An endochitinase A from Vibrio carchariae: cloning, expression, mass and sequence analyses, and chitin hydrolysis

Wipa Suginta; Archara Vongsuwan; Chomphunuch Songsiriritthigul; Heino Prinz; Peter Estibeiro; Rory R Duncan; Jisnuson Svasti; Linda A. Fothergill-Gilmore

doi:10.1016/j.abb.2004.01.017

An endochitinase A from Vibrio carchariae: cloning, expression, mass and sequence analyses, and chitin hydrolysis

Wipa Suginta, Archara Vongsuwan, Chomphunuch Songsiriritthigul, Heino Prinz, Peter Estibeiro, Rory R Duncan, Jisnuson Svasti, Linda A. Fothergill-Gilmore

Research output: Contribution to journal › Article › peer-review

55 Citations (Scopus)

Abstract

We provide evidence that chitinase A from Vibrio carchariae acts as an endochitinase. The chitinase A gene isolated from V. carchariae genome encodes 850 amino acids expressing a 95-kDa precursor. Peptide masses of the native enzyme identified from MALDI-TOF or nanoESIMS were identical with the putative amino acid sequence translated from the corresponding nucleotide sequence. The enzyme has a highly conserved catalytic TIM-barrel region as previously described for Serratia marcescens ChiA. The Mr of the native chitinase A was determined to be 62,698, suggesting that the C-terminal proteolytic cleavage site was located between R597 and K598. The DNA fragment that encodes the processed enzyme was subsequently cloned and expressed in Escherichia coli. The expressed protein exhibited chitinase activity on gel activity assay. Analysis of chitin hydrolysis using HPLC/ESI-MS confirmed the endo characteristics of the enzyme.

Original language	English
Pages (from-to)	171-180
Number of pages	10
Journal	Archives of Biochemistry and Biophysics
Volume	424
Issue number	2
DOIs	https://doi.org/10.1016/j.abb.2004.01.017
Publication status	Published - 15 Apr 2004

Keywords

Amino Acid Sequence
Chitin
Chitinase
Cloning, Molecular
Enzyme Activation
Enzyme Stability
Escherichia coli
Molecular Sequence Data
Sequence Analysis, Protein
Sequence Homology
Vibrio
Amino Acid

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10.1016/j.abb.2004.01.017

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title = "An endochitinase A from Vibrio carchariae: cloning, expression, mass and sequence analyses, and chitin hydrolysis",

abstract = "We provide evidence that chitinase A from Vibrio carchariae acts as an endochitinase. The chitinase A gene isolated from V. carchariae genome encodes 850 amino acids expressing a 95-kDa precursor. Peptide masses of the native enzyme identified from MALDI-TOF or nanoESIMS were identical with the putative amino acid sequence translated from the corresponding nucleotide sequence. The enzyme has a highly conserved catalytic TIM-barrel region as previously described for Serratia marcescens ChiA. The Mr of the native chitinase A was determined to be 62,698, suggesting that the C-terminal proteolytic cleavage site was located between R597 and K598. The DNA fragment that encodes the processed enzyme was subsequently cloned and expressed in Escherichia coli. The expressed protein exhibited chitinase activity on gel activity assay. Analysis of chitin hydrolysis using HPLC/ESI-MS confirmed the endo characteristics of the enzyme.",

keywords = "Amino Acid Sequence, Chitin, Chitinase, Cloning, Molecular, Enzyme Activation, Enzyme Stability, Escherichia coli, Molecular Sequence Data, Sequence Analysis, Protein, Sequence Homology, Vibrio, Amino Acid",

author = "Wipa Suginta and Archara Vongsuwan and Chomphunuch Songsiriritthigul and Heino Prinz and Peter Estibeiro and Duncan, {Rory R} and Jisnuson Svasti and Fothergill-Gilmore, {Linda A.}",

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language = "English",

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T1 - An endochitinase A from Vibrio carchariae

T2 - cloning, expression, mass and sequence analyses, and chitin hydrolysis

AU - Suginta, Wipa

AU - Vongsuwan, Archara

AU - Songsiriritthigul, Chomphunuch

AU - Prinz, Heino

AU - Estibeiro, Peter

AU - Duncan, Rory R

AU - Svasti, Jisnuson

AU - Fothergill-Gilmore, Linda A.

PY - 2004/4/15

Y1 - 2004/4/15

N2 - We provide evidence that chitinase A from Vibrio carchariae acts as an endochitinase. The chitinase A gene isolated from V. carchariae genome encodes 850 amino acids expressing a 95-kDa precursor. Peptide masses of the native enzyme identified from MALDI-TOF or nanoESIMS were identical with the putative amino acid sequence translated from the corresponding nucleotide sequence. The enzyme has a highly conserved catalytic TIM-barrel region as previously described for Serratia marcescens ChiA. The Mr of the native chitinase A was determined to be 62,698, suggesting that the C-terminal proteolytic cleavage site was located between R597 and K598. The DNA fragment that encodes the processed enzyme was subsequently cloned and expressed in Escherichia coli. The expressed protein exhibited chitinase activity on gel activity assay. Analysis of chitin hydrolysis using HPLC/ESI-MS confirmed the endo characteristics of the enzyme.

AB - We provide evidence that chitinase A from Vibrio carchariae acts as an endochitinase. The chitinase A gene isolated from V. carchariae genome encodes 850 amino acids expressing a 95-kDa precursor. Peptide masses of the native enzyme identified from MALDI-TOF or nanoESIMS were identical with the putative amino acid sequence translated from the corresponding nucleotide sequence. The enzyme has a highly conserved catalytic TIM-barrel region as previously described for Serratia marcescens ChiA. The Mr of the native chitinase A was determined to be 62,698, suggesting that the C-terminal proteolytic cleavage site was located between R597 and K598. The DNA fragment that encodes the processed enzyme was subsequently cloned and expressed in Escherichia coli. The expressed protein exhibited chitinase activity on gel activity assay. Analysis of chitin hydrolysis using HPLC/ESI-MS confirmed the endo characteristics of the enzyme.

KW - Amino Acid Sequence

KW - Chitin

KW - Chitinase

KW - Cloning, Molecular

KW - Enzyme Activation

KW - Enzyme Stability

KW - Escherichia coli

KW - Molecular Sequence Data

KW - Sequence Analysis, Protein

KW - Sequence Homology

KW - Vibrio

KW - Amino Acid

U2 - 10.1016/j.abb.2004.01.017

DO - 10.1016/j.abb.2004.01.017

M3 - Article

C2 - 15047189

SN - 0003-9861

VL - 424

SP - 171

EP - 180

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

IS - 2

ER -

An endochitinase A from Vibrio carchariae: cloning, expression, mass and sequence analyses, and chitin hydrolysis

Abstract

Keywords

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