Thermal inactivation kinetics of penicillin g acylase obtained from a mutant derivative of <i>escherichia coli</i> atcc 11105

Erarslan, Altan; Koçer, Halil

doi:10.1002/jctb.280550113

Cited by 32 publications

(3 citation statements)

References 18 publications

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“…The remaining amounts of intact VLPs at fraction #24 were employed to calculate the decay rates; therefore, the Arrhenius plot was used to find slopes for estimating the equivalent instability [24]. …”

Section: Methodsmentioning

confidence: 99%

Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles

Wang

Hsu

et al. 2010

Virus Genes

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The virus-like particle (VLP) assembled from capsid subunits of the dragon grouper nervous necrosis virus (DGNNV) is very similar to its native T = 3 virion. In order to investigate the effects of four cysteine residues in the capsid polypeptide on the assembly/dissociation pathways of DGNNV virions, we recombinantly cloned mutant VLPs by mutating each cysteine to destroy the specific disulfide linkage as compared with thiol reduction to destroy all S–S bonds. The mutant VLPs of C187A and C331A mutations were similar to wild-type VLPs (WT-VLPs); hence, the effects of Cys187 and Cys331 on the particle formation and thermostability were presumably negligible. Electron microscopy showed that either C115A or C201A mutation disrupted de novo VLP formation significantly. As shown in micrographs and thermal decay curves, β-mercaptoethanol-treated WT-VLPs remained intact, merely resulting in lower tolerance to thermal disruption than native WT-VLPs. This thiol reduction broke disulfide linkages inside the pre-fabricated VLPs, but it did not disrupt the appearance of icosahedrons. Small dissociated capsomers from EGTA-treated VLPs were able to reassemble back to icosahedrons in the presence of calcium ions, but additional treatment with β-mercaptoethanol during EGTA dissociation resulted in inability of the capsomers to reassemble into the icosahedral form. These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles.

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Section: Methodsmentioning

confidence: 99%

Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles

Wang

Hsu

et al. 2010

Virus Genes

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“…Several investigations in the past have been focused on enhancing the thermostability of EcPGA, a widely used PGA for the manufacture of β-lactam antibiotics, through cross-linking the enzyme with glutaraldehyde [8] or carrying out sitedirected mutagenesis of few carefully selected amino acid residues [22]. However, these efforts seem to result only in minor enhancement in thermostability.…”

Section: Introductionmentioning

confidence: 99%

Sequence and structure-based comparative analysis to assess, identify and improve the thermostability of penicillin G acylases

Panigrahi

Chand

Mukherji

et al. 2015

Journal of Industrial Microbiology and Biotechnology

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Penicillin acylases are enzymes employed by the pharmaceutical industry for the manufacture of semi-synthetic penicillins. There is a continuous demand for thermostable and alkalophilic enzymes in such applications. We have carried out a computational analysis of known penicillin G acylases (PGAs) in terms of their thermostable nature using various protein-stabilizing factors. While the presence of disulfide bridges was considered initially to screen putative thermostable PGAs from the database, various other factors such as high arginine to lysine ratio, less content of thermolabile amino acids, presence of proline in β-turns, more number of ion-pair and other non-bonded interactions were also considered for comparison. A modified consensus approach designed could further identify stabilizing residue positions by site-specific comparison between mesostable and thermostable PGAs. A most likely thermostable enzyme identified from the analysis was PGA from Paracoccus denitrificans (PdPGA). This was cloned, expressed and tested for its thermostable nature using biochemical and biophysical experiments. The consensus site-specific sequence-based approach predicted PdPGA to be more thermostable than Escherichia coli PGA, but not as thermostable as the PGA from Achromobacter xylosoxidans. Experimental data showed that PdPGA was comparatively less thermostable than Achromobacter xylosoxidans PGA, although thermostability factors favored a much higher stability. Despite being mesostable, PdPGA being active and stable at alkaline pH is an advantage. Finally, several residue positions could be identified in PdPGA, which upon mutation selectively could improve the thermostability of the enzyme.

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“…Ipak, umrežavanjem enzima glutaraldehidom dobija se samo blago povećanje termostabilnosti. Poluvreme dezaktivacije enzima se na 40 ºC povećava sa 30 na 90 minuta, ali na višim temperaturama njegova stabilnost ostaje nepromenjena [61].…”

Section: Stabilizacija Penicilin-acilaze Hemijskom Modifikacijom Enzimaunclassified

Development of immobilized systems with penicillin acylase from Esherichia coli for production of semisyntetic penicillins

Žuža¹

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Thermal inactivation kinetics of penicillin g acylase obtained from a mutant derivative of escherichia coli atcc 11105

Cited by 32 publications

References 18 publications

Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles

Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles

Sequence and structure-based comparative analysis to assess, identify and improve the thermostability of penicillin G acylases

Development of immobilized systems with penicillin acylase from Esherichia coli for production of semisyntetic penicillins

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