A novel alkaline endoglucanase from an alkaliphilic Bacillus isolate: enzymatic properties, and nucleotide and deduced amino acid sequences

Chitinase, as one of the most important extracellular enzymes in the marine environment, has great ecological and applied values. In this study, two chitinases (Chi1557 and Chi4668) with 97.33% amino acid sequences identity were individually found in Vibrio rotiferianus and Vibrio harveyi. They both were encoding by 561 amino acids, but differed in 15 amino acids and showed different enzymatic properties. The optimal temperature and pH ranges were 45–50 °C and pH 5.0–7.0 for Chi1557, while ~50 °C and pH 3.0–6.0 for Chi4668. K+, Mg2+, and EDTA increased the enzymatic activity of Chi4668 significantly, yet these factors were inhibitory to Chi1557. Moreover, Chi1557 degraded colloidal chitin to produce (GlcNAc)2 and minor GlcNAc, whereas Chi4668 produce (GlcNAc)2 with minor (GlcNAc)3 and (GlcNAc)4. The Kcat/Km of Chi4668 was ~4.7 times higher than that of Chi1557, indicating that Chi4668 had stronger catalytic activity than Chi1557. Furthermore, site-directed mutagenesis was performed on Chi1557 focusing on seven conserved amino acid residues of family GH18 chitinases. Chi1557 was almost completely inactive after Glu154, Gln219, Tyr221, or Trp312 was individually mutated, retained ~50% activity after Tyr37 was mutated, and increased two times activity after Asp152 was mutated, indicating that these six amino acids were key sites for Chi1557.

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N-terminal amino acid sequences and enzymatic properties of fibrinolytic/haemorrhagic metalloproteinases purified from snake venom

Toxicon ◽

10.1016/0041-0101(95)99301-i ◽

1995 ◽

Vol 33 (3) ◽

pp. 282

Author(s):

M. Maruyama ◽

K. Anai ◽

M. Sugiki ◽

E. Yoshida ◽

H. Mihara

Keyword(s):

Amino Acid ◽

Snake Venom ◽

Amino Acid Sequences ◽

Enzymatic Properties ◽

Terminal Amino Acid ◽

Terminal Amino

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Enzymatic Properties of Dipeptidyl Aminopeptidase IV Produced by the Periodontal Pathogen Porphyromonas gingivalis and Its Participation in Virulence

Infection and Immunity ◽

10.1128/iai.68.2.716-724.2000 ◽

2000 ◽

Vol 68 (2) ◽

pp. 716-724 ◽

Cited By ~ 47

Author(s):

Yumi Kumagai ◽

Kiyoshi Konishi ◽

Tomoharu Gomi ◽

Hisao Yagishita ◽

Ayako Yajima ◽

...

Keyword(s):

Amino Acid ◽

Porphyromonas Gingivalis ◽

Catalytic Domain ◽

Animal Experiments ◽

Catalytic Triad ◽

Amino Acid Sequences ◽

Enzymatic Properties ◽

Site Directed Mutagenesis ◽

Periodontal Pathogen ◽

Dipeptidyl Aminopeptidase

ABSTRACT Porphyromonas gingivalis is a major pathogen associated with adult periodontitis. We cloned and sequenced the gene (dpp) coding for dipeptidyl aminopeptidase IV (DPPIV) fromP. gingivalis W83, based on the amino acid sequences of peptide fragments derived from purified DPPIV. An Escherichia coli strain overproducing P. gingivalis DPPIV was constructed. The enzymatic properties of recombinant DPPIV purified from the overproducer were similar to those of DPPIV isolated fromP. gingivalis. The three amino acid residues Ser, Asp, and His, which are thought to form a catalytic triad in the C-terminal catalytic domain of eukaryotic DPPIV, are conserved in P. gingivalis DPPIV. When each of the corresponding residues of the enzyme was substituted with Ala by site-directed mutagenesis, DPPIV activity significantly decreased, suggesting that these three residues of P. gingivalis DPPIV are involved in the catalytic reaction. DPPIV-deficient mutants of P. gingivalis were constructed and subjected to animal experiments. Mice injected with the wild-type strain developed abscesses to a greater extent and died more frequently than those challenged with mutant strains. Mice injected with the mutants exhibited faster recovery from the infection, as assessed by weight gain and the rate of lesion healing. This decreased virulence of mutants compared with the parent strain suggests that DPPIV is a potential virulence factor of P. gingivalis and may play important roles in the pathogenesis of adult periodontitis induced by the organism.

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