Hess Matthias, Antranikian Garabed
Institute of Technical Microbiology, Hamburg University of Technology, Kasernenstr. 12, 21073, Hamburg, Germany.
Appl Microbiol Biotechnol. 2008 Jan;77(5):1003-13. doi: 10.1007/s00253-007-1238-8. Epub 2007 Nov 8.
The adhA gene of the extreme thermoacidophilic Archaeon Picrophilus torridus was identified by the means of genome analysis and was subsequently cloned in Escherichia coli. PTO 0846, encoding AdhA, consists of 954 bp corresponding to 317 aa. Sequence comparison revealed that the novel biocatalyst has a low sequence identity (<26%) to previously characterized enzymes. The recombinant alcohol dehydrogenase was purified using hydroxyapatite, and alcohol oxidative activity of the purified AdhA was measured over a wide pH and temperature range with maximal activity at 83 degrees C and pH 7.8. Detailed analysis suggests that the active AdhA is a multimer, consisting of 12 identical subunits, with a molecular mass of 35 kDa each. AdhA represents the first dodecameric alcohol dehydrogenase characterized until to date. AdhA is able to oxidize primary and secondary alcohols with ethanol and 1-phenylalcohol as preferred substrates and NAD(+) as preferred cofactor. In addition, isopropanol, which has been used successfully as cosubstrate in cofactor regeneration, is oxidized as well by AdhA. Besides being thermostable (t (1/2) = 42 min at 70 degrees C), AdhA is also active in the presence of increased concentrations of urea (up to 5 M) and in the presence of organic solvents [up to 50% (v/v)] commonly used for organic synthesis.
通过基因组分析手段鉴定了嗜热嗜酸古菌嗜热栖热菌的adhA基因，随后将其克隆到大肠杆菌中。编码AdhA的PTO 0846由954个碱基对组成，对应317个氨基酸。序列比较显示，这种新型生物催化剂与先前已鉴定的酶的序列同一性较低（<26%）。重组乙醇脱氢酶用羟基磷灰石进行纯化，在较宽的pH和温度范围内测定纯化后的AdhA的乙醇氧化活性，其在83℃和pH 7.8时活性最高。详细分析表明，有活性的AdhA是一种多聚体，由12个相同的亚基组成，每个亚基的分子量为35 kDa。AdhA是迄今为止所鉴定的首个十二聚体乙醇脱氢酶。AdhA能够氧化伯醇和仲醇，以乙醇和1-苯乙醇作为优选底物，以NAD(+)作为优选辅因子。此外，已成功用作辅因子再生共底物的异丙醇也能被AdhA氧化。除了具有热稳定性（70℃下t(1/2)=42分钟）外，AdhA在尿素浓度增加（高达5 M）以及在常用于有机合成的有机溶剂[高达50%（v/v）]存在的情况下也具有活性。
