文献类型： 外文期刊

作者： Wang Nan ¹ ; Wang YueJu ² ; Li GangQiang ¹ ; Sun Ning ¹ ; Liu DeHu ¹ ;

作者机构： 1.Chinese Acad Agr Sci, Biotechnol Res Inst, Beijing 100081, Peoples R China

2.NE State Univ, Dept Nat Sci, Broken Arrow, OK 74014 USA

关键词： T4 lysozyme;antimicrobial activity;Hansenula polymorpha;pGAP;rDNA

期刊名称：SCIENCE CHINA-LIFE SCIENCES （ 影响因子：6.038； 五年影响因子：4.754 ）

ISSN： 1674-7305

年卷期： 2011 年 54 卷 6 期

页码：

收录情况： SCI

摘要： 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.