来源于解酯菌的嗜热耐碱脂肪酶的表达纯化及其酶学性质研究

脂肪酶广泛应用于食品加工、生物柴油制备等领域。为了有效提高微生物脂肪酶的可利用度,将来源于解酯嗜热菌(Thermosyntropha lipolytica DSM 11003)的脂肪酶基因(tll2)克隆到大肠杆菌表达载体pET28a中,获得重组质粒pET28a TLL2,将pET28a TLL2转入大肠杆菌BL21(DE3)宿主菌中进行高效表达。通过热处理和镍柱亲和层析得到电泳纯的蛋白TlLip2,对其进行酶学性质表征。结果显示,TlLip2的亚基分子质量为57 ku,比酶活为31 U/mg,最适反应pH及温度分别为8.0和60℃。在60℃以下、pH 6.0~11.0范围内,TlLip2的稳定性较好,相对酶活均维持在60%以上。金属离子K+和Na+对TlLip2有较大激活作用,而Co^2+、Zn^2+、SDS和Tween 80对TlLip2酶活力抑制作用明显。TlLip2在极性小的有机溶剂中具有较好的耐受性。TlLip2对中等碳链长度的对硝基苯酯显示出更高的活性,其中以对硝基苯己酯为底物时,TlLip2的酶活最高;以对硝基苯棕榈酸酯为底物时,该酶的动力学常数Km为0.29 mmol/L,Vmax为2.28 mmol/(L·min^-1),kcat为6.19 s^-1。此外,将其应用到油酸乙酯的合成研究中,以油酸和乙醇为底物,研究TlLip2催化制备油酸乙酯的转化条件,结果表明在60℃、加酶量为100 U/g的TlLip2,催化摩尔比为1∶2的油酸和乙醇,12 h后油酸乙酯的转化率达到62.1%,表明TlLip2在催化脂肪酸酯化方面具有较大的应用潜能。

Expression,purification and characterization of lipase from Thermosyntropha lipolytica

Lipase is an enzyme that catalyzes the hydrolysis of lipids,which is the subclass of the esterases.Given its excellent functional performance,lipase plays an indispensable role in many industrial areas such as food processing and biodiesel preparation.In this paper,in order to effectively improve the utilization of lipase from microbes,the lipase gene(tll2)of 1569 bp was cloned from thermophilic bacterium.Thermosyntropha lipolytica DSM 11003 that encodes a protein containing 523 amino acid residues was cloned into pET28a.The recombinant plasmid pET28a TLL2 was transformed into Escherichia coli BL21(DE3)and the recombinant TlLip2 was chemically induced in the presence of isopro pyl β D thiogalactoside(IPTG)and heterologously expressed in E.coli.The TlLip2 was purified to homogeneity by a simple two step procedure involving heat treatment and Ni NTA affinity.The enzymatic properties of purified TlLip2 were determined.The results showed that the subunit molecular weight of TlLip2 was 57 ku by SDS PAGE and the specific activity was 31 U/mg.The optimal activity of TlLip2 was at 60℃and a pH of 8.0.TlLip2 was stable when pH was between 6.0-11.0 and temperature was lower than 60℃,and the relative activity of TlLip2 was above 60%.K^+ and Na^+ had positive effects on TlLip2 and Co^2+,Zn^2+,SDS and Tween 80 had a negative effect on TlLip2.TlLip2 exhibited high activity with medium chain length p nitrophenyl esters,especially with p nitrophenyl caproate.Furthermore,TlLip2 exhibited high tolerance among most organic solvents,especially among low polar solvents.When p nitrophenyl palmitate was used as the substrate,the Km,Vmax,and kcat values were 0.29 mmol/L,2.28 mmol/(L·min^-1)and 6.19s^-1,respectively.This study focused on the biosynthesis conditions of ethyl oleate with the recombinant TlLip2.Taking the oleic acid and ethanol as the substrates,with a molar ratio of 1∶2 and the enzyme dosage of 100 U/g,the yield of ethyl oleate was 62.1%at 60℃ for 12 h.These results indicated that TlLip2 could be attractive for potential application in esterification of fatty acid,especially for medium and long chain fatty acids.