High Level Production Of α-arbutin By Whole-cell Biocatalysis

Alpha-arbutin is a kind of efficient, safe, strong compatibility of natural whitening active substances. Arbutin is a natural hydroquinone(HQ) derivative that provides a skin lightening effect by inhibiting tyrosinase activity. Alpha-arbutin’s whitening effect is better and safer than beta-arbutin’s. It is a white needle crystal or powder, it can accelerate the melanin’s decomposition and discharge, thus reduce the deposition of skin melanin, remove spots and freckles, as well as the effect of sterilization, anti-inflammatory, mainly used for the production of cosmetics.Arbutin is a hotspot in the current, producing alpha-arbutin has a variety of ways: natural extraction, plant cell cuLture, organic synthesis and whole cell biocatalysis, etc. The former methods is not commonly used, because there are a lot of disadvantages, such as, it’s difficuLt to separate and purify, low content of product, high costs of production and impractical industrialization.Using the whole cell biocatalysis is a prospective trend, in the reaction, different microbial enzymes play an important role, disaccharide and hydroquinone are converted alpha arbutin. Using whole cell biocatalysis has many advantages compared with other methods, but researches in this field are rare, and it is worth to make our attention.In the experiment, we have constructed a genetic engineering strain, which contained the amylosucrase, and the gene of amylosucrase was amplified from Deinococcus radiodurans CGMCC 1.3828, it contains amylosucrase genes. At the same time, we compared the alpha-arbutin’s transformation rate of two genetic engineering E.coli(TyAS-pet28a-rosetta and DrAS-pet28a-rosetta), the resuLt shows that TyAS-pet28a-rosetta is better than DrAS-pet28a-rosetta, and the transformation rate is 2.92 times. The next step is that optimizing the transformation process to increase the production of alpha-arbutin.Our resuLts indicated that the optimal reaction conditions as follows:the temperature is 30℃,the concentration of hydroquinone is 180mM, the mole ratio of sucrose and hydroquinone was 5.5:1,adding 0.5mM Vc in the reaction buffer, the reaction period is 6h, the rotating speed is 150rpm, the cell concentration is 30 OD/mL, the reaction buffer is 100 mM pH7.0 Tris-HCl buffer.By using this method to produce alpha-arbutin, the convert ratio can reach 99% and the productivity is 7.56g/L/h, this level is better than previously have been reported. Furthermore, in the process of production using sucrose rather than maltose as a substrate, using whole-cell rather than purified enzymes as catalyst, these will greatly reduce the cost of production. The method has many advantages, such as, the raw materials is cheap, the reaction’s method is simple, the product’s purity is higher than others and the production efficiency is highest in all methods, it has great potential in the industrial production.In order to obtain the high purity’s alpha-arbutin,the reaction liquid is separated and purified by following methods, the reaction liquid is firstly purified by macroporous adsorption resin H107, separately collecting eluent of different components, concentrating the eluent of containing alpha-arbutin, the purity of the product reach 98%, analysing the eluent by high performance liquid chromatography (HPLC), it contains a small amount of hydroquinone in the eluent, so the further purification is needed. The second purification is proceeded by using silica gel column, and products were detected by high performance liquid chromatography (HPLC), the purity of the product above 99.9%,through comparing the alpha-arbutin standards’ and product’s figures, product’s appearance time is consist with standards’. We analyzed product’s chemical structure by nuclear magnetic resonance (NMR), and uLtimately confirmed our product as same as alpha-arbutin.