Expression and characterization of the prtV gene encoding a collagenase from Vibrio parahaemolyticus in Escherichia coli

Yu, Mei-Shiuan; Lee, Chia-Yin

doi:10.1099/13500872-145-1-143

Cited by 31 publications

(30 citation statements)

References 45 publications

(16 reference statements)

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“…These results help to explain the specific activity of the two enzymes. Previously, V. parahaemolyticus collagenase has been reported to have a K m value of 1.06 mM toward FALGPA, at pH 8.0 and 25°C (35), and this K m value was comparable to that of the present collagenase. Based on the specific constant, G. hollisae collagenase had a higher specificity for type I collagen and FALGPA than did C. histolyticum collagenase.…”

Section: Resultssupporting

confidence: 51%

“…The collagenase activity of V. alginolyticus collagenase is higher than that of any other bacterial collagenase, and it was found highly efficient in debridement of necrotic burns, ulcers and decubitus. To date, bacterial collagenases have been purified from various species, and their genes have been cloned and sequenced (8,12,18,24,35). However, many collagenases have not yet been both enzymatically and structurally characterized.…”

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confidence: 99%

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Cloning of a Novel Collagenase Gene from the Gram-Negative Bacterium Grimontia (Vibrio) hollisae 1706B and Its Efficient Expression in Brevibacillus choshinensis

et al. 2011

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The collagenase gene was cloned from Grimontia (Vibrio) hollisae 1706B, and its complete nucleotide sequence was determined. Nucleotide sequencing showed that the open reading frame was 2,301 bp in length and encoded an 84-kDa protein of 767 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, the HEXXH motif. G. hollisae collagenase showed 60 and 59% amino acid sequence identities to Vibrio parahaemolyticus and Vibrio alginolyticus collagenase, respectively. In contrast, this enzyme showed <20% sequence identity with Clostridium histolyticum collagenase. When the recombinant mature collagenase, which consisted of 680 amino acids with a calculated molecular mass of 74 kDa, was produced by the Brevibacillus expression system, a major gelatinolytic protein band of ϳ60 kDa was determined by zymographic analysis. This result suggested that cloned collagenase might undergo processing after secretion. Moreover, the purified recombinant enzyme was shown to possess a specific activity of 5,314 U/mg, an ϳ4-fold greater activity than that of C. histolyticum collagenase.Bacterial collagenases are metalloproteases containing a consensus motif for zinc proteases, the HEXXH sequence and are capable of digesting both native and denatured collagen. They make multiple cleavages at the Y-Gly bond in repeating X-Y-Gly sequences within triple helical regions, where proline and hydroxyproline residues are most common in the X and Y positions, respectively (17). Because of their characteristics, bacterial collagenases have been widely used in biological experiment as tissuedispersing enzymes, as well as in medical procedures such as the isolation of pancreatic islet cells for transplantation (14) and the treatment for Dupuytren's disease (6).Much of our knowledge of bacterial collagenases has come from studies of the enzymes produced by Clostridium histolyticum (13,(15)(16)(17)34). Analysis of the primary structure of the gene product from C. histolyticum has revealed that clostridial collagenases consist of three domains (catalytic domain, polycystic kidney disease [PKD] domain, and collagen-binding domain [CBD]) in their molecules. Moreover, CBD has utilized for anchoring molecule that growth factors fused to CBD can be functional to bind to collagen fibrils and maintain biological activities (21). On the other hand, one of the other well-investigated bacterial collagenases is Vibrio alginolyticus collagenase (7, 10, 11, 28). The collagenase activity of V. alginolyticus collagenase is higher than that of any other bacterial collagenase, and it was found highly efficient in debridement of necrotic burns, ulcers and decubitus. To date, bacterial collagenases have been purified from various species, and their genes have been cloned and sequenced (8,12,18,24,35). However, many collagenases have not yet been both enzymatically and structurally characterized.Vibrio hollisae is a Gram-negative bacterium first described in 1982 (4) and recently reclassified ...

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Section: Resultssupporting

confidence: 51%

mentioning

confidence: 99%

Cloning of a Novel Collagenase Gene from the Gram-Negative Bacterium Grimontia (Vibrio) hollisae 1706B and Its Efficient Expression in Brevibacillus choshinensis

et al. 2011

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“…The capability to monitor the concentration change in Ca 2ϩ by CBD is critical for the activation of M9B enzymes against insoluble collagen. Along with Zn 2ϩ at the catalytic center, Vibrio parahaemolyticus M9A enzyme uses Ca 2ϩ as a cofactor (47). However, neither Ca 2ϩ binding residues nor collagen binding residues identified in CBD are found in PPC-M9A.…”

Section: Figmentioning

confidence: 99%

Structural Comparison of ColH and ColG Collagen-Binding Domains from Clostridium histolyticum

et al. 2013

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“…The activity of PrtV was inhibited by EDTA and restored by the addition of various metal ions, indicating that PrtV is a metalloprotease. Since PrtV can degrade both type I collagen and the synthetic substrate, 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which is similar in structure to collagen, it was considered to be a collagenase (48). PrtV seems to differ in many respects from other well-known collagenases.…”

Section: Collagenasesmentioning

confidence: 99%

Metal Content and Biochemical Analyses of a Recombinant Collagenase PrtV from Vibrio parahaemolyticus

Yap

Lee

2000

Microbiology and Immunology

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PrtV is an extracellular metalloprotease of Vibrio parahaemolyticus and regarded as a collagenase. Inductively coupled plasma-optical emission spectrometry analysis indicated that the recombinant PrtV contains 1 mol of zinc per mol of the native enzyme. On the basis of a kinetic study using 2-furanacryloyl-LeuGly-Pro-Ala (FALGPA, the specific substrate for bacterial collagenase) as a substrate, it was suggested that metal ions may play a significant role in the binding and catalytic steps of the substrate. PrtV hydrolyzed type I, II, III, and IV collagens; however, it did not hydrolyze type V. In addition, the hydrolysis of native proteins and synthetic substrates revealed that PrtV possesses higher activity toward collagen and collagenlike sequences. The result of the thermal stability study indicated that PrtV was thermostable up to 40 C; at 50 C, stability gradually decreased. In addition, PrtV showed higher storage stability at -20 and 4 C, respectively, than at 25 C. Compared with collagenases from Clostridium histolyticum and Vibrio alginolyticus, PrtV was immunologically different and had no significant effect on the growth of CHO, HeLa, and Vero cells. Taken together, the results of the studies described in this paper advance our knowledge concerning the metal content and biochemical properties of PrtV.

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Expression and characterization of the prtV gene encoding a collagenase from Vibrio parahaemolyticus in Escherichia coli

Cited by 31 publications

References 45 publications

Cloning of a Novel Collagenase Gene from the Gram-Negative Bacterium Grimontia (Vibrio) hollisae 1706B and Its Efficient Expression in Brevibacillus choshinensis

Cloning of a Novel Collagenase Gene from the Gram-Negative Bacterium Grimontia (Vibrio) hollisae 1706B and Its Efficient Expression in Brevibacillus choshinensis

Structural Comparison of ColH and ColG Collagen-Binding Domains from Clostridium histolyticum

Metal Content and Biochemical Analyses of a Recombinant Collagenase PrtV from Vibrio parahaemolyticus

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