Two-Step Synthesis Of Guanine Arabinoside By Immobolized Enzyme Method

9-beta-D-arabinofuranosyl guanine(ara-G),an active metabolite of nelarabine,is a kind of drug which has anti-leukemia effects.Ara-G can be phosphorylated to ara-G triphosphate(ara-GTP)in the human body.Ara-GTP competes with deoxynucleoside triphosphate to insert into DNA.And then it causes to selectively suppress the synthesis of DNA.The chemical synthesis of ara-G is extremely complex,bringing about different kinds of isomer and hard to separate from isomer.Besides if using free cells or enzymes for biosynthesis,such one-off using will cost too much.In this paper,immobilized enzyme technology was used in the biosynthetic research of ara-G.A continuous catalytic reaction process will be established,which tremendously reduced the production cost.This research provide the reference for the industrialized production of ara-G.In the preparation of immobilized enzyme,enzyme solution was obtained by using ultrasonic,ammonium sulfate fractionation.Immobilized enzyme which can be used for catalytic conversion reaction was obtained by fixing on resin carrier with glutaraldehyde crosslinking method.The immobilized conditions of nucleoside phosphorylase were optimized as following: the materials were set in 25°C,30 rpm and mixed with 0.2% glutaraldehyde for 4h,63.2 u enzyme fixed 1g resin carrier,and the enzyme linked with resin lasting for 12 h.Then resin were washed by distilled water to the neutral pH and preserved in the 25 mmol/L pH 7 PBS buffer.The immobilized conditions of adenosine deaminase were optimized as following: the materials were set in 25°C,30 rpm and mixed with 0.1% glutaraldehyde for 8h,51.4 u enzyme fixed 1g resin carrier,and the enzyme linked with resin lasting for 10 h.Then resin were washed by distilled water to the neutral p H and preserved in the 25 mmol/L pH 7 PBS buffer.At first,biosynthesis system of ara-DA was built: the substrates ara-U and DAP were dissolved in phosphate buffer,using immobilized purine nucleoside phosphorylase(PNPase)as catalyst,intermediate product ara-DA was synthetized in the conical flask.The optimum conditions for the enzymatic reaction were determined: 50 mmol/L ara-U and 50 mmol/L DAP were dissolved in 50 mmol/L pH 7.5 phosphate buffer,added 2g immobilized PNPase,the optimum reaction temperature is 60?,the optimum reaction time was 48 h.Batch operations was designed under the above conditions.The first 5 times it presented a sign of high quality,conversion rate run up to a maximum of 84.93%(averaging 63.9%).At the same time immobilized PNPase enzyme column of continuous reaction was designed as follows:the immobilized PNPase was loaded on the chromatography column which length-diameter ratio was 5.5.The substrates were feeding into the column through constant current pump at the speed of 0.47 ml/min.According to the single-fact experiment,the best conditions were as follows: p H 7.2 45 mmol/L phosphate buffer solution,reaction temperature 58?.Under the optimal conditions,the immobilized PNPase kept high catalytic effect at the first 528 h.The conversion rate of ara-U run up to a maximum of 83.74%(averaging 61.84%).Using the same amount of free cells,the catalytic effect of enzyme column is 45.5 times as much as free cells and16.3 times as much as the batch operations.Then biosynthesis system of ara-G was designed: ara-DA was dissolved in phosphate buffer,using immobilized adenosine deaminase(AMPDAase)as catalyst,the final product ara-G was synthetized in the conical flask.The optimum conditions for the enzymatic reaction were determined: 30 mmol/L ara-DA was dissolved in 150 mmol/L pH 7.5 phosphate buffer,added 1.5g immobilized AMPDAase,the optimum reaction temperature is 58?,the optimum reaction time was 108 h.Batch operations was designed under the above conditions.The first 8 times it presented a sign of high quality,conversion rate run up to amaximum of 82.2%(averaging 64.47%).At the same time immobilized AMPDAase column of continuous reaction was designed as follows:the immobilized enzyme was loaded on the chromatography column which length-diameter ratio was 5.5.The substrates were feeding into the column through constant current pump at the speed of 0.3 ml/min.According to the single-fact experiment,the best conditions were as follows: pH 7.6 160 mmol/L phosphate buffer solution,reaction temperature 54 ?.Under the optimal conditions,the immobilized enzyme kept high capability of catalytic at the first 528 h.The conversion rate of ara-U run up to a maximum of 86.78%(averaging 65.65%).Using the same amount of free cells,the catalytic effect of enzyme column is 21 times as much as free cells and 11 times as much as the batch operations.Through the research above,biotransformation technology of ara-G using immobilized enzyme were established,and the catalytic conversion rate has been improved.Due to the immobilization of enzyme can be reused or immobilized enzyme catalytic reaction performance for a long time.This greatly cuts the production cost is available for ara-G microbial transformation on industrial production.