Simple and efficient enzymatic procedure for p-coumaric acid synthesis: Complete bioconversion and biocatalyst recycling under alkaline condition

p-Coumaric acid (para-hydroxycinnamic acid, p-HCA), as a kind of natural active substance from plant cell walls, is widely used in medicine, food, and cosmetics with its antioxidant, anticancer, and other properties. Biological production of p-HCA can be achieved via one-step deamination of the L-tyrosine by tyrosine ammonia-lyase (TAL). Here, a new highly active TAL (Fc-TAL) from Flavobacterium columnare was identified via gene mining and heterologous expression. The purified Fc-TAL completely converts 22 mM L-tyrosine to p-HCA under alkaline condition (pH 9.5-10.0) at 55 degrees C. To increase enzymatic recyclability and reduce the bioproduction cost, Fc-TAL was immobilized on a silica nanospheres through silica-binding peptides (SiBP2) at pH 10. In comparison to free enzyme, the immobilized TALSiBP2 exhibited higher stability, over 50% activity was retained even after 10 consecutive catalytic cycles. Further experiments revealed that an aliquot of immobilized enzyme (about 200 mg TAL) can transform 20 g L-tyrosine into17.8 g p-HCA with 96% yields and 98% purity through 5 consecutive reaction cycles. Our studies not only provided a strategy for enzyme immobilization in alkaline condition, but also developed a green process for p-coumaric acid biosynthesis with the highest conversion rate and yield re-ported to date.