title: Expression, characterization, and antimicrobial ability of T4 lysozyme from methylotrophic yeast Hansenula polymorpha A16.
abstract: Lysozyme is an enzyme that is essential for protection against bacterial infections. In this study, a T4 lysozyme gene was cloned into the yeast expression vector pPIC9K under the control of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). A Hansenula polymorpha-derived ribosomal DNA (rDNA)-targeting element was inserted into the expression vector and was critical for stable DNA integration into the H. polymorpha chromosome. Recombinant T4 lysozyme was successfully expressed in the yeast H. polymorpha A16; 0.49 g L(-1) secreted recombinant T4 lysozyme was obtained 72 h after incubation in culture broth that had an initial pH of 6.0. Recombinant T4 lysozyme showed lytic activity against the cell walls of the gram positive bacteria, Micrococcus lysodeikticus, and the gram negative bacteria Xanthomonas campestris pv. malvacearum and Xanthomonas oryzae pv. oryzae. The zone of inhibition assay was used to evaluate antimicrobial activity. Mass spectrometry showed the N-terminal sequence of recombinant T4 lysozyme was identical to that of the native enzyme. SDS-PAGE indicated that the molecular mass of recombinant T4 lysozyme was 18.7 kD which corresponds to a monomer of the native enzyme. SDS-PAGE without 0.2 mol L(-1) dithiothreitol treatment detected two bands (15 and 31 kD) suggesting that some recombinant T4 lysozyme formed inter- and intra-molecular disulfide bonds which resulted in loss of enzyme activity.
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.forename: YueJu
authors.initials: Y
authors.lastname: Wang
authors.forename: GangQiang
authors.initials: G
authors.lastname: Li
authors.forename: Ning
authors.initials: N
authors.lastname: Sun
authors.forename: DeHu
authors.initials: D
authors.lastname: Liu
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods; D008826:Microbial Sensitivity Tests; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D000890:Anti-Infective Agents; D004220:Disulfides; D005656:Fungal Proteins; D011994:Recombinant Proteins; D009113:Muramidase