克隆了来自Thermotoga萘酚(TnLip)的超稳定脂肪酶,并在有效的嗜温宿主系统中过表达为可溶性和活性单体蛋白。序列研究表明,TnLip是一个肽酶S9prolyl寡肽酶结构域(乙酰酯酶/脂肪酶样蛋白),属于α/β(α/β)-水解酶超家族,包含保守的α/β-水解酶折叠和五肽(GLSAG)基序。应用各种培养和诱导策略来提高异源表达和细菌生物量,但TnLip细胞内活性在16小时后用IPTG非依赖性自诱导方法增强14.25倍(26°C,150revmin-1)孵育。纯化的TnLip(35kDa)在85°C的McIlvaine缓冲液(pH7.0-8.0)中显示出峰值活性,在很宽的pH范围(5.0-10.0)内具有很好的稳定性,和温度(40-85°C)持续8小时。TnLip对各种商业洗涤剂表现出惊人的抵抗力,化学添加剂,和盐。TnLip活性提高了170.51%,130.67%,127.42%,126.54%,126.61%,120.32%,和116.31%与50%(v/v)的甲醇,乙醇,正丁醇,异丙醇,丙酮,甘油,和乙酸,分别。此外,用3.0M的NaCl,和10mM的Ca2+,Mn2+,Mg2+TnLip活性增加了210%,185.64%,152.03%,和116.26%,分别。TnLip与各种底物(对硝基苯酯和天然油)具有亲和力,但对硝基苯基棕榈酸酯具有最大的水解活性(pNPP,3600Umg-1)和橄榄油(1182.05Umg-1)。Km值(0.576mM),Vmax(4216μmolmg-1min-1),VmaxKm-1(7319.44min-1),kcat(1106.74s-1),使用pNPP底物计算kcatKm-1(1921.42mM-1s-1)。此外,TnLip在3小时和5分钟内降解动物脂肪并去除油渍,分别。所有这些特性使嗜卤-碱-嗜热TnLip成为洗衣洗涤剂(清洁生物添加剂)的吉祥竞争者,脂肪降解,废水处理,并认可生态友好型管理以及其他各种生物技术应用。
A hyperstable lipase from Thermotoga naphthophila (TnLip) was cloned and overexpressed as a soluble and active monomeric protein in an effectual mesophilic host system. Sequence study revealed that TnLip is a peptidase S9 prolyl oligopeptidase domain (acetyl esterase/lipase-like protein), belongs to alpha/beta (α/β)-hydrolase superfamily containing a well-conserved α/β-hydrolase fold and penta-peptide (GLSAG) motif. Various cultivation and induction strategies were applied to improve the heterologous expression and bacterial biomass, but TnLip intracellular activity was enhanced by 14.25- fold with IPTG-independent auto-induction approach after 16 h (26 °C, 150 rev min-1) incubation. Purified TnLip (35 kDa) showed peak activity at 85 °C in McIlvaine buffer (pH 7.0-8.0), and has great stability over a broad range of pH (5.0-10.0), and temperature (40-85 °C) for 8 h. TnLip exhibited prodigious resistance toward various commercial detergents, chemical additives, and salt. TnLip activity was improved by 170.51 %, 130.67 %, 127.42 %, 126.54 %, 126.61 %, 120.32 %, and 116.31 % with 50 % (v/v) of methanol, ethanol, n-butanol, isopropanol, acetone, glycerol, and acetic acid, respectively. Moreover, with 3.0 M of NaCl, and 10 mM of Ca2+, Mn2+, and Mg2+ TnLip activity was augmented by 210 %, 185.64 %, 152.03 %, and 116.26 %, respectively. TnLip has an affinity with various substrates (p-nitrophenyl ester and natural oils) but maximal hydrolytic activity was perceived with p-nitrophenyl palmitate (pNPP, 3600 U mg-1) and olive oil (1182.05 U mg-1). The values of Km (0.576 mM), Vmax (4216 μmol mg-1 min-1), VmaxKm-1 (7319.44 min-1), kcat (1106.74 s-1), and kcatKm-1 (1921.42 mM-1 s-1) were calculated using pNPP substrate. Additionally, TnLip degraded animals\' fats and removed oil stains within 3 h and 5 min, respectively. All these features make halo-alkali-thermophilic TnLip as an auspicious contender for laundry detergents (cleaning bio-additive), fat degradation, wastewater treatment and endorse eco-friendly stewardship along with various other biotechnological applications.