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Kinetic and thermodynamic study of a chemically modified highly active xylanase fromScopulariopsis sp

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An Erratum to this article was published on 23 April 2008

Abstract

The amino groups of purified least acidic xylanase (LAX) isomer and carboxyl groups of purified highly acidic xylanase (HAX) isomer fromScopulariopsis 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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Abbreviations

HAX:

native highly acidic xylanase

HAX-D:

HAX whose thermostability is determined in 50% (v/v) aqueous dioxan

HAX-N:

HAX activated by EDC in presence of methylamine hydrochloride (charge neutralized, − → neutral)

HAX-N-D:

HAX-N whose thermostability is determined in 50% (v/v) aqueous dioxan

HAX-R:

HAX activated by EDC in presence of ethylenediamine dihydrochloride (charge reversed, − → +)

LAX:

native least acidic xylanse

LAX-N:

acetylated LAX (charge neutralized, + → neutral)

LAX-R:

succinylated LAX (charge reversed, + → −)

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Authors and Affiliations

  1. Department of Molecular Biology, Microbiology and Biochemistry, Southern Illinois University at Carbondale, 62901, Carbondale, IL

    Ahmed Jawaard Afzal

  2. Plant Biotechnology and Genome Core Facility, Southern Illinois University, 62901, Carbondale, IL

    Ahmed Jawaard Afzal

  3. SAB, Department of Biological Sciences & Biotechnology, Tsinghua University, 100084, Beijing, P.R. China

    Saleem Ahmed Bokhari

  4. KSS, School of Microbiology and Immunology, University of New South Wales, 2052, Sydney, NSW, Australia

    Khawar Sohail Siddiqui

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  1. Ahmed Jawaard Afzal
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  3. Khawar Sohail Siddiqui
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Existence of an essential amino group

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Correspondence to Ahmed Jawaard Afzal.

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An erratum to this article is available at http://dx.doi.org/10.1007/s12010-008-8157-8.

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Existence of an essential amino group., Afzal, A.J., Bokhari, S.A. et al. Kinetic and thermodynamic study of a chemically modified highly active xylanase fromScopulariopsis sp. Appl Biochem Biotechnol 141, 273–297 (2007). https://doi.org/10.1007/BF02729068

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  • DOI: https://doi.org/10.1007/BF02729068

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