Stabilization of Lysozyme by Introducing N-Glycosylation Signal Sequence

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Abstract
We designed mutant lysozymes with N-glycosylation signal sequences (Asn48 -Gly49-Thr-50 and Asn87-Ile88-Thr89) by substituting Asp to Asn at positions 48 and 87. When these mutant lysozymes were expressed by using yeast (Saccharomyces cerevisiae) in Burkhol-der minimum medium, N-glycosylation occurred in both lysozymes. The mutant lysozyme with the oligosaccharide at Asn87 showed a similar character to a reported polymannosyl lysozyme [Nakamura,Takasaki,Kobayashi, and Kato (1993) J. Biol. Chem. 268, 12706-12712; Kato, Takasaki,and Ban (1994) FEBS Lett 355, 76-80]. As judged from the thermodynamic stabilities of the lysozymes obtained by the guanidine hydrochloride denaturation method, the oligosaccharide-bearing mutant lysozymes were more stable by 0.4-1.6 kcal/mol than the corresponding unglycosylated lysozymes. Therefore, it is suggested that the introduction of an N-glycosylation signal sequence into a protein is an effective means to increase the stability of the protein.

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