Leucine dehydrogenase (LDH) and formate dehydrogenase (FDH) were assembled together based on a high-affinity interaction between two different cohesins in a miniscaffoldin and corresponding dockerins in LDH and FDH. The miniscaffoldin with two enzymes was further absorbed by regenerated amorphous cellulose (RAC) to form a bifunctional enzyme complex (miniscaffoldin with LDH and FDH adsorbed by RAC, RSLF) in vitro. The enzymatic characteristics of the bifunctional enzyme complex and free enzymes mixture were systematically compared. The synthesis of L-tert-leucine by the RSLF and free enzyme mixture were compared under different concentrations of enzymes, coenzyme, and substrates. The initial L-tert-leucine production rate by RSLF was enhanced by 2-fold compared with that of the free enzyme mixture. Ninety-one grams per liter of L-tert-leucine with an enantiomeric purity of 99 % e.e. was obtained by RSLF multienzyme catalysis. The results indicated that the bifuntional enzyme complex based on cohesin-dockerin interaction has great potential in the synthesis of L-tert-leucine.
l‐tert‐leucine, an intermediate in the synthesis of several chiral drugs, is mainly produced by bioconversion, in which leucine dehydrogenase (LeuDH) is the key enzyme. A novel leudh was obtained from the marine bacterium Alcanivorax dieselolei B‐5(T) by PCR. The gene encoded a novel cold‐adapted LeuDH that showed low similarity (less than 50%) to any known proteins; the highest similarity (42.6%) was found for LeuDH from Bacillus cereus. The cold‐adapted LeuDH showed optimal activity at 30℃ and pH 6.5, and was identified to be extremely cold‐adaptive, retaining over 90% activity in the temperature range of 0–37℃. The enzyme exhibited better stability in weak alkali environment (pH 6.0–8.5) than Thermoactinomyces intermedius LeuDH. The best substrate concentration was established, and LeuDH conversion rate in catalyzing trimethylpyruvic acid to l‐tert‐leucine was 54.6%. The cold activity and its ability to produce l‐tert‐leucine with excellent performance of enantiomers of choice make it a promising biocatalyst for industrial application under extreme conditions.
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