Preparation Of Wulti-Enzyme Co-Immobbilized And Chiral Oxidation Of Glucoerucin

Cruciferous vegetables contain compounds associated with protection against cancer, It has been shown that the cancer preventive effects of cruciferous vegetables are related to their unique content in a large variety of isothiocyanate. Sulforaphane, an isothiocyanate derived from glucoraphanin hydrolyzed by myrosinase enzyme, was initially identified as the principal inducer of phase Ⅱ enzymes and has subsequently been shown to possess anticarcinogenic activities. Rocket has its origin in the Mediterranean region but is widely distributed all over the world, belongs to the family of cruciferous plants. It is mainly in nearly pure form and good amount (ca. 3%) in rocket ripe seeds. The conversion of glucoerucin into glucoraphanin is based on the oxidation reaction of sulfides into the corresponding sulfoxides. Both isolated enzymes and whole cells have been used in the enantioselective oxidation of prochiral sulfides. Horseradish peroxidase a isolated heme peroxidases, has been shown to catalyze a broad spectrum of stereoselective epoxidation and sulfoxidation reactions. These hot spots can be avoided by in situ formation of hydrogen peroxide from oxygen and a cosubstrate. In the last few years, several types of nanoparticles have been extensively employed in the enzyme immobilization. The major advantage of multi-enzyme co-immobilization is that the active sites of the enzymes are brought into close proximity with one another in the nanomaterials, and such close confinement minimizes the diffusion of intermediates among the enzymes in the consecutive reactions, thereby enhancing the overall reaction efficiency and specificity。In this paper, we firstly reported the enantioselecive preparation of glucoraphanin by a bienzymatic method using glucose oxidase to produce hydrogen peroxide, which is immediately consumed by horseradish peroxidase to oxidise a sulfide. After binding to the enzymes together by the bis-aryl hydrazone conjugation, it is Encapsulated by TEOS, and used in the enantioselective oxidation of the prochiral sulfides of glucoerucin。(1) Glucoerucin was obtained with high purity (over 95%)。The optimal enzyme catalysis oxidation conditions of Glucoerucin were found to be as follows:the reactive pH, temperature and time were pH=7 28℃ and 240min. The concentration of glucose was 36 mM. The concentration of the two kinds of enzyme are 0.2 mg·mL-1. In this way Chiral sulfoxide groups was obtained。(2) Then, the conjugationed enzymes were Encapsulated. The optimum reaction conditions was:conjugationed enzymes 5mg, Deionized water 0.4mL, Cyclohexane 7.5 mL, Triton-X-100 1.75mL, n-Hexyl alcohol 1.8mL, TEOS 250μL, Reaction of 24 hours. (The embedding rate can reached at 91.6%) The catalytic reaction rate of conjugationed enzymes is 2.12 times higher than free enzymes.(3) The thermal and pH stability of conjugationed enzymes compare favourably with free enzymes. After turnover number of 9, The amount of transformation is 73% of the first time.