Wang Tingting, Xu Yongping, Liu Wenjia, Sun Yongxin, Jin Liji
Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, People's Republic of China.
Protein Expr Purif. 2011 May;77(1):20-5. doi: 10.1016/j.pep.2011.01.002. Epub 2011 Jan 15.
Apostichopus japonicus (sea cucumber) is one of the economically important farmed echinoderm species in Northern China. As a crucial enzyme in innate immunity, lysozyme plays a key role in the overall defense against pathogens in A. japonicus. In the present study, a lysozyme gene from A. japonicus was cloned by PCR and expressed in Pichia pastoris using the expression vector pPIC9K. The expressed lysozyme had a molecular mass of ∼14 kD, as shown by SDS-PAGE and Western-blotting. The expression condition was optimized, and the highest expression level was achieved by induction with 1% methanol at pH 5.0 for 120 h. The recombinant lysozyme was purified by affinity chromatography using a Ni-NTA column. The specific activity of the purified lysozyme was 34,000 U/mg using Micrococcus lysodeikticus as substrates. It exhibited antimicrobial activity toward M.lysodeikticus, as detected by growth inhibition on agar plate and turbidity assay, suggesting a potential application of A. japonicus lysozyme as an antimicrobial agent in A. japonicus aquaculture.
刺参是中国北方具有重要经济价值的养殖棘皮动物之一。作为先天免疫中的一种关键酶，溶菌酶在刺参抵御病原体的整体防御中发挥着关键作用。在本研究中，通过PCR克隆了刺参的溶菌酶基因，并使用表达载体pPIC9K在毕赤酵母中进行表达。SDS-PAGE和Western印迹分析表明，表达的溶菌酶分子量约为14 kD。对表达条件进行了优化，在pH 5.0条件下用1%甲醇诱导120 h时达到最高表达水平。重组溶菌酶通过镍-亚氨基二乙酸（Ni-NTA）柱亲和层析进行纯化。以溶壁微球菌为底物时，纯化溶菌酶的比活性为每毫克34,000 U。通过琼脂平板上的生长抑制和比浊法检测发现，其对溶壁微球菌具有抗菌活性，这表明刺参溶菌酶在刺参水产养殖中作为抗菌剂具有潜在应用价值。
