| Thermostable ketoreductases have great potential in organic synthesis. Two aldo-keto reductases candidate genes tadhl and tadh2from genomes of Thermotoga sp. and Thermotoga maritime were identified based on genome sequence analysis, conserved sequence alignment and homology modeling. The ORF of the two genes contains861bp and825bp, encoding proteins TADH1and TADH2of33.23kDa and31.49kDa, respectively. Prokaryotic expression systems for these two gene were developed with pET28a. An effective recombinant enzyme purification protocol were also developed which include heat treatment (70℃,10min) followed by Ni+-sepharose affinity chromatography. TADH1and TADH2were shown to have some similar chara-cteristics. They were NADPH-dependent with broad substrate specificity and high activity on aldehyde, ketone ester (acid) and aromatic ketones. The optimum temperature and pH for both enzymes were90℃and optimum9.0, respectively. Metal ion Zn2+is a strong inhibitor while K+is an activator for both enzymes. SDS inhibited the activity of both enzymes strongly. Both enzymes showed good tolerance to organic solvents, including10%(v/v) acetonitrile, methanol, isopropyl alcohol, DMSO, etc., However, TADH2was shown to retain63%of its initial activity after15h at85℃and it’s more thermal-stable than TADH1. The presence of EDTA increased the activity of TADH1with89%, indicating it is metal-independent.With TADH1and TADH2as bio-catalysts, asymmetric reduction of trifluoroacetophenone, ethyl2-oxo-4-phenylbutyrate (EOPB) and ethyl4-chloroacetoacetate (COBE) were characterized, in which glucose dehy-drogenase and glucose were used for NADPH recycling. Both enzymes showed excellecnt selectivity on trifluoroacetophenone (e.e.p>99%),while lower selectivity on EOPB (e.e.p were68.85%and69.93%, respectively) and COBE (e.e.p86.76%and no slectivity, respectively). After reaction optimization (coenzyme concentration, temperature and pH), the productivity of trifluoroacetophenone by TADH2was enhanced from9.5%to68%. |
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