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Biocatalytic Synthesis of N-trans-feruloyltyramine Using an Amide Bond Synthetase with an ATP Recycling

Zhao Bingshan,Micklefield Jason,Wang Yonghua,Wang Fanghua

N-trans -feruloyltyramine (FLA) is one kind of phenylpropanoid compound found in various plants. Numerous studies have confirmed that it exhibits a wide range of physiological functions, such as antioxidant, ɑ-glucosidase inhibition, and anti-inflammatory activity. However, the low content of FLA in plants greatly limits its potential use in food and pharmaceutical industries. It is, therefore, very important to establish an effective synthesis of FLA. In this study, a green and efficient method to synthesize FLA was sought using an amide bond synthetase (ABS) biocatalyst. Ten kinds of ABS enzymes, including AlCfaL from Azospirillum lipoferum , were screened as the potential biocatalysts for the production of FLA. To obtain optimum reaction conditions, the effects of various parameters on conversion of FLA were firstly evaluated. Under the optimum conditions using 1 mM N-trans -ferulic acid, 50 mM tyramine (substrate ratio of 1:50), 10 mM MgCl₂, 8 mM ATP, and 35 µM AlCfaL enzyme at 30 °C with a shaking speed of 500 r/min for 48 h, maximum conversion rate of 74% was reached. Given that the amidation reaction is mediated by relative expensive ATP, we further optimized reaction systems to incorporate an ATP recycling system consisting of a polyphosphate kinase enzyme (CHU) and an inexpensive polyphosphate (PolyP) as the phosphate donor. Response surface methodology (RSM) based on five-level, five-variable central composite design (CCD) was used to evaluate the optimal parameters for the production of FLA. The effects of AMP, PolyP, AlCfaL, CHU concentrations, and reaction time on the conversion rate of FLA were analyzed. The optimum conditions derived via RSM were 7.12 mM AMP, 5.96 mg/mL PolyP, 39.72 μM AlCfaL, 27.68 μM CHU, and a reaction time of 36 h. Validation experiments conducted under these optimized conditions yielded an actual conversion rate of 63.5%, which compared well to the maximum predicted value of 64.2%.