α-lactalbumin mutant acting as lysozyme

✍ Scribed by Yuming Xue; Jian-Ning Liu; Ziyong Sun; Zhong Ma; Chunlei Wu; Dexu Zhu

• Book ID: 101324694
• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 188 KB
• Volume: 42
• Category: Article
• ISSN: 0887-3585
• DOI: 10.1002/1097-0134(20010101)42:1<17::aid-prot30>3.0.co;2-a

✦ Synopsis

A mutant of ␣-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 ␣-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 ␣-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.