Lignin is a plentiful and readily accessible renewable resource. Vanillylamine is a crucial raw material used to synthesize pharmaceuticals and high-value furan compounds that can be acquired by aminating lignin-derived vanillin (Van). However, effectually achieving the biocatalytic synthesis of vanillylamine has remained challenging. In this study, a dimethyl sulfoxide (DMSO)-H 2 O (1:9, vol/vol) bioreaction medium was constructed, and a recombinant E. coli ATA1012 carrying ω-transaminase from Caulobacter sp. D5 was used as the ω-transaminase biocatalyst to acquire the effectual biocatalytic synthesis of vanillylamine. Under optimized bioreaction conditions (37 ℃ and pH 7.5) by supplementary of isopropylamine (IPA) (Van/IPA = 1:5, mol/mol), 80–100 mM Van could be effectually converted by ATA1012 whole cells in DMSO-H 2 O (1:9, vol/vol) within 12 h, yielding 91.2 %–95.4 % vanillylamine, with >99 % selectivity. An efficient amination process was developed using ATA1012 with superior transaminase catalytic activity and substrate tolerance to effectively convert Van to vanillylamine in a DMSO-H 2 O medium.
Junhua Di,Yizhen Zhang,Bright Uwse,Paul Arnaud Yao Koffi,Yu-Cai He,Cuiluan Ma
To enhance the application potential of ω-transaminase ATA1012 in the efficient bioamination of biobased aldehydes, this study performed site-directed mutagenesis at key sites in the active center and flexible loop region, resulting in the mutant Q25F with significantly improved thermostability. The half-life of Q25F increased from 4.2 h to 25.1 h at 37 °C and from 0.6 h to 3.3 h at 50 °C. Benefiting from its enhanced stability, Q25F efficiently converted lignin-derived vanillin (260 mM) to vanillylamine within 12 h, achieving a yield of 92.6% and a selectivity >99%. Kinetic analysis revealed a Km of 0.2 mM for vanillin, a kcat of 8.9 s −1 , and a catalytic efficiency (kcat/Km) of 33.1 M −1 s −1 . Furthermore, by optimizing the mutagenesis strategy, engineered strains capable of efficiently catalyzing the transamination of biomass-derived furfural (FAL) and 5-hydroxymethylfurfural (HMF) into biobased amines were constructed. This study establishes a site-directed mutagenesis approach for enhancing the thermostability of ω-transaminase, providing an effective route for the high-value bioconversion of carbohydrates and lignin in lignocellulosic biomass.
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