Characterization of an Immobilized Amino Acid Racemase for Potential Application in Enantioselective Chromatographic Resolution Processes

Enantioselective resolution processes can be improved by integration of racemization. Applying environmentally friendly enzymatic racemization under mild conditions is in particular attractive. Owing to the variety of enzymes and the progress in enzyme engineering, suitable racemases can be found for many chiral systems. An amino acid racemase (AAR) from P. putida KT2440 is capable of processing a broad spectrum of amino acids at fast conversion rates. The focus of this study is the evaluation of the potential of integrating AAR immobilized on Purolite ECR 8309 to racemize L- or D-methionine (Met) within an enantioselective chromatographic resolution process. Racemization rates were studied for different temperatures, pH values, and fractions of organic co-solvents. The long-term stability of the immobilized enzyme at operating and storage conditions was found to be excellent and recyclability using water with up to 5 vol% ethanol at 20 °C could be demonstrated. Packed as an enzymatic fixed bed reactor, the immobilized AAR can be coupled with different resolution processes; for instance, with chromatography or with preferential crystallization. The performance of coupling it with enantioselective chromatography is estimated quantitatively, exploiting parametrized sub-models. To indicate the large potential of the AAR, racemization rates are finally given for lysine, arginine, serine, glutamine, and asparagine

Improvement of acetaldehyde production in Zymomonas mobilis by engineering of Its aerobic metabolism

Acetaldehyde is a valuable product of microbial biosynthesis, which can be used by the chemical industry as the entry point for production of various commodity chemicals. In ethanologenic microorganisms, like yeast or the bacterium Zymomonas mobilis, this compound is the immediate metabolic precursor of ethanol. In aerobic cultures of Z. mobilis, it accumulates as a volatile, inhibitory byproduct, due to the withdrawal of reducing equivalents from the alcohol dehydrogenase reaction by respiration. The active respiratory chain of Z. mobilis with its low energy-coupling efficiency is well-suited for regeneration of NAD+ under conditions when acetaldehyde, but not ethanol, is the desired catabolic product. In the present work, we sought to improve the capacity Z. mobilis to synthesize acetaldehyde, based on predictions of a stoichiometric model of its central metabolism developed herein. According to the model analysis, the main objectives in the course of engineering acetaldehyde producer strains were determined to be: (i) reducing ethanol synthesis via reducing the activity of alcohol dehydrogenase (ADH), and (ii) enhancing the respiratory capacity, either by overexpression of the respiratory NADH dehydrogenase (NDH), or by mutation of other components of respiratory metabolism. Several mutants with elevated respiration rate, decreased alcohol dehydrogenase activity, or a combination of both, were obtained. They were extensively characterized by determining their growth rates, product yields, oxygen consumption rates, ADH, and NDH activities, transcription levels of key catabolic genes, as well as concentrations of central metabolites under aerobic culture conditions. Two mutant strains were selected, with acetaldehyde yield close to 70% of the theoretical maximum value, almost twice the previously published yield for Z. mobilis. These strains can serve as a basis for further development of industrial acetaldehyde producers