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Bum Shik Hong - One of the best experts on this subject based on the ideXlab platform.
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Identification of essential amino acid residues for catalytic activity and thermostability of novel chitosanase by site-directed mutagenesis
Applied Microbiology and Biotechnology, 2001Co-Authors: Ho-geun Yoon, Dong Hoon Shin, Bum Shik HongAbstract:The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues (Glu-50, Glu-62, and Asp-66) was changed to Asp and Gln or Asn and Glu by site-directed mutagenesis, respectively. The Asp-66→Asn and Asp-66→Glu mutation remarkably decreased kinetic parameters such as V max and k cat to approximately 1/1,000 those of the wild-type enzyme, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three Cys residues at positions 49, 72, and 211. The Cys-49→Ser/Tyr and Cys-72→Ser/Tyr mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However, the half-life of the Cys-211→Ser/Tyr mutant enzyme was less than 10 min at 80 °C, while that of the wild-type enzyme was about 90 min. Moreover, the residual activity of Cys-211→Ser/Tyr enzyme was substantially decreased by 8 M urea; and it lost all catalytic activity in 40% ethanol. These results show that the substitution of Cys with any amino acid residues at position 211 seems to affect the conformational stability of the chitosanase.
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Identification of essential amino acid residues for catalytic activity and thermostability of novel chitosanase by site-directed mutagenesis.
Applied microbiology and biotechnology, 2001Co-Authors: Ho-geun Yoon, Dong Hoon Shin, Bum Shik HongAbstract:The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues (Glu-50, Glu-62, and Asp-66) was changed to Asp and Gln or Asn and Glu by site-directed mutagenesis, respectively. The Asp-66-->Asn and Asp-66-->Glu mutation remarkably decreased kinetic parameters such as Vmax and kcat to approximately 1/1,000 those of the wild-type enzyme, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three Cys residues at positions 49, 72, and 211. The Cys-49-->Ser/Tyr and Cys-72-->Ser/Tyr mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However, the half-life of the Cys-211-->Ser/Tyr mutant enzyme was less than 10 min at 80 degrees C, while that of the wild-type enzyme was about 90 min. Moreover, the residual activity of Cys-211-->Ser/Tyr enzyme was substantially decreased by 8 M urea; and it lost all catalytic activity in 40% ethanol. These results show that the substitution of Cys with any amino acid residues at position 211 seems to affect the conformational stability of the chitosanase.
Eiji Ichishima - One of the best experts on this subject based on the ideXlab platform.
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Aspzincin, a family of metalloendopeptidases with a new zinc-binding motif. Identification of new zinc-binding sites (His(128), His(132), and Asp(164)) and three catalytically crucial residues (Glu(129), Asp(143), and Tyr(106)) of deuterolysin from A
Journal of Biological Chemistry, 1999Co-Authors: Naoya Fushimi, Tasuku Nakajima, Eiji IchishimaAbstract:Deuterolysin (EC 3.4.24.39; formerly designated as neutral proteinase II) from Aspergillus oryzae, which contains 1 g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. Active-site determination of the recombinant enzyme expressed in Escherichia coli was performed by site-directed mutagenesis. Substitutions of His(128) and His(132) with Arg, of Glu(129) with Gln or Asp, of Asp(143) with Asn or Glu, of Asp(164) with Asn, and of Tyr(106) with Phe resulted in almost complete loss of the activity of the mutant enzymes. It can be concluded that His(128), His(132), and Asp(164) provide the Zn(2+) ligands of the enzyme according to a (65)Zn binding assay. Based on site-directed mutagenesis experiments, it was demonstrated that the three essential amino acid residues Glu(129), Asp(143), and Tyr(106) are catalytically crucial residues in the enzyme. Glu(129) may be implicated in a central role in the catalytic function. We conclude that deuterolysin is a member of a family of Zn(2+) metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand.
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Five Crucial Carboxyl Residues of 1,2-α-Mannosidase fromAspergillus saitoi(A. phoenicis), a Food Microorganism, Are Identified by Site-Directed Mutagenesis☆
Biochemical and Biophysical Research Communications, 1997Co-Authors: Akiko Fujita, Takashi Yoshida, Eiji IchishimaAbstract:Abstract An acidic 1,2-α-mannosidase from fungus, Aspergillus saitoi (now designated Aspergillus phoenicis ), is highly specific for 1,2-α-mannosidic linkage in the high-mannose type oligosaccharide at pH 5.0. The predicted amino acid sequence of several peptide regions, including aspartic acid and glutamic acid, bears striking similarities to 1,2-α-mannosidases from fungi, yeast and mouse. Active site determination of the enzyme expressed in Saccharomyces cerevisiae cells was performed by site-directed mutagenesis. Substitutions of Asp-269 to Glu and of the Glu-residues, Glu-273, Glu-411, Glu-414 and Glu-474, to Asp altered the drastic decrease of specific activities with Man α 1-2Man-OMe and Man 9 -GlcNAc 2 -PA as substrates and shifted the optimal pH of the mutant enzymes. From the present results, Asp-269 is probably in the ionized COO − form, whereas one of four glutamic acid residues, probably Glu-411, is the un-ionized COOH form according to the analogy of a plausible mechanism for lysozyme catalysis. It is assumed that three glutamic acid residues, Glu-273, Glu-414, and Glu-474, are probably binding sites of substrate.
Klaus Biemann - One of the best experts on this subject based on the ideXlab platform.
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amino acid sequence of a protease inhibitor isolated from sarcophaga bullata determined by mass spectrometry
Protein Science, 2008Co-Authors: Ioannis A Papayannopoulos, Klaus BiemannAbstract:The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.
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Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined w
1992Co-Authors: Klaus BiemannAbstract:The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.
Ho-geun Yoon - One of the best experts on this subject based on the ideXlab platform.
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Identification of essential amino acid residues for catalytic activity and thermostability of novel chitosanase by site-directed mutagenesis
Applied Microbiology and Biotechnology, 2001Co-Authors: Ho-geun Yoon, Dong Hoon Shin, Bum Shik HongAbstract:The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues (Glu-50, Glu-62, and Asp-66) was changed to Asp and Gln or Asn and Glu by site-directed mutagenesis, respectively. The Asp-66→Asn and Asp-66→Glu mutation remarkably decreased kinetic parameters such as V max and k cat to approximately 1/1,000 those of the wild-type enzyme, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three Cys residues at positions 49, 72, and 211. The Cys-49→Ser/Tyr and Cys-72→Ser/Tyr mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However, the half-life of the Cys-211→Ser/Tyr mutant enzyme was less than 10 min at 80 °C, while that of the wild-type enzyme was about 90 min. Moreover, the residual activity of Cys-211→Ser/Tyr enzyme was substantially decreased by 8 M urea; and it lost all catalytic activity in 40% ethanol. These results show that the substitution of Cys with any amino acid residues at position 211 seems to affect the conformational stability of the chitosanase.
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Identification of essential amino acid residues for catalytic activity and thermostability of novel chitosanase by site-directed mutagenesis.
Applied microbiology and biotechnology, 2001Co-Authors: Ho-geun Yoon, Dong Hoon Shin, Bum Shik HongAbstract:The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues (Glu-50, Glu-62, and Asp-66) was changed to Asp and Gln or Asn and Glu by site-directed mutagenesis, respectively. The Asp-66-->Asn and Asp-66-->Glu mutation remarkably decreased kinetic parameters such as Vmax and kcat to approximately 1/1,000 those of the wild-type enzyme, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three Cys residues at positions 49, 72, and 211. The Cys-49-->Ser/Tyr and Cys-72-->Ser/Tyr mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However, the half-life of the Cys-211-->Ser/Tyr mutant enzyme was less than 10 min at 80 degrees C, while that of the wild-type enzyme was about 90 min. Moreover, the residual activity of Cys-211-->Ser/Tyr enzyme was substantially decreased by 8 M urea; and it lost all catalytic activity in 40% ethanol. These results show that the substitution of Cys with any amino acid residues at position 211 seems to affect the conformational stability of the chitosanase.
Ioannis A Papayannopoulos - One of the best experts on this subject based on the ideXlab platform.
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amino acid sequence of a protease inhibitor isolated from sarcophaga bullata determined by mass spectrometry
Protein Science, 2008Co-Authors: Ioannis A Papayannopoulos, Klaus BiemannAbstract:The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.
