【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。更多还原

【Abstract】 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 glutaraldehyde 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-NitD155) 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-1and 1 577 cm-1site,which marks ν C = O,δNH and ν 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-1site,resulting from the overlapping of peaks of C = N bond at 1 660 cm-1and Lys-D155 and nitrilase surface carboxyl at 1707 cm-1. The experimental results indicate that the optimal condition of nitrilase immobilization is:25℃,Lys-D155∶ glutaralde-hyde ∶ nitrilase = 1 ∶ 0. 11 ∶ 2. 12 × 10-4,p H = 5. 25. Furthermore,the activity of the I-NitD155 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.更多还原