Study On Enzymatic Synthesis Of 2-O-?-D-Glucopyranosyl-L-Ascorbic Acid By Marine Microorganism Enzyme

L-ascorbic acid?or Vitamine C?is an essential nutrient to human bodies.It plays an important role in many physiological activities.However,its application is greatly restricted due to instability.2-O-?-D-glucopyranosyl-L-ascorbic acid?AA-2G?is a derivative of vitamin C with good stability,which's biological activity is similar with L-ascorbic acid.AA-2G is synthesized by enzymatic method mainly.And cyclodextrin glucanotransferase?CGTase?is considered the optimum enzyme.In this reserach AA-2G was transformed by marine microorganism CGTase using L-ascorbic acid and?-cyclodextrin as substrate.Firstly,The synthetic condition of AA-2G was established by single factor design,and the synthetic condition of AA-2G was further investigated by response surface method.CGTase was immobilized by adsorption-crosslinking-embedding method,then the enzymatic properties of immobilized CGTase enzymatic properties was studied.At last,we applied immobilized enzyme to the transformation of AA-2G and compared with the free CGTase,the advantages of immobilized CGTase has been discussed.The results of the study are as follows:Optimize conditions of transformation of AA-2G by free CGTase:The synthetic condition of AA-2G was established by single factor design,and three major factors were screened by Plackett-Burman design:pH,enzyme concentration and substrate concentration.Then the transformation conditions were optimized by Box-Behnken design and the optimum conditions were as follows:pH 9,enzyme concentration 90 U/g?-CD,substrate concentration 60 g/L,the ratio of substrate 1:1,reacting for 24h,temperature 45?.Under this condition the yield of AA-2G was 9.86 g/L.The qualitative and quantitative analysis of AA-2G was by LC-MS and HPLC.Optimize the conditions of immoblized CGTase:CGTase was immoblized by adsorption-crosslinking-embedding method.The immoblized conditions?mesoporous molecular sieve SBA-15 concentration,glutaraldehyde concentration,enzyme concen-tration,adsorption time,crosslinking time,CaCl₂ concentration,hardening time?were optimized initially by single factor experiment.Then optimize the conversion conditions by orthogonal experiment.The optimal immoblized conditions were as follows:SBA-152 g/L,glutaraldehyde concentration 0.01%,adsorption time 6 h,crosslinking time 5h,enzyme concentration 2 U/mL,CaCl₂ concentration 4%,hardening time 1 h.Comparative study of property of immoblized CGTase and free CGTase:Compared with the free enzyme and immobilized enzyme properties respectively.The results showed that immobilized enzyme's tolerance of metal ions,organic solvents,strong acid and strong alkalinehas been improved,the thermal stability was slightly increased.After repeated use 4 times,the residual enzyme activity ofimmobilizedenzymewasmorethan50%.Influenceofsubstrate concentration,temperature and pH on the yeild of AA-2G was studied and we compared that to free enzyme.The optimum of immobilized enzyme pH was 9,the optimum substrate concentration was 40g/L,and these were similar to free enzyme.The optimum temperature was 25?,lower than free enzyme.The yeild of AA-2G by immobilized enzyme was 2.54 g/L more than free enzyme.