Kim H K, Park S Y, Lee J K, Oh T K
Korea Research Institute of Bioscience & Biotechnology (KRIBB), Taejon, Korea.
Biosci Biotechnol Biochem. 1998 Jan;62(1):66-71. doi: 10.1271/bbb.62.66.
The gene coding for an extracellular lipase of Bacillus stearothermophilus L1 was cloned in Escherichia coli. Sequence analysis showed an open reading frame of 1254 bp, which encodes a polypeptide of 417 amino acid residues. The polypeptide was composed of a signal sequence (29 amino acids) and a mature protein of 388 amino acids. An alanine replaces the first glycine in the conserved pentapeptide (Gly-X-Ser-X-Gly) around the active site serine. The expressed lipase was purified by hydrophobic interaction and ion exchange chromatography using buffers containing 0.02% (v/v) Triton X-100. The lipase was most active at 60-65 degrees C and in alkaline conditions around pH 9-10. The lipase had highest activity toward p-nitrophenyl caprylate among the synthetic substrates and tripropionin among the triglycerides. It hydrolyzed beef tallow and palm oil more rapidly than olive oil at 50 degrees C.
嗜热脂肪芽孢杆菌L1胞外脂肪酶的编码基因在大肠杆菌中克隆。序列分析显示有一个1254 bp的开放阅读框，编码一个由417个氨基酸残基组成的多肽。该多肽由一个信号序列（29个氨基酸）和一个388个氨基酸的成熟蛋白组成。在活性位点丝氨酸周围保守的五肽（Gly-X-Ser-X-Gly）中，第一个甘氨酸被丙氨酸取代。使用含0.02%（v/v） Triton X-100的缓冲液，通过疏水相互作用和离子交换色谱法对表达的脂肪酶进行纯化。该脂肪酶在60 - 65℃和pH 9 - 10左右的碱性条件下活性最高。在合成底物中，该脂肪酶对辛酸对硝基苯酯的活性最高；在甘油三酯中，对三丙酸甘油酯的活性最高。在50℃时，它水解牛脂和棕榈油的速度比橄榄油快。
