Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa

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Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa. / Brocca, Stefania; Alberghina, Lilia; Lotti, Marina; Persson, Mattias; Wehtje, Ernst; Adlercreutz, Patrick.

In: Protein Science, Vol. 9, No. 5, 01.01.2000, p. 985-990.

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Brocca, Stefania ; Alberghina, Lilia ; Lotti, Marina ; Persson, Mattias ; Wehtje, Ernst ; Adlercreutz, Patrick. / Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa. In: Protein Science. 2000 ; Vol. 9, No. 5. pp. 985-990.

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TY - JOUR

T1 - Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa

AU - Brocca, Stefania

AU - Alberghina, Lilia

AU - Lotti, Marina

AU - Persson, Mattias

AU - Wehtje, Ernst

AU - Adlercreutz, Patrick

PY - 2000/1/1

Y1 - 2000/1/1

N2 - Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N-linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild-type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn35 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect' the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.

AB - Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N-linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild-type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn35 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect' the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.

KW - Candida rugosa

KW - Glycosylation sites

KW - Pichia pastoris

KW - Recombinant lipase

KW - Site-directed mutagenesis

KW - Synthetic gene

KW - Thermostability

KW - Water activity profiles

UR - http://www.scopus.com/inward/record.url?scp=0034114845&partnerID=8YFLogxK

U2 - 10.1110/ps.9.5.985

DO - 10.1110/ps.9.5.985

M3 - Article

C2 - 10850808

AN - SCOPUS:0034114845

VL - 9

SP - 985

EP - 990

JO - Protein Science

JF - Protein Science

SN - 1469-896X

IS - 5

ER -