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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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Phylogenetic anlysis of 16S rRNA gene sequences of all Paenibacillus species showing that the newly isolated CHN26 is most closely related to Paenibacillus lautus. Bootstrap percentages are shown at nodes. The scale bar represents 0.02 changes per nucleotide. The GenBank accession numbers are shown behind the names of the strains, and the strain identified in this study is marked with a solid triangle.
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Figure 1: Phylogenetic anlysis of 16S rRNA gene sequences of all Paenibacillus species showing that the newly isolated CHN26 is most closely related to Paenibacillus lautus. Bootstrap percentages are shown at nodes. The scale bar represents 0.02 changes per nucleotide. The GenBank accession numbers are shown behind the names of the strains, and the strain identified in this study is marked with a solid triangle.

Mentions: We have developed a functional screening assay in which extracellular protease-producing bacteria form clear zones around their colonies on selective skim-milk agar plates (see the Methods). Employing the assay to screen for protease-producing bacteria from a sediment sample of fishery ponds identified several positive colonies, among which the isolate CHN26 produced very large clear zone of hydrolysis on the selective plate, suggesting that it could encode one or multiple highly active protease(s). Its 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the 27 F and 1492R primers, and the gene sequence was determined (GenBank: KF460030). Database searches with the 16S rRNA gene revealed that it displayed 96-99% nucleotide sequence identity with the corresponding genes derived from dozens of Paenibacillus bacteria. The phylogenetic position of the isolate was studied by comparing the 16S rRNA gene sequence with a selected set of Paenibacillus bacteria retrieved from the GenBank databases and a phylogenetic tree was constructed using the MEGA4.0 (Figure 1). Strain CHN26 fell into the clade of P. lautus, suggesting it was closely related to this species.


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)

Phylogenetic anlysis of 16S rRNA gene sequences of all Paenibacillus species showing that the newly isolated CHN26 is most closely related to Paenibacillus lautus. Bootstrap percentages are shown at nodes. The scale bar represents 0.02 changes per nucleotide. The GenBank accession numbers are shown behind the names of the strains, and the strain identified in this study is marked with a solid triangle.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Phylogenetic anlysis of 16S rRNA gene sequences of all Paenibacillus species showing that the newly isolated CHN26 is most closely related to Paenibacillus lautus. Bootstrap percentages are shown at nodes. The scale bar represents 0.02 changes per nucleotide. The GenBank accession numbers are shown behind the names of the strains, and the strain identified in this study is marked with a solid triangle.
Mentions: We have developed a functional screening assay in which extracellular protease-producing bacteria form clear zones around their colonies on selective skim-milk agar plates (see the Methods). Employing the assay to screen for protease-producing bacteria from a sediment sample of fishery ponds identified several positive colonies, among which the isolate CHN26 produced very large clear zone of hydrolysis on the selective plate, suggesting that it could encode one or multiple highly active protease(s). Its 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the 27 F and 1492R primers, and the gene sequence was determined (GenBank: KF460030). Database searches with the 16S rRNA gene revealed that it displayed 96-99% nucleotide sequence identity with the corresponding genes derived from dozens of Paenibacillus bacteria. The phylogenetic position of the isolate was studied by comparing the 16S rRNA gene sequence with a selected set of Paenibacillus bacteria retrieved from the GenBank databases and a phylogenetic tree was constructed using the MEGA4.0 (Figure 1). Strain CHN26 fell into the clade of P. lautus, suggesting it was closely related to this species.

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