MODE OF ACTION, KINETIC-PROPERTIES AND PHYSICOCHEMICAL CHARACTERIZATION OF 2 DIFFERENT DOMAINS OF A BIFUNCTIONAL (1-]4)-BETA-D-XYLANASE FROM RUMINOCOCCUS-FLAVEFACIENS EXPRESSED SEPARATELY IN ESCHERICHIA-COLI

V GARCIACAMPAYO, S I MCCRAE, J X ZHANG, H J FLINT, T M WOOD

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Abstract

Two catalytic domains, A and C, of xylanase A (XYLA) from Ruminococcus flavefaciens were expressed separately as truncated gene products from lacZ fusions in Escherichia coli. The fusion products, referred to respectively as XYLA-A1 and XYLA-C2, were purified to homogeneity by anion-exchange chromatography and chromatofocusing. XYLA-A1 was isoelectric at pH 5.0 and had a molecular mass of 30 kDa, whereas XYLA-C2 had a pI of 5.4 and a molecular mass of 44 kDa. The catalytic activity shown by both domains was optimal at 50-degrees-C, but XYLA-A1 was more sensitive than XYLA-C2 to temperatures higher than the optimum. XYLA-A1 showed a higher sensitivity to pH than XYLA-C2. The enzyme activity of both domains was completely inactivated in the presence of copper or silver ions and partially inactivated by iron or zinc ions. Neither domain was active on xylo-oligosaccharides shorter than xylopentaose: the rate of degradation of longer xylo-oligosaccharides (degree of polymerization 5-10) increased as the chain length increased. Analysis of the products of hydrolysis of xylo-oligosaccharides and xylan (arabinoxylan) polysaccharide showed that the two domains differed in their modes of action: xylobiose was the shortest product of the hydrolysis. With, oat spelt xylan as substrate, XYLA-A1 activity was apparently restricted to regions where xylopyranosyl residues did not carry arabinofuranosyl substituents, whereas XYLA-C2 was able to release hetero-oligosaccharides carrying arabinofuranosyl residues. Neither domain was able to release arabinose from oat spelt xylan.

Original languageEnglish
Pages (from-to)235-243
Number of pages9
JournalBiochemical Journal
Volume296
Publication statusPublished - 15 Nov 1993

Keywords

  • CELLULOSE-BINDING DOMAINS
  • CRYPTOCOCCUS-ALBIDUS
  • CALDOCELLUM-SACCHAROLYTICUM
  • LIMITED PROTEOLYSIS
  • TRICHODERMA-REESEI
  • SUBSTRATE-BINDING
  • XYLANASE
  • GENES
  • PROTEIN
  • ENDO-1,4-BETA-XYLANASE

Cite this

@article{871d798b636f476ca3e65bba694dc310,
title = "MODE OF ACTION, KINETIC-PROPERTIES AND PHYSICOCHEMICAL CHARACTERIZATION OF 2 DIFFERENT DOMAINS OF A BIFUNCTIONAL (1-]4)-BETA-D-XYLANASE FROM RUMINOCOCCUS-FLAVEFACIENS EXPRESSED SEPARATELY IN ESCHERICHIA-COLI",
abstract = "Two catalytic domains, A and C, of xylanase A (XYLA) from Ruminococcus flavefaciens were expressed separately as truncated gene products from lacZ fusions in Escherichia coli. The fusion products, referred to respectively as XYLA-A1 and XYLA-C2, were purified to homogeneity by anion-exchange chromatography and chromatofocusing. XYLA-A1 was isoelectric at pH 5.0 and had a molecular mass of 30 kDa, whereas XYLA-C2 had a pI of 5.4 and a molecular mass of 44 kDa. The catalytic activity shown by both domains was optimal at 50-degrees-C, but XYLA-A1 was more sensitive than XYLA-C2 to temperatures higher than the optimum. XYLA-A1 showed a higher sensitivity to pH than XYLA-C2. The enzyme activity of both domains was completely inactivated in the presence of copper or silver ions and partially inactivated by iron or zinc ions. Neither domain was active on xylo-oligosaccharides shorter than xylopentaose: the rate of degradation of longer xylo-oligosaccharides (degree of polymerization 5-10) increased as the chain length increased. Analysis of the products of hydrolysis of xylo-oligosaccharides and xylan (arabinoxylan) polysaccharide showed that the two domains differed in their modes of action: xylobiose was the shortest product of the hydrolysis. With, oat spelt xylan as substrate, XYLA-A1 activity was apparently restricted to regions where xylopyranosyl residues did not carry arabinofuranosyl substituents, whereas XYLA-C2 was able to release hetero-oligosaccharides carrying arabinofuranosyl residues. Neither domain was able to release arabinose from oat spelt xylan.",
keywords = "CELLULOSE-BINDING DOMAINS, CRYPTOCOCCUS-ALBIDUS, CALDOCELLUM-SACCHAROLYTICUM, LIMITED PROTEOLYSIS, TRICHODERMA-REESEI, SUBSTRATE-BINDING, XYLANASE, GENES, PROTEIN, ENDO-1,4-BETA-XYLANASE",
author = "V GARCIACAMPAYO and MCCRAE, {S I} and ZHANG, {J X} and FLINT, {H J} and WOOD, {T M}",
year = "1993",
month = "11",
day = "15",
language = "English",
volume = "296",
pages = "235--243",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",

}

TY - JOUR

T1 - MODE OF ACTION, KINETIC-PROPERTIES AND PHYSICOCHEMICAL CHARACTERIZATION OF 2 DIFFERENT DOMAINS OF A BIFUNCTIONAL (1-]4)-BETA-D-XYLANASE FROM RUMINOCOCCUS-FLAVEFACIENS EXPRESSED SEPARATELY IN ESCHERICHIA-COLI

AU - GARCIACAMPAYO, V

AU - MCCRAE, S I

AU - ZHANG, J X

AU - FLINT, H J

AU - WOOD, T M

PY - 1993/11/15

Y1 - 1993/11/15

N2 - Two catalytic domains, A and C, of xylanase A (XYLA) from Ruminococcus flavefaciens were expressed separately as truncated gene products from lacZ fusions in Escherichia coli. The fusion products, referred to respectively as XYLA-A1 and XYLA-C2, were purified to homogeneity by anion-exchange chromatography and chromatofocusing. XYLA-A1 was isoelectric at pH 5.0 and had a molecular mass of 30 kDa, whereas XYLA-C2 had a pI of 5.4 and a molecular mass of 44 kDa. The catalytic activity shown by both domains was optimal at 50-degrees-C, but XYLA-A1 was more sensitive than XYLA-C2 to temperatures higher than the optimum. XYLA-A1 showed a higher sensitivity to pH than XYLA-C2. The enzyme activity of both domains was completely inactivated in the presence of copper or silver ions and partially inactivated by iron or zinc ions. Neither domain was active on xylo-oligosaccharides shorter than xylopentaose: the rate of degradation of longer xylo-oligosaccharides (degree of polymerization 5-10) increased as the chain length increased. Analysis of the products of hydrolysis of xylo-oligosaccharides and xylan (arabinoxylan) polysaccharide showed that the two domains differed in their modes of action: xylobiose was the shortest product of the hydrolysis. With, oat spelt xylan as substrate, XYLA-A1 activity was apparently restricted to regions where xylopyranosyl residues did not carry arabinofuranosyl substituents, whereas XYLA-C2 was able to release hetero-oligosaccharides carrying arabinofuranosyl residues. Neither domain was able to release arabinose from oat spelt xylan.

AB - Two catalytic domains, A and C, of xylanase A (XYLA) from Ruminococcus flavefaciens were expressed separately as truncated gene products from lacZ fusions in Escherichia coli. The fusion products, referred to respectively as XYLA-A1 and XYLA-C2, were purified to homogeneity by anion-exchange chromatography and chromatofocusing. XYLA-A1 was isoelectric at pH 5.0 and had a molecular mass of 30 kDa, whereas XYLA-C2 had a pI of 5.4 and a molecular mass of 44 kDa. The catalytic activity shown by both domains was optimal at 50-degrees-C, but XYLA-A1 was more sensitive than XYLA-C2 to temperatures higher than the optimum. XYLA-A1 showed a higher sensitivity to pH than XYLA-C2. The enzyme activity of both domains was completely inactivated in the presence of copper or silver ions and partially inactivated by iron or zinc ions. Neither domain was active on xylo-oligosaccharides shorter than xylopentaose: the rate of degradation of longer xylo-oligosaccharides (degree of polymerization 5-10) increased as the chain length increased. Analysis of the products of hydrolysis of xylo-oligosaccharides and xylan (arabinoxylan) polysaccharide showed that the two domains differed in their modes of action: xylobiose was the shortest product of the hydrolysis. With, oat spelt xylan as substrate, XYLA-A1 activity was apparently restricted to regions where xylopyranosyl residues did not carry arabinofuranosyl substituents, whereas XYLA-C2 was able to release hetero-oligosaccharides carrying arabinofuranosyl residues. Neither domain was able to release arabinose from oat spelt xylan.

KW - CELLULOSE-BINDING DOMAINS

KW - CRYPTOCOCCUS-ALBIDUS

KW - CALDOCELLUM-SACCHAROLYTICUM

KW - LIMITED PROTEOLYSIS

KW - TRICHODERMA-REESEI

KW - SUBSTRATE-BINDING

KW - XYLANASE

KW - GENES

KW - PROTEIN

KW - ENDO-1,4-BETA-XYLANASE

M3 - Article

VL - 296

SP - 235

EP - 243

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

ER -