One-Pot, Two-Step Biocatalytic Cascade Transformation of Biobased Vanillyl Alcohol and <i>p</i> -Anisyl Alcohol into Aromatic Aldehydes and Amines Using Mutant Aryl-Alcohol Oxidase and ω-Transaminase

Aromatic amines such as vanillylamine (VAN) and p-anisylamine (ASN) are widely applied in pharmaceuticals, fine chemicals, food additives, and function materials. However, the synthesis of VAN and ASN is still mainly achieved through traditional chemical methods, which typically suffer from high energy consumption, toxic reagents, and poor selectivity, thereby increasing production costs and limiting scalability. To overcome these issues, this study employs a one-pot, two-step tandem biocatalytic route to efficiently convert biobased vanillyl alcohol (VOL) and p-anisyl alcohol (ASA) into VAN and ASN, respectively. To improve the thermal stability of aryl-alcohol oxidase from Mycobacterium sp. M1601, a site-directed mutagenesis strategy targeting the active center was employed, yielding a mutant (AOYF) with a 6-fold higher half-life (t1/2) than the wild type at 50 °C. In the α-cyclodextrin (α-CD) system, the recombinant E. coli AOYF efficiently catalyzed the oxidation of high concentrations of VOL (400 mM) and ASA (100 mM) to yield 86.6% vanillin (VAL) and 100% p-anisaldehyde (ASD), respectively. Furthermore, in a 5 mL reaction system, recombinant Escherichia coli CV whole cells harboring ω-transaminase catalyzed the efficient amination of vanillyl alcohol-derived vanillin (0.116 g) and p-anisyl alcohol-derived p-anisaldehyde (0.069 g), affording 0.115 g of vanillylamine and 0.069 g of p-anisylamine, respectively. Under mild and green conditions, a one-pot, two-step whole-cell biocatalytic system was developed for the efficient conversion of VOL and ASA into high-value VAN and ASN, respectively. This catalytic strategy provides an efficient and sustainable route for upgrading biobased VOL and ASA into high-value chemical products.