高效乳酸生物传感器的制备及其在发酵组分检测中的应用  

Preparation of high-efficiency amperometric lactic acid biosensor for determination of lactic acid in microbial fermentation

作　　者：徐欣[1] 杨璐 马婕妤 陈敏皎 周杰 XU Xin;YANG Lu;MA Jieyu;CHEN Minjiao;ZHOU Jie(Productivity Promotion Center of Jiangsu Province,Nanjing 210018,China;College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University,Nanjing 211800,China;State Key Laboratory of Materials-Oriented Chemical Engineering,Nanjing Tech University,Nanjing 211800,China)

机构地区：[1]江苏省生产力促进中心,江苏南京210018 [2]南京工业大学生物与制药工程学院,江苏南京211800 [3]南京工业大学材料化学工程国家重点实验室,江苏南京211800

出　　处：《生物加工过程》2022年第3期284-291,共8页Chinese Journal of Bioprocess Engineering

基　　金：国家重点研发计划(2021YFC2101100);国家自然科学基金(21727818)。

摘　　要：为了制备一种高效检测发酵过程中乳酸含量的电化学生物传感器,首先,以来源于绿色气球菌的乳酸氧化酶(AvLOX)作为检测酶元件,对其进行重组异源表达与制备研究,经发酵诱导产酶、蛋白纯化获得乳酸氧化酶纯酶,其酶活和比酶活分别达到34.0 U/mL和28.8 U/mg。酶学性质研究表明AvLOX在30~60℃和pH 6.0~7.5时具有良好的稳定性;进一步从生物相容性、固定化率和酶负载量3个方面优化确定了最佳酶固定化方式,即12.5 g/L壳聚糖(CS)与1.0 U AvLOX等体积混合。其次,通过层层组装,将制备获得的乳酸氧化酶与氧化石墨烯(GO)固载于玻碳(GC)电极上,构建的AvLOX/CS/GO/GC乳酸电化学酶生物传感器具有优异的乳酸传感检测性能,其灵敏度为45μA·L/(mmol·cm^(2)),线性响应范围为0.1~0.9 mmol/L,响应时间为4 s。该乳酸生物传感器具有良好的检测稳定性,存放96 h后仍可保持95%以上的检测活性。最后,利用制备的AvLOX/CS/GO/GC乳酸电化学酶生物传感器对模拟和真实微生物发酵体系中乳酸含量进行了检测,其相对误差分别小于6.5%和10%。因此,本研究制备的乳酸生物传感器具有高效的检测灵敏度以及良好的稳定性,为发酵过程中乳酸含量的检测提供了一种可行的方案。An electrochemical biosensor was developed to detect lactic acid content in fermentation process.First,the recombinant lactate oxidase derived from Aerococcus viridans(AvLOX) with enzyme activity of 34.0 U/mL and specific enzyme activity of 28.8 U/mg was obtained.Moreover,the optimal immobilization strategy of 12.5 g/L chitosan(CS) mixed with 1.0 U AvLOX in equal volume was optimized from the three aspects of biocompatibility,immobilization rate and enzyme loading.Further,a highly sensitive and fast response AvLOX/CS/GO/GC lactate electrode was constructed by fixing the enzyme layer and graphene oxide(GO) on the glassy carbon(GC) electrode,with a sensitivity of 45 μA·L/(mmol·cm~2) with a linear range of 0.1-0.9 mmol/L,as well as 4 s of response time.Especially,95% sensitivity of this biosensor is still remained after 96-hour storage.Notably,the lactic acid biosensor AvLOX/CS/GO/GC detected lactic acid in simulated and real fermentation systems with a relative error of less than 6.5% and 10%,respectively,showing excellent application potential.

关 键 词：乳酸生物传感器 乳酸氧化酶 壳聚糖 发酵过程检测 

分 类 号：TP212.3[自动化与计算机技术—检测技术与自动化装置]