Rationally re-designed mutation of NAD-independent l-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli

Jiang Tianyi, Gao Chao, Dou Peipei, Ma Cuiqing, Kong Jian, Xu, Xu Ping

Description

NAD-independent l -lactate dehydrogenase ( l -iLDH) from Pseudomonas stutzeri SDM can potentially be used for the kinetic resolution of small aliphatic 2-hydroxycarboxylic acids. However, this enzyme showed rather low activity towards aromatic 2-hydroxycarboxylic acids. Results Val-108 of l -iLDH was changed to Ala by rationally site-directed mutagenesis. The l -iLDH mutant exhibited much higher activity than wide-type l -iLDH towards l -mandelate, an aromatic 2-hydroxycarboxylic acid. Using the engineered Escherichia coli expressing the mutant l -iLDH as a biocatalyst, 40 g·L -1 of dl -mandelic acid was converted to 20.1 g·L -1 of d -mandelic acid (enantiomeric purity higher than 99.5%) and 19.3 g·L -1 of benzoylformic acid. Conclusions A new biocatalyst with high catalytic efficiency toward an unnatural substrate was constructed by rationally re-design mutagenesis. Two building block intermediates (optically pure d -mandelic acid and benzoylformic acid) were efficiently produced by the one-pot biotransformation system.

Sujets

• Site-directed mutagenesis
• Optical resolution

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 21
• Langue: English

Extrait

Jianget al. Microbial Cell Factories2012,11:151 http://www.microbialcellfactories.com/content/11/1/151

R E S E A R C H

Open Access

Rationally redesigned mutation of NADindependent Llactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineeredEscherichia coli 1 1 1,2 1* 1 2,1* Tianyi Jiang , Chao Gao , Peipei Dou , Cuiqing Ma , Jian Kong and Ping Xu

Abstract Background:NADindependent Llactate dehydrogenase (LiLDH) fromPseudomonas stutzeriSDM can potentially be used for the kinetic resolution of small aliphatic 2hydroxycarboxylic acids. However, this enzyme showed rather low activity towards aromatic 2hydroxycarboxylic acids. Results:Val108 of LiLDH was changed to Ala by rationally sitedirected mutagenesis. The LiLDH mutant exhibited much higher activity than widetype LiLDH towards Lmandelate, an aromatic 2hydroxycarboxylic acid. Using the 1 engineeredEscherichia coliexpressing the mutant LiLDH as a biocatalyst, 40 g∙L of DLmandelic acid was 1 1 converted to 20.1 g∙L of Dmandelic acid (enantiomeric purity higher than 99.5%) and 19.3 g∙L of benzoylformic acid. Conclusions:A new biocatalyst with high catalytic efficiency toward an unnatural substrate was constructed by rationally redesign mutagenesis. Two building block intermediates (optically pure Dmandelic acid and benzoylformic acid) were efficiently produced by the onepot biotransformation system. Keywords:NADindependent Llactate dehydrogenase, Sitedirected mutagenesis, Optical resolution, Dmandelic acid

Background DMandelic acid, an aromatic 2hydroxycarboxylic acid, is a valuable chiral building block for the synthesis of various pharmaceuticals, such as antiobesity agents, antitumor agents, penicillins, and semisynthetic cepha losporins [13]. Chemical processes for mandelic acid production result in the racemic mixture of both stereo specific forms. Several biocatalytic methods, including lipase catalyzed enantioselective esterification [4], oxi doreductase catalyzed enantioselective oxidation, and microbial mediated enantioselective degradation [510], have been developed to prepare Dmandelic acid from racemic mandelic acid. Among these routes, oxidative resolution of racemic mandelic acid is much more

* Correspondence: [e-mail]; [e-mail] 1 State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China Full list of author information is available at the end of the article

promising because of its easy manipulation, exclusion of cosubstrate addition, and high yield. The NADindependent Llactate dehydrogenase (LiLDH) ofPseudomonas stutzeriSDM is located on the cell membrane, and quinine, as its electron acceptor, could be directly regenerated by the membrane electron trans port chain [11]. So it may exhibit higher catalytic effi ciency than the soluble FMNdependentαhydroxyacid dehydrogenases. Previous report showed that it exhibits high catalytic efficiency and enantioselectivity toward small aliphatic 2hydroxycarboxylic acids such as Llactate and L2hydroxybutanoate [12]. Cells of P. stutzeriSDM have been used in the kinetic resolution of lactate and 2hydroxybutanoate racemic mixtures to produce Dlactate and D2hydroxybutanoate [13,14]. Considering the similar structures of lactic acid and mandelic acid, LiLDH might also be able to catalyze the kinetic resolution of racemic mandelic acid (Figure 1).

© 2012 Jiang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.