Directed Evolution of Enzymes for Industrial Biocatalysis

Porter, Joanne L.; Rusli, Rukhairul A.; Ollis, David L.

doi:10.1002/cbic.201500280

Cited by 195 publications

(125 citation statements)

References 111 publications

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“…[2] The characterization of more and more enzymes over the years, with diverse substrate scopes,h as enabled researchers to devise novel and elegant reaction combinations.Ina ddition, tailoringa nd optimizing biocatalysts through enzyme engineering is becoming faster and more efficient. [3] The multienzyme strategy is deemed to be ac riticals tepping stone on the way to the large-scale industrial applicationofb iocatalysis.…”

Section: Introductionmentioning

confidence: 99%

Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes

2018

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One approach to bringing enzymes together for multienzyme biocatalysis is genetic fusion. This enables the production of multifunctional enzymes that can be used for whole-cell biotransformations or for in vitro (cascade) reactions. In some cases and in some aspects, such as expression and conversions, the fused enzymes outperform a combination of the individual enzymes. In contrast, some enzyme fusions are greatly compromised in activity and/or expression. In this Minireview, we give an overview of studies on fusions between two or more enzymes that were used for biocatalytic applications, with a focus on oxidative enzymes. Typically, the enzymes are paired to facilitate cofactor recycling or cosubstrate supply. In addition, different linker designs are briefly discussed. Although enzyme fusion is a promising tool for some biocatalytic applications, future studies could benefit from integrating the findings of previous studies in order to improve reliability and effectiveness.

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Section: Introductionmentioning

confidence: 99%

Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes

2018

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Section: Discussionmentioning

confidence: 99%

“…However, wild-type enzymes often show defects in biochemical properties, which limit their application in some industries [17]. Directed evolution is usually regarded as an effective means to improve the biochemical properties of enzyme, even when lack of structure information impedes the use of rational design [17]. The GH family 5 β-mannanase ( Rm Man5A) from R. meihei CAU432 shows high specific activity and can hydrolyze many mannan substrates into manno-oligosaccharides, which makes this β-mannanase a suitable candidate for many industry applications [30].…”

Section: Discussionmentioning

confidence: 99%

“…In alkaline condition, β-mannanases from subfamily 8 display higher activity than subfamilies 7 and 10, which usually act at acidic and neutral pH [16]. Even though wild-type enzymes have various desirable properties, their biochemical properties also display many limitations, which make them not suitable for industrial applications in some cases [17]. After the pretreatments of biomass, such as acidic hydrolysis, hydrothermolysis, and steam explosion, the materials are usually tested to be acidic during biorefinery process [5, 18, 19].…”

Section: Introductionmentioning

confidence: 99%

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Directed evolution of a β-mannanase from Rhizomucor miehei to improve catalytic activity in acidic and thermophilic conditions

Yan

et al. 2017

Biotechnol Biofuels

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Backgroundβ-Mannanase randomly cleaves the β-1,4-linked mannan backbone of hemicellulose, which plays the most important role in the enzymatic degradation of mannan. Although the industrial applications of β-mannanase have tremendously expanded in recent years, the wild-type β-mannanases are still defective for some industries. The glycoside hydrolase (GH) family 5 β-mannanase (RmMan5A) from Rhizomucor miehei shows many outstanding properties, such as high specific activity and hydrolysis property. However, owing to the low catalytic activity in acidic and thermophilic conditions, the application of RmMan5A to the biorefinery of mannan biomasses is severely limited.ResultsTo overcome the limitation, RmMan5A was successfully engineered by directed evolution. Through two rounds of screening, a mutated β-mannanase (mRmMan5A) with high catalytic activity in acidic and thermophilic conditions was obtained, and then characterized. The mutant displayed maximal activity at pH 4.5 and 65 °C, corresponding to acidic shift of 2.5 units in optimal pH and increase by 10 °C in optimal temperature. The catalytic efficiencies (k cat/K m) of mRmMan5A towards many mannan substrates were enhanced more than threefold in acidic and thermophilic conditions. Meanwhile, the high specific activity and excellent hydrolysis property of RmMan5A were inherited by the mutant mRmMan5A after directed evolution. According to the result of sequence analysis, three amino acid residues were substituted in mRmMan5A, namely Tyr233His, Lys264Met, and Asn343Ser. To identify the function of each substitution, four site-directed mutations (Tyr233His, Lys264Met, Asn343Ser, and Tyr233His/Lys264Met) were subsequently generated, and the substitutions at Tyr233 and Lys264 were found to be the main reason for the changes of mRmMan5A.ConclusionsThrough directed evolution of RmMan5A, two key amino acid residues that controlled its catalytic efficiency under acidic and thermophilic conditions were identified. Information about the structure–function relationship of GH family 5 β-mannanase was acquired, which could be used for modifying β-mannanases to enhance the feasibility in industrial application, especially in biorefinery process. This is the first report on a β-mannanase from zygomycete engineered by directed evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0833-x) contains supplementary material, which is available to authorized users.

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“…Protein engineers have been successfully improving enzymes for more than 30 years using a variety of experimental and computational techniques. Tremendous advances have been made including the design and engineering of enzymes with activities not found in nature . Metabolic engineering and some synthetic biology applications focus on assembling enzymes from different organisms to perform novel multi‐step chemical reactions, in vitro or in vivo .…”

Section: Protein Engineering Scaffold Structuresmentioning

confidence: 99%

Extreme makeover: Engineering the activity of a thermostable alcohol dehydrogenase (AdhD) from Pyrococcus furiosus

Solanki

Abdallah

Banta

2016

Biotechnology Journal

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Alcohol dehydrogenase D (AdhD) is a monomeric thermostable alcohol dehydrogenase from the aldo-keto reductase (AKR) superfamily of proteins. We have been exploring various strategies of engineering the activity of AdhD so that it could be employed in future biotechnology applications. Driven by insights made in other AKRs, we have made mutations in the cofactor-binding pocket of the enzyme and broadened its cofactor specificity. A pre-steady state kinetic analysis yielded new insights into the conformational behavior of this enzyme. The most active mutant enzyme concomitantly gained activity with a non-native cofactor, nicotinamide mononucleotide, NMN(H), and an enzymatic biofuel cell was demonstrated with this enzyme/cofactor pair. Substrate specificity was altered by grafting loop regions near the active site pocket from a mesostable human aldose reductase (hAR) onto the thermostable AdhD. These moves not only transferred the substrate specificity of hAR but also the cofactor specificity of hAR. We have added alpha-helical appendages to AdhD to enable it to self-assemble into a thermostable catalytic proteinaceous hydrogel. As our understanding of the structure/function relationship in AdhD and other AKRs advances, this ubiquitous protein scaffold could be engineered for a variety of catalytic activities that will be useful for many future applications.

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Directed Evolution of Enzymes for Industrial Biocatalysis

Cited by 195 publications

References 111 publications

Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes

Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes

Directed evolution of a β-mannanase from Rhizomucor miehei to improve catalytic activity in acidic and thermophilic conditions

Extreme makeover: Engineering the activity of a thermostable alcohol dehydrogenase (AdhD) from Pyrococcus furiosus

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