Jo Jin Chul, Kim Soon-Ja, Kim Hyung Kwoun
Department of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi 420-743, Republic of Korea.
Department of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi 420-743, Republic of Korea.
Enzyme Microb Technol. 2014 Dec;67:32-9. doi: 10.1016/j.enzmictec.2014.08.014. Epub 2014 Sep 6.
Staphylococcus haemolyticus L62 (SHL62) lipase was displayed on the outer membrane of Escherichia coli using the OmpA signal peptide and the autotransporter EstAβ8 protein. Localization of SHL62 lipase on the outer membrane of E. coli was confirmed using immunofluorescence microscopy and flow cytometry analysis. Lipase activity of the displayed SHL62 lipase was also measured using spectrophotometry and pH titration. SHL62 lipase activity of whole cells reached 2.0U/ml culture (OD600nm of 10) when it was measured by the p-nitrophenyl caprylate assay after being induced with 1mM IPTG for 24h. The optimum temperature and pH for the lipase was 45°C and 10, respectively. Furthermore, it maintained more than 90% of maximum lipase activity at up to 50°C and in a pH range of 5-9. The hydrolytic activity assay conduted with various substrates confirmed that p-nitrophenyl caprylate and corn oil were preferred substrates among various synthetic and natural substrates, respectively. The displayed SHL62 lipase produced fatty acid esters from various alcohols and plant oils through transesterification.
利用OmpA信号肽和自转运蛋白EstAβ8将溶血葡萄球菌L62（SHL62）脂肪酶展示在大肠杆菌的外膜上。通过免疫荧光显微镜和流式细胞术分析证实了SHL62脂肪酶在大肠杆菌外膜上的定位。还使用分光光度法和pH滴定法测量了展示的SHL62脂肪酶的脂肪酶活性。在用1mM IPTG诱导24小时后，通过对硝基苯辛酸酯测定法测量，全细胞的SHL62脂肪酶活性在培养物（OD600nm为10）中达到2.0U/ml。该脂肪酶的最适温度和pH分别为45°C和10。此外，在高达50°C的温度和5-9的pH范围内，它保持了超过90%的最大脂肪酶活性。用各种底物进行的水解活性测定证实，对硝基苯辛酸酯和玉米油分别是各种合成底物和天然底物中优选的底物。展示的SHL62脂肪酶通过酯交换反应从各种醇类和植物油中产生脂肪酸酯。
