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Youichi Tamai - One of the best experts on this subject based on the ideXlab platform.
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Purification and Characterization of <B>PhospholipaseB> B from Candida utilis
Bioscience biotechnology and biochemistry, 2006Co-Authors: Shuji Fujino, Yasuo Watanabe, Daigo Akiyama, Satoko Akaboshi, Tomonari Fujita, Youichi TamaiAbstract:<B>PhospholipaseB> B (PLB) from the asporogenous yeast Candida utilis was purified to homogeneity from a culture Broth. The apparent molecular mass was 90–110 kDa By SDS–PAGE. The enzyme had two pH optima, one acidic (pH 3.0) and the other alkaline (pH 7.5). At acidic pH the enzyme hydrolyzed all phospholipids tested without metal ions. On the other hand, the PLB showed suBstrate specificity and required metal ions for alkaline activity.The cDNA sequence of the PLB was analyzed By a comBination of several PCR procedures. The PLB encoded a protein consisting of 643 amino acids. The amino acid sequence contained a lipase consensus sequence (GxSxG) and catalytic arginine and aspartic acid motifs which were identified as the catalytic triad in the PLB from Kluyveromyces lactis, suggesting that the catalytic mechanism of the PLB is similar to that of cytosolic <B>PhospholipaseB> A2 (cPLA2), found in mammalian tissues.
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Purification and Characterization of <B>PhospholipaseB> B from Kluyveromyces lactis, and Cloning of <B>PhospholipaseB> B gene
Bioscience biotechnology and biochemistry, 1999Co-Authors: Hideki Oishi, Yasuo Watanabe, Takahiro Morimoto, Youichi TamaiAbstract:<B>PhospholipaseB> B (PLB) from the yeast Kluyveromyces lactis was purified to homogeneity from culture medium. The enzyme was highly glycosylated with apparent molecular mass of 160-250 kDa, and had two pH optima, at pH 2.0 and pH 7.5. At acidic pH the enzyme hydrolyzed all phospholipid suBstrates tested here without metal ion. On the other hand, at alkaline pH the enzyme showed suBstrate specificity for phosphatidylcholine and lysophosphatidylcholine and required Ca2+, Fe3+, or Al3+ for the activity. The alkaline activity was increased more than 20-fold in the presence of Al3+ compared to that in the presence of Ca2+.cDNA sequence of PLB (KlPLB) was analyzed By a comBination of several PCR procedures. KlPLB encoded a protein consist of 640 amino acids and the deduced amino acid sequence showed 66.7% similarity with the T. delBrueckii PLB. The amino acid sequence contained the lipase consensus sequence (G-X-S-X-G) and the catalytic aspartic acid motif. Replacement of Arg-112 or Asp-406 with alanine caused los...
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Disruption of <B>PhospholipaseB> B gene, PLB1, increases the survival of Baker's yeast Torulaspora delBrueckii
FEMS microbiology letters, 1996Co-Authors: Yasuo Watanabe, Kenji Imai, Hideki Oishi, Youichi TamaiAbstract:ABstract An uracil auxotrophic mutant of Baker's yeast Torulaspora delBrueckii , which is resitant to 5-fluoro-orotic acid, was complemented By transformation with YEp24 which harBors 2 μm origin and URA3 derived from Saccharomyces cerevisiae . The <B>PhospholipaseB> B in T. delBrueckii cells is active in Both acidic and alkaline conditions. However, activity of <B>PhospholipaseB> B gene ( PLB1 ) in cells of disruption mutant ( plBI : : URA3 ) was lost in Both conditions, which indicates that all <B>PhospholipaseB> B activity is encoded By a single gene (or a single polypeptide) in these yeast cells. Over-expression of PLB1 with YEp plasmid vector in T. delBrueckii cells showed ≈ 2.5-fold increase in <B>PhospholipaseB> B activity, comparing with that in wild-type cells. Cells of plB1 Δ mutant showed increased survival when cells of plB1 Δ mutant and wild-type strain were incuBated in water at 30 °C. Cells of PLB1 -over-expressed strain died rapidly even during the cultivation period, indicating that <B>PhospholipaseB> B activity may Be a determinant for the survival of this yeast.
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Purification and Some Properties of <B>PhospholipaseB> B from Schizosaccharomyces pomBe
Bioscience biotechnology and biochemistry, 1996Co-Authors: Hideki Oishi, Yasuo Watanabe, Shinji Tsuda, Youichi TamaiAbstract:<B>PhospholipaseB> B from Schizosaccharomyces pomBe was purified By ammonium sulfate fractionation and chromatographed on phenyl-Sepharose CL-4B, DEAE-Toyopearl 650M, and TSK gel G4000SW columns. The pu...
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Cloning and sequencing of <B>PhospholipaseB> B gene from the yeast Torulaspora delBrueckii
FEMS microbiology letters, 1994Co-Authors: Yasuo Watanabe, Masafumi Maruyama, Y. Yashiki, Gazi Nurun-nahar Sultana, Kenji Kangawa, Youichi TamaiAbstract:The extracellular <B>PhospholipaseB> B gene from Baker's yeast Torulaspora delBrueckii was cloned and sequenced. Analysis of DNA sequence data revealed an open reading frame (ORF) encoding a 649-amino acid protein, that included amino acid sequences oBtained from the purified enzyme. Comparison of these sequence data with the N-terminal amino acid sequence of the enzyme indicated that this secreted protein is synthesized as a large precursor with a 21-amino acid N-terminal extension to the mature enzyme of 628 amino acids. A homology search was carried out Between <B>PhospholipaseB> B from T. delBrueckii and Penicillium notatum. The deduced amino acid sequence of the cloned <B>PhospholipaseB> B was homologous (aBout 50% identity) to <B>PhospholipaseB> B from P. notatum, and contained six conserved regions. The transcriptional level of the <B>PhospholipaseB> B gene in different growth phases of the cells was investigated By Northern Blot analysis. The level of mRNA of the <B>PhospholipaseB> B gene was higher in the cells from early exponential and stationary phases.
Yasuo Watanabe - One of the best experts on this subject based on the ideXlab platform.
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In vitro synthesis of phospholipids with yeast <B>PhospholipaseB> B, a phospholipid deacylating enzyme.
Biotechnology reports (Amsterdam Netherlands), 2018Co-Authors: Yasuo Watanabe, Itsuki Kobayashi, Takanori Ohnaka, Seiya WatanabeAbstract:ABstract The gene encoding the Saccharomyces cerevisiae phospholipid deacylation enzyme, <B>PhospholipaseB> B (ScPLB1), was successfully expressed in E. coli. The enzyme (ScplB1p) was engineered to have a histidine-tag at the C-terminal end and was purified By metal (Ni) affinity chromatography. Enzymatic properties, optimal pH, and suBstrate specificity were similar to those reported previously. For example, deacylation activity was oBserved in acidic pH in the aBsence of Ca2+ and was additive in neutral pH in the presence of Ca2+, and the enzyme had the same suBstrate priority as reported previously, with the exception of PE, suggesting that yeast <B>PhospholipaseB> B could Be produced in its native structure in Bacterial cells. ScplB1p retained transacylation activity in aqueous medium, and esterified lysophosphatidylcholine with free fatty acid to form phosphatidylcholine in a non-aqueous, glycerin medium. We propose that <B>PhospholipaseB> B could serve as an additional tool for in vitro enzyme-mediated phospholipid synthesis.
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Purification and Characterization of <B>PhospholipaseB> B from Candida utilis
Bioscience biotechnology and biochemistry, 2006Co-Authors: Shuji Fujino, Yasuo Watanabe, Daigo Akiyama, Satoko Akaboshi, Tomonari Fujita, Youichi TamaiAbstract:<B>PhospholipaseB> B (PLB) from the asporogenous yeast Candida utilis was purified to homogeneity from a culture Broth. The apparent molecular mass was 90–110 kDa By SDS–PAGE. The enzyme had two pH optima, one acidic (pH 3.0) and the other alkaline (pH 7.5). At acidic pH the enzyme hydrolyzed all phospholipids tested without metal ions. On the other hand, the PLB showed suBstrate specificity and required metal ions for alkaline activity.The cDNA sequence of the PLB was analyzed By a comBination of several PCR procedures. The PLB encoded a protein consisting of 643 amino acids. The amino acid sequence contained a lipase consensus sequence (GxSxG) and catalytic arginine and aspartic acid motifs which were identified as the catalytic triad in the PLB from Kluyveromyces lactis, suggesting that the catalytic mechanism of the PLB is similar to that of cytosolic <B>PhospholipaseB> A2 (cPLA2), found in mammalian tissues.
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Purification and Characterization of <B>PhospholipaseB> B from Kluyveromyces lactis, and Cloning of <B>PhospholipaseB> B gene
Bioscience biotechnology and biochemistry, 1999Co-Authors: Hideki Oishi, Yasuo Watanabe, Takahiro Morimoto, Youichi TamaiAbstract:<B>PhospholipaseB> B (PLB) from the yeast Kluyveromyces lactis was purified to homogeneity from culture medium. The enzyme was highly glycosylated with apparent molecular mass of 160-250 kDa, and had two pH optima, at pH 2.0 and pH 7.5. At acidic pH the enzyme hydrolyzed all phospholipid suBstrates tested here without metal ion. On the other hand, at alkaline pH the enzyme showed suBstrate specificity for phosphatidylcholine and lysophosphatidylcholine and required Ca2+, Fe3+, or Al3+ for the activity. The alkaline activity was increased more than 20-fold in the presence of Al3+ compared to that in the presence of Ca2+.cDNA sequence of PLB (KlPLB) was analyzed By a comBination of several PCR procedures. KlPLB encoded a protein consist of 640 amino acids and the deduced amino acid sequence showed 66.7% similarity with the T. delBrueckii PLB. The amino acid sequence contained the lipase consensus sequence (G-X-S-X-G) and the catalytic aspartic acid motif. Replacement of Arg-112 or Asp-406 with alanine caused los...
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Disruption of <B>PhospholipaseB> B gene, PLB1, increases the survival of Baker's yeast Torulaspora delBrueckii
FEMS microbiology letters, 1996Co-Authors: Yasuo Watanabe, Kenji Imai, Hideki Oishi, Youichi TamaiAbstract:ABstract An uracil auxotrophic mutant of Baker's yeast Torulaspora delBrueckii , which is resitant to 5-fluoro-orotic acid, was complemented By transformation with YEp24 which harBors 2 μm origin and URA3 derived from Saccharomyces cerevisiae . The <B>PhospholipaseB> B in T. delBrueckii cells is active in Both acidic and alkaline conditions. However, activity of <B>PhospholipaseB> B gene ( PLB1 ) in cells of disruption mutant ( plBI : : URA3 ) was lost in Both conditions, which indicates that all <B>PhospholipaseB> B activity is encoded By a single gene (or a single polypeptide) in these yeast cells. Over-expression of PLB1 with YEp plasmid vector in T. delBrueckii cells showed ≈ 2.5-fold increase in <B>PhospholipaseB> B activity, comparing with that in wild-type cells. Cells of plB1 Δ mutant showed increased survival when cells of plB1 Δ mutant and wild-type strain were incuBated in water at 30 °C. Cells of PLB1 -over-expressed strain died rapidly even during the cultivation period, indicating that <B>PhospholipaseB> B activity may Be a determinant for the survival of this yeast.
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Purification and Some Properties of <B>PhospholipaseB> B from Schizosaccharomyces pomBe
Bioscience biotechnology and biochemistry, 1996Co-Authors: Hideki Oishi, Yasuo Watanabe, Shinji Tsuda, Youichi TamaiAbstract:<B>PhospholipaseB> B from Schizosaccharomyces pomBe was purified By ammonium sulfate fractionation and chromatographed on phenyl-Sepharose CL-4B, DEAE-Toyopearl 650M, and TSK gel G4000SW columns. The pu...
Raghuvir K. Arni - One of the best experts on this subject based on the ideXlab platform.
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Modeling and molecular dynamics indicate that snake venom <B>PhospholipaseB> B-like enzymes are Ntn-hydrolases
Toxicon, 2018Co-Authors: M. Adrian E Coronado, Danilo S. Olivier, Raphael Josef Eberle, Marcos Serrou Do Amaral, Raghuvir K. ArniAbstract:ABstract <B>PhospholipaseB>-B-like (SVPLB-like) enzymes are present in relatively small amounts in a numBer of venoms, however, their Biological function and mechanisms of action are un-clear. A three-dimensional model of the SVPLB-like enzyme from Crotalus adamanteus was generated By homology modeling Based on the crystal structures of Bovine Ntn-hydrolyases and the modeled protein possesses conserved domains characteristic of Ntn-hydrolases. Molecular dynamics simulations indicate that activation By autocatalytic cleavage results in the removal of 25 amino acids which increases accessiBility to the active site. SVPLB-like enzymes possess a highly reactive cysteine and are hence amidases that to Belong to the N-terminal nucleophile (Ntn) hydrolase family. The Ntn-hydrolases (N-terminal nucleophile) form a superfamily of diverse enzymes that are activated autocatalytically; wherein the N-terminal catalytic nucleophile is implicated in the cleavage of the amide Bond.
Jacinta L Farn - One of the best experts on this subject based on the ideXlab platform.
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expression purification and characterization of recomBinant <B>PhospholipaseB> B from moraxella Bovis with anomalous electrophoretic Behavior
Protein Expression and Purification, 2007Co-Authors: Brian J Shiell, Mary Tachedjian, Kerri Bruce, Gary Beddome, Jacinta L Farn, Peter A Hoyne, Wojtek P MichalskiAbstract:ABstractMoraxella Bovis is the causative agent of infectious Bovine keratoconjunctivitis (IBK) also known as pinkeye, a highly contagious andpainful eye disease that is common in cattle throughout the world. Vaccination appears to Be a reasonaBle and cost-effective means ofcontrol of pinkeye. Identification of genes encoding novel secreted antigens have Been reported, and these antigens are Being assessed foruse in a vaccine. One of the genes encodes <B>PhospholipaseB> B, which can Be expressed with high purity and yield in recomBinant Escherichiacoli as a secreted, soluBle, non-tagged, mature construct (less signal peptide with predicted mass 63 kDa). The recomBinant phospholi-pase B exhiBited anomalous electrophoretic moBility that was dependent on the temperature of the denaturing process, with BandsoBserved at either 52 or 63 kDa. Analysis By in-gel digestion and liquid chromatography–mass spectrometry revealed these two distinctforms most likely had identical sequences. <B>PhospholipaseB> B is a compact, gloBular protein with a predicted structure typical of a con-ventional autotransporter. It is suggested that high temperature is required to unfold the protein (to denature the B-Barrel-rich trans-porter domain) and to ensure accessiBility of the reducing agent. Interestingly, the two forms of the enzyme, differing in size andisoelectric points, were also detected in cell-free supernatants of M. Bovis cultures, indicating that native <B>PhospholipaseB> B may existin two differentially folded states possiBly also differing in oxidation status of cysteine residues.Crown copyright 2007 PuBlished By Elsevier Inc. All rights reserved.
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molecular characterization of a secreted enzyme with <B>PhospholipaseB> B activity from moraxella Bovis
Journal of Bacteriology, 2001Co-Authors: Jacinta L Farn, Peter A Hoyne, Wojtek P Michalski, Richard A Strugnell, Jan TennentAbstract:A candidate for a vaccine against infectious Bovine keratoconjunctivitis (IBK) has Been cloned and characterized fromMoraxella Bovis. The plB gene encodes a protein of 616 amino acids (molecular mass of ∼65.8 kDa) that expresses <B>PhospholipaseB> B activity. Amino acid sequence analysis revealed that PLB is a new memBer of the GDSL (Gly-Asp-Ser-Leu) family of lipolytic enzymes.
Tania C. Sorrell - One of the best experts on this subject based on the ideXlab platform.
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Cryptococcal <B>PhospholipaseB> B antigen is not detected in serum of patients infected with Cryptococcus neoformans using a sandwich enzyme-linked immunosorBent assay.
FEMS yeast research, 2007Co-Authors: Sharon C.-a. Chen, Rosemary T. Santangelo, Patricia Martin, Richard Malik, Tania C. SorrellAbstract:Extracellular <B>PhospholipaseB> B (PLB) is a virulence determinant of Cryptococcus neoformans and Cryptococcus gattii. In this study, we developed a sensitive enzyme-linked immunosorBent assay (ELISA) for PLB antigen with a detection limit of 3.9 ng mL(-1). PLB was detected in culture supernatants of C. neoformans and C. gattii. PLB, however, was not detected in sera of seven human patients and 10 feline patients with active cryptococcosis. Furthermore, none of five rats with extensive pulmonary C. gattii infection had a positive ELISA test result. In conclusion, cryptococcal PLB could not Be detected in serum using a PLB antigen-Based ELISA. Despite its sensitivity, this ELISA is of limited diagnostic value. Exploration of further extracellular molecules suitaBle for serodiagnosis of active cryptococcal infection is warranted.
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<B>PhospholipaseB> B activity enhances adhesion of cryptococcus neoformans to a human lung epithelial cell line
Microbes and Infection, 2006Co-Authors: Ranjini Ganendren, Tania C. Sorrell, Elizabeth A. Carter, Fred Widmer, Lesley C. WrightAbstract:Secreted <B>PhospholipaseB> B (PLB1), which contains three enzyme activities in the one protein, is necessary for the initiation of pulmonary infection By Cryptococcus neoformans and for dissemination from the lung via the lymphatics and Blood. Adhesion to lung epithelium is the first step in this process, therefore we investigated the role of PLB1 in adhesion to a human lung epithelial cell line, A549, using C. neoformans var. gruBii wild-type strain H99, a PLB1 deletion mutant (deltaplB1), and a reconstituted strain (deltaplB1rec). Adhesion of H99 and deltaplB1rec was approximately 69% greater than deltaplB1 at 4 h. Adhesion of deltaplB1 significantly increased after killing By chemicals or heat, and Fourier-transformed analysis By FTIR spectroscopy indicated this was due to changes in capsular and/or cell wall polysaccharides and proteins. InhiBition By specific PLB1 antiBodies, or inhiBitors of <B>PhospholipaseB> B (PLB), But not lyso<B>PhospholipaseB> (LPL) or lyso<B>PhospholipaseB> transacylase (LPTA) activities decreased the adhesion of H99 and deltaplB1rec By 33-58%. Growth under conditions of osmotic stress and high glucose concentration increased Both PLB secretion and suBsequent cryptococcal adhesion. Dose-dependent increases (to 67%) in adhesion of live deltaplB1 were oBserved in the presence of 0.1-2 mM palmitic acid. We conclude that PLB1 plays a role in the Binding of C. neoformans to host lung epithelial cells, possiBly due to production of fatty acids from plasma memBranes and/or surfactant By PLB activity.
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<B>PhospholipaseB> B activity enhances adhesion of Cryptococcus neoformans to a human lung epithelial cell line.
Microbes and infection, 2006Co-Authors: Ranjini Ganendren, Tania C. Sorrell, Elizabeth A. Carter, Fred Widmer, Lesley C. WrightAbstract:ABstract Secreted <B>PhospholipaseB> B (PLB1), which contains three enzyme activities in the one protein, is necessary for the initiation of pulmonary infection By Cryptococcus neoformans and for dissemination from the lung via the lymphatics and Blood. Adhesion to lung epithelium is the first step in this process, therefore we investigated the role of PLB1 in adhesion to a human lung epithelial cell line, A549, using C. neoformans var. gruBii wild-type strain H99, a PLB1 deletion mutant (Δ plB1), and a reconstituted strain (Δ plB1 rec ). Adhesion of H99 and Δ plB1 rec was approximately 69% greater than Δ plB1 at 4 h. Adhesion of Δ plB1 significantly increased after killing By chemicals or heat, and Fourier-transformed analysis By FTIR spectroscopy indicated this was due to changes in capsular and/or cell wall polysaccharides and proteins. InhiBition By specific PLB1 antiBodies, or inhiBitors of <B>PhospholipaseB> B (PLB), But not lyso<B>PhospholipaseB> (LPL) or lyso<B>PhospholipaseB> transacylase (LPTA) activities decreased the adhesion of H99 and Δ plB1 rec By 33–58%. Growth under conditions of osmotic stress and high glucose concentration increased Both PLB secretion and suBsequent cryptococcal adhesion. Dose-dependent increases (to 67%) in adhesion of live Δ plB1 were oBserved in the presence of 0.1–2 mM palmitic acid. We conclude that PLB1 plays a role in the Binding of C. neoformans to host lung epithelial cells, possiBly due to production of fatty acids from plasma memBranes and/or surfactant By PLB activity.
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Detection of antiBodies to <B>PhospholipaseB> B in patients infected with Cryptococcus neoformans By enzyme-linked immunosorBent assay (ELISA).
Medical mycology, 2005Co-Authors: Rosemary T. Santangelo, Tania C. Sorrell, Sharon C.-a. Chen, Lesley C. WrightAbstract:Secreted <B>PhospholipaseB>s are virulence factors of several fungi, including Cryptococcus neoformans. We descriBe for the first time the detection By ELISA of antiBodies to cryptococcal <B>PhospholipaseB> B in the serum of patients infected with C. neoformans or C. gattii. Sixty-nine sera from 25 patients with cryptococcosis, 23 patients with Candida infections and 26 with Bacterial or viral infections were tested. The sensitivity of the ELISA in patients with cryptococcosis was 100% in immunocompetent hosts and 64.3% in immunosuppressed patients with cryptococcal meningitis. ABsorBance readings were significantly higher in immunocompetent patients (P
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PCR-Restriction Fragment Length Polymorphism Analysis of the <B>PhospholipaseB> B (PLB1) Gene for SuBtyping of Cryptococcus neoformans Isolates
Applied and environmental microbiology, 2003Co-Authors: G. Nicolas Latouche, Tania C. Sorrell, Matthew Huynh, Wieland MeyerAbstract:Cryptococcus neoformans is a pathogenic yeast that is currently divided into three varieties, five serotypes, and eight molecular types. The following report descriBes the use of PCR-restriction fragment length polymorphism (RFLP) analysis of the <B>PhospholipaseB> B gene (PLB1) as a simple tool to differentiate Between C. neoformans suBgroups. A PLB1 fragment, 1,970 Bp, was amplified and digested with either AvaI or HindIII. Both sets of profiles grouped the isolates into their respective varieties, But only the AvaI profiles allowed for the identification of the eight molecular types via the corresponding RFLP profiles A1 to A8. Digestion of the same fragments with HindIII resulted in RFLP profiles H1 to H5, which distinguished only Between serotype A, AD, D, and B/C. Neither enzyme distinguished serotype B from serotype C. The serotype AD profile was a composite of the serotype A and D profiles. Further investigation showed that the serotype AD isolates used in this study are heterozygous, with one allele of PLB1 originating from a serotype A parent and the other from a serotype D parent.