测蔗糖复合酶电极的研究

Study on Multiple-enzyme Electrode for Sucrose Determination

采用酶电极流动注射分析系统(EFIA),由固定化酶膜包括蔗糖转化酶(INV)、葡萄糖变旋酶(MUT)及葡萄糖氧化酶(GOD)与氧电极共同组成的复合酶电极用于蔗糖的快速测定。实验确定每张酶膜的最适酶量(IU比)为1NV:MUT:GOD=72:48:2.4。酶经固定化后,INV与MUT的综合回收活力>42.9%。其最适ph为5.8—6.5。最适温度范围是35—45℃。动态法和稳态法测试的线性范围分别为:5×10^(-4)—10^(-1)和10^(-5)—2×10^(-3)mol/L,响应时间分别<20s和<2min。实验的重复性良好,变异系数<1.7%。用此酶电极测定以蔗糖为碳源的发酵液中的蔗糖含量,平均回收率达到98%。发酵液中的蔗糖分解产生的葡萄糖对本电极的干扰可通过平行运行的GOD电极来校正。连续使用的寿命至少为120h。比前年报道的14h有了显著的提高。酶膜显示了较好的保存稳定性(30天,保存于4℃蒸馏水中)和一定的抗热性(50℃,30min)。

Invertase (INV), mutarotase (MUT), glucose oxidase, (GOD) andBSA were coimmobilised via glutaraldehyde-bridged covalent bonding,and directly absorbed on the teflon membrane.This membrane was cove-red with a nylon mesh and placed over an oxygen electrode.An enzymeelectrode flow injection analyze system (EFIA) was adopted. The opti-mun enzyme composition (IU) for immobilisation on the teflon membraneof INV-MUT-GOD was found to be in the ratio 72: 48: 2.4, with a reco-very activity (INV+MUT) more than 42.9%. pH 5.8--6.5 was the mostsuitable for the sensor activity. The optimum temperature was 35--40℃.The system exhibited good linearity in the range 5×10^(-4)--10^(-1) mol/Lsucrose (kinetic mothod) and 10^(-5)--2×10^(-3) mol/L sucrose (steady statemethod), short response time (<20s for kinetic method,<2 min forsteady state method). CV=1.7% (kinetic method). The sensor had beenused for determination of sucrose concentration in fermentation broth,with an average recovery rate of 98%.