从黑曲霉基因组中克隆了一种新的脂肪酶基因tglE,并在毕赤酵母中成功表达。重组酶具有典型的脂肪酶活性,其最适pH为7.0,最适温度为40 ℃;在30~60 ℃或pH 6.5~9.0该酶的酶活力保持在60%以上;在pH 6.0~8.0或20~30 ℃均较为稳定。Cu2+、Mn2+、Ca2+、K+、Mg2+和Sn2+对tglE有明显的激活作用;Fe3+、Zn2+和EDTA对重组酶的酶活抑制作用明显,特别是EDTA可抑制重组酶90%的活。tglE对橄榄油和C4底物的作用最强,对棕果油和C16底物的作用最弱。以C4为底物,tglE的Km值为14.40 mmol/L,Vmax为46.72 mmol/(mL·h)。tglE具有催化辛酸和乙醇生成辛酸乙酯的酯化活性。
The novel lipase gene (tglE) from Aspergillus niger was cloned and expressed in Pichia pastoris. The recombinant TglE had typical lipase activity with the maximal activity at 40 ℃ and pH 7.0. The enzyme retained over 60% activities at 30 ℃-60 ℃ or pH 6.5-9.0 and was stable at 20-30 ℃ or pH 6.0-8.0. The catalytic activity of TglE was significantly improved by Cu2+, Mn2+, Ca2+, K+, Mg2+, and Sn2+ but inhibited by Fe3+, Zn2+ and EDTA. The prefered substrates for tglE were chemically synthesized pNPB and natural olive oil. The Km and Vmax for pNPB were 14.40 mmol/L and 46.72 mmol/(mL·h), respectively. In addition to the hydrolysis activity, tglE exhibited significant esterification efficiency for ethyl caprylate synthesis from octanoic acid and ethanol.
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