Purification and characterization of thermostable H2O2-forming NADH oxidase from 2-phenylethanol-assimilating Brevibacterium sp. KU1309
Purification and characterization of thermostable H2O2-forming NADH oxidase from 2-phenylethanol-assimilating Brevibacterium sp. KU1309
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DOI：
10.1007/s00253-008-1535-x
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发表时间：
2008-06
影响因子：
5
通讯作者：
J. Hirano;K. Miyamoto;H. Ohta
中科院分区：
文献类型：
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作者：
J. Hirano;K. Miyamoto;H. Ohta
从土壤细菌短杆菌（Brevibacteriumsp.）KU 1309，其能够在有氧条件下在含有2-苯基乙醇作为唯一碳源的培养基中生长。该酶催化NADH氧化为NAD+，涉及O2到H2 O2的双电子还原。凝胶过滤法测得该酶的分子量为102 kDa，SDS-PAGE法测得该酶的分子量为57 kDa，表明该酶是由一个亚基组成的同源二聚体。该酶在高达70°C和7至11的广泛pH范围内稳定。铵盐的加入使酶活力提高了10倍左右，而Zn ~（2+）、Cu ~（2+）、Hg ~（2+）、Ag ~+对酶活力的抑制率分别为39%、41%、72%、37%。该酶作用于NADH，但不作用于NADPH。利用该酶再生NAD+，使扁桃酸或L-苯丙氨酸的选择性氧化成为可能。这种热稳定的酶有望作为一种有用的生物催化剂用于NAD+的回收。
A cytoplasmic NADH oxidase (NOX) was purified from a soil bacteria,Brevibacteriumsp. KU1309, which is able to grow in the medium containing 2-phenylethanol as the sole source of carbon under an aerobic condition. The enzyme catalyzed the oxidation of NADH to NAD+involving two-electron reduction of O2to H2O2. The molecular weight of the enzyme was estimated to be 102 kDa by gel filtration and 57 kDa by SDS-PAGE, which indicates that the NOX was a homodimer consisting of a single subunit. The enzyme was stable up to 70°C at a broad range of pH from 7 to 11. The enzyme activity increased about ten-fold with the addition of ammonium salt, while it was inhibited by Zn2+(39%), Cu2+(41%), Hg2+(72%) and Ag+(37%). The enzyme acts on NADH, but not on NADPH. The regeneration of NAD+utilizing this enzyme made selective oxidation of mandelic acid orl-phenylalanine possible. This thermostable enzyme is expected to be applicable as a useful biocatalyst for NAD+recycling.