂0.2 g/L,NaH₂PO₄·2H₂O 1.3 g/L,Na₂HPO₄·12H₂O 3.82 g/L,tempreture 18oC,initial pH 6.5,inculation volume 2%,liquid volume 70 mL.Meanwhile,the three mediums were reduced comparing with the basal mediums.The biomass reached 1.4,which was 80.4%over initial biomass?0.776?.Carrageenase activity reached 40.8 U/mL,and enzyme activity was 52.2%higher than initial carrageenase activity?26.8 U/mL?.Then,the experiment of culture conditions in 5 L bioreactor was carried out.The results showed that high rotation speed can promote the rate of producing enzyme and short the ferment time,280 rpm was the optimal rotation speed,and that additional medium and control pH had not an obvious effect on improving enzyme production.Finally,the pilot enlarge experiment of 20 L,75 L and 200 L were conducted based on the ferment results of shaking flask and 5 L bioreactor.The maximum carrageenase activitives of 20 L,75 L and 200 L bioreactor reached 33.9 U/mL,34.7 U/mL and 36.1 U/mL,respectively.And the maximum enzyme activity of 200 L bioreactor was 88.5%of the maximum enzyme activity of shaking flask,and 85.7%of the enzyme activity of 5 L bioreactor.Shaddock peel was a novel supporter which possessed excellent performance.In this present work,carrageenase was immobilized with supporter which was made up of polymine and shaddock peel.Immobilized conditions and the properties of immobilized carrageenase were investigated by single factor experiment.And the optimal immobilized conditions were obtained with 0.1 mg/mL polymine for 1 h,pH 7.0,enzyme dosage 10 U,immobilized time 8 h,immobilized tempreture 4oC.Under the optimal immobilized conditions,the enzyme recovery of immobilized carrageenase reached 50.0%,which was1 time higher than that of initial condition?24.7%?and increased 4.1 times than that of only shaddock peel used?9.8%?.The optimal temperature and pH of immobilized enzyme were 60oC and 7.5,respectively,and had no change comparing with that of free enzyme.The temperature stability of immobilized enzyme was decreased,while the immobilized carrageenase showed a better pH stability than the free enzyme.Under the optimal conditions,the enzyme recovery of immobilized carrageenase remained 53.2%after 3 cycles.The Michaelis constant of immobilized enzyme was smaller than that of free enzyme.And the result demonstrated that the affinity of immobilized carrageenase with substrate was increased.After storing 14 weeks at 4oC,the residul activities of immobilized enzyme and free enzyme were 76.0%and 82.5%,respectively.And FTIR indicated that carrageenase was successful fixed onto the shaddock peel carrier.The solid enzyme preparation of carrageenase was prepared by spray drying.The process parameters were investigated by single factor experiment and then response surface ananlysis was conducted based on the result of single factor test.Under the optimal process parameters?maltodextrin 27%,inlet air temperature 131oC,hot air flow rate 4.5 m³/min,feeding speed 456mL/h,atomizer pressure 0.2 MPa?,the enzyme recovery of carrageenase reached 56.9%,which was increased by 12.7%comparing to the result of single factor test.The results of storage stability showed that the residul activities of carrageenase solid enzyme preparation were both about 93.0%after storing 13 weeks at 4oC and 28oC,and that the retained rates of the enzyme preparation activity,which were preserved in drying condition for 13 weeks and ultraviolet irradiation for 120 min,were 94.6%and 55.4%,respectively.But the carrageenase solid enzyme preparation can not be exposed to humid environment in the preservation process.