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1. The biogenesis of CLEL peptides involves several processing events in consecutive compartments of the secretory pathway

Author

Nils Stührwohldt, Stefan Scholl, Lisa Lang, Julia Katzenberger, Karin Schumacher, and Andreas Schaller

Subjects
post-translational modification, precursor processing, peptide hormone, subtilase, subtilisin-like proteinase, peptide signaling, Medicine, Science, Biology (General), QH301-705.5
Abstract

Post-translationally modified peptides are involved in many aspects of plant growth and development. The maturation of these peptides from their larger precursors is still poorly understood. We show here that the biogenesis of CLEL6 and CLEL9 peptides in Arabidopsis thaliana requires a series of processing events in consecutive compartments of the secretory pathway. Following cleavage of the signal peptide upon entry into the endoplasmic reticulum (ER), the peptide precursors are processed in the cis-Golgi by the subtilase SBT6.1. SBT6.1-mediated cleavage within the variable domain allows for continued passage of the partially processed precursors through the secretory pathway, and for subsequent post-translational modifications including tyrosine sulfation and proline hydroxylation within, and proteolytic maturation after exit from the Golgi. Activation by subtilases including SBT3.8 in post-Golgi compartments depends on the N-terminal aspartate of the mature peptides. Our work highlights the complexity of post-translational precursor maturation allowing for stringent control of peptide biogenesis.

Published
2020
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3. From structure to function – a family portrait of plant subtilases.

Author

Schaller, Andreas, Stintzi, Annick, Rivas, Susana, Serrano, Irene, Chichkova, Nina V., Vartapetian, Andrey B., Martínez, Dana, Guiamét, Juan J., Sueldo, Daniela J., van der Hoorn, Renier A. L., Ramírez, Vicente, and Vera, Pablo

Subjects
*SUBTILISINS, *PROTEOLYTIC enzyme structure, *PLANT enzymes, *PLANT defenses, *PLANT physiology, *PLANT development, *PROTEOLYSIS, *PLANTS
Abstract

Contents Summary 901 I. Introduction 901 II. Biochemistry and structure of plant SBTs 902 III. Phylogeny of plant SBTs and family organization 903 IV. Physiological roles of plant SBTs 905 V. Conclusions and outlook 911 Acknowledgements 912 References 912 Summary: Subtilases (SBTs) are serine peptidases that are found in all three domains of life. As compared with homologs in other Eucarya, plant SBTs are more closely related to archaeal and bacterial SBTs, with which they share many biochemical and structural features. However, in the course of evolution, functional diversification led to the acquisition of novel, plant‐specific functions, resulting in the present‐day complexity of the plant SBT family. SBTs are much more numerous in plants than in any other organism, and include enzymes involved in general proteolysis as well as highly specific processing proteases. Most SBTs are targeted to the cell wall, where they contribute to the control of growth and development by regulating the properties of the cell wall and the activity of extracellular signaling molecules. Plant SBTs affect all stages of the life cycle as they contribute to embryogenesis, seed development and germination, cuticle formation and epidermal patterning, vascular development, programmed cell death, organ abscission, senescence, and plant responses to their biotic and abiotic environments. In this article we provide a comprehensive picture of SBT structure and function in plants. [ABSTRACT FROM AUTHOR]

Published
2018
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4. Subtilisin-like proteinase secreted by the Bacillus pumilus KMM 62 strain at different growth stages.

Author

Mardanova, A., Malikova, L., Balaban, N., Zamatlyutdinova, N., and Sharipova, M.

Subjects
*PROTEINASES, *BACILLUS pumilus, *HYDROLYSIS, *SERINE proteinases, *CHROMATOGRAPHIC analysis
Abstract

Proteinase secreted in the environment by bacilli on different growth stages was isolated by ion chromatography from the culture medium of Bacillus pumilus KMM 62. According to the hydrolysis character of specific chromogenic substrates and inhibition type, the enzyme belongs to subtilisin-like serine proteinases. The isolated proteinase with the molecular mass of 30 kDa displays maximum activity on hydrolysis of the peptide substrate Z-Ala-Ala-Leu-pNA at pH 8.0-8.5 and temperature 30°C. The protein is stable in the range of pH 7.5-10.0. It was shown that subtilisin-like serine proteinase from B. pumilus KMM 62 possessed thrombolytic activity. [ABSTRACT FROM AUTHOR]

Published
2012
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5. A NaCl-stable serine proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce

Author

Sinsuwan, Sornchai, Rodtong, Sureelak, and Yongsawatdigul, Jirawat

Subjects
*SERINE proteinases, *HYDROPHOBIC surfaces, *HYDROXYAPATITE, *SAUCES, *PROTEIN folding, *MOLECULAR weights, *MICROBIAL enzymes, *MATRIX-assisted laser desorption-ionization
Abstract

Abstract: An extracellular proteinase from Virgibacillus sp. SK33, isolated from 1month-old fish sauce, was purified to electrophoretic homogeneity, using hydrophobic interaction chromatography and hydroxyapatite with purification fold of 2.5 and 7% yield. The anomalous molecular weight (MW) of 19kDa was obtained from SDS–PAGE, whereas a MW of 33.7kDa was determined by MALDI-TOF. Optimum conditions for catalytic activity were 55°C and pH 7.5. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferentially hydrolysed Suc-Ala-Ala-Pro-Phe-AMC, indicating a serine proteinase with subtilisin-like characteristics. K m and k cat of the purified proteinase were 27μM and 12s−1, respectively. Proteinase activity, toward both synthetic and anchovy substrates, increased with NaCl up to 25%. The proteinase exhibited high stability in both the absence and presence of NaCl up to 25%. Approximately 2.5-fold increase in activity was observed in the presence of divalent cations, including Ca2+, Mg2+ and Sr2+ at 100mM. MALDI-TOF MS and LC–ESI-MS/MS analyses, as well as N-terminal sequences, revealed that the purified enzyme did not match microbial proteinases in the database, indicating it to be a novel proteinase. [Copyright &y& Elsevier]

Published
2010
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6. Effect of the regulation system of metabolic nitrogen exchange on biosynthesis of serine proteinases from Bacillus intermedius.

Author

Kayumov, A. R., Shamsutdinov, T. R., Sabirova, A. R., and Sharipova, M. R.

Subjects
*BACILLUS (Bacteria), *PROTEINASES, *ENDOPEPTIDASES, *GENE expression, *GENETIC mutation
Abstract

The regulatory link between biosynthesis of Bacillus intermedius subtilisin-like serine proteinase and nitrogen metabolism in B. intermedius cells was determined. The level of the enzyme biosynthesis by the recombinant strain of Bacillus subtilis in the medium containing ammonium ions was three- to fivefold less than that in the medium with poorly utilized sodium nitrate. Accumulation of glutamyl endopeptidase in a culture liquid of this microorganism did not depend on the source of nitrogen present in the medium. During cultivation in the rich medium, the productivity of subtilisin-like proteinase in the recombinant B. subtilis strain carrying a mutation in the NrgB sensor protein was demonstrated to increase threefold compared to that of the control strain. In the minimal culture medium, mutation in the nrgB gene abolished the effect of a nitrogen source on the level of the subtilisin-like proteinase gene expression. At the same time, this mutation did not affect glutamyl endopeptidase biosynthesis. Thus, expression of the gene coding for subtilisin-like proteinase from B.intermedius is suggested to be positively regulated by the regulatory system of nitrogen metabolism. [ABSTRACT FROM AUTHOR]

Published
2009
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7. Purification of a subtilisin-like serine proteinase from recombinant Bacillus subtilis during different phases of growth.

Author

Mikhailova, Ekaterina, Balaban, Nelly, Mardanova, Ayslu, Rudakova, Nataliya, Ilyinskaya, Olga, Rudenskaya, Galina, Rizvanov, Albert, and Sharipova, Margarita

Abstract

Subtilisin-like proteinase Bacillus intermedius which is secreted at different stages of bacterial growth (at 28 h and 48 h) were purified from the culture media of recombinant strain Bacillus subtilis JB 20–36(pCS9) by chromatography on CM-cellulose and MonoS columns. MALDI-TOF mass spectroscopy of purified enzymes demonstrated that they were identical in regard to amino acid sequence. The molecular weights of both proteins were 27 kDa. Biochemical analysis revealed differences in K m values for proteinase isolated at different growth stages (1.85 and 0.86 mM for first and second fractions respectively), and in substrate specificity and sensitiveness to Ca2+ ions. Gel-filtration experiments demonstrated that subtilisin-like proteinase B. Intermedius was produced as an active monomer (27 kDa) during early stationary phase (28 h of growth) and as a dimer (54 kDa) during the late stationary phase (48 h). [ABSTRACT FROM AUTHOR]

Published
2009
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8. Hydrogen sulfide inhibits the abscission of tomato pedicel through reconstruction of a basipetal auxin gradient.

Author

Liu, Danmei, Guo, Ting, Li, Jianing, Hao, Yuan, Zhao, Dan, Wang, Longdan, Liu, Zhiqiang, Zhang, Liping, Jin, Zhuping, and Pei, Yanxi

Subjects
*HYDROGEN sulfide, *TOMATOES, *PEPTIDES, *AGRICULTURAL processing, *CELLULAR signal transduction
Abstract

Abscission is an important developmental process and an essential agricultural trait. Auxin and ethylene are two phytohormones with important roles in the complex, but still elusive signaling network of abscission. Here, we found that hydrogen sulfide (H 2 S), a newly identified gasotransmitter, inhibits the initiation of tomato pedicel abscission. The underlying mechanism was explored through transcriptome profile analysis in various pedicel tissues with or without H 2 S treatment in the early abscission stage. The data suggested that H 2 S strongly influences the global transcription of pedicel tissues, exerts differential expression regulation along the pedicel, and markedly influences both the auxin and ethylene signaling pathways. Computational analysis revealed that H 2 S reconstructs a basipetal auxin gradient along the pedicel at 4 h after treatment; this finding was further substantiated by the GUS-staining results of DR5 :: GUS pedicels. The inhibitory effect of H 2 S to the ethylene signaling pathway might be an indirect action. Moreover, the subtilisin-like proteinase family members involved in the release of peptide signal molecules are critical components of the abscission signaling network downstream of auxin and ethylene. • Hydrogen sulfide (H 2 S) inhibited the initiation of the tomato pedicel abscission process. • H 2 S reconstructed a basipetal auxin gradient along the treated tomato pedicel. • H 2 S played an important role in the transcription level. • The ligand-receptor and the ROX systems are potential regulatory nodes downstream auxin in tomato pedicel abscission. [ABSTRACT FROM AUTHOR]

Published
2022
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9. Biochemical properties of Bacillus intermedius subtilisin-like proteinase secreted by a Bacillus subtilis recombinant strain in its stationary phase of growth.

Author

Mikhailova, E. O., Mardanova, A. M., Balaban, N. P., Rudenskaya, G. N., Ilyinskaya, O. N., and Sharipova, M. R.

Subjects
*BACILLUS subtilis, *PROTEINASES, *BACTERIAL growth, *OXIDIZING agents, *CALCIUM ions
Abstract

Biochemical properties of Bacillus intermedius subtilisin-like proteinase (AprBi) secreted by a B. subtilis recombinant strain in the early and late stationary phases of growth have been determined. Protein structure was analyzed and its stability estimated. It was noted that the enzyme corresponding to different phases of bacterial growth retains activity in the presence of reducing and oxidizing agents (C2H5OH and H2O2). Different effects of bivalent metal ions on activity of two proteinase fractions were found. Calcium ions more efficiently activate proteinase secreted in the late stationary phase. Unlike the first enzyme fraction, the second forms catalytically active dimers. [ABSTRACT FROM AUTHOR]

Published
2009
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10. Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by Bacillus pumilus KMM 62.

Author

Malikova, L., Mardanova, A., Sokolova, O., Balaban, N., Rudenskaya, G., and Sharipova, M.

Subjects
*BIOSYNTHESIS, *EXTRACELLULAR enzymes, *SUBTILISINS, *SERINE proteinases, *BACILLUS (Bacteria), *BACTERIAL growth, *AMINO acids
Abstract

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46–48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46–48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The involvement of different regulatory mechanisms of the synthesis of this proteinase is assumed in the early and late stationary phases of growth. [ABSTRACT FROM AUTHOR]

Published
2007
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11. Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species.

Author

Arnórsdóttir, Jóhanna, Smáradóttir, Rúna B., Magnússon, Ólafur Th., Thorbjarnardóttir, Sigrídur H., Eggertsson, Gudmundur, and Kristjánsson, Magnús M.

Subjects
*SUBTILISINS, *SERINE proteinases, *ESCHERICHIA coli, *COLD adaptation
Abstract

The gene encoding a subtilisin-like serine proteinase in the psychrotrophic Vibrio sp. PA44 has been successfully cloned, sequenced and expressed in Escherichia coli . The gene is 1593 basepairs and encodes a precursor protein of 530 amino acid residues with a calculated molecular mass of 55.7 kDa. The enzyme is isolated, however, as an active 40.6-kDa proteinase, without a 139 amino acid residue N-terminal prosequence. Under mild conditions the enzyme undergoes a further autocatalytic cleavage to give a 29.7-kDa proteinase that retains full enzymatic activity. The deduced amino acid sequence of the enzyme has high homology to proteinases of the proteinase K family of subtilisin-like proteinases. With respect to the enzyme characteristics compared in this study the properties of the wild-type and recombinant proteinases are the same. Sequence analysis revealed that especially with respect to the thermophilic homologues, aqualysin I from Thermus aquaticus and a proteinase from Thermus strain Rt41A, the cold-adapted Vibrio -proteinase has a higher content of polar/uncharged amino acids, as well as aspartate residues. The thermophilic enzymes had a higher content of arginines, and relatively higher number of hydrophobic amino acids and a higher aliphatic index. These factors may contribute to the adaptation of these proteinases to different temperature conditions. [ABSTRACT FROM AUTHOR]

Published
2002
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12. The biogenesis of CLEL peptides involves several processing events in consecutive compartments of the secretory pathway

Author

Andreas Schaller, Stefan Scholl, Karin Schumacher, Nils Stührwohldt, Julia Katzenberger, and Lisa Lang

Subjects
0106 biological sciences, 0301 basic medicine, Tyrosine sulfation, Signal peptide, QH301-705.5, Science, Arabidopsis, Plant Biology, Peptide, 01 natural sciences, peptide signaling, General Biochemistry, Genetics and Molecular Biology, Subtilase, 03 medical and health sciences, symbols.namesake, precursor processing, Biology (General), Secretory pathway, chemistry.chemical_classification, Secretory Pathway, General Immunology and Microbiology, peptide hormone, Arabidopsis Proteins, Chemistry, General Neuroscience, Endoplasmic reticulum, General Medicine, Golgi apparatus, Cell biology, 030104 developmental biology, subtilisin-like proteinase, post-translational modification, A. thaliana, symbols, Medicine, subtilase, Protein Processing, Post-Translational, Biogenesis, Research Article, 010606 plant biology & botany
Abstract

Post-translationally modified peptides are involved in many aspects of plant growth and development. The maturation of these peptides from their larger precursors is still poorly understood. We show here that the biogenesis of CLEL6 and CLEL9 peptides in Arabidopsis thaliana requires a series of processing events in consecutive compartments of the secretory pathway. Following cleavage of the signal peptide upon entry into the endoplasmic reticulum (ER), the peptide precursors are processed in the cis-Golgi by the subtilase SBT6.1. SBT6.1-mediated cleavage within the variable domain allows for continued passage of the partially processed precursors through the secretory pathway, and is a prerequisite for subsequent post-translational modifications including tyrosine sulfation and proline hydroxylation within, and proteolytic maturation after exit from the Golgi. Activation by subtilase SBT3.8 in post-Golgi compartments depends on the N-terminal aspartate of the mature peptides. Our work highlights the complexity of post-translational precursor maturation allowing for stringent control of peptide biogenesis.

Published
2018
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13. From structure to function - a family portrait of plant subtilases

Author

Nina V. Chichkova, Andrey B. Vartapetian, Andreas Schaller, Susana Rivas, Renier A. L. van der Hoorn, Daniela J. Sueldo, Vicente Ramírez, Annick Stintzi, Dana Ethel Martinez, Irene Serrano, Pablo Vera, Juan José Guiamet, Institute of Plant Physiology and Biotechnology, University of Hohenheim, Laboratoire des interactions plantes micro-organismes (LIPM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University (MSU), Instituto de Fisiología Vegetal [La Plata] (INFIVE), Facultad de Ciencias Naturales y Museo [La Plata] (FCNyM), Universidad Nacional de la Plata [Argentine] (UNLP)-Universidad Nacional de la Plata [Argentine] (UNLP), The Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Institute for Plant Cell Biology and Biotechnology, Heinrich-Heine-Universität Düsseldorf [Düsseldorf], Institute for Plant Molecular and Cell Biology, Universitat Politecnica de Valencia (UPV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), University of Oxford [Oxford], Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf], Universitat Politècnica de València (UPV), Institut National de la Recherche Agronomique (INRA), French Laboratory of Excellence project 'TULIP' : ANR-10-LABX-41, ANR-11-IDEX-0002-02, Spanish MINECO : BFU2015-68199-R, Generalitat Valenciana : PrometeoII/2014/024, Russian Science Foundation : 16-14-10043, Russian Foundation for Basic Research : 14-04-00232, 14-04-00256, 14-04-91330, ERC consolidator grant : 616449, German Research Foundation (DFG) : SCHA 591/8-1, and European Project: 267196,EC:FP7:PEOPLE,FP7-PEOPLE-2010-COFUND,AGREENSKILLS(2012)

Subjects
0301 basic medicine, Physiology, Biología, Plant Science, peptide signaling, Structure–function relationship, purl.org/becyt/ford/1 [https], Organism, PEPTIDE SIGNALING, 2. Zero hunger, Vegetal Biology, PLANT DEVELOPMENT, food and beverages, structure-function relationship, Cell biology, SUBTILISIN-LIKE PROTEINASE, cell death, plant development, CIENCIAS NATURALES Y EXACTAS, Cell signaling, Proteases, proteolysis, STRUCTURE–FUNCTION RELATIONSHIP, Otras Ciencias Biológicas, Biology, CELL DEATH, Subtilase, Cell wall, Ciencias Biológicas, 03 medical and health sciences, physiologie végétale, Abscission, Phylogenetics, plant defense, precursor processing, Ciencias Naturales, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, purl.org/becyt/ford/1.6 [https], PROTEOLYSIS, biochimie végétale, Ciencias Exactas, subtilisin-like proteinase, fungi, PRECURSOR PROCESSING, 030104 developmental biology, PLANT DEFENSE, Function (biology), Biologie végétale
Abstract

Subtilases (SBTs) are serine peptidases that are found in all three domains of life. As compared with homologs in other Eucarya, plant SBTs are more closely related to archaeal and bacterial SBTs, with which they share many biochemical and structural features. However, in the course of evolution, functional diversification led to the acquisition of novel, plant-specific functions, resulting in the present-day complexity of the plant SBT family. SBTs are much more numerous in plants than in any other organism, and include enzymes involved in general proteolysis as well as highly specific processing proteases. Most SBTs are targeted to the cell wall, where they contribute to the control of growth and development by regulating the properties of the cell wall and the activity of extracellular signaling molecules. Plant SBTs affect all stages of the life cycle as they contribute to embryogenesis, seed development and germination, cuticle formation and epidermal patterning, vascular development, programmed cell death, organ abscission, senescence, and plant responses to their biotic and abiotic environments. In this article we provide a comprehensive picture of SBT structure and function in plants., Instituto de Fisiología Vegetal

Published
2017
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15. Действие бациллярных протеиназ на матриксные металлопротеиназы разных клеточных линий

Subjects
Bacillus pumilus, глутамилэндопептидаза, субтилизиноподобная протеиназа, адамализиноподобная металлоэндопептидаза, матриксные металлопротеиназы, Bacillus pumilus, glutamyl endopeptidase, subtilisin-like proteinase, adams-like metalloproteinase, matrix metalloproteinases
Abstract

Исследовали действие различных внеклеточных протеиназ Bacillus pumilus 7P на матриксные металлопротеиназы фибробластов мыши (3T3BalbSV40), мультипотентных мезенхимальных стромальных клеток и клеток Hela-M. Глутамилэндопептидаза инактивировала матриксные металлопротеиназы всех клеточных линий. В отличие от высокоспецифичной протеиназы субтилизиноподобная протеиназа и металлоэндопептидаза B. pumilus не изменяли активность матриксных металлопротеиназ клеток культуры ткани разных клеточных линий., It was investigated the effect of secreted proteinases Bacillus pumilus 7P belonging to different classes of proteolytic proteins on matrix metalloproteinases of mouse fibroblast (3T3BalbSV40), multipotent mesenchymal stromal and cells Hela-M. Matrix metalloproteinases of all cell lines was inactivated by glutamyl-specific endopeptidase. Subtilisin-like proteinase and adams-like metalloproteinase of B. pumilus did not change the activity of matrix metalloproteinases of differentcell lines.

Published
2014

16. Thrombolytic and fibrinolytic activity of bacterial proteases

Author

Danilova J., Cheremin A., Zamaleeva A., Mardanova A., and Sharipova M.

Subjects
Thrombolytic and fibrinolytic properties, Glutamyl endopeptidase, Subtilisin-like proteinase, Anticoagulant activity, Metalloproteinase
Abstract

In medical practice is increasingly use proteolytic enzymes of microorganisms. Particular attention of researchers is attracted proteases, which have fibrinolytic properties, and can lyse clots. Previously had been isolated and purified to homogeneity glutamyl endopeptidase, subtilisin-like proteinase and metalloendopeptidase of Bacillus pumilis 3-19, secreted by B. subtilis JB 2036 recombinant strain. The analysis of thrombolytic, fibrinolytic, and anticoagulant properties of the recombinant enzymes was conducted. It is shown that all investigated proteinases are able to efficiently lyse the clot. In an in vitro subtilisin and glutamyl endopeptidase of B.subtilis recombinant strain have anticoagulant activity. Metalloproteinase is not able to influence the process clot formation. Subtilisinlike proteinase and glutamyl endopeptidase possess fibrinolytic activity and the ability of the activator relative to plasminogen. Metalloproteinase shows no fibrinolytic properties. Due to the high incidence of cardiovascular diseases is urgent search for new enzymes with high biological activity, specificity and low toxicity.

Published
2012

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

Author

Mardanova A., Malikova L., Balaban N., Zamatlyutdinova N., and Sharipova M.

Subjects
Thrombolytic activity, Subtilisin-like proteinase, fungi, Properties, Bacillus pumilus, Isolation
Abstract

Proteinase secreted in the environment by bacilli on different growth stages was isolated by ion chromatography from the culture medium of Bacillus pumilus KMM 62. According to the hydrolysis character of specific chromogenic substrates and inhibition type, the enzyme belongs to subtilisin-like serine proteinases. The isolated proteinase with the molecular mass of 30 kDa displays maximum activity on hydrolysis of the peptide substrate Z-Ala-Ala-Leu-pNA at pH 8.0 - 8.5 and temperature 30°C. The protein is stable in the range of pH 7.5 - 10.0. It was shown that subtilisin-like serine proteinase from B. pumilus KMM 62 possessed thrombolytic activity. © Pleiades Publishing, Ltd., 2012.

Published
2012

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

Author

Mikhailova E., Mardanova A., Balaban N., Rudenskaya G., Ilyinskaya O., and Sharipova M.

Subjects
Subtilisin-like proteinase, AprBi, Dimers
Abstract

Biochemical properties of Bacillus intermedius subtilisin-like proteinase (AprBi) secreted by a B. subtilis recombinant strain in the early and late stationary phases of growth have been determined. Protein structure was analyzed and its stability estimated. It was noted that the enzyme corresponding to different phases of bacterial growth retains activity in the presence of reducing and oxidizing agents (C2H5OH and H 2O2). Different effects of bivalent metal ions on activity of two proteinase fractions were found. Calcium ions more efficiently activate proteinase secreted in the late stationary phase. Unlike the first enzyme fraction, the second forms catalytically active dimers. © 2009 Pleiades Publishing, Ltd.

Published
2009

19. Effect of the regulation system of metabolic nitrogen exchange on biosynthesis of serine proteinases from Bacillus intermedius

Author

Kayumov A., Shamsutdinov T., Sabirova A., and Sharipova M.

Subjects
Glutamyl endopeptidase, Subtilisin-like proteinase, fungi, Nitrogen catabolite repression, Bacillus intermedius
Abstract

The regulatory link between biosynthesis of Bacillus intermedius subtilisin-like serine proteinase and nitrogen metabolism in B. intermedius cells was determined. The level of the enzyme biosynthesis by the recombinant strain of Bacillus subtilis in the medium containing ammonium ions was three- to fivefold less than that in the medium with poorly utilized sodium nitrate. Accumulation of glutamyl endopeptidase in a culture liquid of this microorganism did not depend on the source of nitrogen present in the medium. During cultivation in the rich medium, the productivity of subtilisin-like proteinase in the recombinant B. subtilis strain carrying a mutation in the NrgB sensor protein was demonstrated to increase threefold compared to that of the control strain. In the minimal culture medium, mutation in the nrgB gene abolished the effect of a nitrogen source on the level of the subtilisin-like proteinase gene expression. At the same time, this mutation did not affect glutamyl endopeptidase biosynthesis. Thus, expression of the gene coding for subtilisin-like proteinase from B. intermedius is suggested to be positively regulated by the regulatory system of nitrogen metabolism. © Pleiades Publishing, Ltd., 2009.

Published
2009

20. Purification of a subtilisin-like serine proteinase from recombinant Bacillus subtilis during different phases of growth

Author

Mikhailova E., Balaban N., Mardanova A., Rudakova N., Ilyinskaya O., Rudenskaya G., Rizvanov A., and Sharipova M.

Subjects
Subtilisin-like proteinase, Protein dimer, Bacillus intermedius, Bacillus subtilis
Abstract

Subtilisin-like proteinase Bacillus intermedius which is secreted at different stages of bacterial growth (at 28 h and 48 h) were purified from the culture media of recombinant strain Bacillus subtilis JB 20-36(pCS9) by chromatography on CM-cellulose and MonoS columns. MALDI-TOF mass spectroscopy of purified enzymes demonstrated that they were identical in regard to amino acid sequence. The molecular weights of both proteins were 27 kDa. Biochemical analysis revealed differences in Km values for proteinase isolated at different growth stages (1.85 and 0.86 mM for first and second fractions respectively), and in substrate specificity and sensitiveness to Ca2+ ions. Gel-filtration experiments demonstrated that subtilisin-like proteinase B. intermedius was produced as an active monomer (27 kDa) during early stationary phase (28 h of growth) and as a dimer (54 kDa) during the late stationary phase (48 h).

Published
2009

21. Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by Bacillus pumilus KMM 62

Author

Malikova L., Mardanova A., Sokolova O., Balaban N., Rudenskaya G., and Sharipova M.

Subjects
Subtilisin-like proteinase, Conditions of growth and biosynthesis, Biosynthesis, Bacillus pumilus
Abstract

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46-48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46-48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The involvement of different regulatory mechanisms of the synthesis of this proteinase is assumed in the early and late stationary phases of growth. © Nauka/Interperiodica 2007.

Published
2007

23. Thrombolytic and fibrinolytic activity of bacterial proteases

Author

Danilova J., Cheremin A., Zamaleeva A., Mardanova A., Sharipova M., Danilova J., Cheremin A., Zamaleeva A., Mardanova A., and Sharipova M.

Abstract

In medical practice is increasingly use proteolytic enzymes of microorganisms. Particular attention of researchers is attracted proteases, which have fibrinolytic properties, and can lyse clots. Previously had been isolated and purified to homogeneity glutamyl endopeptidase, subtilisin-like proteinase and metalloendopeptidase of Bacillus pumilis 3-19, secreted by B. subtilis JB 2036 recombinant strain. The analysis of thrombolytic, fibrinolytic, and anticoagulant properties of the recombinant enzymes was conducted. It is shown that all investigated proteinases are able to efficiently lyse the clot. In an in vitro subtilisin and glutamyl endopeptidase of B.subtilis recombinant strain have anticoagulant activity. Metalloproteinase is not able to influence the process clot formation. Subtilisinlike proteinase and glutamyl endopeptidase possess fibrinolytic activity and the ability of the activator relative to plasminogen. Metalloproteinase shows no fibrinolytic properties. Due to the high incidence of cardiovascular diseases is urgent search for new enzymes with high biological activity, specificity and low toxicity.

24. Purification of a subtilisin-like serine proteinase from recombinant Bacillus subtilis during different phases of growth

Author

Mikhailova E., Balaban N., Mardanova A., Rudakova N., Ilyinskaya O., Rudenskaya G., Rizvanov A., Sharipova M., Mikhailova E., Balaban N., Mardanova A., Rudakova N., Ilyinskaya O., Rudenskaya G., Rizvanov A., and Sharipova M.

Abstract

Subtilisin-like proteinase Bacillus intermedius which is secreted at different stages of bacterial growth (at 28 h and 48 h) were purified from the culture media of recombinant strain Bacillus subtilis JB 20-36(pCS9) by chromatography on CM-cellulose and MonoS columns. MALDI-TOF mass spectroscopy of purified enzymes demonstrated that they were identical in regard to amino acid sequence. The molecular weights of both proteins were 27 kDa. Biochemical analysis revealed differences in Km values for proteinase isolated at different growth stages (1.85 and 0.86 mM for first and second fractions respectively), and in substrate specificity and sensitiveness to Ca2+ ions. Gel-filtration experiments demonstrated that subtilisin-like proteinase B. intermedius was produced as an active monomer (27 kDa) during early stationary phase (28 h of growth) and as a dimer (54 kDa) during the late stationary phase (48 h).

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

Author

Mardanova A., Malikova L., Balaban N., Zamatlyutdinova N., Sharipova M., Mardanova A., Malikova L., Balaban N., Zamatlyutdinova N., and Sharipova M.

Abstract

Proteinase secreted in the environment by bacilli on different growth stages was isolated by ion chromatography from the culture medium of Bacillus pumilus KMM 62. According to the hydrolysis character of specific chromogenic substrates and inhibition type, the enzyme belongs to subtilisin-like serine proteinases. The isolated proteinase with the molecular mass of 30 kDa displays maximum activity on hydrolysis of the peptide substrate Z-Ala-Ala-Leu-pNA at pH 8.0 - 8.5 and temperature 30°C. The protein is stable in the range of pH 7.5 - 10.0. It was shown that subtilisin-like serine proteinase from B. pumilus KMM 62 possessed thrombolytic activity. © Pleiades Publishing, Ltd., 2012.

26. Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by Bacillus pumilus KMM 62

Author

Malikova L., Mardanova A., Sokolova O., Balaban N., Rudenskaya G., Sharipova M., Malikova L., Mardanova A., Sokolova O., Balaban N., Rudenskaya G., and Sharipova M.

Abstract

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46-48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46-48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The involvement of different regulatory mechanisms of the synthesis of this proteinase is assumed in the early and late stationary phases of growth. © Nauka/Interperiodica 2007.

27. Effect of the regulation system of metabolic nitrogen exchange on biosynthesis of serine proteinases from Bacillus intermedius

Author

Kayumov A., Shamsutdinov T., Sabirova A., Sharipova M., Kayumov A., Shamsutdinov T., Sabirova A., and Sharipova M.

Abstract

The regulatory link between biosynthesis of Bacillus intermedius subtilisin-like serine proteinase and nitrogen metabolism in B. intermedius cells was determined. The level of the enzyme biosynthesis by the recombinant strain of Bacillus subtilis in the medium containing ammonium ions was three- to fivefold less than that in the medium with poorly utilized sodium nitrate. Accumulation of glutamyl endopeptidase in a culture liquid of this microorganism did not depend on the source of nitrogen present in the medium. During cultivation in the rich medium, the productivity of subtilisin-like proteinase in the recombinant B. subtilis strain carrying a mutation in the NrgB sensor protein was demonstrated to increase threefold compared to that of the control strain. In the minimal culture medium, mutation in the nrgB gene abolished the effect of a nitrogen source on the level of the subtilisin-like proteinase gene expression. At the same time, this mutation did not affect glutamyl endopeptidase biosynthesis. Thus, expression of the gene coding for subtilisin-like proteinase from B. intermedius is suggested to be positively regulated by the regulatory system of nitrogen metabolism. © Pleiades Publishing, Ltd., 2009.

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

Author

Mikhailova E., Mardanova A., Balaban N., Rudenskaya G., Ilyinskaya O., Sharipova M., Mikhailova E., Mardanova A., Balaban N., Rudenskaya G., Ilyinskaya O., and Sharipova M.

Abstract

Biochemical properties of Bacillus intermedius subtilisin-like proteinase (AprBi) secreted by a B. subtilis recombinant strain in the early and late stationary phases of growth have been determined. Protein structure was analyzed and its stability estimated. It was noted that the enzyme corresponding to different phases of bacterial growth retains activity in the presence of reducing and oxidizing agents (C2H5OH and H 2O2). Different effects of bivalent metal ions on activity of two proteinase fractions were found. Calcium ions more efficiently activate proteinase secreted in the late stationary phase. Unlike the first enzyme fraction, the second forms catalytically active dimers. © 2009 Pleiades Publishing, Ltd.

29. Thrombolytic and fibrinolytic activity of bacterial proteases

Author

Danilova J., Cheremin A., Zamaleeva A., Mardanova A., Sharipova M., Danilova J., Cheremin A., Zamaleeva A., Mardanova A., and Sharipova M.

Abstract

In medical practice is increasingly use proteolytic enzymes of microorganisms. Particular attention of researchers is attracted proteases, which have fibrinolytic properties, and can lyse clots. Previously had been isolated and purified to homogeneity glutamyl endopeptidase, subtilisin-like proteinase and metalloendopeptidase of Bacillus pumilis 3-19, secreted by B. subtilis JB 2036 recombinant strain. The analysis of thrombolytic, fibrinolytic, and anticoagulant properties of the recombinant enzymes was conducted. It is shown that all investigated proteinases are able to efficiently lyse the clot. In an in vitro subtilisin and glutamyl endopeptidase of B.subtilis recombinant strain have anticoagulant activity. Metalloproteinase is not able to influence the process clot formation. Subtilisinlike proteinase and glutamyl endopeptidase possess fibrinolytic activity and the ability of the activator relative to plasminogen. Metalloproteinase shows no fibrinolytic properties. Due to the high incidence of cardiovascular diseases is urgent search for new enzymes with high biological activity, specificity and low toxicity.

30. Purification of a subtilisin-like serine proteinase from recombinant Bacillus subtilis during different phases of growth

Author

Mikhailova E., Balaban N., Mardanova A., Rudakova N., Ilyinskaya O., Rudenskaya G., Rizvanov A., Sharipova M., Mikhailova E., Balaban N., Mardanova A., Rudakova N., Ilyinskaya O., Rudenskaya G., Rizvanov A., and Sharipova M.

Abstract

Subtilisin-like proteinase Bacillus intermedius which is secreted at different stages of bacterial growth (at 28 h and 48 h) were purified from the culture media of recombinant strain Bacillus subtilis JB 20-36(pCS9) by chromatography on CM-cellulose and MonoS columns. MALDI-TOF mass spectroscopy of purified enzymes demonstrated that they were identical in regard to amino acid sequence. The molecular weights of both proteins were 27 kDa. Biochemical analysis revealed differences in Km values for proteinase isolated at different growth stages (1.85 and 0.86 mM for first and second fractions respectively), and in substrate specificity and sensitiveness to Ca2+ ions. Gel-filtration experiments demonstrated that subtilisin-like proteinase B. intermedius was produced as an active monomer (27 kDa) during early stationary phase (28 h of growth) and as a dimer (54 kDa) during the late stationary phase (48 h).

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

Author

Mardanova A., Malikova L., Balaban N., Zamatlyutdinova N., Sharipova M., Mardanova A., Malikova L., Balaban N., Zamatlyutdinova N., and Sharipova M.

Abstract

Proteinase secreted in the environment by bacilli on different growth stages was isolated by ion chromatography from the culture medium of Bacillus pumilus KMM 62. According to the hydrolysis character of specific chromogenic substrates and inhibition type, the enzyme belongs to subtilisin-like serine proteinases. The isolated proteinase with the molecular mass of 30 kDa displays maximum activity on hydrolysis of the peptide substrate Z-Ala-Ala-Leu-pNA at pH 8.0 - 8.5 and temperature 30°C. The protein is stable in the range of pH 7.5 - 10.0. It was shown that subtilisin-like serine proteinase from B. pumilus KMM 62 possessed thrombolytic activity. © Pleiades Publishing, Ltd., 2012.

32. Effect of the regulation system of metabolic nitrogen exchange on biosynthesis of serine proteinases from Bacillus intermedius

Author

Kayumov A., Shamsutdinov T., Sabirova A., Sharipova M., Kayumov A., Shamsutdinov T., Sabirova A., and Sharipova M.

Abstract

The regulatory link between biosynthesis of Bacillus intermedius subtilisin-like serine proteinase and nitrogen metabolism in B. intermedius cells was determined. The level of the enzyme biosynthesis by the recombinant strain of Bacillus subtilis in the medium containing ammonium ions was three- to fivefold less than that in the medium with poorly utilized sodium nitrate. Accumulation of glutamyl endopeptidase in a culture liquid of this microorganism did not depend on the source of nitrogen present in the medium. During cultivation in the rich medium, the productivity of subtilisin-like proteinase in the recombinant B. subtilis strain carrying a mutation in the NrgB sensor protein was demonstrated to increase threefold compared to that of the control strain. In the minimal culture medium, mutation in the nrgB gene abolished the effect of a nitrogen source on the level of the subtilisin-like proteinase gene expression. At the same time, this mutation did not affect glutamyl endopeptidase biosynthesis. Thus, expression of the gene coding for subtilisin-like proteinase from B. intermedius is suggested to be positively regulated by the regulatory system of nitrogen metabolism. © Pleiades Publishing, Ltd., 2009.

33. Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by Bacillus pumilus KMM 62

Author

Malikova L., Mardanova A., Sokolova O., Balaban N., Rudenskaya G., Sharipova M., Malikova L., Mardanova A., Sokolova O., Balaban N., Rudenskaya G., and Sharipova M.

Abstract

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46-48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46-48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The involvement of different regulatory mechanisms of the synthesis of this proteinase is assumed in the early and late stationary phases of growth. © Nauka/Interperiodica 2007.

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

Author

Mikhailova E., Mardanova A., Balaban N., Rudenskaya G., Ilyinskaya O., Sharipova M., Mikhailova E., Mardanova A., Balaban N., Rudenskaya G., Ilyinskaya O., and Sharipova M.

Abstract

Biochemical properties of Bacillus intermedius subtilisin-like proteinase (AprBi) secreted by a B. subtilis recombinant strain in the early and late stationary phases of growth have been determined. Protein structure was analyzed and its stability estimated. It was noted that the enzyme corresponding to different phases of bacterial growth retains activity in the presence of reducing and oxidizing agents (C2H5OH and H 2O2). Different effects of bivalent metal ions on activity of two proteinase fractions were found. Calcium ions more efficiently activate proteinase secreted in the late stationary phase. Unlike the first enzyme fraction, the second forms catalytically active dimers. © 2009 Pleiades Publishing, Ltd.

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