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 glyceral...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 interand intra-molecular disulfide bonds which resulted in loss of enzyme activity.展开更多
基金supported by the National High Technology Research & Development Program of China (Grant No. 2007AA02Z111)National Technology for the 10th Five-year Plan of China (Grant No. 2006BAD31B01-04)+1 种基金National Biotechnology Development Plan (Grant Nos. 2008ZX08005-004 and 2009ZX08005-004B)the Researcher Foundation of the Chinese Academy of Agricultural Sciences
文摘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 interand intra-molecular disulfide bonds which resulted in loss of enzyme activity.
