Optimization of a reduced enzymatic reaction cascade for the production of L-alanine

Gmelch, Tobias Johann; Sperl, Josef; Sieber, Volker

doi:10.1038/s41598-019-48151-y

Cited by 28 publications

(41 citation statements)

References 46 publications

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“…In addition to the observation that product concentration and reaction rate of a cascade are not automatically dependent on each other, the stability and activity of enzymes do not necessarily correlate. This was demonstrated for a reaction cascade for the synthesis of L-alanine, in which one enzyme of the five-enzyme cascade was exchanged with a 40-fold more active enzyme, but at the expense of lower thermostability and simultaneously reduced TTNs [21]. Therefore, a careful selection of requirements and a ranking of the optimization goals must be made, which need to be adjusted during the process.…”

Section: Optimization Goalsmentioning

confidence: 99%

“…Enzyme homologues originating from different organisms may differ in their catalytic properties [64,65], but their careful selection can substantially increase the performance of the reaction system. This was demonstrated during the development of an L-alanine producing cascade from glucose by five enzymes [21]. The previously selected enzymes [61] were sought to be replaced with mostly more active homologues and variants, which led to an improved economy in terms of enzyme loading and stability.…”

Section: Enzyme-selection and -Optimizationmentioning

confidence: 99%

“…What performance parameter do we optimize for? The literature provides many approaches ranging from experimental-based optimizations [19][20][21] to in silico approaches [22,23] as well as combinations of both [24,25]. This review aims to give an overview on the concepts for optimizing enzyme cascade reactions, and covers both experimental as well as in silico approaches.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Getting the Most Out of Enzyme Cascades: Strategies to Optimize In Vitro Multi-Enzymatic Reactions

et al. 2021

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In vitro enzyme cascades possess great benefits, such as their synthetic capabilities for complex molecules, no need for intermediate isolation, and the shift of unfavorable equilibria towards the products. Their performance, however, can be impaired by, for example, destabilizing or inhibitory interactions between the cascade components or incongruous reaction conditions. The optimization of such systems is therefore often inevitable but not an easy task. Many parameters such as the design of the synthesis route, the choice of enzymes, reaction conditions, or process design can alter the performance of an in vitro enzymatic cascade. Many strategies to tackle this complex task exist, ranging from experimental to in silico approaches and combinations of both. This review collates examples of various optimization strategies and their success. The feasibility of optimization goals, the influence of certain parameters and the usage of algorithm-based optimizations are discussed.

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Section: Optimization Goalsmentioning

confidence: 99%

Section: Enzyme-selection and -Optimizationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Getting the Most Out of Enzyme Cascades: Strategies to Optimize In Vitro Multi-Enzymatic Reactions

et al. 2021

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“…Pyruvate was then further converted to produce ethanol and isobutanol. The core of this cascade has recently been adopted and modified to also produce other products like D-lactate and L-alanine [16,17]. Besides starting from D-glucose, the dehydratation of D-glycerate is also a critical step for cascades that convert glycerol into amino acids, for example [18].…”

Section: Introductionmentioning

confidence: 99%

Development of an Improved Peroxidase-Based High-Throughput Screening for the Optimization of D-Glycerate Dehydratase Activity

Begander

Huber

Döring

et al. 2020

IJMS

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Successful directed evolution examples span a broad range of improved enzyme properties. Nevertheless, the most challenging step for each single directed evolution approach is an efficient identification of improved variants from a large genetic library. Thus, the development and choice of a proper high-throughput screening is a central key for the optimization of enzymes. The detection of low enzymatic activities is especially complicated when they lead to products that are present in the metabolism of the utilized genetic host. Coupled enzymatic assays based on colorimetric products have enabled the optimization of many of such enzymes, but are susceptible to problems when applied on cell extract samples. The purpose of this study was the development of a high-throughput screening for D-glycerate dehydratase activity in cell lysates. With the aid of an automated liquid handling system, we developed a high-throughput assay that relied on a pre-treatment step of cell extract prior to performing the enzymatic and assay reactions. We could successfully apply our method, which should also be transferable to other cell extract-based peroxidase assays, to identify an improved enzyme for the dehydration of D-glycerate.

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“…Aspartate 4-decarboxylase (EC 4.1.1.12, ASD) has been employed industrially for the production of L-alanine and the resolution of DL-aspartic acid (Gmelch et al, 2019;Takamatsu & Tosa, 1993).…”

mentioning

confidence: 99%

Characterization and rational modification of aspartate 4‐decarboxylase from Acinetobacter radioresistens for the production of l‐alanine

Liu

Wang

et al. 2021

Biotech & Bioengineering

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Enzymatic synthesis of L-alanine has the advantages of less byproducts, strong stereoselectivity, and high catalytic efficiency. Aspartate 4-decarboxylase (ASD) is used industrially in DL-aspartic acid resolution and L-alanine production because it catalyzes the decarboxylation of L-aspartic acid. In this study, the ASD gene from Acinetobacter radioresistens (ArASD) was cloned, and its enzymatic properties were analyzed. ArASD is a dodecamer and has the highest enzyme activity ever reported to date. The optimal conditions for ArASD catalysis are 50°C and pH 4.5.Site-directed mutagenesis was used to improve ArASD stability under acidic conditions to compensate for its weak acid resistance, and the variant N35D with higher catalytic ability was obtained. The conversion by N35 recombinant cells of L-aspartic acid to L-alanine was 92.5% at pH 4.5% and 99.9% at pH 6.0, whereas that of the wild-type recombinant cells was 29.7% and 31.4%, respectively. Aspartase from Escherichia coli (AspA) was employed with ArASD to construct a dual-enzyme system that catalyzes fumaric acid to L-alanine, and the conversion reached 97.1% using recombinant cells harboring the dual-enzyme system. This study explored the enzymatic properties of ArASD and an effective strategy for the acidic resistance modification of ASD. Moreover, the strain expressing the ArASD variant and AspA engineered in this study has great potential application for the L-alanine production industry, especially in the case of high optical purity requirements.

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Optimization of a reduced enzymatic reaction cascade for the production of L-alanine

Cited by 28 publications

References 46 publications

Getting the Most Out of Enzyme Cascades: Strategies to Optimize In Vitro Multi-Enzymatic Reactions

Getting the Most Out of Enzyme Cascades: Strategies to Optimize In Vitro Multi-Enzymatic Reactions

Development of an Improved Peroxidase-Based High-Throughput Screening for the Optimization of D-Glycerate Dehydratase Activity

Characterization and rational modification of aspartate 4‐decarboxylase from Acinetobacter radioresistens for the production of l‐alanine

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