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Alkaline serine proteases from Helicoverpa armigera: potential candidates for industrial applications

机译:棉铃虫的碱性丝氨酸蛋白酶:工业应用的潜在候选人

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摘要

We characterized trypsin- and chymotrypsin-like serine alkaline proteases from cotton bollworm, Helicoverpa armigera, for their probable potential application as additives in various bio-formulations. Purification was achieved by using hydroxylapatite, DEAE sephadex and CM sephadex columns, which resulted in increased enzyme activity by 13.76- and 14.05-fold for trypsin and chymotrypsin, respectively. Michaelis–Menten constants (Km) for substrates of trypsin and chymotrypsin, BApNA and SAAPFpNA, were found to be 1.25 and 0.085 mM, correspondingly. Fluorescent zymogram analysis indicated the presence of five trypsin bands with molecular masses of ∼21, 25, 38, 40, and 66 kDa and two chymotrypsin bands with molecular masses of ∼29 and 34 kDa in SDS-PAGE. The optimum pH was 10.0 and optimum temperature was 50°C for proteolytic activity for the purified proteases. The proteases were inhibited by synthetic inhibitors such as PMSF, aprotonin, leupeptin, pefabloc, and antipain. TLCK and TPCK inhibited about 94 and 90% of trypsin and chymotrypsin activity, respectively, while EDTA, EGTA, E64, pepstatin, idoacetamide, and bestatin did not affect the enzymes. The purified enzymes exhibited high stability and compatibility with metal ions; oxidizing, reducing, and bleaching agents; organic solvents; and commercial detergents. Short life cycles, voracious feeding behavior, and production of multiple forms of proteases in the midgut with rapid catalytic activity and chemostability can serve H. armigera as an excellent alternative source of industrially important proteases for use as additives in stain removers, detergents, and other bio-formulations. Identification of enzymes with essential industrial properties from insect species could be a bioresource.
机译:我们对棉铃虫Helicoverpa armigera的胰蛋白酶和胰凝乳蛋白酶样丝氨酸碱性蛋白酶进行了表征,因为它们可能作为添加剂在各种生物制剂中应用。通过使用羟基磷灰石,DEAE葡聚糖和CM葡聚糖色谱柱进行纯化,胰蛋白酶和胰凝乳蛋白酶的酶活性分别提高了13.76倍和14.05倍。胰蛋白酶和胰凝乳蛋白酶的底物,BApNA和SAAPFpNA的Michaelis-Menten常数(Km)分别为1.25和0.085 mM。荧光酶谱分析表明在SDS-PAGE中存在5个分子量约为21、25、38、40和66 kDa的胰蛋白酶带,和2个分子量约为29和34 kDa的胰凝乳蛋白酶带。对于纯化的蛋白酶的蛋白水解活性,最适pH为10.0,最适温度为50℃。蛋白酶被合成抑制剂(例如PMSF,抑肽酶,亮蛋白,pefabloc和抗痛药)抑制。 TLCK和TPCK分别抑制约94%和90%的胰蛋白酶和胰凝乳蛋白酶活性,而EDTA,EGTA,E64,胃蛋白酶抑制素,氨基乙酰胺和Bestatin则不影响该酶。纯化后的酶具有很高的稳定性和与金属离子的相容性。氧化剂,还原剂和漂白剂;有机溶剂;和商业洗涤剂。短生命周期,旺盛的进食行为以及中肠中多种形式的蛋白酶的产生,具有快速的催化活性和化学稳定性,可将棉铃虫作为工业上重要的蛋白酶的极佳替代来源,用作去污剂,洗涤剂和其他产品中的添加剂生物制剂。从昆虫物种中鉴定具有重要工业特性的酶可能是一种生物资源。

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    Akbar, S M; Sharma, H C;

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  • 年度 2017
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