高活性酪氨酸解氨酶的表征及其在对香豆酸生物合成中应用

Characterization of highly active tyrosine ammonia lyase and its application in biosynthesis of p-coumaric acid

摘要:

对香豆酸(p-coumaric acid)具有抗菌、抗氧化和预防心血管疾病的作用，也是许多重要化合物的前体或中间体，被广泛应用于食品、化妆品和医药等领域。酪氨酸解氨酶(tyrosine ammonia-lyase,TAL)能直接催化酪氨酸脱氨生成对香豆酸。然而，缺少高活性和高底物耐受性的酪氨酸解氨酶限制了对香豆酸的高效生物合成。为了提高对香豆酸的合成能力，本研究挖掘了2个黄杆菌来源的酪氨酸解氨酶，分别是柱状黄杆菌(Flavobacterium columnare)来源的Fc-TAL2和顺天黄杆菌(Flavobacterium suncheonense)来源的Fs-TAL。异源表达纯化表征分析显示，Fc-TAL2和Fs-TAL的最适温度和最适pH相同，分别为55℃、pH9.5。在最适条件下，Fs-TAL的比酶活为82.47U/mg，而Fc-TAL2的比酶活为13.27U/mg。结构模拟和比对分析显示，内盖环上保守的Y50残基酚羟基朝向和到底物的距离是造成Fs-TAL活性高于Fc-TAL2的主要原因。全细胞催化研究进一步证实Fs-TAL具有较高活性和特异性，能够催化10g/L酪氨酸生成6.2g/L对香豆酸，产率达到67.9%。该研究丰富了酪氨酸解氨酶的酶资源，促进了对香豆酸及其衍生物的生产。

Abstract:

p-coumaric acid is one of the aromatic compounds that are widely used in food, cosmetics and medicine due to its properties of antibacterium, antioxidation and cardiovascular disease prevention. Tyrosine ammonia-lyase (TAL) catalyzes the deamination of tyrosine to p-coumaric acid. However, the lack of highly active and specific tyrosine ammonia lyase limits cost-effective microbial production of p-coumaric acid. In order to improve biosynthesis efficiency of p-coumaric acid, two tyrosine ammonia-lyases, namely Fc-TAL2 derived from Flavobacterium columnare and Fs-TAL derived from Flavobacterium suncheonense, were selected and characterized. The optimum temperature (55℃) and pH (9.5) for Fs-TAL and Fc-TAL2 are the same. Under optimal conditions, the specific enzyme activity of Fs-TAL and Fc-TAL2 were 82.47 U/mg and 13.27 U/mg, respectively. Structural simulation and alignment analysis showed that the orientation of the phenolic hydroxyl group of the conserved Y50 residue on the inner lid loop and its distance to the substrate were the main reasons accounting for the higher activity of Fs-TAL than that of Fc-TAL2. The higher activity and specificity of Fs-TAL were further confirmed via whole-cell catalysis using recombinant Escherichia coli, which could convert 10 g/L tyrosine into 6.2 g/L p-coumaric acid with a yield of 67.9%. This study provides alternative tyrosine ammonia-lyases and may facilitate the microbial production of p-coumaric acid and its derivatives.