Cloning And Expression Of A Gene Of Catalytic Synthesis Of ?-arbutin And Its Enzymatic Properties And Industrial Application

?-Arbutin is a glucosylated compound of hydroquinone,?hydroquinone-O-?-D-glucopyranoside?,linked by the glycosidic bonds,and it is a safe,green and common tyrosinase inhibitor.It also has antibacterial,antitumor,anti-inflammatory,whitening activity and effect in the treatment of urinary tract infection.By searching in NCBI website,the transglucosidase gene from Xanthomonas campestris genomic was found.The transglucosidase gene was amplified by PCR,connected with the plasmid vector pET-28a?+?,and transfered into the expression host E.coli BL21 Gold?DE3?plysS for catalytic synthesis of?-arbutin.The recombinant Escherichia coli was optimized by lactose induction.The enzymatic properties of recombinant E.coli were also studied for the following whole cell catalysts.Finally macroporous resin was used to isolation and purification for?-arbutin.The transglucosidase gene from Xanthomonas campestris pv.campestris was ligated with plasmids pET28a?+?,transformed into different competent cells.The resulting recombinants E.coli BL21 Gold?DE3?plysS pET28a-agl successfully expressed transglucosidase and apparent high enzymatic activity of catalyzing enzymatic synthesis of?-arbutin.In this work the recombinants E.coli induced with lactose for optimization method,The optimal inducing condition of lactose was as following:0.5 g/L lactose was added to induce 8 h at 28?when recombinants E.coli grew to a degree.The auto-induction medium for expression had significant advantages,the biomass and protein expression was 2 times higher than that induced by IPTG and lactose,and the glucosidase activity was 33 times and 15 times higher than that induced by IPTG and lactose separately,which has potential application.The catalysis efficiency was best when the transglucosidase was at the concentration of 0.2775 mg/mL in reactiom solution.The temperature stability of the enzyme was quite stable between 4?and 30?.The optimum reaction temperature and pH of transglucosidase was 30?and6.5,respectively.In addition,when the concentration of hydroquinone in the reaction solution was 11 g/L,the most of?-arbutin was accumulated in the reaction solution at this time.The results also showed that the enzymatic activity of catalyzing enzymatic synthesis of?-arbutin was negatively influenced by the accumulation of?-arbutin in the reaction solution.The metal ion of Cu²⁺almost completely inhibited the enzyme activity.K⁺significantly activated the enzyme activity.Under the above conditions,the accumulation of?-arbutin reached 23 g/L,the molar yield of the hydroquinone reached 88%.For the study on the whole-cell catalyst technology,0.5‰CTAB could improve 3 times of the effective enzymatic catalysis;with batch addition of substrate in the catalytic process of 30 h,a total of the hydroquinone additive amount was 2.23%,the final conversion rate was 90.9%,the final yield of ?-arbutin was 50.31 g/L.Studies on separation and purification of ?-arbutin with the purolite macroporous adsorption resin MN202,the optimization of macroporous resin was performed by taking static and dynamic adsorption parameters of ?-arbutin respectively.the best conditions of static adsorption parameters were as following:20 g/L of ?-arbutin,25? of temperature,adsorption time for 30 min,finally the resin reached the saturation adsorption,resin MN202 could adsorp 80 mg?-arbutin per gram rein;at 25?,the Freundlich equation is:ln Q_e=0.2616 x ln C_e-9.168;the concentration of?-arbutin was 18 g/L,the optimum amount of sample was 5 column volumes;after loading the sample,it was eluted by 10% and 20% ethanol.