摘要
将来源于解脂嗜热互营杆菌(Thermosyntrophalipolytica)的脂肪酶(TlLipA)基因tll1导入大肠杆菌BL21(DE3)中表达,通过热处理和镍柱亲和层析获得纯酶,并对其酶学性质进行研究。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)显示TlLipA分子量为53×103,其最适反应温度为65℃,最适反应pH为8.0。在55~65℃范围内酶活较高且比较稳定;在pH7.0~11.0于室温保存1h后,残留相对酶活仍达80%以上。1mmol/L金属离子Zn2+、Fe3+和试剂SDS,0.05%(质量分数)Tween80,对酶活力具有强烈的抑制作用,残留相对酶活皆低于15%;1mmol/LMg2+、Mn2+对酶活力表现出轻微的激活作用。由底物专一性实验可得,该酶对辛酸对硝基苯酯(C8)和癸酸对硝基苯酯(C10)偏好明显。以棕榈酸对硝基苯酯(p-NPP)为底物,该酶动力学参数Km值为0.23mmol/L,Vmax为33.50mmol/(L·min),kcat为22.83S-1。以重组脂肪酶为催化剂在无溶剂体系中制备生物柴油,含水率20%,酶加量200U/g油,醇油比为4∶1的条件下,在55℃催化大豆油反应48h,收率可达91.75%。
In order to excavate thermo-alkaline lipases from bacterial living in extreme conditions,we try to express new gene from Thermosyntropha lipolytica DSM 11003,an anaerobic,thermophilic,alkali-tolerant bacterium which grows in alkaline hot springs Lake Bogoria in Kenya and explore its application in biodiesel production.The lipase gene(tll1)of 1434 bp were ligated at the Nco I/EcoR I sites of the expression vectors pET28a to yield the construct of pET28a-TLL1.The strain harboring pET28a-TLL1 was cultivated for expression at 25℃,the specific activity of 1.99 U/mg protein were detected in disrupted cells.The recombinant lipase TlLipA was purified by a simple twostep procedure involving heat treatment and Ni-chelating affinity chromatography.The subunit of purified TlLipA showed a molecular mass of 53×103 on 12%sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDSPAGE).The purified TlLipA exhibited optimal activity at 65℃and pH 8.0 and it was stable from 55℃to 65℃.The enzyme remained above 80%of its original activity at pH ranging from 7.0 to 11.0 and at room temperature for 1 h.The activity of TlLipA was little unaffected by Co2+,K+,Na+,and Ni2+,and a little activated by Mg2+and Mn2+,but were significantly inhibited by Zn2+,Fe3+,and SDS,and Tween 80 under the assay conditions.The purified recombinant TlLipA had a specific activity of 22.11 U/mg protein using p-nitrophenyl palmitate(p-NPP)as substrate.Determined by Sigma-Plot of reaction rate on p-NPP,the Km was 0.23 mmol/L,the Vmax was 33.50 mmol/(L·min),and the kcat was 22.83 s-1.The enzyme was also active towards p-NPP,p-nitrophenyl laurate(p-NPL),pnitrophenyl myristate(p-NPM)and p-nitrophenyl caproate(p-NPC),moreover TlLipA exhibited a strong preference for p-nitrophenol decanoate(p-NPD)and p-nitrophenyl octoate(p-NPO).Using recombinant lipase as a catalyst to prepare biodiesel in a solvent-free system,with a water content of 20%,an enzyme dosage of 200 U/g oil,and an alcohol-to-oil ratio of 4∶1,catalyzed soybean oil reaction at 55℃for 48 h,t
作者
张昕怡
许蕊
王钰棋
张瑜
王飞
李迅
ZHANG Xinyi;XU Rui;WANG Yuqi;ZHANG Yu;WANG Fei;LI Xun(College of Chemical Engineering,Nanjing Forestry University,Nanjing 210037,Jiangsu,China;Jiangsu Key Laboratory of Biomass Based Green Fuels and Chemicals,Nanjing 210037,Jiangsu,China)
出处
《化工学报》
EI
CAS
CSCD
北大核心
2020年第11期5246-5255,共10页
CIESC Journal
基金
国家重点研发计划(2019YFB1504002)。
