A new bacterial L-amino acid oxidase with a broad substrate specificity: purification and characterization
A new bacterial L-amino acid oxidase with a broad substrate specificity: purification and characterization
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DOI：
10.1016/s0141-0229(02)00072-8
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发表时间：
2002-07-01
影响因子：
3.4
通讯作者：
Hummel, W
中科院分区：
文献类型：
--
作者：
Geueke, B;Hummel, W
革兰氏阳性细菌不透明红球菌DSM 43250产生具有非常广泛的底物特异性的L-氨基酸氧化酶（L-AAO）。该酶已被纯化至均一，并进行了详细的生化表征。测定了L-AAO基因的全序列，并推测了L-AAO的一级结构。凝胶过滤法测得天然酶的分子量为99 kDa，MALDI-TOF/MS测得的分子量为54.2/108.5 kDa，SDS/PAGE后计算的亚基分子量为53.2 kDa。辅酶结合基序G-X-G-X-X-G是已知的所有L-AAO被发现非常接近的N-末端的蛋白质。L-AAO氧化43种测试的L-氨基酸中的39种。测定了这些L-氨基酸中的16种的动力学数据，揭示了底物如L-苯丙氨酸、L-亮氨酸、L-瓜氨酸和L-赖氨酸的Km值在15-30 μ M范围内。L-AAO的稳定性可以通过在甘氨酸/NaOH缓冲液中储存或孵育酶来增加。该蛋白质的pI为4.8，对作为底物的L-丙氨酸、L-苯丙氨酸和L-亮氨酸测得的最适pH值为pH 8-9。考察了该方法拆分外消旋体的能力，得到了对映体过量>99%的D-氨基酸。(C)2002年由Elsevier Science Inc.出版
The Gram-positive bacterium Rhodococcus opacus DSM 43250 produces an L-amino acid oxidase (L-AAO) with a very broad substrate specificity. This enzyme has been purified to homogeneity and a detailed biochemical characterization was carried out. The complete nucleotide sequence of the L-AAO gene was determined and the primary structure Of L-AAO was deduced. The molecular mass of the native enzyme was 99 kDa determined by gel filtration, 54.2/108.5 kDa measured by MALDI-TOF/MS, 53.2 kDa for the subunit calculated after SDS/PAGE. The coenzyme-binding motif G-X-G-X-X-G which is known for all L-AAOs was found very close to the N-terminus of the protein. L-AAO oxidized 39 out of 43 tested L-amino acids. The kinetic data for 16 of these L-ammo acids were determined revealing K-m-values in the range of 15-30 muM for substrates like L-phenylalanine, L-leucine, L-citrulline and L-lysine. The stability Of L-AAO can be increased by storage or incubation of the enzyme in glycine/NaOH buffer. The protein has a pI of 4.8 and a slightly basic pH-optimum at pH 8-9 measured for L-alanine, L-phenylalanine and L-leucine as substrates. The ability for resolution of racemic mixtures was investigated and D-amino acids with an enantiomeric excess of >99% were obtained. (C) 2002 Published by Elsevier Science Inc.