Cofactor engineering of <i>Lactobacillus brevis</i> alcohol dehydrogenase by computational design

Machielsen, Ronnie; Looger, Loren L.; Raedts, John; Dijkhuizen, Sjoerd; Hummel, Werner; Hennemann, Hans-Georg; Daußmann, Thomas; Oost, John van der

doi:10.1002/elsc.200800046

Cited by 24 publications

(23 citation statements)

References 24 publications

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“…6). For the measurements in cuvettes the obtained temperature optimum for the LbADH wild-type of about 55°C is in good agreement with reported data [48,49]. However, activity measurements at temperatures above 60°C were not reliable due to fast cofactor deactivation showing half-lives of about 30 min [44].…”

Section: Enzyme Activities Deviate Due To Spatial and Temporal Tempersupporting

confidence: 89%

Enzyme activity deviates due to spatial and temporal temperature profiles in commercial microtiter plate readers

Grosch

Sieben

Lattermann

et al. 2016

Biotechnology Journal

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Microtiter plates (MTP) and automatized techniques are increasingly applied in the field of biotechnology. However, the susceptibility of MTPs to edge effects such as thermal gradients can lead to high variation of measured enzyme activities. In an effort to enhance experimental reliability, to quantify, and to minimize instrument-caused deviations in enzyme kinetics between two MTP-readers, we comprehensively quantified temperature distribution in 96-well MTPs. We demonstrated the robust application of the absorbance dye cresol red as easily applicable temperature indicator in cuvettes and MTPs and determined its accuracy to ±0.16°C. We then quantified temperature distributions in 96-well MTPs revealing temperature deviations over single MTP of up to 2.2°C and different patterns in two commercial devices (BioTek Synergy 4 and Synergy Mx). The obtained liquid temperature was shown to be substantially controlled by evaporation. The temperature-induced enzyme activity variation within MTPs amounted to about 20 %. Activity deviations between MTPs and to those in cuvettes were determined to 40 % due to deviations from the set temperature in MTPs. In conclusion, we propose a better control of experimental conditions in MTPs or alternative experimental systems for reliable determination of kinetic parameters for bioprocess development.

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Section: Enzyme Activities Deviate Due To Spatial and Temporal Tempersupporting

confidence: 89%

Enzyme activity deviates due to spatial and temporal temperature profiles in commercial microtiter plate readers

Grosch

Sieben

Lattermann

et al. 2016

Biotechnology Journal

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“…A number of reactions were found to be involved in more than one pathway such as reactions catalyzed by alcohol dehydrogenase (ADH) enzymes (EC 1.1.1.1) that can be found in carbohydrate, lipid and amino acid metabolisms in agreement with literature [69]. 37.3% of the total reactions were unique to C. salexigens i OA584 most of which were from amino acid (38 reactions) metabolism followed by carbohydrate metabolism (31 reactions).…”

Section: Resultssupporting

confidence: 73%

Genome-scale reconstruction of metabolic network for a halophilic extremophile, Chromohalobacter salexigens DSM 3043

2011

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BackgroundChromohalobacter salexigens (formerly Halomonas elongata DSM 3043) is a halophilic extremophile with a very broad salinity range and is used as a model organism to elucidate prokaryotic osmoadaptation due to its strong euryhaline phenotype.ResultsC. salexigens DSM 3043's metabolism was reconstructed based on genomic, biochemical and physiological information via a non-automated but iterative process. This manually-curated reconstruction accounts for 584 genes, 1386 reactions, and 1411 metabolites. By using flux balance analysis, the model was extensively validated against literature data on the C. salexigens phenotypic features, the transport and use of different substrates for growth as well as against experimental observations on the uptake and accumulation of industrially important organic osmolytes, ectoine, betaine, and its precursor choline, which play important roles in the adaptive response to osmotic stress.ConclusionsThis work presents the first comprehensive genome-scale metabolic model of a halophilic bacterium. Being a useful guide for identification and filling of knowledge gaps, the reconstructed metabolic network iOA584 will accelerate the research on halophilic bacteria towards application of systems biology approaches and design of metabolic engineering strategies.

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“…[5] Overexpression of the transhydrogenase in an engineered E. coli strain might cause ah igherm etabolic load and more energy requirements. [8] The enzyme l-aspartate-b-Scheme1.Three cascade reactionsf or the production of l-homoserine. [7] In addition, somes tudies suggested that NAD(H)-dependent enzymes are preferable in industry,d ue to lower costs and greater stability.…”

Section: Introductionmentioning

confidence: 99%

Mutagenesis of Key Residues in the Binding Center of l‐Aspartate‐β‐Semialdehyde Dehydrogenase from Escherichia coli Enhances Utilization of the Cofactor NAD(H)

Wang

Duan

et al. 2015

ChemBioChem

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L-Aspartate-β-semialdehyde dehydrogenase (ASADH) is a key enzyme in the aspartate pathway. In bacteria, ASADH is highly specific for the cofactor NADP(+) rather than NAD(+). Limited information on cofactor utilization is available, and neither the wild-type protein nor the available mutants could utilize NAD(+) efficiently. In this study, we identified several residues crucial for cofactor utilization by Escherichia coli ASADH (ecASADH) by mutating residues within the cofactor binding center. Among the investigated mutants, ecASADH-Q350N and ecASADH-Q350N/H171A, which exhibited markedly improved NAD(+) utilization, were further investigated by various biochemical approaches and molecular modeling. Relative to the wild type, the two mutants showed approximately 44-fold and 66-fold increases, respectively, in the constant kcat /Km of NAD(+). As desired, they could also utilize NADH efficiently to synthesize l-homoserine in cascade reactions in vitro.

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Cofactor engineering of Lactobacillus brevis alcohol dehydrogenase by computational design

Cited by 24 publications

References 24 publications

Enzyme activity deviates due to spatial and temporal temperature profiles in commercial microtiter plate readers

Enzyme activity deviates due to spatial and temporal temperature profiles in commercial microtiter plate readers

Genome-scale reconstruction of metabolic network for a halophilic extremophile, Chromohalobacter salexigens DSM 3043

Mutagenesis of Key Residues in the Binding Center of l‐Aspartate‐β‐Semialdehyde Dehydrogenase from Escherichia coli Enhances Utilization of the Cofactor NAD(H)

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