硅藻土吸附固定化Bacillus sp.DL-2胞内蛋白酶拆分制备(S)-乙酸苏合香酯  

Immobilization of Intracellular Proteases of Bacillus sp.DL-2 Using Diatomite to Prepare(S)-Phenylethyl Acetate Through Kinetic Resolution

作　　者：王晓敏 董璐 徐湘薇 张继福[3] 张云[1,4] 胡云峰 WANG Xiaomin;DONG Lu;XU Xiangwei;ZHANG Jifu;ZHANG Yun;HU Yunfeng(South China Sea Institute of Oceanology,CAS Key Laboratory of Tropical Marine Bio-resources and Ecology,Guangdong Key Laboratory of Marine Materia Medical,Chinese Academy of Sciences,Guangzhou 510301,China;University of Chinese Academy of Sciences,Beijing 100049,China;Guangdong Provincial Hospital of Chinese Medicine,Guangzhou 510120,China;Southern Marine Science and Engineering Guangdong Laboratory(Guangzhou),Guangzhou 511458,China)

机构地区：[1]中国科学院南海海洋研究所/中国科学院热带海洋生物资源与生态重点实验室/广东省海洋药物重点实验室,广东广州510301 [2]中国科学院大学,北京100049 [3]广东省中医院,广东广州510120 [4]南方海洋科学与工程广东省实验室(广州),广东广州511458

出　　处：《江西农业大学学报》2021年第2期415-425,共11页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然科学基金项目(21302199);南方海洋科学与工程广东省实验室(广州)人才团队引进重大专项(GML2019ZD0406);广东省自然科学基金项目(2018A030313151);广东省海洋渔业科技攻关与研发方向项目(A201701C12);中国科学院战略性先导科技专项(XDA11030404);中国科学院“科学”号高端用户项目(KEXUE2018G05)。

摘　　要：【目的】(S)-乙酸苏合香酯是手性药物合成的关键手性砌块,其在多种手性化合物的合成及医药、香料的工业生产中具有重要的作用。传统的化学合成手性化合物的方法需要有毒有机溶剂及重金属的参与,对环境及人类具有严重的危害,同时得到的手性化合物的光学纯度较低。与传统的化学合成相比,酶法选择性拆分消旋体化合物,具有高立体专一性和区域选择性、副反应少、产率高、产物光学纯度好以及反应条件温和的优点,是一种被广泛认可的拆分方法。实验表明,Bacillus sp.DL-2胞内蛋白游离酶可拆分制备(S)-乙酸苏合香酯,然而游离酶不易回收且稳定性差,将Bacillus sp.DL-2胞内蛋白游离酶制备成固定化制剂,克服了游离酶对环境敏感,稳定性不高的缺点,为工业化生产(S)-乙酸苏合香酯奠定了基础。【方法】酶的固定化方法主要有物理吸附法、离子结合法、共价结合法、交联法和包埋法。以吸附法固定酶,酶的构象很少改变,因此,酶的催化活力损失较少;且吸附法固定化操作过程简单,因此吸附法在经济上是最具吸引力的固定化方法。硅藻土由硅藻的细胞壁沉积而成,硅藻土表面的多孔性与负电性使其呈现明显表面吸附性,因而被常用做吸附载体。以硅藻土作为固定化载体,对Bacillus sp.DL-2胞内蛋白酶进行固定化,用以拆分制备高光学纯的(S)-乙酸苏合香酯。利用单因素实验优化,确定制备固定化酶的最佳固定化条件及制备(S)-乙酸苏合香酯的最佳拆分条件,并测定了制备的固定化酶对金属离子的敏感度及储存稳定性。【结果】最佳固定化条件为:温度40℃,pH为7,固定化时间10 h,载体添加量100 g/L。在最佳固定化条件下制备了固定化酶,优化确定了制备(S)-乙酸苏合香酯的最佳拆分条件为:固定化酶用量为160 mg/mL,拆分时间7 h,拆分温度30℃,缓冲液pH为7。【结论】在最佳固[Objective](S)-phenylethyl acetate is a key chiral building block in the synthesis of chiral drugs,and plays important roles in the synthesis of many chiral compounds and in the industrial production of medicines and spices.The traditional method for producing chiral compounds is chemical synthesis.But it requires the participation of toxic organic solvents and heavy metals,which has serious harm to the environment and human beings.At the same time,the obtained chiral compounds have low optical purity.Compared with traditional chemical synthesis,enzymatic selective separation of racemic compounds is a widely accepted method,which has many advantages,such as high stereoscopic specificity,regional selectivity,fewer side reactions,high yield,good optical purity of products and mild reaction conditions,which is a widely recognized method of resolution.Experiments show that the intracellular proteases from strain Bacillus sp.DL-2 could asymmetrically hydrolyze(±)-1-phenylethyl acetate to obtain(S)-phenylethyl acetate.However,free enzymes are not easy to recover and are less stable.The intracellular proteases were used to prepare immobilized preparations,which overcame the disadvantages of the free enzyme’s being sensitive to the environment and low stability,and laid a foundation for the industrial production of(S)-phenylethyl acetate[Method]The immobilization methods of enzyme mainly include physical adsorption,ion binding,covalent binding,crosslinking and embedding.When the enzyme is immobilized by adsorption,the conformation of the enzyme seldom changes,so the enzyme catalytic activity loss is less.In addition,the carrier selection range is wide and the price is low.And the operation process of adsorption immobilization is simple,so adsorption is the most attractive immobilization method in economy.Diatomite is deposited by the cell wall of diatoms.The porous and electronegative properties of the surface of diatomite make it show obvious surface adsorption,so it is commonly used as adsorption carrier.In order to

关 键 词：硅藻土 固定化酶 手性拆分 (S)-乙酸苏合香酯 

分 类 号：TQ424.22[化学工程] TQ925.2[轻工技术与工程—发酵工程]