Highly active metallocarboxypeptidase from newly isolated Geobacillus strain SBS-4S : Cloning and characterization(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
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概要
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The carboxypeptidase gene from Geobacillus SBS-4S was cloned and sequenced. The sequence analysis displayed the gene consists of an open reading frame of 1503 nucleotides encoding a protein of 500 amino acids (CBP_<SBS>). The amino acid sequence comparison revealed that CBP_<SBS> exhibited a highest homology of 41.6% (identity) with carboxypeptidase Taq from Thermus aquaticus among the characterized proteases. CBP_<SBS> contained an active site motif ^<265>HEXXH^<269> which is conserved in family-M32 of carboxypeptidases. The gene was expressed with His-Tag utilizing Escherichia coli expression system and purified to apparent homogeneity. The purified CBP_<SBS> showed highest activity at pH 7.5 and 70℃. The enzyme activity was metal ion dependent. Among metal ions highest activity was found in the presence of Co^<2+>. Thermostability studies of CBP_<SBS> by circular dichroism spectroscopy demonstrated the melting temperature of the protein around 77℃. The enzyme exhibited K_m and V_<max> values of 14mM and 10526 μmol min^<-1>mg^<-1> when carbobenzoxy-alanine-arginine was used as substrate. k_<cat> and k_<cat>/K_m valves were 10175s^<-1> and 726mM^<-1>s^<-1>. To our knowledge this is the highest ever reported enzyme activity of a metallocarboxypeptidase and the first characterization of a metallocarboxypeptidase from genus Geobacillus.
著者
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KANAYA Shigenori
Department of Material and Life Science, Osaka University
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Rashid Naeem
School Of Biological Sciences University Of The Punjab
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Akhtar Muhammad
School Of Biological Sciences University Of The Punjab:school Of Biological Sciences University Of S
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Akhtar Muhammad
School Of Biological Sciences University Of The Punjab Quaid-e-azam Campus
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Kanaya Shigenori
Department Of Material And Life Sciences Graduate School Of Engineering Osaka University
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Angkawidjaja Clement
Department Of Material And Life Sciences Graduate School Of Engineering Osaka University
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Angkawidjaja Clement
Department Of Material And Life Science Osaka University
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Tayyab Muhammad
School of Biological Sciences, University of the Punjab
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Tayyab Muhammad
School Of Biological Sciences University Of The Punjab
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Akhtar Muhammad
School Of Biological Sciences University Of The Punjab Quaid-e-azam Campus:school Of Biological Scie
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Kanaya Shigenori
Department of Material and Life Science, Graduate School of Engineering, Osaka University
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