Biochemical properties of Bacillus intermedius subtilisin-like proteinase secreted by a Bacillus subtilis recombinant strain in its stationary phase of growth

Михайлова, Е. О.; Марданова, А. М.; Balaban, N. P.; Rudenskaya, G. N.; Ilyinskaya, O. N.; Шарипова, М. Р.

doi:10.1134/s0006297909030109

Cited by 9 publications

(7 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although microbial serine proteases are mainly monomeric enzymes, such parallel models of inactivation (Eqs. [3][4][5] were evaluated since the occurence of proteolytic enzymes with quaternary structures is reported [30] and, particularly, a subtilisin-like protease from Bacillus intermedius secreted by a Bacillus subtilis recombinant strain was reported to form dimers, possibly through interactions mediated by calcium ions [31].…”

Section: Resultsmentioning

confidence: 99%

Kinetic Stability Modelling of Keratinolytic Protease P45: Influence of Temperature and Metal Ions

Daroit

Sant’Anna

Brandelli

2011

Appl Biochem Biotechnol

View full text Add to dashboard Cite

The activity and kinetic stability of a keratinolytic subtilisin-like protease from Bacillus sp. P45 was investigated in 100 mM Tris-HCl buffer (pH 8.0; control) and in buffer with addition of Ca(2+) or Mg(2+) (1-10 mM), at different temperatures. Addition of 3 mM Ca(2+) or 4 mM Mg(2+) resulted in a 26% increment on enzyme activity towards azocasein when compared to the control (100%; without added Ca(2+) or Mg(2+)) at 55 °C. Optimal temperature for activity in the control (55 °C) was similar with Mg(2+); however, temperature optimum was increased to 60 °C with 3 mM Ca(2+), displaying an enhancement of 42% in comparison to the control at 55 °C. Stability of protease P45 in control buffer and with Mg(2+) addition was assayed at 40-50 °C, and at 55-62 °C with Ca(2+) addition. Data were fitted to six kinetic inactivation models, and a first-order equation was accepted as the best model to describe the inactivation of protease P45 with and without metal ions. The kinetic and thermodynamic parameters obtained showed the crucial role of calcium ions for enzyme stability. As biocatalyst stability is fundamental for commercial/industrial purposes, the stabilising effect of calcium could be exploited aiming the application of protease P45 in protein hydrolysis.

show abstract

Section: Resultsmentioning

confidence: 99%

Kinetic Stability Modelling of Keratinolytic Protease P45: Influence of Temperature and Metal Ions

Daroit

Sant’Anna

Brandelli

2011

Appl Biochem Biotechnol

View full text Add to dashboard Cite

show abstract

“…For this purpose, AprBp and GseBp proteins were digested (trypsinized) in silico and peptides (precursors) with their transitions were selected. The uncleaved sequences of AprBp and GseBp have 381 and 303 residues with a theoretical molecular weight of 27 and 23 kDa, respectively [ 21 , 22 ]. Amino acids 1–29 (for AprBp) and 1–26 (for GseBp) comprise signal peptides which are normally removed from the mature proteins by cleavage prior to secretion into cultivation media.…”

Section: Resultsmentioning

confidence: 99%

“…A calibration curve using a highly intense transition for each target protein is vital for accurate protein quantification by MRM analysis. For this purpose, purified serine proteases with a degrees of protein purity of 757.11 for AprBp and 1257 for GseBp, were used to construct the calibration curve, basing on a range that us suitable for the protein concentration in the samples [ 21 , 22 ]. Purified enzymes (AprBp and GseBp) were added to the gel and separated via a one-dimensional SDS-PAGE.…”

Section: Resultsmentioning

confidence: 99%

Label-Free Multiple Reaction Monitoring, a Promising Method for Quantification Analyses of Specific Proteins in Bacteria

Toymentseva¹,

Koryagina²,

Laikov³

et al. 2020

IJMS

View full text Add to dashboard Cite

Bacillus subtilis produces eight industrially important exo-proteases. For the detection of proteases, the activity- and antibody-based assays are normally used. Current activity-based assays require expensive multiplex chemical substrates which allow specificity determination of each enzyme. In this study, we provide evidences pertaining to the usefulness of the label-free multiple reaction monitoring (MRM) assay for a rapid identification and quantitation of specific proteins in bacteria. We used wild-type B. pumilus cells producing at least two serine proteases, subtilisin-like protease (AprBp) and glutamyl endopeptidase (GseBp), as well as optimized recombinant B. subtilis cells containing the same protease genes under control of the LIKE expression system. The Skyline software was used for the selection of three specific proteotypic peptides and their fragment ions for quantification and confirmation of AprBp and GseBp in complex mixtures. MRM indicated that the production of AprBp and GseBp exo-enzymes were respectively 0.9- and 26.6-fold higher in the culture medium of B. pumilus strain in comparison to the recombinant B. subtilis strains carrying optimized LIKE expression systems under identical conditions. The developed procedure in this study is fast, easy to perform and dependable. Additionally, it achieves accurate proteins identification and quantification in complex mixture.

show abstract

“…The MALDI TOF analysis data and the results of the enzymatic proper ties study suggest that the B. pumilus enzymes secreted at the early and late stationary phase of the culture growth are expression products of one gene. Insignifi cant differences in physico chemical properties could be explained by the differences in proteinase secretion conditions from the cell at different stages of culture development, which could define modulation in the tertiary structure of the protein globe [28]. B. pumilus proteinases have pronounced thrombolytic properties, which hold much promise for application of these enzymes as thrombolytic agents.…”

Section: Resultsmentioning

confidence: 99%

Subtilisin-like proteinase secreted by the Bacillus pumilus KMM 62 strain at different growth stages

et al. 2012

Self Cite

View full text Add to dashboard Cite

A protease secreted in Bacillus pumilus KMM 62 culture liquid on different growth stages was isolated using ion-exchange chromatography. On the basis of pattern of specific chromogenic substrates hydrolysis and inhibitory analysis the protease was classified as subtilisin like serine protease. The molecular weight ofprotease is 31 kDa. Proteolytic activity towards Z-Ala-Ala-Leu-pNa substrate was maximal at pH 8-8.5. The optimal temperature for proteolytic activity was observed at a temperature of 30 degrees C, and the protein was stable within the pH range of 7.5-10.0. Bacillus pumilus KMM 62 subtilisin like serine protease was shown to have thrombolytic activity.

show abstract

Biochemical properties of Bacillus intermedius subtilisin-like proteinase secreted by a Bacillus subtilis recombinant strain in its stationary phase of growth

Cited by 9 publications

References 30 publications

Kinetic Stability Modelling of Keratinolytic Protease P45: Influence of Temperature and Metal Ions

Kinetic Stability Modelling of Keratinolytic Protease P45: Influence of Temperature and Metal Ions

Label-Free Multiple Reaction Monitoring, a Promising Method for Quantification Analyses of Specific Proteins in Bacteria

Subtilisin-like proteinase secreted by the Bacillus pumilus KMM 62 strain at different growth stages

Contact Info

Product

Resources

About