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Alpha-lactalbumin mutant acting as lysozyme

Academic Article
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Overview

authors

  • Xue, Y. M.
  • Liu, J. N.
  • Sun, Z. Y.
  • Ma, Z.
  • Wu, Chunlei
  • Zhu, D. X.

publication date

  • January 2001

journal

  • Proteins-Structure Function and Genetics  Journal

abstract

  • A mutant of alpha-lactalbumin was expressed and purified, in which His32, Thr33, Glu49, Ile59, Val99, and Tyr103 were substituted by Leu32, Glu33, Asp49, Trp59, Asn99, and Ala103, respectively, to create a catalytic site of lysozyme in alpha-lactalbumin. The mutant catalyzed hydrolysis of the synthetic substrate, pNP-(NAcGlc)(3), with a K(M) and k(cat) of 0.160 +/- 0.00986 mmol/L and 3.39 +/- 0. 0456 x10(-5) min(-1), respectively, which was comparable with those of chicken lysozyme of 0.137 +/- 0.0153 mmol/L and 5.25 +/- 0.115 x10(-4) min(-1). By using the Isothermal Titration Calorimetre (ITC), the average binding enthalpy of the mutant or chicken lysozyme with the substrate (chitopentaose) was measured, which was 49.22 KJ/mol for the mutant and 105.47 KJ/mol for chicken lysozyme. In conclusion, the six point mutations occurring in alpha-lactalbumin could be converted into an enzyme that was 17.5-fold less efficient than chicken lysozyme but nevertheless capable of hydrolyzing the glycosidic bond.

subject areas

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Calorimetry, Differential Scanning
  • Catalytic Domain
  • Cattle
  • Chickens
  • Evolution, Molecular
  • Hydrolysis
  • Kinetics
  • Lactalbumin
  • Molecular Sequence Data
  • Muramidase
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Sequence Alignment
  • Substrate Specificity
  • Thermodynamics
  • Trisaccharides
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Research

keywords

  • alpha-lactalbumin
  • chicken lysozyme
  • chitopentaose
  • glycosidic bond
  • isothermal titration calorimeter
  • pNP-(NAcGlc)(3)
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Identity

International Standard Serial Number (ISSN)

  • 0887-3585

Digital Object Identifier (DOI)

  • 10.1002/1097-0134(20010101)42:1<17::aid-prot30>3.0.co;2-a

PubMed ID

  • 11093257
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Additional Document Info

start page

  • 17

end page

  • 22

volume

  • 42

issue

  • 1

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