scholarly journals In Silico Analysis of Glucose Oxidase from Aspergillus niger: Potential Cysteine Mutation Sites for Enhancing Protein Stability

2021 ◽  
Vol 8 (11) ◽  
pp. 188
Author(s):  
Sirawit Ittisoponpisan ◽  
Itthipon Jeerapan
Keyword(s):  
Thermal Stability ◽  
Aspergillus Niger ◽  
Glucose Oxidase ◽  
Disulfide Bond ◽  
In Silico ◽  
Disulfide Bonds ◽  
Grand Challenge ◽  
Analysis Tool ◽  
Bond Quality ◽  
Protein Database

Glucose oxidase (GOx) holds considerable advantages for various applications. Nevertheless, the thermal instability of the enzyme remains a grand challenge, impeding the success in applications outside the well-controlled laboratories, particularly in practical bioelectronics. Many strategies to modify GOx to achieve better thermal stability have been proposed. However, modification of this enzyme by adding extra disulfide bonds is yet to be explored. This work describes the in silico bioengineering of GOx from Aspergillus niger by judiciously analyzing characteristics of disulfide bonds found in the Top8000 protein database, then scanning for amino acid residue pairs that are suitable to be replaced with cysteines in order to establish disulfide bonds. Next, we predicted and assessed the mutant GOx models in terms of disulfide bond quality (bond length and α angles), functional impact by means of residue conservation, and structural impact as indicated by Gibbs free energy. We found eight putative residue pairs that can be engineered to form disulfide bonds. Five of these are located in less conserved regions and, therefore, are unlikely to have a deleterious impact on functionality. Finally, two mutations, Pro149Cys and His158Cys, showed potential for stabilizing the protein structure as confirmed by a structure-based stability analysis tool. The findings in this study highlight the opportunity of using disulfide bond modification as a new alternative technique to enhance the thermal stability of GOx.

Effects of metal ions on simultaneous production of glucose oxidase and catalase by Aspergillus niger

2001 ◽  
Vol 32 (1) ◽  
pp. 16-19 ◽  
Author(s):  
J.-Z. Liu ◽  
Y.-Y. Huang ◽  
J. Liu ◽  
L.-P. Weng ◽  
L.-N. Ji
Keyword(s):  
Aspergillus Niger ◽  
Metal Ions ◽  
Glucose Oxidase ◽  
Simultaneous Production

scholarly journals A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hitomi Nakamura ◽  
Moeka Yoshikawa ◽  
Naoko Oda-Ueda ◽  
Tadashi Ueda ◽  
Takatoshi Ohkuri
Keyword(s):  
Thermal Stability ◽  
Pichia Pastoris ◽  
Protein Stability ◽  
Disulfide Bond ◽  
Binding Activity ◽  
Comprehensive Analysis ◽  
The Other ◽  
Constant Domain ◽  
Intermolecular Disulfide Bond ◽  
Sds Page

AbstractGenerally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

scholarly journals Predicting Absorption-Distribution Properties of Neuroprotective Phosphine-Borane Compounds Using In Silico Modeling and Machine Learning

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2505
Author(s):  
Raheem Remtulla ◽  
Sanjoy Kumar Das ◽  
Leonard A. Levin
Keyword(s):  
Machine Learning ◽  
Neural Networks ◽  
In Silico ◽  
Disulfide Bonds ◽  
Oral Absorption ◽  
In Vivo Studies ◽  
Drug Candidates ◽  
In Silico Methods

Phosphine-borane complexes are novel chemical entities with preclinical efficacy in neuronal and ophthalmic disease models. In vitro and in vivo studies showed that the metabolites of these compounds are capable of cleaving disulfide bonds implicated in the downstream effects of axonal injury. A difficulty in using standard in silico methods for studying these drugs is that most computational tools are not designed for borane-containing compounds. Using in silico and machine learning methodologies, the absorption-distribution properties of these unique compounds were assessed. Features examined with in silico methods included cellular permeability, octanol-water partition coefficient, blood-brain barrier permeability, oral absorption and serum protein binding. The resultant neural networks demonstrated an appropriate level of accuracy and were comparable to existing in silico methodologies. Specifically, they were able to reliably predict pharmacokinetic features of known boron-containing compounds. These methods predicted that phosphine-borane compounds and their metabolites meet the necessary pharmacokinetic features for orally active drug candidates. This study showed that the combination of standard in silico predictive and machine learning models with neural networks is effective in predicting pharmacokinetic features of novel boron-containing compounds as neuroprotective drugs.

15N- AND 13C-NMR INVESTIGATIONS OF GLUCOSE OXIDASE FROM ASPERGILLUS NIGER

1991 ◽  
pp. 209-212
Author(s):  
Christoph Sanner ◽  
Peter Macheroux ◽  
Heinz Rüterjans ◽  
Franz Müller ◽  
Adalbert Bacher
Keyword(s):  
Aspergillus Niger ◽  
Glucose Oxidase ◽  
13C Nmr

Diauxic production of glucose oxidase by Aspergillus niger in submerged culture

2002 ◽  
Vol 31 (5) ◽  
pp. 615-620 ◽  
Author(s):  
J. Mirón ◽  
M.P. González ◽  
L. Pastrana ◽  
M.A. Murado
Keyword(s):  
Aspergillus Niger ◽  
Glucose Oxidase ◽  
Submerged Culture

scholarly journals Characterization of Soluble, Disulfide Bond-Stabilized, Prokaryotically Expressed Human Thyrotropin Receptor Ectodomain*

Endocrinology ◽  
1997 ◽  
Vol 138 (2) ◽  
pp. 588-593 ◽  
Author(s):  
Y. Bobovnikova ◽  
P. N. Graves ◽  
H. Vlase ◽  
T. F. Davies
Keyword(s):  
Amino Acids ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Thioredoxin Reductase ◽  
Biologically Active ◽  
Receptor Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fusion Partner ◽  
E Coli ◽  
Recombinant Receptor

Abstract To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein, it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) products. To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21–415) was inserted into the vector pGEX-2TK by directional cloning and used to transform the thioredoxin reductase mutant strain of E. coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm. After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR amino acids 21–35. This showed that over 50% of the expressed hTSHR-ecd was soluble in contrast to expression in a wild-type E. coli (strain αF′), where the majority of the recombinant receptor was insoluble. The soluble recombinant receptor was affinity purified and characterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein. This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes. Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant.

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