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المصدر: SC10201804160065
NARO成果DBa
Green open Access
This journal has an embargo period of 12 months.
著者版依頼中／Applying for the author's permission.مصطلحات موضوعية: 0301 basic medicine, Proline, Aspergillus oryzae, 030106 microbiology, Bioengineering, medicine.disease_cause, Aminopeptidases, Applied Microbiology and Biotechnology, Aminopeptidase, Substrate Specificity, 03 medical and health sciences, Escherichia coli, medicine, Histidine, Amino Acid Sequence, chemistry.chemical_classification, Metalloproteinase, biology, Hydrolysis, Temperature, Dipeptides, Hydrogen-Ion Concentration, biology.organism_classification, Molecular biology, Enzyme assay, Amino acid, Molecular Weight, 030104 developmental biology, Enzyme, chemistry, Biochemistry, biology.protein, Electrophoresis, Polyacrylamide Gel, Oligopeptides, Biotechnology
الوصف: Xaa-Pro aminopeptidases are peptidases responsible for the cleavage of any amino acid N-terminally adjacent to a proline residue. We identified a gene encoding a putative Xaa-Pro aminopeptidase in the genome of the filamentous fungus Aspergillus oryzae (genome database number: AO090701000720) and named this gene xpmA . We produced its enzyme in a C-terminally His 6 -tag-fused form in an Escherichia coli expression system and purified it. The purified recombinant XpmA (rXpmA) showed hydrolysis activity toward Xaa-Pro-oligopeptides, especially the two dipeptides Ala-Pro and Phe-Pro. The molecular weight of rXpmA was estimated to be 69 kDa by SDS-PAGE and 126 kDa by gel filtration, suggesting that it is a homodimer. The enzyme was activated by various divalent metal ions such as Mn 2+ , Co 2+ , and Mg 2+ ; in particular, the enzyme activity was increased 27.6-times relative to the no-addition control by 1 mM Mn 2+ . Additionally, 10 mM EDTA suppressed its activity to 0.26-times of the control level. Therefore, rXpmA was a metalloprotease. Optimal hydrolytic activity of rXpmA was observed at 50°C and pH 8.5–9.0. The enzyme was stable up to 50°C and from pH 4.0 to 11.0. rXpmA showed substrate inhibition by Leu-Pro, Ser-Pro and Arg-Pro at concentrations over 4 mM, 10 mM, and 3 mM, respectively. NaCl increased the enzyme activity in the concentration range 0.5–3.0 M, suggesting that the enzyme is halophilic.
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المؤلفون: Seiichi Mizuno, Takanobu Gotou, Tadashi Shinoda, Naoyuki Yamamoto
المصدر: Journal of Bioscience and Bioengineering. 107:615-619
مصطلحات موضوعية: Chromatography, biology, Chemistry, Aspergillus oryzae, Molecular Sequence Data, Proteolytic enzymes, Caseins, Angiotensin-Converting Enzyme Inhibitors, Bioengineering, Aspergillus sojae, biology.organism_classification, Applied Microbiology and Biotechnology, Aminopeptidase, Endopeptidase, Fungal Proteins, Biochemistry, Casein, Endopeptidases, Amino Acid Sequence, Oligopeptides, Leucyl aminopeptidase, Peptide sequence, Antihypertensive Agents, Biotechnology
الوصف: Two proteolytic enzymes capable of releasing the angiotensin I-converting enzyme (ACE) inhibitor Ile-Pro-Pro from casein were identified by purification of an Aspergillus oryzae extract by three-step column chromatography. First, proteins capable of producing Ile-Pro-Pro from β-casein were eluted using a DEAE-sepharose FF column with a linear sodium chloride gradient. An endopeptidase capable of releasing Pro-Ile-Pro-Gln-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro from Pro-Ile-Pro-Gln-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln and an aminopeptidase producing Ile-Pro-Pro from Gln-Asn-Ile-Pro-Pro were separated from the resultant fraction using a hydroxyapatite column. Each active enzyme was then loaded onto a Develosil 300Diol gel filtration column for high performance liquid chromatography (HPLC) and purified to homogeneity. The endopeptidase had a molecular mass of approximately 46,000 Da and exhibited an N-terminal amino acid sequence identical to that of neutral protease I (NP I) of A. oryzae. Meanwhile, the aminopeptidase had a molecular mass of 36,000 Da and an N-terminal amino acid sequence similar to that of Leucine aminopeptidase (LAP), as reported in Aspergillus sojae and A. oryzae. The eluted endopeptidase and aminopeptidase were thus identified as NP I and LAP, respectively. Analysis of peptide production using synthetic proteins containing an Ile-Pro-Pro sequence showed that NP I processed the C-terminal end and LAP processed the N terminus to produce Ile-Pro-Pro. While Ile-Pro-Pro was successfully produced from casein by the addition of these two purified enzymes, it was not generated with the addition of only a single enzyme. Based on our experimental findings, we suggest that NP I and LAP are key proteolytic enzymes in the release of Ile-Pro-Pro from casein in A. oryzae.
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المؤلفون: Masahiro Ogawa, Toshiyuki Nishio, Tadatake Oku, Takurou Yagi, Keiko Yoshikawa, Takako Hirano, Wataru Hakamata, Yasuko Kumaki, Naoya Iwasawa
المصدر: Journal of Bioscience and Bioengineering. 111:134-139
مصطلحات موضوعية: Molecular Sequence Data, Bioengineering, Molecular cloning, Biology, medicine.disease_cause, Aminopeptidases, Applied Microbiology and Biotechnology, Aminopeptidase, Substrate Specificity, law.invention, Open Reading Frames, Bacterial Proteins, law, Escherichia coli, medicine, Amino Acid Sequence, Cloning, Molecular, Gene, Bacteria, Thermophile, Symbiobacterium thermophilum, Molecular biology, Recombinant Proteins, Open reading frame, Biochemistry, Recombinant DNA, Biotechnology
الوصف: The chromosomal DNA of the syntrophic thermophile Symbiobacterium thermophilum contains open reading frames of the genes encoding family M42 aminopeptidases, Pep1079, Pep1080, and Pep1081. To characterize these peptidases, the genes were cloned into Escherichia coli and overexpressed. Our experiments using the recombinant proteins confirmed that Pep1079, Pep1080, and Pep1081 are components of arginyl or lysinyl aminopeptidases that require Co2+ for enzymatic activity. Coexistence of Pep1079 and Pep1080 is necessary for expressing high peptidase activity. Pep1081 enhances the activity of Pep1079 and Pep1080.
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المؤلفون: Atsushi Nagasawa, Mikio Fujii, Keitarou Kimura, Yoshifumi Itoh
المصدر: Journal of Bioscience and Bioengineering. 93:589-594
مصطلحات موضوعية: Lactobacillus helveticus, biology, Permease, Nucleic acid sequence, Bioengineering, Oligopeptide transport, Molecular cloning, biology.organism_classification, Applied Microbiology and Biotechnology, Molecular biology, Aminopeptidase, Plasmid, Biochemistry, Gene, Biotechnology
الوصف: X-prolyl dipeptidyl aminopeptidase (X-PDAP) from Lactobacillus helveticus IF03809 expressed nearly full activity under high salt conditions, such as 2 M NaCl. We cloned and sequenced the pepX gene for X-PDAP. The calculated M, of deduced X-PDAP (803 amino acids) was 90,847 and the protein was distantly related (35 to 44% identity) to known X-PDAPs of Lactobacillus sp. including L. helveticus CNRZ32 (40% identity). Native and recombinant X-PDAP were purified to homogeneity from both L. helveticus IF03809 and Escherichia coli DH5alpha harboring the pepX gene on a plasmid, respectively. The native enzyme appeared to be a dimer of 220 kDa, as estimated by gel filtration column chromatography. It hydrolyzed an X-prolyl-linkage, but not prolyl- or X-prolyl-X-peptide bonds, and tolerated up to 2 M NaCl as well as some other chlorides of monovalent cations. Determination of the flanking sequences revealed two divergent genes. The upstream region of the pepX gene encodes oppA gene for a putative oligopeptide permease, while the downstream region encodes tnp gene specifying a possible transposase of the IS3 family. The oppA gene shares a 176 bp-promoter region with pepX in the intergenic region, implying a relationship between this oligopeptide transport system and X-PDAP.
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6دورية أكاديمية
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المؤلفون: Kwanghyun Nam, Sung Gyun Kang, Jung-Hyun Lee, Hyun Sook Lee, Yun Jae Kim, Yona Cho
المصدر: Journal of bioscience and bioengineering. 104(3)
مصطلحات موضوعية: chemistry.chemical_classification, biology, Molecular Sequence Data, Bioengineering, biology.organism_classification, Applied Microbiology and Biotechnology, Aminopeptidase, Aminopeptidases, Hyperthermophile, Enzyme assay, Substrate Specificity, Enzyme Activation, Thermococcus, Pyrococcus horikoshii, Enzyme activator, Enzyme, Biochemistry, chemistry, Species Specificity, Enzyme Stability, Pyrococcus furiosus, biology.protein, Amino Acid Sequence, Biotechnology
الوصف: A genomic analysis of the hyperthermophilic archaeon Thermoccoccus onnurineus NA1 (TNA1) revealed the presence of a deblocking aminopeptidase (DAP) gene with high similarity to the genes of DAPs from Pyrococcus furiosus (86%) and Pyrococcus horikoshii (83% identity). The optimum aminopeptidase activity of the recombinant enzyme was observed at pH 7.5 and in the range of 90 degrees C to 100 degrees C. The specific aminopeptidase and deblocking activities of the enzyme toward Leu-pNA and Ac-Leu-pNA were 18- and 3-fold higher than those of a P. horikoshii DAP (DAP2), respectively. The enzyme activity was significantly increased by Co(2+) ions. The presence of Co(2+) ions induced the activation of the enzyme with heating and changed the large oligomer to a dimer. The enzyme activated by Co(2+) ions appeared to eventually be inactivated by autodegradation, which was confirmed by mass spectrometry.
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8دورية أكاديمية
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المؤلفون: Furuta Michio, Toshikazu Nishiwaki, Satoshi Yoshimizu, Kiyoshi Hayashi
المصدر: Journal of bioscience and bioengineering. 93(1)
مصطلحات موضوعية: Chemistry, food and beverages, Bioengineering, Phenylalanine, Applied Microbiology and Biotechnology, Aminopeptidase, Hydrolysate, stomatognathic system, Biochemistry, Valine, Enzymatic hydrolysis, Casein, Soy protein, Grifola frondosa, Biotechnology
الوصف: Bitter peptide solutions, prepared by the enzymatic hydrolysis of soy protein and milk casein, were treated with an aminopeptidase from the edible basidiomycete Grifola frondosa. As the incubation time elapsed, the amount of free amino acids released increased and the bitterness of the enzyme reaction mixtures decreased. However, the debittering of the milk casein hydrolysate by the aminopeptidase was less effective than that observed for the soy protein hydrolysate. Hydrophobic amino acids such as valine, leucine, phenylalanine, tyrosine, and isoleucine were preferentially released from the bitter solutions by the action of the aminopeptidase.
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10دورية أكاديمية
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