Bakir Zehra Burcu, Metin Kubilay
Department of Biology, Faculty of Science and Arts, Adnan Menderes University, 09010 Aydn, Turkey.
J Microbiol Biotechnol. 2016 Jun 28;26(6):1087-97. doi: 10.4014/jmb.1512.12056.
An intracellular lipase from Anoxybacillus flavithermus HBB 134 was purified to 7.4-fold. The molecular mass of the enzyme was found to be about 64 kDa. The maximum activity of the enzyme was at pH 9.0 and 50°C. The enzyme was stable between pH 6.0 and 11.0 at 25°C, 40°C, and 50°C for 24 h. The Km and Vmax of the enzyme for pNPL substrate were determined as 0.084 mM and 500 U/mg, respectively. Glycerol, sorbitol, and mannitol enhanced the enzyme thermostability. The enzyme was found to be highly stable against acetone, ethyl acetate, and diethyl ether. The presence of PMSF, NBS, DTT and β-mercaptoethanol inhibited the enzyme activity. Hg(2+), Fe(3+), Pb(2+), Al(3+), and Zn(2+) strongly inhibited the enzyme whereas Li(+), Na(+), K(+), and NH4(+) slightly activated it. At least 60% of the enzyme activity and stability were retained against sodium deoxycholate, sodium taurocholate, n-octyl-β-D-glucopyranoside, and CHAPS. The presence of 1% Triton X-100 caused about 34% increase in the enzyme activity. The enzyme is thought to be a true lipase since it has preferred the long-chain triacylglycerols. The lipase of HBB 134 cleaved triolein at the 1- or 3-position.
从嗜热栖热放线菌HBB 134中纯化得到一种细胞内脂肪酶，纯化倍数达到7.4倍。该酶的分子量约为64 kDa。酶的最大活性出现在pH 9.0和50°C条件下。在25°C、40°C和50°C时，该酶在pH 6.0至11.0之间保持稳定24小时。该酶对对硝基苯棕榈酸酯（pNPL）底物的Km和Vmax分别测定为0.084 mM和500 U/mg。甘油、山梨醇和甘露醇可增强该酶的热稳定性。发现该酶对丙酮、乙酸乙酯和乙醚具有高度稳定性。苯甲基磺酰氟（PMSF）、N-溴代琥珀酰亚胺（NBS）、二硫苏糖醇（DTT）和β-巯基乙醇的存在会抑制酶活性。汞离子（Hg(2+)）、铁离子（Fe(3+)）、铅离子（Pb(2+)）、铝离子（Al(3+)）和锌离子（Zn(2+)）强烈抑制该酶，而锂离子（Li(+)）、钠离子（Na(+)）、钾离子（K(+)）和铵离子（NH4(+)）则对其有轻微激活作用。在脱氧胆酸钠、牛磺胆酸钠、正辛基-β-D-葡萄糖苷和3-[(3-胆酰胺丙基)二甲基铵]-1-丙磺酸（CHAPS）存在的情况下，至少保留了60%的酶活性和稳定性。1% Triton X-100的存在使酶活性提高了约34%。由于该酶偏好长链三酰甘油，因此被认为是一种真正的脂肪酶。HBB 134的脂肪酶在1-或3-位切割三油酸甘油酯。
