超嗜热酯酶EST2在不同宿主中的异源高效表达研究

来源于超嗜热古菌Alicyclobacillus acidocaldarius的酯酶EST2是目前报道的活性最高的超嗜热酯酶,具有极大的工业应用价值。为促进EST2的生产应用,将其分别在大肠杆菌及毕赤酵母中进行异源表达,并就不同宿主对表达情况和重组酶酶学性质的影响进行了分析。在大肠杆菌和毕赤酵母中重组表达的EST2酶学性质基本一致:最适温度分别为75℃和77.5℃,最适pH均为8.0,比活力分别为4656.6 U/mg和4078.3 U/mg,70℃水浴保温4.5 h,残余活力均在70%以上。在摇瓶发酵的基础上,于5 L发酵罐中进行了重组大肠杆菌及毕赤酵母的高密度发酵。毕赤酵母高密度发酵120 h菌体干重达68 g/L,最大表达酶活力为959.6 U/ml。大肠杆菌高密度发酵25 h菌体干重达60.8 g/L,最大酶活力14825.6 U/ml,表达量是毕赤酵母的15.4倍,单位时间产量是酵母的74.2倍。结果表明大肠杆菌发酵周期短、表达量高,更适合进行嗜热酯酶EST2的高效生产,这为促进嗜热酯酶在工业生物技术产业的应用奠定了基础。

High-level Heterogenous Expression of a Hyperthermophilic Esterase in Different Hosts

Thermophilic Esterase 2 (EST2) from Alicyclobacillus acidocaldarius belongs to the HSL group of the esterase/lipase superfamily. It has been reported to be the most active hyperthermophilic esterase with excellent thermostability, which makes it a good choice for many industrial applications, such as food industry, fine chemical industry, pulp and paper industry and environmental bio-mediation. However, the biomass yield and the basal enzyme expression level are remarkably low in the cultivation of hyperthermophiles, which results in the high cost for the large-scale production of EST2 in its natural host. Heterogenous production of EST2 in a proper host organism holds great potential to solve this problem. E. coli and Pichia pastoris are the most widely used hosts for the production of heterologous proteins. In order to establish the high-level expression system, the gene encoding EST2 was cloned and expressed in Pichia pastoris and E.coli, and the enzymatic properities of the recombinant proteins were systematically characterized and compared. The recombinant EST2 expressed in E.coli and Pichia pastoris showed very similar properties: the optimal temperatures were 75℃ and 77.5℃, the optimal pH were both 8.0, the specific activities were 4656.6 U/mg and 4078.3 U/mg, and the enzymes were stable up after incubation at 70℃ for 4.5h. After the optimized cultivation conditions in the shake flake, the expression of EST2 in different hosts was conducted in a 5 L fermentor by high cell-density fermentation. In Pichia pastoris, the recombinant EST2 was predominantly expressed extracellularly with activity of about 960 U/ml and a cell dry weight reached 68g/L after induction with methanol for 90h. On the other hand, the EST2 activity increased to 14825.6U/ml after 25h of fermentation in E.coli, with a cell dry weight of 60g/L. The expression level and productivity in E.coli are 14.4-fold and 73.2-fold higher than that of Pichia pastoris, respectively. The results illustrated that E.coli is more suited for the high-level expression of EST2 than Pichia pastoris, which will further promote wider application of EST2 in biotech industries.