Thermostability engineering of industrial enzymes through structure modification

Nezhad, Nima Ghahremani; Rahman, Raja Noor Zaliha Raja Abd; Normi, Yahaya M.; Oslan, Siti Nurbaya; Shariff, Fairolniza Mohd; Leow, Adam Thean Chor

doi:10.1007/s00253-022-12067-x

Cited by 54 publications

(19 citation statements)

References 174 publications

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“…The immobilized PGA raised the temperature to 50°C, thereby revealing the immobilized PGA had better thermal stability. When free PGA was immobilized, the entropy and enthalpy of the protein molecule would be changed such that the thermal stability increased ( Nezhad et al, 2022 ).…”

Section: Resultsmentioning

confidence: 99%

Immobilization and property of penicillin G acylase on amino functionalized magnetic Ni0.3Mg0.4Zn0.3Fe2O4 nanoparticles prepared via the rapid combustion process

Chen

Yue

et al. 2023

Front. Bioeng. Biotechnol.

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Penicillin G acylase plays an important role in the biocatalytic process of semi-synthetic penicillin. In order to overcome the disadvantages of free enzymes and improve the catalytic performance of enzymes, it is a new method to immobilize enzymes on carrier materials. And magnetic materials have the characteristics of easy separation. In the present study, the Magnetic Ni0.3Mg0.4Zn0.3Fe2O4 nanoparticles were successfully prepared by a rapid-combustion method and calcined at 400°C for 2 h. The surface of the nanoparticles was modified with sodium silicate hydrate, and the PGA was covalently bound to the carrier particles through the cross-linking of glutaraldehyde. The results showed that the activity of immobilized PGA reached 7121.00 U/g. The optimum pH for immobilized PGA was 8 and the optimum temperature was 45°C, the immobilized PGA exhibited higher stability against changes in pH and temperature. The Michaelis–Menten constant (Km) values of the free and immobilized PGA were 0.00387 and 0.0101 mol/L and the maximum rate (Vmax) values were 0.387 and 0.129 μmol/min. Besides, the immobilized PGA revealed excellent cycling performance. The immobilization strategy presented PGA had the advantages of reuse, good stability, cost saving and had considerable practical significance for the commercial application of PGA.

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Section: Resultsmentioning

confidence: 99%

Immobilization and property of penicillin G acylase on amino functionalized magnetic Ni0.3Mg0.4Zn0.3Fe2O4 nanoparticles prepared via the rapid combustion process

Chen

Yue

et al. 2023

Front. Bioeng. Biotechnol.

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“…Hydrogen bonds and hydrophobic interactions are crucial for protein stability, [11,33] which help maintain its conformational stability. [34] The total number of hydrogen bonds was increased in KdAKR M10 compared to that of KdAKR M6 (Table S4), contributing to its enhanced stability.…”

Section: Mechanism Elucidation For Stability Enhancementmentioning

confidence: 99%

Computer‐aided design to enhance the stability of aldo‐keto reductase KdAKR

Dai,

Tian,

Chen

et al. 2024

Biotechnology Journal

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The aldo‐keto reductase (AKR) KdAKR from Kluyvermyces dobzhanskii can reduce t‐butyl 6‐chloro‐(5S)‐hydroxy‐3‐oxohexanoate ((5S)‐CHOH) to t‐butyl 6‐chloro‐(3R,5S)‐dihydroxyhexanoate ((3R,5S)‐CDHH), which is the key chiral intermediate of rosuvastatin. Herein, a computer‐aided design that combined the use of PROSS platform and consensus design was employed to improve the stability of a previously constructed mutant KdAKRM6. Experimental verification revealed that S196C, T232A, V264I and V45L produced improved thermostability and activity. The “best” mutant KdAKRM10 (KdAKRM6‐S196C/T232A/V264I/V45L) was constructed by combining the four beneficial mutations, which displayed enhanced thermostability. Its T5015 and Tm values were increased by 10.2 and 10.0°C, respectively, and half‐life (t1/2) at 40°C was increased by 17.6 h. Additionally, KdAKRM10 demonstrated improved resistance to organic solvents compared to that of KdAKRM6. Structural analysis revealed that the increased number of hydrogen bonds and stabilized hydrophobic core contributed to the rigidity of KdAKRM10, thus improving its stability. The results validated the feasibility of the computer‐aided design strategy in improving the stability of AKRs.

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“…As a further validation, we benchmarked the predictions of our method with published experimental data for generic proteins (not antibodies). Since stability and solubility are crucial properties of recombinant proteins, there are many reports of experimentally characterised mutations that improve at least one of these properties 62,75,76 . However, our method automatically identifies specific mutations that should be performed, which are thus unlikely to be those that were characterised in previous studies.…”

Section: Experimental Validationmentioning

confidence: 99%

Automated optimisation of solubility and conformational stability of antibodies and proteins

et al. 2023

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Biologics, such as antibodies and enzymes, are crucial in research, biotechnology, diagnostics, and therapeutics. Often, biologics with suitable functionality are discovered, but their development is impeded by developability issues. Stability and solubility are key biophysical traits underpinning developability potential, as they determine aggregation, correlate with production yield and poly-specificity, and are essential to access parenteral and oral delivery. While advances for the optimisation of individual traits have been made, the co-optimization of multiple traits remains highly problematic and time-consuming, as mutations that improve one property often negatively impact others. In this work, we introduce a fully automated computational strategy for the simultaneous optimisation of conformational stability and solubility, which we experimentally validate on six antibodies, including two approved therapeutics. Our results on 42 designs demonstrate that the computational procedure is highly effective at improving developability potential, while not affecting antigen-binding. We make the method available as a webserver at www-cohsoftware.ch.cam.ac.uk.

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Thermostability engineering of industrial enzymes through structure modification

Cited by 54 publications

References 174 publications

Immobilization and property of penicillin G acylase on amino functionalized magnetic Ni0.3Mg0.4Zn0.3Fe2O4 nanoparticles prepared via the rapid combustion process

Immobilization and property of penicillin G acylase on amino functionalized magnetic Ni0.3Mg0.4Zn0.3Fe2O4 nanoparticles prepared via the rapid combustion process

Computer‐aided design to enhance the stability of aldo‐keto reductase KdAKR

Automated optimisation of solubility and conformational stability of antibodies and proteins

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