Improving and repurposing biocatalysts via directed evolution

Denard, Carl A.; Ren, Hengqian; Zhao, Huimin

doi:10.1016/j.cbpa.2014.12.036

Cited by 227 publications

(105 citation statements)

References 67 publications

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“…The entire processes can be improved with some biotechnological methods, i.e., enzyme engineering and process engineering 18 . The strategies of rational design, directed evolution, or a combination offer efficient and powerful tools to improve and optimize natural enzymes for generating robust biocatalysts for practical applications 29, 30 . Enzyme co-immobilization technologies insure the stability, recovery and reuse of the cell-free biosystem for several consecutive batches 31 .…”

Section: Discussionmentioning

confidence: 99%

A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate

Jia

Liu

Han

2017

Sci Rep

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Acetoin (3-hydroxy-2-butanone) is an important bio-based platform chemical with wide applications. In vitro enzyme catalysed synthesis exhibits great feasibility in the production of chemicals with high purity. In the present work, a synthetic pathway involving a two-step continuous reaction was constructed in vitro for acetoin production from pyruvate at improved temperature. Thermostable candidates, acetolactate synthase (coAHASL1 and coAHASL2 from Caldicellulosiruptor owensensis OL) and α-acetolactate decarboxylase (bsALDC from Bacillus subtilis IPE5-4) were cloned, heterologously expressed, and characterized. All the enzymes showed maximum activities at 65–70 °C and pH of 6.5. Enzyme kinetics analysis showed that coAHASL1 had a higher activity but lower affinity against pyruvate than that of coAHASL2. In addition, the activities of coAHASL1 and bsALDC were promoted by Mn2+ and NADPH. The cascade enzymatic reaction was optimized by using coAHASL1 and bsALDC based on their kinetic properties. Under optimal conditions, a maximum concentration of 3.36 ± 0.26 mM acetoin was produced from 10 mM pyruvate after reaction for 24 h at 65 °C. The productivity of acetoin was 0.14 mM h−1, and the yield was 67.80% compared with the theoretical value. The results confirmed the feasibility of synthesis of acetoin from pyruvate with a cell-free enzyme catalysed system at improved temperature.

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Section: Discussionmentioning

confidence: 99%

A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate

Jia

Liu

Han

2017

Sci Rep

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“…The result can be a significant reduction in industrial waste and cost of production compared to stoichiometric syntheses and other catalytic processes. The importance of directed evolution and other protein engineering tools for tuning enzyme activity, selectivity or stability to meet industrial process requirements has been thoroughly established [1,2]. However, a significant hurdle is presented by the fact that a number of valuable chemical transformations, including many catalyzed by synthetic catalysts, do not have biocatalytic counterparts.…”

Section: Introductionmentioning

confidence: 99%

Exploiting and engineering hemoproteins for abiological carbene and nitrene transfer reactions

Brandenberg

Fasan

Arnold

2017

Current Opinion in Biotechnology

292

221

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The surge in reports of heme-dependent proteins as catalysts for abiotic, synthetically valuable carbene and nitrene transfer reactions dramatically illustrates the evolvability of the protein world and our nascent ability to exploit that for new enzyme chemistry. We highlight the latest additions to the hemoprotein-catalyzed reaction repertoire (including carbene Si–H and C–H insertions, Doyle-Kirmse reactions, aldehyde olefinations, azide-to-aldehyde conversions, and intermolecular nitrene C–H insertion) and show how different hemoprotein scaffolds offer varied reactivity and selectivity. Preparative-scale syntheses of pharmaceutically relevant compounds accomplished with these new catalysts are beginning to demonstrate their biotechnological relevance. Insights into the determinants of enzyme lifetime and product yield are providing generalizable cues for engineering heme-dependent proteins to further broaden the scope and utility of these non-natural activities.

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“…The production of biocatalysts has benefited from advances in protein science and the availability of genetic engineering techniques to develop new enzymes with improved or altered properties. Traditionally, enzyme engineering methods comprise three main strategies for improving enzyme stability and catalytic properties: rational design, directed evolution, and a combination of both methods ('semi-rational') [3][4][5]. Although these methods yield reliable results, being limited to using the side chains of natural amino acids in such engineered enzymes restricts the scope of possible applications.…”

Section: Advent Of Novel Enzyme Engineering Methodsmentioning

confidence: 99%