Preparation Of Tyrosinase Cross-linked Aggregates And Their Use In Drug Synthesis

Tyrosinase is an oxidoreductase which does not require a coenzyme.It catalyzes the hydroxylation of a variety of monophenols to o-diphenols and their subsequent oxidation to o-quinones.As a new type of enzyme immobilization methodology combining purification and immobilization into a single operation,CLEA(cross-linked enzyme aggregates)technology offers many advantages such as easy preparation,low cost,no need for enzyme purification,and excellent catalytic performance.Therefore it has been successfully applied to the immobilization of various enzymes.Deep eutectic solvent(DES)is a new generation of green reactio n medium,which has been proven to be able to work as a co-solvent to effectively enhance the activity and stability of an enzyme.However,DES has not been reported to be introduced to the preparation of immobilized enzyme.In this paper,DES and CLEA were combined for the first time,by introducing DES into the preparation of the tyrosinase CLEAs.The preparation conditions were studied,the effects of DES on CLEA's activity and stability were examined,and the use of tyrosinase CLEAs as catalyst for synthesis of 3,4-dihydroxyphenyl acetic acid,piceatannol and 3'-hydroxypterostilbene were investigated.The major contents are given below:1.Preparation of DES-CLEA and its catalytic properties24 DESs were prepared by mixing 2 choline salts(choline chloride and choline acetate)with 4 hydrogen-bond donors(HBDs)(e.g.,urea,acetamide,glycerol and ethylene glycol)at 3 molar ratios(1:2,1:1,2:1).The impacts of the DES compositions(such as the use of different anions and HBDs and different HBD/salt molar ratios)on the catalytic activity and stability of DES-CLEAs have been studied.The results showed that addition of DES could effectively improve the catalytic activity of tyrosinase CLEAs.Among the 24 DES tested,Ch Cl-based DESs were more likely to increase the activity of CLEA than the Ch Ac-based ones.Ch Cl / U(1: 2)gave the highest CLEA activity,which was 2.2 times as active as the one exhibited by the DES-free CLEAs.DES has shown little effect on the stability of tyrosinase CLEAs.2.Use of tyrosinase CLEAs for enzymatic synthesis of polyhydroxy compoundsIn this paper,three polyhydroxy compounds,namely3,4-dihydroxyphenyl acetic acid,piceatannoland 3'-hydroxypterostilbene,were synthesized by utilizing tyrosinase CLEAs as the catalyst,in comparison with the use of free enzyme solution and DES-CLEA.1)Synthesis of 3,4-dihydroxyphenylacetic acid from 4-hydroxyphenylacetic acid: The optimum reaction conditions were determined.The maximum yields were 48.9%(catalyzed by free enzyme solution)and 40.5%(catalyzed by tyrosinase CLEAs).When DES-CLEAs were used as the catalyst,a yield of 37.3% was obtained under optimum conditions.The productivity obtained in this study(340.0 mg/L/h)was much higher than those reported in literature.2)Synthesis of piceatannol from resveratrol: Reaction conditions have been optimized,and a maximal yield of 74% was obtained under optimal conditions.The product was identified as piceatannol by 1H-NMR.The productivity obtained in this study(903.7 mg /L/h)was much higher than those reported in literature.This is the first time that mushroom tyrosinase was used as the catalyst for the synthesis of piceatannol with a high yield,indicating that this enzyme has great potential for this application.3)Synthesis of 3'-hydroxypterostilbene from pterostilbene: Under the optimal conditions,a yield of 61.7% was obtained.The product was identified as 3'-hydroxylpterostilbene by 1H-NMR.This is the first report on biological synthesis of 3'-hydroxylpterostilbene,which has been demonstrated to be feasible.In summary,DES has shown its ability in enhancing the activity of tyrosinase CLEAs by introducing it into the CLEA preparation.Tyrosinase CLEAs have been demonstrated to possess a great potential in working as catalyst for synthesis of 3,4-dihydroxyphenylacetic acid,piceatannol,and 3'-hydroxypterostilbene.