Abstract
Muramidases/lysozymes are important bio-molecules, which cleave the glycan backbone in the peptidoglycan polymer found in bacterial cell walls. The glycoside hydrolase (GH) family 22 C-type lysozyme, from the folivorous bird Opisthocomus hoazin (stinkbird), was expressed in Aspergillus oryzae, and a set of variants was produced. All variants were enzymatically active, including those designed to probe key differences between the Hoatzin enzyme and Hen Egg White lysozyme. Four variants showed improved thermostability at pH 4.7, compared to the wild type. The X-ray structure of the enzyme was determined in the apo form and in complex with chitin oligomers. Bioinformatic analysis of avian GH22 amino acid sequences showed that they separate out into three distinct subgroups (chicken-like birds, sea birds and other birds). The Hoatzin is found in the “other birds” group and we propose that this represents a new cluster of avian upper-gut enzymes.
Highlights
Peptidoglycans are unique to prokaryotic organisms and consist of a glycan backbone of muramic acid and glucosamine, cross-linked with peptide chains
Archer et al previously concluded that proteolysis occurs in HEWL between Gly49 and Ser50 when it is expressed in A. niger [20]
Our results show that OhLys can be expressed at a reasonable level in A. oryzae, with about 30 variants being produced and shown to be active in the turbidity assay
Summary
Peptidoglycans are unique to prokaryotic organisms and consist of a glycan backbone of muramic acid and glucosamine (both N-acetylated), cross-linked with peptide chains. In Gram-positive bacteria (e.g., Staphylococcus aureus) the glycan backbone is highly cross-linked, while it is only partially cross-linked in Gram-negative bacteria, such as Escherichia coli. The cross-linking amino acid chain contains L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanine in E. coli, or L-alanine, D-glutamine, L-lysine, and D-alanine, with a five-glycine interbridge between tetrapeptides, in the case of S. aureus [1]. The unique composition of both the carbohydrate polymer and the peptide cross-linker means that only specialised enzymes can hydrolyse peptidoglycans. Lysins (amidases, lysozymes/muramidases and peptidases) are such specialised bio-molecules. The term lysozyme, or muramidase, is broadly used to describe the enzymes that cleave the β-1,4-glycosidic bond between
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