荚膜红细菌来源的酪氨酸酚裂解酶的酶学性质

Purification and enzymatic properties of tyrosine phenol lyase from Rhodobacter capsulatus

L-酪氨酸作为人体非必需氨基酸之一,是合成苯丙素类化合物等多种高价值化学产品的重要前体物质。因其可作为营养补充剂,以及多肽类激素、抗生素、L-多巴、黑色素、对羟基肉桂酸等产品的原料,被广泛应用于食品、化工、饲料和医药等行业。为了促进L-酪氨酸的高效生产,在大肠杆菌(Escherichia coli)中异源表达来源于荚膜红细菌(Rhodobacter capsulatus)的酪氨酸酚裂解酶(Tyrosine phenol lyase,TPL),并进行了诱导条件优化、酶活测定和酶学性质分析。结果表明,来源于荚膜红细菌的TPL最适pH为8.5;最适温度为40℃;酶活和比酶活分别为0.29 U/m L和0.29 U/mg;体外全细胞催化的酶活为0.41 U/g;反应10 h生成L-酪氨酸产量为0.30 g/L。结果表明,来源于R.capsulatus的TPL在大肠杆菌中成功表达,为后续提高表达量及分子改造提高催化性能的研究奠定基础。

L-tyrosine,one of the non-essential amino acids for human,is an important precursor for numerous high-value chemical products,such as phenylpropanoids. L-tyrosine is widely used in food,chemical,feed and pharmaceutical industries as the nutritional supplements and the raw materials for peptide hormones,antibiotics,L-DOPA,melanin,p-hydroxycinnamic acid. The tyrosine phenol lyase（ TPL） derived from Rhodobacter capsulatus was expressed in Escherichia coli（ named pET28 a RcTPL） to promote the efficient production of L-tyrosine. Optimization of induction conditions,determination of enzyme activity and analysis of enzymatic properties were performed. The optimum catalytic p H of TPL determined by purified Rc TPL was 8. 5 and the optimum catalytic temperature was determined to be 40 ℃. Furthermore,enzyme activity of purified enzyme and whole-cell catalysis were analyzed. The enzyme activity and the specific enzyme activity of purified Rc TPL was 0. 29 U/mL and 0. 29 U/mg,respectively. The enzyme activity of whole-cell catalysis was 0. 41 U/g,while the titer of L-tyrosine was 0. 30 g/L after reaction for 10 hours. The results showed that TPL from Rhodobacter capsulatus was successfully expressed in E. coli,which laid the foundation for further studies on increasing the expression level and improving the catalytic performance based on molecular transformation.