Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

F.C Cançado,W.R Terra,P Chimoy Effio,S.R Marana

doi:10.1590/s0100-879x2008001100005

Abstract

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (mu = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (mu = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotriozide (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

Highlights

Lysozymes (EC 3.2.1.17) catalyze the hydrolysis of β1,4 glycosidic linkages between N-acetylglucosamine and N-acetylmuramic acid residues in the peptidoglycan of the bacterial cell wall
The present study reports on the molecular cloning, sequencing, expression as recombinant protein, and purification of a new digestive lysozyme from M. domestica (MdL2)
MdL1 and MdL2 were classified in the glycoside hydrolase family 22 [1], and the E32 and D50 residues were identified as catalytic acid/base and nucleophile of these lysozymes, respectively

Summary

Introduction

Lysozymes (EC 3.2.1.17) catalyze the hydrolysis of β1,4 glycosidic linkages between N-acetylglucosamine and N-acetylmuramic acid residues in the peptidoglycan of the bacterial cell wall These enzymes are classified in the glycoside hydrolase families 22, 23, 24, 25, and 73 [1]. Of the c-type lysozymes, found in family 22, the hen egg white lysozyme (HEWL) is the most extensively studied This lysozyme has 129 amino acid residues that form an ellipsoidal structure containing two domains. The HEWL catalytic mechanism has two steps: the first, called glycosylation, results in the formation of a glycosyl-enzyme intermediate, whereas www.bjournal.com.br the second is the hydrolysis of that intermediate [6]. Carboxylic groups of the side chain of E35 and D52 (HEWL numbering) act as a catalytic acid/base and a nucleophile, respectively

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Conclusion