Conjugation of d-glucosamine to bovine trypsin increases thermal stability and alters functional properties

Gizurarson, Jóhann Grétar Kröyer; Filippusson, Hörður

doi:10.1016/j.enzmictec.2015.04.005

Cited by 6 publications

(4 citation statements)

References 37 publications

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“…As shown in Fig. 7, the spectrum of the wild-type trypsin showed a single minimum near 210 nm, indicating a βsheet secondary structures, comparable to what has been reported for trypsin [12,25]. Besides this, the average CD spectra of N84S and R104S were similar to that of the wild-type trypsin, suggesting that the mutations did not disrupt the secondary structure of trypsin and maintained a conformation close to the native protein.…”

Section: Spectroscopy Analysissupporting

confidence: 79%

A Novel Strategy for Thermostability Improvement of Trypsin Based on N-Glycosylation within the ��-Loop Region

Guo¹,

Liu²,

Yu³

et al. 2016

Journal of Microbiology and Biotechnology

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The Ω-loop is a nonregular and flexible structure that plays an important role in molecular recognition, protein folding, and thermostability. In the present study, molecular dynamics simulation was carried out to assess the molecular stability and flexibility profile of the porcine trypsin structures. Two Ω-Loops (fragment 57-67 and fragment 78-91) were confirmed to represent the flexible region. Subsequently, glycosylation site-directed mutations (A73S, N84S, and R104S) were introduced within the Ω-loop region and its wing chain based on its potential N-glycosylation sites (Asn-Xaa-Ser/Thr consensus sequences) and structure information to improve the thermostability of trypsin. The result demonstrated that the halflife of the N84S mutant at 50°C increased by 177.89 min when compared with that of the wildtype enzyme. Furthermore, the significant increase in the thermal stability of the N84S mutant has also been proven by an increase in the Tm values determined by circular dichroism. Additionally, the optimum temperatures of the wild-type enzyme and the N84S mutant were 75°C and 80°C, respectively. In conclusion, we obtained the thermostability-improved enzyme N84S mutant, and the strategy used to design this mutant based on its structural information and N-linked glycosylation modification could be applied to engineer other enzymes to meet the needs of the biotechnological industry.

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Section: Spectroscopy Analysissupporting

confidence: 79%

A Novel Strategy for Thermostability Improvement of Trypsin Based on N-Glycosylation within the ��-Loop Region

Guo¹,

Liu²,

Yu³

et al. 2016

Journal of Microbiology and Biotechnology

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“…The results discussed in this paper demonstrate a strong stabilizing effect. While the conjugation with polymers or particles is a well-known approach to improve stability of enzymes, − our results show the stability of the conjugates for up to 115–150 °C, which significantly exceeds the thermal stability limits achieved with previously proposed stabilizing methods, ,, including the examples of enzyme conjugation methods tested on lysozyme, trypsin, organophosphorus hydrolase, human carbonic anhydrase II, and sarcosine oxidase …”

Section: Introductionmentioning

confidence: 63%

“…The immobilization of enzymes is well-known approach for encapsulation, storage, delivery, separation, and biocatalytic synthesis . Many synthetic methods have been developed to protect the three-dimensional conformation of the enzymes via immobilization/encapsulation or conjugation on solid surfaces, in porous structures, ceramic and polymer matrixes, − particles, , capsules, imprints, inorganic hybrids, nanofibers, , by cyclization of protein backbone, and through various protein–polymer conjugation techniques. − Encapsulation of enzymes by tethering a single polymer molecule shell has advantages due to a combination of some conformational freedom and, at the same time, structural stabilization with a moderately stiff polymer shell. − …”

Section: Introductionmentioning

confidence: 99%

Thermal Stabilization of Enzymes with Molecular Brushes

et al. 2017

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Herein, we report a conjugation strategy, where we utilize a poly(ethylene oxide) cylindrical molecular brush architecture to design a self-assembled structure for thermal stabilization of enzymes. We demonstrate that the proposed architecture of the moderately stiff polymer ligand results in a significant improvement of biocatalytic activity and thermal stability of lysozyme and trypsin that retain their activity, even upon heating to 100 °C and above. The molecular brush is bound via epoxy functional groups to the amino groups of the lysine on the surface of the enzyme globule, promoting the formation of stiff and crowded cages around the enzymes and preventing the water molecules access to the enzyme and enzymes agglomeration. The molecular dynamic simulations show that the high concentration of poly(ethylene oxide) in the vicinity of the enzyme is critical for their stability. Monitoring of lysozyme–molecular brush conjugates for 6 and 12 months in lyophilized form and in solution, respectively, has shown that the conjugation does not compromise the shelf life of the enzyme.

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“…Enzyme stabilization is typically achieved either via the genetic mutations , or via conjugation of enzymes with solid substrates or other molecules. − In latter scenarios, the stability and activity of the resulting conjugate depends on the choice of chemical functionality of the monomer units, molecular weight of the polymer, and conjugation sites on the protein. For example, conjugating cationic polymers with lysozyme increases its activity, and this effect is attributed to the electrostatic attraction between the negatively charged cell wall and the positively charged lysozyme–polymer conjugate .…”

Section: Introductionmentioning

confidence: 99%

Designing Highly Thermostable Lysozyme–Copolymer Conjugates: Focus on Effect of Polymer Concentration

Choudhury

Luzinov

et al. 2018

Biomacromolecules

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Designing biomaterials capable of functioning in harsh environments is vital for a range of applications. Using molecular dynamics simulations, we show that conjugating lysozymes with a copolymer [poly(GMA- stat-OEGMA)] comprising glycidyl methacrylate (GMA) and oligo(ethylene glycol) methyl ether methacrylate (OEGMA) results in a dramatic increase of stability of these enzymes at high temperatures provided that the concentration of the copolymer in the close vicinity of the enzyme exceeds a critical value. In our simulations, we use triads containing the same ratio of GMA to OEGMA units as in our recent experiments (N. S. Yadavalli et al., ACS Catalysis, 2017, 7, 8675). We focus on the dynamics of the conjugate at high temperatures and on its structural stability as a function of the copolymer/water content in the vicinity of the enzyme. We show that the dynamics of phase separation in the water-copolymer mixture surrounding the enzyme is critical for the structural stability of the enzyme. Specifically, restricting water access promotes the structural stability of the lysozyme at high temperatures. We identified critical water concentration below which we observe a robust stabilization; the phase separation is no longer observed at this low fraction of water so that the water domains promoting unfolding are no longer formed in the vicinity of the enzyme. This understanding provides a basis for future studies on designing a range of enzyme-copolymer conjugates with improved stability.

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Conjugation of d-glucosamine to bovine trypsin increases thermal stability and alters functional properties

Cited by 6 publications

References 37 publications

A Novel Strategy for Thermostability Improvement of Trypsin Based on N-Glycosylation within the ��-Loop Region

A Novel Strategy for Thermostability Improvement of Trypsin Based on N-Glycosylation within the ��-Loop Region

Thermal Stabilization of Enzymes with Molecular Brushes

Designing Highly Thermostable Lysozyme–Copolymer Conjugates: Focus on Effect of Polymer Concentration

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