Centre for Biodiscovery, School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.
Protein Sci. 2023 Sep;32(9):e4743. doi: 10.1002/pro.4743.
l-Malate is a key flavor enhancer and acidulant in the food and beverage industry, particularly winemaking. Enzyme-based amperometric biosensors offer convenience for monitoring its concentration. However, only a small number of off-the-shelf malate-oxidizing enzymes have been used in previous devices. These typically have linear ranges poorly suited for the l-malate concentrations found in fruit processing and winemaking, making it necessary to use precisely diluted samples. Here, we describe a pipeline of database-mining, gene synthesis, recombinant expression, and spectrophotometric assays to characterize previously untested enzymes for their suitability in biosensors. The pipeline yielded a bespoke biocatalyst-the Ascaris suum malic enzyme carrying mutation R181Q [AsME(R181Q)]. Our first prototype with AsME(R181Q) had an ultra-wide linear range of 50-200 mM l-malate, corresponding to concentrations found in undiluted fruit juices (including grape). Changing the dication from Mg to Mn increased sensitivity five-fold and adding citrate (100 mM) increased it another six-fold, albeit decreasing the linear range to 1-10 mM. To our knowledge, this is the first time an l-malate biosensor with a tuneable combination of sensitivity and linear range has been described. The sensor response was also tested in the presence of various molecules abundant in juices and wines, with ascorbate shown to be a potent interferent. Interference was mitigated by the addition of ascorbate oxidase, allowing for differential measurements on an undiluted, untreated wine sample that corresponded well with commercial l-malate testing kits. Overall, this work demonstrates the power of an enzyme-centric approach for designing electrochemical biosensors with improved operational parameters and novel functionality.
L-苹果酸是食品和饮料行业（特别是酿酒）的一种重要风味增强剂和酸化剂。基于酶的安培生物传感器为监测其浓度提供了便利。然而，以前的设备中只使用了少数现成的苹果酸氧化酶。这些酶通常具有不适合水果加工和酿酒中发现的 L-苹果酸浓度的线性范围，因此需要使用精确稀释的样品。在这里，我们描述了一个数据库挖掘、基因合成、重组表达和分光光度测定的流水线，以表征以前未经过测试的酶，评估其在生物传感器中的适用性。该流水线产生了一种定制的生物催化剂——携带 R181Q 突变的蛔虫苹果酸酶[AsME（R181Q）]。我们的第一个带有 AsME（R181Q）的原型具有超宽的线性范围，为 50-200mM L-苹果酸，相当于未稀释果汁（包括葡萄）中的浓度。将二价阳离子从 Mg 改为 Mn 可将灵敏度提高五倍，而添加柠檬酸（100mM）可再提高六倍，但线性范围降低至 1-10mM。据我们所知，这是首次描述具有可调节灵敏度和线性范围组合的 L-苹果酸生物传感器。该传感器的响应也在果汁和葡萄酒中丰富的各种分子存在的情况下进行了测试，结果表明抗坏血酸是一种很强的干扰物。通过添加抗坏血酸氧化酶可以减轻干扰，从而可以对未经稀释、未经处理的葡萄酒样品进行差分测量，与商业 L-苹果酸测试试剂盒的结果非常吻合。总的来说，这项工作展示了以酶为中心的方法设计具有改进操作参数和新功能的电化学生物传感器的强大功能。
