Gao Qianqian, Tan Xiaoming, Lü Xuefeng
Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, Shandong, China.
Sheng Wu Gong Cheng Xue Bao. 2012 Dec;28(12):1473-81.
Free fatty acid profiles of wild type and fatty acyl-ACP synthase deletion mutant strain of Synechocystis sp. PCC6803 indicated that one origin of these fatty acids is the process of lipid remodeling or lipid degradation. Lipase is the key enzyme involved in this process. The gene sll1969 is the sole gene encodes a putative lipase in Synechocystis sp. PCC6803. To identify the function of this gene and its role in fatty acid metabolism, we cloned the sll1969 from genomic DNA, overexpressed it in Escherichia coli BL21 (DE3) using pET expression system and purified this recombinant enzyme with Nickel-nitrilotriacetic acid affinity chromatography. The enzyme activity was assayed by spectrophotometric with p-nitro-phenylbutyrate as substrate. The K(m) and k(cat) of the enzyme is (1.16 +/- 0.01) mmol/L and (332.8 +/- 10.0)/min, respectively toward p-nitro-phenylbutyrate at 30 degrees C. The optimal temperature of the enzyme is 55 degrees C. To investigate the biological role of Sll1969 in fatty acid metabolism in cyanobacteria, we constructed sll1969 deletion and overexpression mutant strains in the background of fatty acyl-ACP synthase deletion mutant of Synechocystis sp. PCC6803. The analyses of the content of free fatty acids in different mutant strains showed that the contents of Sll1969 and free fatty acid are positively correlated. The free fatty acid profiles of the sll1969 mutant strains suggested this enzyme is not the sole enzyme for degrading lipid in Synechocystis sp. PCC6803.
集胞藻PCC6803野生型和脂肪酸酰基-ACP合酶缺失突变株的游离脂肪酸谱表明，这些脂肪酸的一个来源是脂质重塑或脂质降解过程。脂肪酶是参与此过程的关键酶。基因sll1969是集胞藻PCC6803中唯一编码假定脂肪酶的基因。为了鉴定该基因的功能及其在脂肪酸代谢中的作用，我们从基因组DNA中克隆了sll1969，使用pET表达系统在大肠杆菌BL21(DE3)中过表达它，并通过镍-次氮基三乙酸亲和色谱法纯化该重组酶。以对硝基苯丁酸为底物，通过分光光度法测定酶活性。在30℃下，该酶对硝基苯丁酸的K(m)和k(cat)分别为(1.16±0.01)mmol/L和(332.8±10.0)/min。该酶的最适温度为55℃。为了研究Sll1969在蓝藻脂肪酸代谢中的生物学作用，我们在集胞藻PCC6803脂肪酸酰基-ACP合酶缺失突变体的背景下构建了sll1969缺失和过表达突变株。对不同突变株中游离脂肪酸含量的分析表明，Sll1969含量与游离脂肪酸含量呈正相关。sll1969突变株的游离脂肪酸谱表明该酶不是集胞藻PCC6803中唯一的脂质降解酶。
