The performance of different enzymes towards the bioprocessing of aroma-related compounds was investigated and a strategy based on GC-FID analysis was developed to facilitate assessment of the stages of characterisation, screening and optimisation, including chiral ratio determination. Characterisation included activity assays (UV–Vis and GC-FID), protein quantification (NanoDrop spectrophotometry) and molar mass estimation (SDS-PAGE electrophoresis). Screening experiments assessed different enzymes, substrates, solvents, acyl donors or mediators. Aroma-related substrates comprised terpene and phenolic compounds. The enzymes tested included the lipases CALA (Sigma-Aldrich), NZ-435, LZ-TLIM, NC-ADL, LZ-CALBL and the laccases NZ-51003 and DL-IIS (all from Novozymes). Among those, NZ-435 and NZ-51003 had the highest activities in the characterisation stage and, along with CALA, achieved conversions above 70% for citronellol (lipases) or 50% for eugenol (laccases) at the screening stage. The lipases had preference for the primary alcohol and laccases for phenolic compounds, among the tested substrates. The transesterification reaction between the lipase CALA and the standards mixture (citronellol, menthol, linalool) was used to demonstrate the optimisation stage, where the best levels of temperature, enzyme and acyl donor concentrations were investigated. Optimum conditions were found to be 37–40 °C, 3–4 mg/mL of enzyme and 58–60% (v/v) vinyl acetate. Additional confirmation experiments using the same terpene standards mixture and citronella oil sample, gave a conversion of > 95% for citronellol after 1 h (for both, standards mixture and sample), and 20% or 74% for menthol after 1 h or 24 h, respectively. None of the tested enzymes demonstrated significant enantioselectivity under the tested conditions. The GC-FID approach demonstrated here was suitable to determine the reaction profiles and chiral ratio variations for biocatalysed reactions with aroma compounds in low complexity samples. Advanced separations will be applied to more complex samples in the future.

研究了不同酶对芳香相关化合物的生物加工的性能，并开发了一种基于 GC-FID 分析的策略，以促进对表征、筛选和优化阶段的评估，包括手性比率的确定。表征包括活性测定（UV-Vis 和 GC-FID）、蛋白质定量（NanoDrop 分光光度法）和摩尔质量估计（SDS-PAGE 电泳）。筛选实验评估了不同的酶、底物、溶剂、酰基供体或介质。与香气相关的底物包括萜烯和酚类化合物。测试的酶包括脂肪酶 CALA (Sigma-Aldrich)、NZ-435、LZ-TLIM、NC-ADL、LZ-CALBL 和漆酶 NZ-51003 和 DL-IIS（均来自 Novozymes）。在这些，NZ-435 和 NZ-51003 在表征阶段具有最高的活性，并且与 CALA 一起在筛选阶段实现了香茅醇（脂肪酶）的 70% 以上或丁香酚（漆酶）的 50% 以上的转化率。在测试的底物中，脂肪酶对酚类化合物的伯醇和漆酶具有偏好。脂肪酶 CALA 和标准混合物（香茅醇、薄荷醇、芳樟醇）之间的酯交换反应用于证明优化阶段，其中研究了温度、酶和酰基供体浓度的最佳水平。最佳条件为 37–40 °C、3–4 mg/mL 酶和 58–60% (v/v) 醋酸乙烯酯。使用相同的萜烯标准混合物和香茅油样品进行额外的确认实验，1 小时后香茅醇的转化率 > 95%（对于两者，标准混合物和样品），薄荷醇在 1 小时或 24 小时后分别为 20% 或 74%。在测试条件下，没有一种测试的酶表现出显着的对映选择性。这里展示的 GC-FID 方法适用于确定低复杂度样品中与芳香化合物的生物催化反应的反应曲线和手性比变化。未来，高级分离将应用于更复杂的样品。