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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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A multiple sequence alignment of P. lautus CtpA and a selected set of bacterial CtpA proteins. Numbers above the alignments indicate relative positions of the entirely aligned sequences. Identical amino acid residues as well as the conserved ones (>50% of the sequences) are highlighted in black and in grey, respectively, with the consensus sequence shown below the alignment. The catalytic dyad and PDZ motif are boxed. The CtpA sequences chosen for the analysis are obtained from GenBank and NCBI databases: BAA00577.1: E. coli K-12; AAB61766: B. bacilliformis KC583; NP_698817.1: B. suis 1330; AAC66729.1: B. burgdorferi B31; YP_104685.1: B. mallei ATCC23344; YP_005982591.1: P. aeruginosa NCGM2.S1; Q55669.1: Synechocystis sp. PCC 6803 substr. GT-1; CtpAp (GenBank: KF169841): P. lautus CHN26, obtained in this study.
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Figure 2: A multiple sequence alignment of P. lautus CtpA and a selected set of bacterial CtpA proteins. Numbers above the alignments indicate relative positions of the entirely aligned sequences. Identical amino acid residues as well as the conserved ones (>50% of the sequences) are highlighted in black and in grey, respectively, with the consensus sequence shown below the alignment. The catalytic dyad and PDZ motif are boxed. The CtpA sequences chosen for the analysis are obtained from GenBank and NCBI databases: BAA00577.1: E. coli K-12; AAB61766: B. bacilliformis KC583; NP_698817.1: B. suis 1330; AAC66729.1: B. burgdorferi B31; YP_104685.1: B. mallei ATCC23344; YP_005982591.1: P. aeruginosa NCGM2.S1; Q55669.1: Synechocystis sp. PCC 6803 substr. GT-1; CtpAp (GenBank: KF169841): P. lautus CHN26, obtained in this study.

Mentions: The ctpA gene from P. lautus CHN26 was amplified using the primer pair ctp1-F and ctp1-R (see the Methods) targeting the corresponding gene sequence of Paenibacillus sp.Y412MC10 (GenBank: NC_013406.1), which was re-classed as P. lautus recently [24,25]. A PCR product of the expected size (1.4 kb) was obtained from P. lautus CHN26, and the ctpA gene sequence of P. lautus CHN26 was determined (GenBank: KF169841): an open reading frame of 1,473 bp encoding a protein of 490 aa with a calculated molecular mass of 53.92 KDa. The deduced amino acid sequence displayed high sequence identities to putative C-terminal proteases encoded in Paenibacillus species (53-99% identity), all of which have not been characterized. Furthermore, P. lautus CtpA showed relatively lower sequence similarities (29-38% identities) to all characterized bacterial CtpA proteins, including those encoded in E. coli, Bartonella bacilliformis, B. burgdorferi, B. suis, B. mallei, and Pseudomonas aeruginosa[4,12,13,27-29]. These CtpA sequences were retrieved from the GenBank database and analyzed by multiple sequence alignments. This analysis revealed that CtpA of the P. lautus (CtpAp) contains the catalytic dyad (Ser309-Lys334) and substrate-binding B domain, also designated as PDZ motif [2,3], both of which are highly conserved in all known CtpA proteins (Figure 2).


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)

A multiple sequence alignment of P. lautus CtpA and a selected set of bacterial CtpA proteins. Numbers above the alignments indicate relative positions of the entirely aligned sequences. Identical amino acid residues as well as the conserved ones (>50% of the sequences) are highlighted in black and in grey, respectively, with the consensus sequence shown below the alignment. The catalytic dyad and PDZ motif are boxed. The CtpA sequences chosen for the analysis are obtained from GenBank and NCBI databases: BAA00577.1: E. coli K-12; AAB61766: B. bacilliformis KC583; NP_698817.1: B. suis 1330; AAC66729.1: B. burgdorferi B31; YP_104685.1: B. mallei ATCC23344; YP_005982591.1: P. aeruginosa NCGM2.S1; Q55669.1: Synechocystis sp. PCC 6803 substr. GT-1; CtpAp (GenBank: KF169841): P. lautus CHN26, obtained in this study.
© Copyright Policy - open-access
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Figure 2: A multiple sequence alignment of P. lautus CtpA and a selected set of bacterial CtpA proteins. Numbers above the alignments indicate relative positions of the entirely aligned sequences. Identical amino acid residues as well as the conserved ones (>50% of the sequences) are highlighted in black and in grey, respectively, with the consensus sequence shown below the alignment. The catalytic dyad and PDZ motif are boxed. The CtpA sequences chosen for the analysis are obtained from GenBank and NCBI databases: BAA00577.1: E. coli K-12; AAB61766: B. bacilliformis KC583; NP_698817.1: B. suis 1330; AAC66729.1: B. burgdorferi B31; YP_104685.1: B. mallei ATCC23344; YP_005982591.1: P. aeruginosa NCGM2.S1; Q55669.1: Synechocystis sp. PCC 6803 substr. GT-1; CtpAp (GenBank: KF169841): P. lautus CHN26, obtained in this study.
Mentions: The ctpA gene from P. lautus CHN26 was amplified using the primer pair ctp1-F and ctp1-R (see the Methods) targeting the corresponding gene sequence of Paenibacillus sp.Y412MC10 (GenBank: NC_013406.1), which was re-classed as P. lautus recently [24,25]. A PCR product of the expected size (1.4 kb) was obtained from P. lautus CHN26, and the ctpA gene sequence of P. lautus CHN26 was determined (GenBank: KF169841): an open reading frame of 1,473 bp encoding a protein of 490 aa with a calculated molecular mass of 53.92 KDa. The deduced amino acid sequence displayed high sequence identities to putative C-terminal proteases encoded in Paenibacillus species (53-99% identity), all of which have not been characterized. Furthermore, P. lautus CtpA showed relatively lower sequence similarities (29-38% identities) to all characterized bacterial CtpA proteins, including those encoded in E. coli, Bartonella bacilliformis, B. burgdorferi, B. suis, B. mallei, and Pseudomonas aeruginosa[4,12,13,27-29]. These CtpA sequences were retrieved from the GenBank database and analyzed by multiple sequence alignments. This analysis revealed that CtpA of the P. lautus (CtpAp) contains the catalytic dyad (Ser309-Lys334) and substrate-binding B domain, also designated as PDZ motif [2,3], both of which are highly conserved in all known CtpA proteins (Figure 2).

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