Rational backbone redesign of a fructosyl peptide oxidase to widen its active site access tunnel

Rigoldi, Federica; Donini, Stefano; Torretta, Archimede; Carbone, Anna; Redaelli, Alberto; Bandiera, Tiziano; Parisini, Emilio; Gautieri, Alfonso

doi:10.1002/bit.27535

Cited by 11 publications

(19 citation statements)

References 55 publications

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“…With the cross-application of computer science, computational chemistry, and other disciplines in protein engineering, enzymatic catalysis has reached unprecedented new heights and has contributed to a boom in in vitro synthetic biology. − Cell-free production offers many advantages, such as high product yields and conversion, fast reaction rates, ease of control, no accumulation of intermediates, and even the ability to create unnatural pathways. , All of these advantages have led to the growth of in vitro synthetic biology as a core technology for the green manufacturing of chemicals. − …”

Section: Introductionmentioning

confidence: 99%

Rational Design to Enhance Enzyme Activity for the Establishment of an Enzyme–Inorganic Hybrid Nanoflower Co-Immobilization System for Efficient Nucleotide Production

Lou

2022

J. Agric. Food Chem.

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Increasing yields while reducing costs is one of the ultimate pursuits of industrial production. To achieve this goal in the enzymatic production of multiple nucleotides, in this study, a co-immobilized polyphosphate kinase–nucleoside kinase hybridized nanoflower system (PPK@NK) was constructed. To improve the productivity, the nucleoside kinase (NK) used was rationally designed, and a variant with significantly increased activity compared to the wild type was obtained. The polyphosphate kinase (PPK) and NK could be sequentially adsorbed on the surface of hybrid nanoflowers at room temperature (25 °C) through the interaction of Cu2+ and proteins without any other chemical pretreatment. The optimal preparation conditions and reaction parameters of PPK@NK hybrid nanoflowers were investigated. Under optimal reaction conditions, the newly prepared co-immobilization system could catalyze the conversion of 100 mM uridine, cytidine, and inosine to the corresponding nucleotides completely within 4 h and could be reused at least six times. The storage stability of the co-immobilized system was more than 2-fold higher than that of the free enzyme, and there was no significant difference in thermostability. PPK@NK hybridized nanoflowers have properties such as easy preparation and storage and low cost, indicating their suitability for the efficient production of nucleotides.

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Section: Introductionmentioning

confidence: 99%

Rational Design to Enhance Enzyme Activity for the Establishment of an Enzyme–Inorganic Hybrid Nanoflower Co-Immobilization System for Efficient Nucleotide Production

Lou

2022

J. Agric. Food Chem.

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“…Protein conformational dynamics has become one of the most important areas of research in the life sciences. The combination of MD simulations and conformational dynamics analysis allows the identification of regions where residues that have an impact on activity or thermostability are located, and has successfully guided the modification of many protein properties (Han et al, 2016; Rigoldi et al, 2020).…”

Section: Resultsmentioning

confidence: 99%

A cocktail of protein engineering strategies: Breaking the enzyme bottleneck one by one for high UTP production in vitro

Sun

Lou

et al. 2022

Biotech & Bioengineering

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The pyrimidine metabolic pathway is tightly regulated in microorganisms, allowing limited success in metabolic engineering for the production of pathway‐related substances. Here, we constructed a four‐enzyme coupled system for the in vitro production of uridine triphosphate (UTP). The enzymes used include nucleoside kinase, uridylate kinase, nucleoside diphosphate kinase, and polyphosphate kinase for energy regeneration. All these enzymes are derived from extremophiles. To increase the total and unit time yield of the product, three enzymes other than polyphosphate kinase were modified separately by multiple protein engineering strategies. A nucleoside kinase variant with increased specific activity from 2.7 to 36.5 U/mg, a uridylate kinase variant (specific activity of 37.1 U/mg) with a 5.2‐fold increase in thermostability, and a nucleoside diphosphate kinase variant with a 2‐fold increase in a specific activity to over 900 U/mg were obtained, respectively. The reaction conditions of the coupled system were further optimized, and a two‐stage method was taken to avoid the problem of enzymatic pH adaptation mismatch. Under optimal conditions, this system can produce more than 65 mM UTP (31.5 g/L) in 3.0 h. The substrate conversion rate exceeded 98% and the maximum UTP productivity reached 40 mM/h.

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“…For example, in the case of enzyme redesign, rational engineering of the catalytic center is quite crucial for the activity. 29 Inversely, fabrication of porous protein delivery materials should give more attention to the edge surface redesign of the protein scaffold, 30,31 and conversion of protein structure is largely associated with the intramolecular interface redesign. 32 Remarkably, to start the protein redesign, three practical concerns need to be questioned: (1) Which amino acids should be mutated, deleted, or inserted in the protein sequence?…”

Section: Overview Of the Basic Principles And Strategies Of Protein D...mentioning

confidence: 99%

Advances in Rational Protein Engineering toward Functional Architectures and Their Applications in Food Science

Chen

Dai

et al. 2022

J. Agric. Food Chem.

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Protein biomolecules including enzymes, cagelike proteins, and specific peptides have been continuously exploited as functional biomaterials applied in catalysis, nutrient delivery, and food preservation in food-related areas. However, natural proteins usually function well in physiological conditions, not industrial conditions, or may possess undesirable physical and chemical properties. Currently, rational protein design as a valuable technology has attracted extensive attention for the rational engineering or fabrication of ideal protein biomaterials with novel properties and functionality. This article starts with the underlying knowledge of protein folding and assembly and is followed by the introduction of the principles and strategies for rational protein design. Basic strategies for rational protein engineering involving experienced protein tailoring, computational prediction, computation redesign, and de novo protein design are summarized. Then, we focus on the recent progress of rational protein engineering or design in the application of food science, and a comprehensive summary ranging from enzyme manufacturing to cagelike protein nanocarriers engineering and antimicrobial peptides preparation is given. Overall, this review highlights the importance of rational protein engineering in food biomaterial preparation which could be beneficial for food science.

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Rational backbone redesign of a fructosyl peptide oxidase to widen its active site access tunnel

Cited by 11 publications

References 55 publications

Rational Design to Enhance Enzyme Activity for the Establishment of an Enzyme–Inorganic Hybrid Nanoflower Co-Immobilization System for Efficient Nucleotide Production

Rational Design to Enhance Enzyme Activity for the Establishment of an Enzyme–Inorganic Hybrid Nanoflower Co-Immobilization System for Efficient Nucleotide Production

A cocktail of protein engineering strategies: Breaking the enzyme bottleneck one by one for high UTP production in vitro

Advances in Rational Protein Engineering toward Functional Architectures and Their Applications in Food Science

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