Kinetic and thermodynamic study of a chemically modified highly active xylanase from Scopulariopsis sp.

Ahmed Afzal, Saleem Ahmed Bokhari, Khawar Sohail Siddiqui

Research output: Contribution to journalArticle

Abstract

The amino groups of purified least acidic xylanase (LAX) isomer and carboxyl groups of purified highly acidic xylanase (HAX) isomer from Scopulariopsis sp. were chemically modified, resulting in charge neutralization and reversal. Modification of the second amino group was accompanied by the complete loss of enzyme activity in both the absence and presence of xylose. Multiple alignments of family 10 and 11 xylanases revealed that there is a pair of fully conserved Lys residues only in family 10 members. Xylanase structures from family 10 members showed that one of the conserved Lys residues is found near the active-site cleft that makes an H-bond with the substrate. The LAX and HAX isoenzymes in which one amino and three to four carboxyl groups were modified were subjected to kinetic and thermodynamic characterization. There were no differences in pH optima between the native and modified HAX, but there was a broadening of pH optimum toward the alkaline range for charge-neutralized LAX and a double pH optimum for charge-reversed LAX. TheV max/K m of both modified LAX and HAX decreased relative to the native species. The thermodynamics of xylan hydrolysis showed that the decrease in the catalytic activity of modified LAX enzymes was entropically driven. When compared with native enzyme, the thermostabilities of modified LAX enzymes increased in the presence and decreased in the absence of substrate. The thermodynamics of kinetic stability for modified LAX enzymes revealed that this increase in thermolability was owing to the decrease in ΔH# with a concomitant increase in ΔS# compared with native LAX. The thermostabilities of all the modified HAX species decreased except that of charge-neutralized HAX, whose half-life significantly increased in 50% (v/v) aqueous dioxan. These results suggest that the altered properties of the modified enzymes were a result of the conformational changes brought about by chemical modification.

Original languageEnglish (US)
Pages (from-to)273-297
Number of pages25
JournalApplied Biochemistry and Biotechnology
Volume141
Issue number2-3
DOIs
StatePublished - Jun 1 2007

Fingerprint

Scopulariopsis
Thermodynamics
Enzymes
Kinetics
Isomers
Isoenzymes
Xylans
Enzyme kinetics
Xylose
Chemical modification
Enzyme activity
Substrates
Hydrolysis
Catalyst activity
Half-Life
Catalytic Domain

Keywords

  • Chemical modification
  • Family 10 xylanases
  • Fungus
  • Structure-function-stability relationship
  • Thermostability

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology

Cite this

Kinetic and thermodynamic study of a chemically modified highly active xylanase from Scopulariopsis sp. / Afzal, Ahmed; Bokhari, Saleem Ahmed; Siddiqui, Khawar Sohail.

In: Applied Biochemistry and Biotechnology, Vol. 141, No. 2-3, 01.06.2007, p. 273-297.

Research output: Contribution to journalArticle

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abstract = "The amino groups of purified least acidic xylanase (LAX) isomer and carboxyl groups of purified highly acidic xylanase (HAX) isomer from Scopulariopsis sp. were chemically modified, resulting in charge neutralization and reversal. Modification of the second amino group was accompanied by the complete loss of enzyme activity in both the absence and presence of xylose. Multiple alignments of family 10 and 11 xylanases revealed that there is a pair of fully conserved Lys residues only in family 10 members. Xylanase structures from family 10 members showed that one of the conserved Lys residues is found near the active-site cleft that makes an H-bond with the substrate. The LAX and HAX isoenzymes in which one amino and three to four carboxyl groups were modified were subjected to kinetic and thermodynamic characterization. There were no differences in pH optima between the native and modified HAX, but there was a broadening of pH optimum toward the alkaline range for charge-neutralized LAX and a double pH optimum for charge-reversed LAX. TheV max/K m of both modified LAX and HAX decreased relative to the native species. The thermodynamics of xylan hydrolysis showed that the decrease in the catalytic activity of modified LAX enzymes was entropically driven. When compared with native enzyme, the thermostabilities of modified LAX enzymes increased in the presence and decreased in the absence of substrate. The thermodynamics of kinetic stability for modified LAX enzymes revealed that this increase in thermolability was owing to the decrease in ΔH# with a concomitant increase in ΔS# compared with native LAX. The thermostabilities of all the modified HAX species decreased except that of charge-neutralized HAX, whose half-life significantly increased in 50{\%} (v/v) aqueous dioxan. These results suggest that the altered properties of the modified enzymes were a result of the conformational changes brought about by chemical modification.",
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