Engineering Therapeutic Enzymes

Lutz, Stefan; Williams, Elizabeth; Muthu, Pravin

doi:10.1007/978-3-319-50413-1_2

Cited by 4 publications

(4 citation statements)

References 226 publications

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“…Finally, our study showed that residues with strong ECs are very tolerant to mutations, and that a second mutation following a mutation which results in largely reduced activity can restore activity to nearly WT levels. Overall, our findings support the current view that coevolution of two residues most often relies on a first mutation resulting in unchanged or reduced fitness, which is then followed by a second mutation resulting in increased fitness.…”

Section: Discussionsupporting

confidence: 88%

“…We showed that the calculation of evolutionary couplings is effective for identifying residue pairs for protein engineering by saturation mutagenesis. Even for large proteins without known 3D structures, the ECSM approach allows one to focus on a small number of top‐scoring residue pairs in order to generate small saturation mutagenesis libraries . Importantly, coevolution analysis enables the identification of (peripheral) distant sites influencing enzyme activity, that is , information which cannot be derived with structure‐based engineering protocols focusing on the active site and its vicinity.…”

Section: Discussionmentioning

confidence: 99%

“…Based on these properties of ECs, we hypothesized that coevolution analysis of protein sequence and prediction of evolutionary couplings could be effective for identifying productive contacts across the entire enzyme molecule for protein engineering and to create a focused library providing positive variants with desired properties, even for large proteins without known 3D structures. Here, using the industrially important 926‐residue pullulanase from Bacillus naganoensis as a target protein, we describe a new approach called ‘evolutionary coupling saturation mutagenesis’ (ECSM), to engineer the enzyme guided by coevolution analysis.…”

mentioning

confidence: 99%

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Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity

et al. 2019

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Pullulanases are well‐known debranching enzymes hydrolyzing α‐1,6‐glycosidic linkages. To date, engineering of pullulanase is mainly focused on catalytic pocket or domain tailoring based on structure/sequence information. Saturation mutagenesis‐involved directed evolution is, however, limited by the low number of mutational sites compatible with combinatorial libraries of feasible size. Using Bacillus naganoensis pullulanase as a target protein, here we introduce the ‘evolutionary coupling saturation mutagenesis’ (ECSM) approach: residue pair covariances are calculated to identify residues for saturation mutagenesis, focusing directed evolution on residue pairs playing important roles in natural evolution. Evolutionary coupling (EC) analysis identified seven residue pairs as evolutionary mutational hotspots. Subsequent saturation mutagenesis yielded variants with enhanced catalytic activity. The functional pairs apparently represent distant sites affecting enzyme activity.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity

et al. 2019

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“…As such, a potent anti-DENV protease could also be developed into a therapeutic agent to treat viral infection in humans. Often overlooked as a class of therapeutics, proteases have been approved by the U.S. FDA to treat conditions such as hemophilia, acute myocardial infarction or muscle spasms since the 1980s [ 29 , 30 ]. While their antiviral potential has only been explored in topical applications against cold-causing rhinoviruses and, more recently, herpes simplex virus [ 31 ], the great amount of research on alternative-to-parenteral delivery methods for biologics brings promise on the ability to deliver therapeutic proteins through non-invasive routes such as orally or intranasally in the near future [ 32 – 34 ].…”

Section: Discussionmentioning

confidence: 99%

Aminopeptidase secreted by Chromobacterium sp. Panama inhibits dengue virus infection by degrading the E protein

et al. 2018

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Dengue virus (DENV) is the most prevalent and burdensome arbovirus transmitted by Aedes mosquitoes, against which there is only a limited licensed vaccine and no approved drug treatment. A Chromobacterium species, C. sp. Panama, isolated from the midgut of A. aegypti is able to inhibit DENV replication within the mosquito and in vitro. Here we show that C. sp. Panama mediates its anti-DENV activity through secreted factors that are proteinous in nature. The inhibitory effect occurs prior to virus attachment to cells, and is attributed to a factor that destabilizes the virion by promoting the degradation of the viral envelope protein. Bioassay-guided fractionation, coupled with mass spectrometry, allowed for the identification of a C. sp. Panama-secreted neutral protease and an aminopeptidase that are co-expressed and appear to act synergistically to degrade the viral envelope (E) protein and thus prevent viral attachment and subsequent infection of cells. This is the first study characterizing the anti-DENV activity of a common soil and mosquito-associated bacterium, thereby contributing towards understanding how such bacteria may limit disease transmission, and providing new tools for dengue prevention and therapeutics.

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Enzyme engineering and its industrial applications

Amatto

Rosa-Garzon

Simões

et al. 2021

Biotech and App Biochem

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Recently, there has been an increase in the demand for enzymes with modified activity, specificity, and stability. Enzyme engineering is an important tool to meet the demand for enzymes adjusted to different industrial processes. Knowledge of the structure and function of enzymes guides the choice of the best strategy for engineering enzymes. Each enzyme engineering strategy, such as rational design, directed evolution, and semi-rational design, has specific applications, as well as limitations, which must be considered when choosing a suitable strategy. Engineered enzymes can be optimized for different industrial applications by choosing the appropriate strategy. This review features engineered enzymes that have been applied in food, animal feed, pharmaceuticals, medical applications, bioremediation, biofuels, and detergents.

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Engineering Therapeutic Enzymes

Cited by 4 publications

References 226 publications

Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity

Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity

Aminopeptidase secreted by Chromobacterium sp. Panama inhibits dengue virus infection by degrading the E protein

Enzyme engineering and its industrial applications

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