Kaur Gagandeep, Singh Amninder, Sharma Rohit, Sharma Vinay, Verma Swati, Sharma Pushpender K
Department of Biotechnology, Sri Guru Granth Sahib World University, Fatehgarh Sahib, Punjab, India.
NABI, Mohali, Punjab, India.
3 Biotech. 2016 Jun;6(1):49. doi: 10.1007/s13205-016-0369-y. Epub 2016 Feb 8.
In the present investigation, a gene encoding extracellular lipase was cloned from a Bacillus licheniformis. The recombinant protein containing His-tag was expressed as inclusion bodies in Esherichia coli BL21DE3 cells, using pET-23a as expression vector. Expressed protein purified from the inclusion bodies demonstrated ~22 kDa protein band on 12 % SDS-PAGE. It exhibited specific activity of 0.49 U mg and % yield of 8.58. Interestingly, the lipase displayed activity at wide range of pH and temperature, i.e., 9.0-14.0 pH and 30-80 °C, respectively. It further demonstrated ~100 % enzyme activity in presence of various organic solvents. Enzyme activity was strongly inhibited in the presence of β-ME. Additionally, the serine and histidine modifiers also inhibited the enzyme activities strongly at all concentrations that suggest their role in the catalytic center. Enzyme could retain its activity in presence of various detergents (Triton X-100, Tween 20, Tween 40, SDS). Sequence and structural analysis employing in silico tools revealed that the lipase contained two highly conserved sequences consisting of ITITGCGNDL and NLYNP, arranged as parallel β-sheet in the core of the 3D structure. The function of these conserve sequences have not fully understood.
在本研究中，从地衣芽孢杆菌中克隆了一个编码细胞外脂肪酶的基因。使用pET-23a作为表达载体，含有His标签的重组蛋白在大肠杆菌BL21DE3细胞中以包涵体形式表达。从包涵体中纯化的表达蛋白在12% SDS-PAGE上显示出22 kDa的蛋白条带。其比活性为0.49 U/mg，产率为8.58%。有趣的是，该脂肪酶在较宽的pH和温度范围内均有活性，即pH为9.0 - 14.0，温度为30 - 80°C。在各种有机溶剂存在下，它进一步显示出100%的酶活性。在β-巯基乙醇存在下，酶活性受到强烈抑制。此外，丝氨酸和组氨酸修饰剂在所有浓度下也强烈抑制酶活性，这表明它们在催化中心发挥作用。该酶在各种洗涤剂（Triton X-100、吐温20、吐温40、SDS）存在下仍能保持其活性。利用计算机工具进行的序列和结构分析表明，该脂肪酶包含两个高度保守的序列，分别为ITITGCGNDL和NLYNP，在三维结构的核心区域排列成平行β-折叠。这些保守序列的功能尚未完全了解。
