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α‐lactalbumin mutant acting as lysozyme
Author(s) -
Xue Yuming,
Liu JianNing,
Sun Ziyong,
Ma Zhong,
Wu Chunlei,
Zhu Dexu
Publication year - 2000
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/1097-0134(20010101)42:1<17::aid-prot30>3.0.co;2-a
Subject(s) - lysozyme , mutant , chemistry , lactalbumin , computational biology , biology , biochemistry , gene
A mutant of α‐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 α‐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 ×10 −5 min −1 , respectively, which was comparable with those of chicken lysozyme of 0.137 ± 0.0153 mmol/L and 5.25 ± 0.115 ×10 ‐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 α‐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. Proteins 2001;42:17–22. © 2000 Wiley‐Liss, Inc.