Redesigning Robust Biocatalysts by Engineering Enzyme Microenvironment and Enzyme Immobilization

Riaz, Roha; Ashraf, Mubeen; Hussain, N.; Baqar, Zulqarnain; Bilal, Muhammad; Iqbal, Hafiz M.N.

doi:10.1007/s10562-022-04137-6

Cited by 15 publications

(2 citation statements)

References 92 publications

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“…Furthermore, microenvironment adjacent to catalytic residues may also have an effect on enzyme activity. [36] Glutamine at site 179 in the substrate binding pocket was located above the carbonyl group of the substrate and classified as an uncharged amino acid (Fig. 4F).…”

Section: Modification Of the Microenviroment To Optimize The Substrat...mentioning

confidence: 99%

Rational design of short-chain dehydrogenase DHDR for efficient synthesis of (S)-equol

qin,

zhang,

zhou

et al. 2024

Preprint

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(S)-equol, the most influential metabolite of daidzein in vivo, has aroused great attention due to the excellent biological activities. Although existing studies have accomplished the construction of its heterologous synthetic pathway in the context of anaerobicity and inefficiency of natural strains, the low productivity of (S)-equol limits its industrial application. Here, rational design strategies based on decreasing the pocket steric hindrance and fine-tuning the pocket microenvironment to systematically redesign the binding pocket of enzyme were developed and processed to the rate-limiting enzyme DHDR in (S)-equol synthesis. After iterative combinatorial mutagenesis, an effective mutant S118G/T169A capable of significantly increasing (S)-equol yield was obtained. Computational analyses illustrated that the main reason of the increased activity relied on the decreased critical distance and more stable interacting conformation. Then, the reaction optimization was performed, and the recombinant Escherichia coli whole-cell biocatalyst harboring S118G/T169A enabled the efficient conversion of 2 mM daidzein to (S)-equol, achieving yields of 84.5%, which was 2.9 times higher than that of the parental strain expressing wide type DHDR. This study provides an effective idea and a feasible method for enzyme modification and whole-cell catalytic synthesis of (S)-equol, and will greatly accelerate the process of industrial production.

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Section: Modification Of the Microenviroment To Optimize The Substrat...mentioning

confidence: 99%

Rational design of short-chain dehydrogenase DHDR for efficient synthesis of (S)-equol

qin,

zhang,

zhou

et al. 2024

Preprint

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“…[46] A massive hydrophobic tunnel like this aids the tryptophan synthase complex in binding intermediate indole and transporting it between active sites. [47]…”

Section: Engineering Cofactor Preference Substrate Recognition and Sp...mentioning

confidence: 99%

Protein engineering for natural product biosynthesis: expanding diversity for therapeutic applications

Otun,

Lerma-Escalera,

Ntushelo

et al. 2023

J Bio-X Res

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In this dispensation of the fourth industrial revolution, protein engineering has become a popular approach for increasing enzymatic activity, stability, and titer in the biosynthesis of natural products. This is attributed to its numerous advantages (over direct isolation from plants or via chemical synthesis), including decreasing or eliminating reaction byproducts, high precision, moderate handling of intricate and chemically unstable chemicals, overall reusability, and cost efficiency. Recently, protein engineering tools have advanced to redesign and enhance natural product biosynthesis. These methods include direct evolution, substrate engineering, medium engineering, enzyme engineering and immobilization, structure-assisted protein engineering, and advanced computational. Recent successes in implementing these emerging protein engineering technologies were critically discussed in this article. Also, the advantages, limitations, and applications in industrial and medical biotechnology were discussed. Last, future research directions and potential were also highlighted. Graphical Abstract: Plants are an excellent reservoir of enzymes with compound production applications. However, they can have several production restrictions like low enzyme activity, narrow substrate range, poor stability and loss of function in heterologous hosts. Extracting new enzymes is a popular approach against these restrictions. Nevertheless, this approach involves highly time-consuming and labor-intensive. In this article, we are exploring the protein engineering approach against these restrictions.

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Exploring the Potential of Various Cyclodextrin‐Based Derivatives in Enzyme Supramolecular Engineering

Foroutan Kalourazi,

Amirabbas Nazemi,

Unniram Parambil

et al. 2024

ChemBioChem

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Enzyme stability and activity are pivotal factors for their implementation in different industrial applications. Enzyme supramolecular engineering relies on the fabrication of a tailor‐made enzyme nano‐environment to ensure enzyme stability without impairing activity. Cyclodextrins (CDs), cyclic oligomers of glucose, act as protein chaperones and stabilize, upon interaction with hydrophobic amino acid residues exposed at the protein surface, its three‐dimensional structure. When used to build an organosilica layer shielding an enzyme, they enhance the protective effect of this layer. In the present study, we systematically assessed the protective effects of three organosilane derivatives based on ɑ‐, β‐ and γ‐CDs. A model lipase enzyme was immobilized at the surface of silica nanoparticles and shielded in an organosilica layer containing these organosilanes. Besides layer thickness optimization, the effect of different stressors (i. e., temperature, SDS, urea) was tested. Our results showed that organosilica layers produced with CDs improve enzyme thermal stability. They also support enzyme refolding after denaturation under chaotic conditions. Additionally, we demonstrated that the protective effect of the smallest CD derivative tested, namely ɑ‐CD, surpassed the other macrocycles studied for conferring the immobilized enzyme with higher resistance to stress conditions. This protection strategy was also applied to a thermostable β‐galactosidase enzyme.

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Redesigning Robust Biocatalysts by Engineering Enzyme Microenvironment and Enzyme Immobilization

Cited by 15 publications

References 92 publications

Rational design of short-chain dehydrogenase DHDR for efficient synthesis of (S)-equol

Rational design of short-chain dehydrogenase DHDR for efficient synthesis of (S)-equol

Protein engineering for natural product biosynthesis: expanding diversity for therapeutic applications

Exploring the Potential of Various Cyclodextrin‐Based Derivatives in Enzyme Supramolecular Engineering

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