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A novel carboxyl-terminal protease derived from Paenibacillus lautus CHN26 exhibiting high activities at multiple sites of substrates.

Li Y, Pan Y, She Q, Chen L - BMC Biotechnol. (2013)

Bottom Line: CtpAp was expressed as a recombinant protein and characterized.The purified enzyme showed an endopeptidase activity, which effectively cleaved α S1- and β- casein substrates at carboxyl-terminus as well as at multiple internal sites.We have demonstrated that CtpAp is a novel endopeptidase with distinct cleavage specificities, showing a strong potential in biotechnology and industry applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), China Ministry of Agriculture, Engineering Centre for Quality Control and Risk Assessment of Aquatic Products, College of Food Science and Technology, Shanghai Ocean University, 999 Hu Cheng Huan Road, Shanghai 201306, People's Republic of China. lmchen@shou.edu.cn.

ABSTRACT

Background: Carboxyl-terminal protease (CtpA) plays essential functions in posttranslational protein processing in prokaryotic and eukaryotic cells. To date, only a few bacterial ctpA genes have been characterized. Here we cloned and characterized a novel CtpA. The encoding gene, ctpAp (ctpA of Paenibacillus lautus), was derived from P. lautus CHN26, a Gram-positive bacterium isolated by functional screening. Recombinant protein was obtained from protein over-expression in Escherichia coli and the biochemical properties of the enzyme were investigated.

Results: Screening of environmental sediment samples with a skim milk-containing medium led to the isolation of a P. lautus CHN26 strain that exhibited a high proteolytic activity. A gene encoding a carboxyl-terminal protease (ctpAp) was cloned from the isolate and characterized. The deduced mature protein contains 466 aa with a calculated molecular mass of 51.94 kDa, displaying 29-38% amino acid sequence identity to characterized bacterial CtpA enzymes. CtpAp contains an unusual catalytic dyad (Ser₃₀₉-Lys₃₃₄) and a PDZ substrate-binding motif, characteristic for carboxyl-terminal proteases. CtpAp was expressed as a recombinant protein and characterized. The purified enzyme showed an endopeptidase activity, which effectively cleaved α S1- and β- casein substrates at carboxyl-terminus as well as at multiple internal sites. Furthermore, CtpAp exhibited a high activity at room temperature and strong tolerance to conventional protease inhibitors, demonstrating that CtpAp is a novel endopeptidase.

Conclusions: Our work on CtpA represents the first investigation of a member of Family II CtpA enzymes. The gene was derived from a newly isolated P. lautus CHN26 strain exhibiting a high protease activity in the skim milk assay. We have demonstrated that CtpAp is a novel endopeptidase with distinct cleavage specificities, showing a strong potential in biotechnology and industry applications.

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Effect of metal ions, detergents and inhibitors on P. lautus CtpA enzyme stability. Aliquots of the enzyme were incubated with each metal ion (10 mM), detergent (0.5%) and inhibitors (10 mM) at 30°C for 1 h, the residual activity of CtpAp was determined using β-casein as the substrate. All determinants were performed in triplicate.
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Figure 8: Effect of metal ions, detergents and inhibitors on P. lautus CtpA enzyme stability. Aliquots of the enzyme were incubated with each metal ion (10 mM), detergent (0.5%) and inhibitors (10 mM) at 30°C for 1 h, the residual activity of CtpAp was determined using β-casein as the substrate. All determinants were performed in triplicate.

Mentions: To address the effect of metal ions on CtpA activity, aliquotes of the purified enzyme were individually incubated with ZnCl2, CaCl2, MgCl2, BaCl2, CuCl2, MnCl2 and KCl at 1-10 mM concentration at 30°C for 1 h, and residual activities were measured under the standard assay condition (Figure 8). Comparing to the control, Mn2+ and Ca2+ elevated CtpA activity by 59.6% and 32.8%, respectively, whereas Ba2+, Cu2+ and Zn2+ strongly inhibited enzyme activity, reducing the activity by 30-50%. These results resemble those reported for E. coli CtpA [5].


A novel carboxyl-terminal protease derived from Paenibacillus lautus CHN26 exhibiting high activities at multiple sites of substrates.

Li Y, Pan Y, She Q, Chen L - BMC Biotechnol. (2013)

Effect of metal ions, detergents and inhibitors on P. lautus CtpA enzyme stability. Aliquots of the enzyme were incubated with each metal ion (10 mM), detergent (0.5%) and inhibitors (10 mM) at 30°C for 1 h, the residual activity of CtpAp was determined using β-casein as the substrate. All determinants were performed in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231363&req=5

Figure 8: Effect of metal ions, detergents and inhibitors on P. lautus CtpA enzyme stability. Aliquots of the enzyme were incubated with each metal ion (10 mM), detergent (0.5%) and inhibitors (10 mM) at 30°C for 1 h, the residual activity of CtpAp was determined using β-casein as the substrate. All determinants were performed in triplicate.
Mentions: To address the effect of metal ions on CtpA activity, aliquotes of the purified enzyme were individually incubated with ZnCl2, CaCl2, MgCl2, BaCl2, CuCl2, MnCl2 and KCl at 1-10 mM concentration at 30°C for 1 h, and residual activities were measured under the standard assay condition (Figure 8). Comparing to the control, Mn2+ and Ca2+ elevated CtpA activity by 59.6% and 32.8%, respectively, whereas Ba2+, Cu2+ and Zn2+ strongly inhibited enzyme activity, reducing the activity by 30-50%. These results resemble those reported for E. coli CtpA [5].

Bottom Line: CtpAp was expressed as a recombinant protein and characterized.The purified enzyme showed an endopeptidase activity, which effectively cleaved α S1- and β- casein substrates at carboxyl-terminus as well as at multiple internal sites.We have demonstrated that CtpAp is a novel endopeptidase with distinct cleavage specificities, showing a strong potential in biotechnology and industry applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), China Ministry of Agriculture, Engineering Centre for Quality Control and Risk Assessment of Aquatic Products, College of Food Science and Technology, Shanghai Ocean University, 999 Hu Cheng Huan Road, Shanghai 201306, People's Republic of China. lmchen@shou.edu.cn.

ABSTRACT

Background: Carboxyl-terminal protease (CtpA) plays essential functions in posttranslational protein processing in prokaryotic and eukaryotic cells. To date, only a few bacterial ctpA genes have been characterized. Here we cloned and characterized a novel CtpA. The encoding gene, ctpAp (ctpA of Paenibacillus lautus), was derived from P. lautus CHN26, a Gram-positive bacterium isolated by functional screening. Recombinant protein was obtained from protein over-expression in Escherichia coli and the biochemical properties of the enzyme were investigated.

Results: Screening of environmental sediment samples with a skim milk-containing medium led to the isolation of a P. lautus CHN26 strain that exhibited a high proteolytic activity. A gene encoding a carboxyl-terminal protease (ctpAp) was cloned from the isolate and characterized. The deduced mature protein contains 466 aa with a calculated molecular mass of 51.94 kDa, displaying 29-38% amino acid sequence identity to characterized bacterial CtpA enzymes. CtpAp contains an unusual catalytic dyad (Ser₃₀₉-Lys₃₃₄) and a PDZ substrate-binding motif, characteristic for carboxyl-terminal proteases. CtpAp was expressed as a recombinant protein and characterized. The purified enzyme showed an endopeptidase activity, which effectively cleaved α S1- and β- casein substrates at carboxyl-terminus as well as at multiple internal sites. Furthermore, CtpAp exhibited a high activity at room temperature and strong tolerance to conventional protease inhibitors, demonstrating that CtpAp is a novel endopeptidase.

Conclusions: Our work on CtpA represents the first investigation of a member of Family II CtpA enzymes. The gene was derived from a newly isolated P. lautus CHN26 strain exhibiting a high protease activity in the skim milk assay. We have demonstrated that CtpAp is a novel endopeptidase with distinct cleavage specificities, showing a strong potential in biotechnology and industry applications.

Show MeSH
Related in: MedlinePlus