title: Enzymatic cell wall degradation of Chlorella               vulgaris and other microalgae for biofuels production.
abstract: Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae.
authors.affiliation: National Renewable Energy Laboratory, National Bioenergy Center, 15013 Denver West Parkway, Golden, CO 80401, USA.
authors.forename: Henri G
authors.initials: HG
authors.lastname: Gerken
authors.forename: Bryon
authors.initials: B
authors.lastname: Donohoe
authors.forename: Eric P
authors.initials: EP
authors.lastname: Knoshaug
mesh_terms: D001483:Base Sequence; D056804:Biofuels; D002463:Cell Membrane Permeability; D002473:Cell Wall / Q000378:metabolism* / Q000648:ultrastructure; D044602:Cellulases / Q000378:metabolism; D002688:Chitinases / Q000378:metabolism; D048408:Chlorella vulgaris / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D004798:Enzymes / Q000378:metabolism*; D005434:Flow Cytometry; D005966:Glucuronidase / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D058086:Microalgae / Q000235:genetics / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism; D011096:Polygalacturonase / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013429:Sulfatases / Q000378:metabolism
chemical_list: D056804:Biofuels; D004798:Enzymes; D006023:Glycoproteins; D011134:Polysaccharides; D012337:RNA, Ribosomal, 18S; D013429:Sulfatases; D044602:Cellulases; D002688:Chitinases; D011096:Polygalacturonase; D009113:Muramidase; D005966:Glucuronidase