恒电位自动滴定法分析脂肪酶水解活性影响因素  

Factors Affecting Lipase Activity Assay by Automatic pH-Stat Potentiometric Titration Method

作　　者：赵炜[1] 黄卓楠[1] 李娜[1] 

机构地区：[1]西安交通大学能动学院化工系,西安710049

出　　处：《分析化学》2011年第3期351-355,共5页Chinese Journal of Analytical Chemistry

基　　金：国家重点基础研究计划(973项目)项目(No.2009CB2199006);国家自然科学基金项目(No.20406014)资助

摘　　要：利用恒电位自动滴定法分析了脂肪酶(Candida rugosa lipase,CRL)催化橄榄油水解活性的影响因素,确定了适宜的酶活性测定条件,对比了传统表面活性剂和咪唑类离子液体对酶活性的影响。37℃时,与酶蛋白浓度呈线性关系的酶活性测定范围为1～14 mmol/min;酶蛋白浓度范围为0.01～0.07 g/L;水解反应的米氏常数Km=4.828×10-4mol/L,最大反应速率3.498×10-3mol/min;缓冲液pH值7.2、温度37℃,搅拌速度为1200 r/min;PVA、AOT和卵磷脂对酶活的促进和抑制作用与其浓度有关,最大相对酶活力分别为297%,255%和154%;离子液体对酶活的最大促进效果为[OMIM][Br]>[BMIM][Br]>[EMIM][Br]>[OMIM][BF4]>[BMIM][BF4]>[EMIM][BF4];最大相对酶活力分别可达到1734%,1709%,1579%,1063%,1030%和960%;阴离子种类对酶活性的影响大于阳离子种类的影响;离子液体的烷基链长对酶活性的影响与其浓度密切相关,烷基链越长,其达到最大酶活促进效果的浓度越低。本研究为筛选合适的离子液体种类及浓度提供了快速有效的分析方法。An accurate quantitative analytical method of lipase activity was established. The activity of Candida rugosa lipase （CRL） in the hydrolysis reaction of olive oil was analyzed by automatic pH-stat potentiometric titration method. Various factors influencing lipase activity assay were discussed and appropriate analysis conditions were selected. The results showed that the hydrolytic activity of CRL was linearly proportional to enzyme protein concentration only within 0.01--0.07 g/L range of protein concentration at 37 ℃. The corresponding range of enzyme activity was 1--14 μmol/min. Kinetics studies showed that the corresponding values of Km and Vmax were 4. 828× 10-4 mol/L and 3. 498X 10-a mol/min, respectively. The optimum temperature, pH value and stirring speed were 37 ℃ 7.2 and 1200 r/rain, respectively. Low content of polyvingl alcohol （PVA） and lecithin promoted CRL activity but high content of them inhibited CRL activity. The maximum relative activity of CRL reached to 297% at 2.0 g/L of PVA or 154 % at 5.0 g/L of lecithin in the system. PVA and lecithin showed inhibiting effect on CRL activity at more than 4.0 g/L and 10.0 g/L of content, respectively. In contrast, low concentration of AOT restrained CRL activity, while high concentration of it （more than 12.5 g/L） enhanced CRL activity. The maximum relative activity of CRL reached to 255% at 50 g/L of AOT in the system. Tween-80 significantly inhibited CRL activity. A several of imidazole ionic liquids improved CRL activity greatly in sequence of [-OMIM][Br]〉[BMIM][Br]〉[EMIM][Br]〉[OMIM][-BF4]〉[BMIM][BF4]〉[EMIM][BF4]. The maximum relative activity of CRL reached to1734%, 1709%, 1579%, 1063%, 1030% and 960% with 12, 43, 90, 5, 45 and 88 g/L of corresponding concentration of the ionic liquids, respectively. The promotion effect of the ionic liquids on CRL activity were much better than those of PVA, lecithin and bis （2-ethylhexyl）sulfosuccinate sodium （AOT）. The impact of anions in ionic liquids on enzyme activity was gre

关 键 词：恒电位滴定 假丝酵母脂肪酶 酶活性 离子液体 表面活性剂 

分 类 号：Q814[生物学—生物工程]