Ali Shaukat, Ren Shunxiang, Huang Zhen
Engineering Research Center of Bhological Control, Ministry of Education, College of Natural Resource and Environment, South China Agricultural University. Wushan Road, Guangzhou City, P. R., China.
J Basic Microbiol. 2014 Nov;54(11):1148-59. doi: 10.1002/jobm.201300813. Epub 2014 Mar 27.
Lipases play an important role in the infection process of entomopathogenic fungi by hydrolyzing the ester bonds of lipoproteins, fats and waxes present on the insect surface and in the body. Here we report the purification and characterization of an extracellular lipase from Isaria fumosorosea. The enzyme was purified (138.46-fold) in three steps using (NH4 )2 SO4 precipitation followed by DEAE-cellulose and Sephadex G-100 column chromatography. The molecular weight of purified enzyme was determined to be 31 KDa by SDS-PAGE. The optimum temperature and pH for enzyme activity were 35 °C and 7.0, respectively, using p-nitrophenylpalmitate as the substrate. Lipolytic activity was enhanced in the presence of Ca(+2) , Mg(+2) , Na(+) , and NH4 (+) salts, while Zn(+2) , Fe(+2) , and Cu(+2) inhibited enzyme activity. The enzyme displayed broad substrate specificity with the highest activity observed for coconut oil and p-nitrophenyl carprate. Topical co-application of purified lipase with fungal conidial suspensions decreased the median survival time (ST50 ) of Dysmicoccus neobrevipes nymphs as compared to the fungus alone. Our results indicate that an extracellular lipase produced by I. fumosorosea can be exploited for development of enzyme-based insect management.
脂肪酶通过水解昆虫体表和体内存在的脂蛋白、脂肪及蜡质的酯键，在昆虫病原真菌的感染过程中发挥重要作用。在此，我们报告了玫烟色棒束孢一种胞外脂肪酶的纯化及特性研究。该酶通过三步纯化（纯化倍数为138.46倍），首先采用硫酸铵沉淀，随后进行DEAE - 纤维素和葡聚糖G - 100柱层析。通过SDS - PAGE测定纯化酶的分子量为31 kDa。以对硝基苯基棕榈酸酯为底物时，酶活性的最适温度和pH分别为35 °C和7.0。在Ca(+2)、Mg(+2)、Na(+)和NH4(+)盐存在时，脂解活性增强，而Zn(+2)、Fe(+2)和Cu(+2)抑制酶活性。该酶表现出广泛的底物特异性，对椰子油和对硝基苯基癸酸酯的活性最高。与单独使用真菌相比，将纯化的脂肪酶与真菌分生孢子悬浮液局部联合应用可缩短新菠萝灰粉蚧若虫的中位存活时间（ST50）。我们的结果表明，玫烟色棒束孢产生的一种胞外脂肪酶可用于开发基于酶的昆虫防治方法。
