Lipases are versatile biocatalysts with many biotechnological applications and the necessity of screening, production and characterization of new lipases from diverse microbial strains to meet industrial needs is constantly emerging. In this study, the lipase gene (gklip) from a thermophilic bacterium, Geobacillus kaustophilus DSM 7263 ^T was cloned into the pET28a ( +) vector with N-terminal 6xHis-tag. The recombinant gklip gene was heterologously expressed in host E. coli BL21 (DE3) cells and purified by Ni–NTA affinity chromatography. Histidine tag was removed from the purified 6xHistag-Gklip enzyme with thrombin enzyme and the molecular mass was determined to be approximately 43 kDa by SDS-PAGE. Gklip showed optimal activity at pH 8.0 and 50 °C. The specific hydrolytic activities against substrates were significantly increased by the removal of the His-tag. K_m and k_cat values of Gklip against p-nitrophenyl palmitate (pNPP, 4-nitrophenyl palmitate) as the target substrate were found to be as 1.22 mM and 417.1 min⁻¹, respectively. Removing His-tag changed the substrate preference of the enzyme leading to maximum lipolytic activity towards C10 and C12 lipids. Similarly, the activity against coconut oil that containing 62% medium-chain fatty acids was significantly higher than other oils. Furthermore, preservation of activity in the presence of inhibitors, organic solvents support the effect of lid structure of the enzyme.

脂肪酶是具有许多生物技术应用的多功能生物催化剂，并且不断出现从不同微生物菌株中筛选、生产和表征新脂肪酶以满足工业需求的必要性。在这项研究中，来自嗜热菌嗜热地芽孢杆菌DSM 7263  ^T的脂肪酶基因 ( gklip )被克隆到 pET28a ( +) 载体中，其 N 端带有 6xHis 标签。重组gklip基因在宿主大肠杆菌BL21 (DE3) 细胞中异源表达，并通过 Ni-NTA 亲和层析纯化。从纯化的 6xHistag- Gk 中去除组氨酸标签lip酶与凝血酶酶和分子量通过SDS-PAGE测定为大约43kDa。Gk lip 在 pH 8.0 和 50 °C 下表现出最佳活性。通过去除 His 标签，对底物的特定水解活性显着增加。ķ_米和ķ_猫的值Gk中对唇p硝基苯基棕榈酸酯（p发现NPP，4-硝基苯基棕榈酸酯）作为目标基板是如1.22 mM和417.1分钟^-1， 分别。去除 His 标签改变了酶的底物偏好，导致对 C10 和 C12 脂质的最大脂解活性。同样，对含有 62% 中链脂肪酸的椰子油的活性显着高于其他油。此外，在抑制剂存在下保持活性，有机溶剂支持酶的盖结构的作用。