| Biocatalysis exploits the versatility of enzymes to catalyse a variety of processes for the production of novel compounds and natural products. Because of this feature, the number of biocatalysts used in organic synthesis has rapidly increased during the last decades, especially for the production of chiral compounds. Among them, alcohol dehydrogenase(ADH) are of particular interest ,which is one kind of oxido-reductase and have the capacity to catalyse a wide rang of reduction of prochiralβ–keto ester to corresponding second alcohol with very high regioselectivity and stereoselectivity, while the latter is a useful chiral building blocks for the synthesis of pharmaceuticals, agrochemicals and fragrances.Although free enzyme used in the biocatalysis reaction have high catalytic activity, the processes of extraction and separation of product are complecated. In the immobilized state, the stability of enzyme is improved , separation from reaction mixtures is easy, and also it can be recycled for further use to reduce production costs. Therefore, immobilization in recent years is of particular interest.Accordingly, a lot of methods have been developed, including adsorption,covalent binding or binding to a support (carrier) and entrapment et.al. The support (carrier) used include a synthetic organic polymer, a biopolymer or an inorganic solid. To overcome the problem of low pH, thermodynamic stability of organic materials, we isolated the alcohol dehydrogenase (ADH) from the permeabilized brewer's yeast cells (Saccharomyces cerevisiae) and immobilized it on the derivatization inorganic materials, via glutaraldehyde covalent binding on the basis of references.Making using of the acid resistance , thermodynamic stability and mechanical strength of attapulgite ,attapulgite is used for immobilization of alcohol dehydrogenase via glutaraldehyde as cross-linking agent. The purpose is to improve the pH , thermodynamics stability and reusability of enzyme. In this study, Selecting ethyl acetoacetate as a model substrate to prepare immobilization of alcohol dehydrogenase and biocatalysis asymmetric synthesis of ethyl(S)-3-hydroxybutyrate. The attapulgite derivative preparation, extraction from brewer's yeast alcohol dehydrogenase in the process, immobilization of alcohol dehydrogenase,as well as its reduction reaction in the catalytic asymmetric synthesis of ethyl (S)-3-hydroxybutyrate are discussed in this article. Moreover,the activity of free and immobilized enzyme, yield and ee of the product in reaction were used to evaluate separation of alcohol dehydrogenase, stability of immobilized alcohol dehydrogenase and and other factors on the impact of catalytic reduction.The experimental results show that, alcohol dehydrogenase was successfully immobilised on modified attapulgite via covalent binding. When extraction the alcohol dehydrogenase from permeabilized brewer's yeast, the optimum preparation was: cell : buffer(m/v)=5:20,T=5℃, pH7.5 phosphate buffer ultrasound broken cells, the concertrate of ammonium sulfate was 35%, the extraction of ADH enzyme activity up to 105U/ml. Stability experiments show that, optimum pH and temperature of ADH clude extract were 7.0 and 25℃,respectively.Preparation of the immobilized ADH, the best temperature,pH and concentration of glutaraldehyde were 30℃, 6.8 and 0.5%,respectively. And the highest activity of immobilized enzyme could reach 0.074U/mg. The stability of the immobilized ADH experiments show that the stability of enzyme had been improved , compared to free counterpart. The optimum pH and temperature of immobilized were 7.5 and 35℃,respectively, and 58% of its original activity was retented after incubated at 35℃for 32 h.Asymmetric reduction of experiments show that in bioreduction of EOB mediated by immobilized ADH, the yield and enantiomeric excess (ee) of product reached 88% and 99.2%, respectively, within 2 h and retained about 42% of the initial activity after 8 cycles.
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