Practical applications of hydrogenase I from Pyrococcus furiosus for NADPH generation and regeneration

被引:74
作者
Mertens, R
Greiner, L
van den Ban, ECD
Haaker, HBCM
Liese, A
机构
[1] Forschungszentrum Julich, Inst Biotechnol 2, D-52425 Julich, Germany
[2] Wageningen Univ Agr, Dept Biochem, NL-6703 HA Wageningen, Netherlands
关键词
Pyrococcus furiosus hydrogenase; NADPH; cofactor regeneration; dihydrogen; enantioselective reduction;
D O I
10.1016/S1381-1177(03)00071-7
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The soluble hydrogenase I (H(2):NADP(+) oxidoreductase, EC 1.18.99.1) from the marine hyperthermophilic strain of the archaeon Pyrococcus furiosus was partially purified by anion-exchange chromatography. This P furiosus hydrogenase I preparation (PF H(2)ase I) has been used as biocatalyst in the enzymatic production and regeneration of beta-1,4-nicotinamide adenindinucleotide phosphate, reduced form (NADPH), utilizing cheap molecular hydrogen and forming protons as the only side-product. Any excess of dihydrogen can be removed easily. It could be demonstrated, that this hyperthermophilic hydrogenase exhibits a high stability under reaction conditions. Generation as well as regeneration of NADPH were performed in batch and repetitive batch experiments with recyclisation of the biocatalyst. In two repetitive batch-series 6.2 g l(-1) NADPH could be produced with a total turnover number (ttn: mol produced NADPH/mol consumed enzyme) of 10,000. Utilizing the thermophilic NADPH-dependent alcohol dehydrogenase from Thermoanaerobium spec. (ADH M) coupled to the PF H(2)ase I in situ NADPH-regenerating system, two prochiral model substrates, acetophenone and (2S)-hydroxy-1-phenyl-propanone (HPP), were quantitatively reduced to the corresponding (S)-alcohol and (1R,2S)-diol. An e.e. >99.5% and d.e. >98%, respectively, with total turnover numbers (ttn: mol product/mol consumed cofactor NADP(+)) of 100 and 160 could be reached. (C) 2003 Elsevier B.V. All rights reserved.
引用
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页码:39 / 52
页数:14
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