Vanillin is a popular flavoring compound and an important food additive. Owing to the consumer preference for inexpensive natural aroma flavors, vanillin production through a biotechnological pathway has become of great interest and commercial value in recent years. In this study, an enzymatic synthetic system for vanillin using a coenzyme-independent decarboxylase (FDC) and oxygenase (CSO2) cascade was reconstituted and optimized. This system produces a slightly higher production yield (40.20%) than the largest yield reported for immobilized FDC and CSO2 (35.00%) with ferulic acid as a substrate. It was previously reported that the low catalytic activity and thermal instability of CSO2 restrict the overall productivity of vanillin. In present study, site-directed mutagenesis was applied to rate-limiting oxygenase CSO2 to generate positive mutants. The production yields of mutants A49P (58.44%) and Q390A (65.29%) were 1.45- and 1.62-fold that of CSO2 wild type, respectively. The potential mechanism for enhanced vanillin production using A49P involved increased thermostability and catalytic efficiency, while that using Q390A was probably associated with a better thermostable performance and increased catalytic efficiency resulting from a larger entrance channel.

香兰素是一种流行的调味化合物和重要的食品添加剂。由于消费者偏爱廉价的天然芳香香料，近年来通过生物技术途径生产香兰素已引起人们极大的兴趣和商业价值。在这项研究中，重组和优化了使用辅酶非依赖性脱羧酶 (FDC) 和加氧酶 (CSO2) 级联的香草醛酶合成系统。该系统产生的产量 (40.20%) 略高于固定化 FDC 和 CSO2 (35.00%) 的最大产量 (35.00%)，以阿魏酸为底物。之前有报道称，CSO2 的低催化活性和热不稳定性限制了香草醛的整体生产率。在本研究中，定点诱变应用于限速加氧酶 CSO2 以产生阳性突变体。突变体 A49P (58.44%) 和 Q390A (65.29%) 的产量分别是 CSO2 野生型的 1.45 倍和 1.62 倍。使用 A49P 提高香草醛产量的潜在机制涉及增加热稳定性和催化效率，而使用 Q390A 可能与更好的热稳定性能和更大的入口通道导致的催化效率增加有关。