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Sun Chul Kang - One of the best experts on this subject based on the ideXlab platform.
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Staphylococcus haemolyticus lipase: biochemical properties, substrate specificity and gene cloning
FEMS microbiology letters, 1999Co-Authors: Hyung Kwoun Kim, Jung Kee Lee, Sun Chul KangAbstract:Lipase of Staphylococcus haemolyticus L62 was purified from culture supernatant and its molecular mass was estimated to be 45 kDa by SDS-PAGE. Its optimum temperature and pH for the hydrolysis of olive oil was 28 degrees C and pH 8.5, respectively. The enzyme was stable up to 50 degrees C in the presence of Ca(2+)and over the pH range 5-11. It had high hydrolytic activity against tributyrin, tripropionin, and trimyristin among various triglycerides. The gene encoding the lipase was cloned in Escherichia coli. Sequence analysis showed an open reading frame of 2136 bp, which encodes a Preproenzyme of 711 amino acids. The Preproenzyme is composed of a signal peptide (60 aa), a pro-peptide (259 aa), and a mature enzyme (392 aa). The mature enzyme has 49-67% amino acid sequence homology with other staphylococcal lipases.
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Staphylococcus haemolyticus lipase: biochemical properties, substrate specificity and gene cloning
Wiley-Blackwell, 1999Co-Authors: Hyung Kwoun Kim, Jung Kee Lee, Sun Chul KangAbstract:Lipase of Staphylococcus haemolyticus L62 was purified from culture supernatant and its molecular mass was estimated to be 45 kDa by SDS-PAGE. Its optimum temperature and pH for the hydrolysis of olive oil was 28°C and pH 8.5, respectively. The enzyme was stable up to 50°C in the presence of Ca2+and over the pH range 5-11. It had high hydrolytic activity against tributyrin, tripropionin, and trimyristin among various triglycerides. The gene encoding the lipase was cloned in Escherichia coli. Sequence analysis showed an open reading frame of 2136 bp, which encodes a Preproenzyme of 711 amino acids. The Preproenzyme is composed of a signal peptide (60 aa), a pro-peptide (259 aa), and a mature enzyme (392 aa). The mature enzyme has 49-67% amino acid sequence homology with other staphylococcal lipases.ope
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Staphylococcus haemolyticus lipase: biochemical properties, substrate speci¢city and gene cloning
1999Co-Authors: Hyung Kwoun Kim, Jung Kee Lee, Sun Chul KangAbstract:Lipase of Staphylococcus haemolyticus L62 was purified from culture supernatant and its molecular mass was estimated to be 45 kDa by SDS-PAGE. Its optimum temperature and pH for the hydrolysis of olive oil was 28‡C and pH 8.5, respectively. The enzyme was stable up to 50‡C in the presence of Ca 2a and over the pH range 5^11. It had high hydrolytic activity against tributyrin, tripropionin, and trimyristin among various triglycerides. The gene encoding the lipase was cloned in Escherichia coli. Sequence analysis showed an open reading frame of 2136 bp, which encodes a Preproenzyme of 711 amino acids. The Preproenzyme is composed of a signal peptide (60 aa), a pro-peptide (259 aa), and a mature enzyme (392 aa). The mature enzyme has 49^67% amino acid sequence homology with other staphylococcal lipases. fl 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Reinhard Rüchel - One of the best experts on this subject based on the ideXlab platform.
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molecular cloning of an extracellular aspartic proteinase from rhizopus microsporus and evidence for its expression during infection
Medical Mycology, 2002Co-Authors: Christoph Schoen, Utz Reichard, Michel Monod, Hartmut Kratzin, Reinhard RüchelAbstract:An extracellular aspartic proteinase (Rmap) from Rhizopus microsporus var. rhizopodiformis was detected in the culture supernatant of a fungal isolate from a case of rhinocerebral mucormycosis (case HA). The proteinase was purified to near homogeneity by ion exchange and affinity chromatography on pepstatin agarose. Based on its N-terminus the RMAP gene was cloned and found to code for 388 amino acids. The Preproenzyme has an aminoterminal leader sequence of 65 amino acids, whereas the mature enzyme consists of 323 amino acids. The deduced amino-acid sequence of the Preproenzyme was 82% homologous to an extracellular aspartic proteinase of Rhizopus niveus. Low stringency Southern blot analysis of R. microsporus DNA suggested the presence of other homologous genes. Expression of Rmap in Pichia pastoris was achieved, and the recombinant enzyme was active in the yeast culture supernatant. Both enzyme preparations exhibited a similar optimum of activity in the pH 2·5 region. Furthermore, Rmap was shown to act...
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Molecular cloning of an extracellular aspartic proteinase from Rhizopus microsporus and evidence for its expression during infection
Medical mycology, 2002Co-Authors: Christoph Schoen, Utz Reichard, Michel Monod, Hartmut Kratzin, Reinhard RüchelAbstract:An extracellular aspartic proteinase (Rmap) from Rhizopus microsporus var. rhizopodiformis was detected in the culture supernatant of a fungal isolate from a case of rhinocerebral mucormycosis (case HA). The proteinase was purified to near homogeneity by ion exchange and affinity chromatography on pepstatin agarose. Based on its N-terminus the RMAP gene was cloned and found to code for 388 amino acids. The Preproenzyme has an aminoterminal leader sequence of 65 amino acids, whereas the mature enzyme consists of 323 amino acids. The deduced amino-acid sequence of the Preproenzyme was 82% homologous to an extracellular aspartic proteinase of Rhizopus niveus. Low stringency Southern blot analysis of R. microsporus DNA suggested the presence of other homologous genes. Expression of Rmap in Pichia pastoris was achieved, and the recombinant enzyme was active in the yeast culture supernatant. Both enzyme preparations exhibited a similar optimum of activity in the pH 2.5 region. Furthermore, Rmap was shown to activate bovine blood coagulation factor X at slightly acidic pH in vitro. Expression of the proteinase during mycosis was proven by a specific immune response of patient HA.
Charles Gerday - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the C-Terminal Propeptide Involved in Bacterial Wall Spanning of Alpha-Amylase from the Psychrophile Alteromonas Haloplanctis
The Journal of biological chemistry, 1998Co-Authors: Georges Feller, Salvino D'amico, A. M. Benotmane, F. Joly, J. Van Beeumen, Charles GerdayAbstract:The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- and Cl--dependent alpha-amylase. The nucleotide sequence of the amy gene and the amino acid sequences of the gene products indicate that the alpha-amylase precursor is a Preproenzyme composed by the signal peptide (24 residues), the mature alpha-amylase (453 residues, 49 kDa), and a long C-terminal propeptide or secretion helper (192 residues, 21 kDa). In cultures of the wild-type strain, the 70-kDa precursor is secreted at the mid-exponential phase and is cleaved by a nonspecific protease into the mature enzyme and the propeptide. The purified C-terminal propeptide displays several features common to beta-pleated transmembrane proteins. It has no intramolecular chaperone function because active alpha-amylase is expressed by Escherichia coli in the absence of the propeptide coding region. In E. coli, the 70-kDa precursor is directed toward the supernatant. When the alpha-amylase coding region is excised from the gene, the secretion helper can still promote its own membrane spanning. It can also accept a foreign passenger, as shown by the extracellular routing of a beta-lactamase-propeptide fusion protein.
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Sequence of the subtilisin-encoding gene from an antarctic psychrotroph Bacillus TA41.
Gene, 1992Co-Authors: Stephane Davail, Georges Feller, Emmanuel Narinx, Charles GerdayAbstract:The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph, Bacillus TA41, was determined. The primary structure of the subtilisin precursor corresponds to a Preproenzyme of 419 amino acids. Asp144, His181 and Ser359 are the proposed catalytic residues of the protease active site.
Hyung Kwoun Kim - One of the best experts on this subject based on the ideXlab platform.
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Biochemical properties and substrate specificity of lipase from Staphylococcus aureus B56
Journal of Microbiology and Biotechnology, 2002Co-Authors: Woo Hyuk Jung, Hyung Kwoun Kim, Chan Yong LeeAbstract:A lipase of Staphylococcus aureus B56 was purified from a culture supernatant and its molecular weight was estimated to be 45 kDa by SDS-PAGE. The optimum temperature and pH for the hydrolysis of olive oil was 42°C and pH 8-8.5, respectively. The enzyme was stable up to 55°C in the presence of Ca ++ at pHs 5-11. The lipase gene was cloned using the PCR amplification method. The sequence analysis showed an open reading frame of 2,076 bp, which encoded a Preproenzyme of 691 amino acids. The Preproenzyme was composed of a signal sequence (37 aa), propeptide (255 aa), and mature enzyme (399 aa). Based on a sequence comparison, lipase B56 constituted of a separate subgroup among the staphylococcal lipase groups, such as S. aureus PS54 and S. haemolyticus L62 lipases, and was distinct from other lipases in their optimum pH and substrate specificity.
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Staphylococcus haemolyticus lipase: biochemical properties, substrate specificity and gene cloning
FEMS microbiology letters, 1999Co-Authors: Hyung Kwoun Kim, Jung Kee Lee, Sun Chul KangAbstract:Lipase of Staphylococcus haemolyticus L62 was purified from culture supernatant and its molecular mass was estimated to be 45 kDa by SDS-PAGE. Its optimum temperature and pH for the hydrolysis of olive oil was 28 degrees C and pH 8.5, respectively. The enzyme was stable up to 50 degrees C in the presence of Ca(2+)and over the pH range 5-11. It had high hydrolytic activity against tributyrin, tripropionin, and trimyristin among various triglycerides. The gene encoding the lipase was cloned in Escherichia coli. Sequence analysis showed an open reading frame of 2136 bp, which encodes a Preproenzyme of 711 amino acids. The Preproenzyme is composed of a signal peptide (60 aa), a pro-peptide (259 aa), and a mature enzyme (392 aa). The mature enzyme has 49-67% amino acid sequence homology with other staphylococcal lipases.
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Staphylococcus haemolyticus lipase: biochemical properties, substrate specificity and gene cloning
Wiley-Blackwell, 1999Co-Authors: Hyung Kwoun Kim, Jung Kee Lee, Sun Chul KangAbstract:Lipase of Staphylococcus haemolyticus L62 was purified from culture supernatant and its molecular mass was estimated to be 45 kDa by SDS-PAGE. Its optimum temperature and pH for the hydrolysis of olive oil was 28°C and pH 8.5, respectively. The enzyme was stable up to 50°C in the presence of Ca2+and over the pH range 5-11. It had high hydrolytic activity against tributyrin, tripropionin, and trimyristin among various triglycerides. The gene encoding the lipase was cloned in Escherichia coli. Sequence analysis showed an open reading frame of 2136 bp, which encodes a Preproenzyme of 711 amino acids. The Preproenzyme is composed of a signal peptide (60 aa), a pro-peptide (259 aa), and a mature enzyme (392 aa). The mature enzyme has 49-67% amino acid sequence homology with other staphylococcal lipases.ope
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Staphylococcus haemolyticus lipase: biochemical properties, substrate speci¢city and gene cloning
1999Co-Authors: Hyung Kwoun Kim, Jung Kee Lee, Sun Chul KangAbstract:Lipase of Staphylococcus haemolyticus L62 was purified from culture supernatant and its molecular mass was estimated to be 45 kDa by SDS-PAGE. Its optimum temperature and pH for the hydrolysis of olive oil was 28‡C and pH 8.5, respectively. The enzyme was stable up to 50‡C in the presence of Ca 2a and over the pH range 5^11. It had high hydrolytic activity against tributyrin, tripropionin, and trimyristin among various triglycerides. The gene encoding the lipase was cloned in Escherichia coli. Sequence analysis showed an open reading frame of 2136 bp, which encodes a Preproenzyme of 711 amino acids. The Preproenzyme is composed of a signal peptide (60 aa), a pro-peptide (259 aa), and a mature enzyme (392 aa). The mature enzyme has 49^67% amino acid sequence homology with other staphylococcal lipases. fl 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
M.a. Wells - One of the best experts on this subject based on the ideXlab platform.
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PURIFICATION, CHARACTERIZATION AND CDNA SEQUENCE OF AN ALKALINE CHYMOTRYPSIN FROM THE MIDGUT OF MANDUCA SEXTA
Insect biochemistry and molecular biology, 1995Co-Authors: A.m. Peterson, Germain J. P. Fernando, M.a. WellsAbstract:The chymotrypsin in the midgut of Manduca sexta has been purified, characterized and the cDNA encoding the protein has been cloned. The enzyme exists as a monomer of approx. 24 kDa and shows maximal activity between pH 10.5 and 11.0. Kinetic studies reveal that the Michaelis constant (Km) for the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide varies only slightly between pH 7.5 and 11.5 and the Dixon plot shows a kinetically significant pKa at 9.2. The specificity of the purified enzyme was determined to be the peptide bond on the carboxyl side of tyrosine, phenylalanine, tryptophan, histidine, leucine, threonine and glycine. The protease is inhibited by TPCK, PMSF, chymostatin and DFP. A 1 kilobase chymotrypsin cDNA clone was isolated and sequenced. The cDNA sequence encodes a Preproenzyme with a putative 17 amino acid signal sequence, a 41 amino acid activation peptide and a mature enzyme of 235 amino acids. The isolated clone encodes the highly conserved active site residues (His, Asp, Ser) and specificity pocket residues present in bovine chymotrypsinogen B. Northern analysis localizes the mRNA for the chymotrypsin to the anterior and middle third of the midgut.
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Sequence of three cDNAs encoding an alkaline midgut trypsin from Manduca sexta.
Insect biochemistry and molecular biology, 1994Co-Authors: A.m. Peterson, Carolina V. Barillas-mury, M.a. WellsAbstract:We have purified trypsin from the midgut of Manduca sexta and shown it has an alkaline pH optimum of 10.5. In order to clone the midgut trypsin, a DNA probe was generated using the polymerase chain reaction (PCR) with template isolated from a midgut cDNA library phage stock, a mixture of degenerate primers synthesized to code for the highly conserved region around the active site serine found in trypsins, and the T7 sequencing primer. Three different trypsin cDNAs were isolated each of which encodes a Preproenzyme of 256 amino acids with a putative signal sequence of 17 amino acids, an activation peptide of seven amino acids and a mature trypsin of 232 amino acids. The encoded midgut trypsins contain the highly conserved residues, Asp, His, Ser, involved in catalysis in serine proteases, along with the residues which define the trypsin specificity pocket. Sequence comparisons show that all sequences are similar to other invertebrate and vertebrate serine proteases, but they differ in that two of the three encoded trypsins have an odd number of cysteines. Northern analysis localizes the trypsin mRNA to the middle third of the midgut. A large number of arginines (19, 20 and 21) are encoded by the three cDNAs which may stabilize the trypsin, by remaining protonated, in the alkaline midgut of M. sexta.