Purification and partial characterization of Lactobacillus species SK007 lactate dehydrogenase (LDH) catalyzing phenylpyruvic acid (PPA) conversion into phenyllactic acid (PLA)
Purification and partial characterization of Lactobacillus species SK007 lactate dehydrogenase (LDH) catalyzing phenylpyruvic acid (PPA) conversion into phenyllactic acid (PLA)
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乳酸菌 SK007 乳酸脱氢酶 (LDH) 催化苯丙酮酸 (PPA) 转化为苯乳酸 (PLA) 的纯化和部分表征
DOI：
10.1021/jf0731503
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发表时间：
2008-04-09
影响因子：
6.1
通讯作者：
Zhang, Tao
中科院分区：
文献类型：
--
作者：
Li, Xingfeng;Jiang, Bo;Zhang, Tao
苯乳酸（PLA）是一种由乳酸菌（LAB）合成的新型抗菌化合物，而以苯丙氨酸（PPA）为原料生产PLA是一条有效的途径。在这项工作中，乳酸脱氢酶（LDH），它催化PPA到PLA的还原，已被纯化到同质从无细胞提取的乳杆菌属SK 007的沉淀与硫酸铵，离子交换，凝胶过滤色谱。纯化的酶具有分子量为78 kDa（尺寸排阻色谱法）或39 kDa（SIDS-PAGE）的二聚体形式。在每个纯化步骤中，PPA与丙酮酸的酶活性的比率几乎不变，这表明在乳杆菌SK 007中，LDH负责PPA转化为PLA。研究了乳酸杆菌SK 007的生长细胞、无细胞提取物和纯化LDH对PPA转化为PLA的HPLC图谱。结果表明，NADH的存在被发现是必要的酶法生产聚乳酸从PPA。纯化的LDH在pH6.0和40 ℃下对PPA显示最佳活性。酶对PPA和丙酮酸的Km值分别为1.69和0.32 mM。此外，由于其他筛选的LAB菌株表现出相对较高的。乳酸脱氢酶（LDH）对PPA的活性也能产生大量的聚乳酸，因此可作为筛选产聚乳酸菌的一个指标。
Phenyllactic acid (PLA) is a novel antimicrobial compound synthesized by lactic acid bacteria (LAB), and its production from phenylpyruvic acid (PPA) is an effective approach. In this work, a lactate dehydrogenase (LDH), which catalyzes the reduction of PPA to PLA, has been purified to homogeneity from a cell-free extract of Lactobacillus sp. SK007 by precipitation with ammonium sulfate, ion exchange, and gel filtration chromatography. The purified enzyme had a dimeric form with a molecular mass of 78 kDa (size exclusion chromatography) or 39 kDa (SIDS-PAGE). The ratio of enzyme activity with PPA to that with pyruvate being almost invariable at every purification step indicated that, in Lactobacillus sp. SK007, LDH is responsible for the conversion of PPA into PLA. HPLC profiles of PPA transformation into PLA by growing cells, cell-free extract, and purified LDH of Lactobacillus sp. SK007 were also investigated. Results showed that the presence of NADH was found to be necessary for the enzymatic production of PLA from PPA. The purified LDH displayed optimal activity for PPA at pH 6.0 and 40 degrees C. The K-m values of the enzyme for PPA and pyruvate were 1.69 and 0.32 mM, respectively. Moreover, because other screened LAB strains exhibiting relatively high. LDH activity toward PPA produced also considerable amounts of PLA, LDH activity for PPA could be therefore used as a screening marker for PLA-producing LAB.