接枝赖氨酸D155树脂固定化腈水解酶及其性质  

Immobilization of Nitrilase on Lys-D155 Resin and its Application in the Synthesis of R-mandelic Acid

作　　者：陶仁友[1] 江文[1] 石业新 徐扬[1] 康宏宽[1] 周小华[1] 周志明[1] TAO Ren-you JIANG Wen SHI Ye-xin XU Yang KANG Hong-kuan ZHOU Xiao-hua ZHOU Zhi-ming(School of Chemistry and Chemical Engineering of Chongqing University, Chongqing 400044, Chin)

机构地区：[1]重庆大学化学化工学院,四川重庆400044

出　　处：《药物生物技术》2016年第6期471-476,共6页Pharmaceutical Biotechnology

基　　金：国家自然科学基金(Grant 21106191;21206175);材料化学工程国家重点实验室(Grant KL14-11);中央高校基本业务费(project No.CQDXWL-2013-019)资助

摘　　要：以赖氨酸（Lys）修饰阳离子交换树脂D155合成出赖氨酸修饰两性阳离子交换树脂（Lys-D155）,再以该树脂为载体,制备出固定化腈水解酶（I-Nit-D155）,最后以I-Nit-D155为催化剂水解R,S-扁桃腈研究制备固定化腈水解酶的优化条件、主要酶学性质并对水解产物进行表征。红外分析表明,经赖氨酸修饰的D155成功制备出固定化腈水解酶I-Nit-D155。制备I-Nit-D155的优化条件为：室温、p H 5.25、载体游离氨基/戊二醛/腈水解酶摩尔比1∶0.11∶2.12×10-4,I-Nit-D15活力为10~15 U/mg、偶联率43.2%、Km值23.11 mmol/L,重复使用15次后活力仍可保持50%以上;I-Nit-D15水解R,S-扁桃腈的产物的熔点、比旋光度等分别为133℃、-155.57°,与R-扁桃酸一致,经红外、1H-和13C-核磁共振波谱表征确证为R-扁桃酸。This study is aimed at optimizing the preparation method of I-Nit-D155,investigating its enzymatic properties and characterizing the hydrolysis products of mandelonitrile. With glntaraldehyde as the crosslinker,nitrilase（Nit） was immobilized onto D155- lysine resin（ Lys-D155 ） ,which was porous and grafted with a flexible and hydrophilic amino acid. The immobilized nitrilase （I-Nit- D155 ） was then applied to hydrolyze mandelonitrile. The results of infrared spectra analysis show that the Lys-D155 resin has been synthesized since two absorption peaks appear respectively at 1 627 cm-1 and 1 577 cm-1 site, which marks v C = O,δNH and v CN of amide bond,and additionally nitrilase was successfully immobilized on Lys-D155 resin under the fixation of Schiff base by the reason that an absorption peak occurs at 1 671 cm-l site,resulting from the overlapping of peaks of C = N bond at 1 660 cm-1 and Lys-D155 and nitrilase surface carboxyl at 1707 cm-1. The experimental results indicate that the optimal condition of nitrilase immo- bilization is：25℃, Lys-D155： glutaralde-hyde： nitrilase = 1： 0.11：2.12 × 10-4, pH = 5.25. Furthermore, the activity of the I-Nit- D155 reached 10 - 15 U/mg and the coupling ratio was 43.2% under the conditions mentioned above. It is worth noting that the activity of I-Nit-D155 remains up to 50% after reusing 15 times;hydrolysate of R, S-mandelonitrile was proved to be R-mandelic acid by the detection of IR, NMR and some other physical index.

关 键 词：腈水解酶 固定化 D-155树脂 赖氨酸 R-扁桃酸 水解 

分 类 号：TQ460.6[化学工程—制药化工]