Purification and some properties of dipeptidyl carboxypeptidase from Bacillus pumilus.

Nagamori, Yoichi; Fujishima, Noboru; Okada, Shigetaka

doi:10.1271/bbb1961.54.999

Cited by 8 publications

(3 citation statements)

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“…The serine, cysteine, and aspartic protease inhibitors (PMSF, NBS, and WRK) had almost no effect, whereas the group selective modification regents DEPC (for histidine residues), EAM (for lysine residues), and CHD (for arginine residues) reduced the activity of His 6 -EcDCP to between 6 and 66.9% of its maximum. In agreement with other studies of microbial DCPs (Henrich et al 1993;Miyoshi et al 1992;Nagamori et al 1990;Ogasawara et al 1997), His 6 -EcDCP exhibited a strong susceptibility toward the chelating agents EDTA and 1,10-phenanthroline. Together with the presence of a potential Zn 2?…”

Section: Effects Of Metal Ions and Other Compounds On His 6 -Ecdcp Acsupporting

confidence: 91%

The dipeptidyl carboxypeptidase of Escherichia coli novablue: overproduction and molecular characterization of the recombinant enzyme

Chen

Chang

Lin

et al. 2008

World J Microbiol Biotechnol

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To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His 6 -tagged EcDCP (His 6 -EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity by Ni 2? -NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His 6 -EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His 6 -EcDCP was stimulated by 1.5 fold. The K M and k cat values of the enzyme for N-benzoyl-L-glycyl-L-histidyl-L-leucine were 1.83 mM and 168.3 s -1 , respectively. His 6 -EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His 6 -EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17% a-helix, 35% b-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme.

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Section: Effects Of Metal Ions and Other Compounds On His 6 -Ecdcp Acsupporting

confidence: 91%

The dipeptidyl carboxypeptidase of Escherichia coli novablue: overproduction and molecular characterization of the recombinant enzyme

Chen

Chang

Lin

et al. 2008

World J Microbiol Biotechnol

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“…The HIS CPXAC temperature test was performed at various temperatures, and the remaining activity of the cysteine peptidases was measured (Nagamori et al, 1990).…”

Section: Optimal Temperaturementioning

confidence: 99%

Recombinant expression and characterization of a cysteine peptidase from Xanthomonas citri subsp citri

Soares-Costa¹,

Silveira²,

Novo³

et al. 2012

Genet. Mol. Res.

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ABSTRACT. Xanthomonas citri subsp citri (Xac) is the bacterium responsible for citrus canker disease in citrus plants. The aim of this study was to describe the recombinant expression, purification, and characterization of a cysteine peptidase from Xac strain 306, which is a candidate for involvement in the pathogenicity of this bacterium. The gene was cloned and expressed in Pichia pastoris, and the cysteine peptidase was successfully expressed, secreted, and purified using affinity chromatography with a yield of approximately 10 mg/L. A polyclonal antibody produced against cysteine peptidase from X. citri subsp citri fused with HIS tag ( HIS CPXAC) recognized the purified recombinant cysteine peptidase HIS CPXAC, confirming the correct production of this protein in P. pastoris. The same antibody detected the protein in the culture supernatant of Xac grown in pathogenicityinducing medium. Kinetic analysis revealed that HIS CPXAC hydrolyzed the carbobenzoxy-Leu-Arg-7-amido-4-methylcoumarin substrate with a catalytic efficiency (k cat /K m ) of 47 μM -1 •s -1 . The purified HIS CPXAC displayed maximal catalytic activity at pH 5.5 and 30°C. The recombinant enzyme was inhibited by the specific cysteine peptidase inhibitor E-64, as well as by the recombinant cysteine peptidase inhibitors CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4, with K i values of 1.214, 84.64, 0.09, 0.09, and 0.012 nM, respectively. Finally, the N-terminal sequencing of the purified protein enabled the identification of the first 5 amino acid residues (AVHGM) immediately after the putative signal peptide, thereby enabling the identification of the cleavage point and corroborating previous studies that have identified this sequence in a secreted protein from Xanthomonas spp.

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“…Both were declared metallopeptidases. Dipeptidyl CP, a 155 kDa metalloprotease from Bacillus pumilus, was completely inhibited by 1 mM EDTA but 38% inhibited by 1 mM diisopropyl fluorophosphate (DFP), a serine protease inhibitor (40). Like squid CP-I (Table 8), it is activated by Co 2+ but inhibited by Cu 2+ .…”

Section: Effect Of Znso 4 and Nacl On The Activity Of Cpmentioning

confidence: 99%

Purification and Characterization of a Carboxypeptidase from Squid Hepatopancreas (Illex illecebrosus)

Raksakulthai

Haard

2001

J. Agric. Food Chem.

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The hepatopancreas of squid (Illex illecebrosus) extract contains a wide range of carboxypeptidase (CP) activities based on hydrolysis of N-CBZ-dipeptide substrates. SDS-PAGE zymograms with N-CBZ-Phe-Leu substrate revealed three activity zones (CP-I, 23 kDa; CP-II, 29 kDa; CP-III, 42 kDa). CP-I was purified 225-fold with 86.20% recovery based on N-CBZ-Ala-Phe activity by chromatography on DEAE-cellulose, gel filtration, and chromatofocusing. The purified enzyme had broad specificity toward N-CBZ-dipeptides; however, it preferred substrates with a hydrophobic amino acid at the C terminus. CP-I had greatest activity with N-CBZ-Ala-Phe (specific activity = 7104 units/mg of protein, K(m) = 0.40 mM, and physiological efficiency = 22863). CP-I had a pI of 3.4 and is a metalloprotease that is activated by Co(2+) and partially inhibited by Pefabloc, a serine protease inhibitor. With N-CBZ-Ala-Phe and Gly-Phe, it had optimum activity at pH 8 and 70 degrees C. The amino acid composition of squid CP-I is similar to that of CP A from other species.

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Purification and some properties of dipeptidyl carboxypeptidase from Bacillus pumilus.

Cited by 8 publications

References 0 publications

The dipeptidyl carboxypeptidase of Escherichia coli novablue: overproduction and molecular characterization of the recombinant enzyme

The dipeptidyl carboxypeptidase of Escherichia coli novablue: overproduction and molecular characterization of the recombinant enzyme

Recombinant expression and characterization of a cysteine peptidase from Xanthomonas citri subsp citri

Purification and Characterization of a Carboxypeptidase from Squid Hepatopancreas (Illex illecebrosus)

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