Purification and characterization of novel thermo-alkaline lipase and its application

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 two-step 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 (SDS-PAGE). 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), p-nitrophenyl 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, the yield can reach 91.75%. © 2020, Chemical Industry Press Co., Ltd. All right reserved.