Extracellular trypsin-like proteases produced by Cordyceps militaris
Extracellular trypsin-like proteases produced by Cordyceps militaris
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DOI：
10.1263/jbb.100.631
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发表时间：
2005-12-01
影响因子：
2.8
通讯作者：
Hara, A
中科院分区：
文献类型：
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作者：
Hattori, M;Isomura, S;Hara, A
采用（NH4）(2)、SO4沉淀、DEAE Bio-Gel Agarose、TSKgel CM-5PW层析、HiLoad 26/60 Superdex 75 pg凝胶过滤等方法，从真菌cordyeps militaris培养上清中纯化了胰蛋白酶样蛋白酶P-1-1，并对其性能进行了检测。纯化后的P-1-1通过SDS-PAGE显示为单带，通过基质辅助激光解吸/电离质谱（MALDI-MS）估计其分子质量为23,405。酶的最适pH为8.5 ~ 12.0。lepeptin和二异丙基氟磷酸（DFP）对其有较强的抑制作用，n - α - toyl -l -赖氨酸氯甲基酮盐酸盐（TLCK）、苯基甲基磺酰氟（PMSF）和chymostatin对其有一定的抑制作用。结果表明，P-1-1是一种胰蛋白酶型丝氨酸蛋白酶，其裂解位点为精氨酸和赖氨酸的羰基侧。P-1-1的n端氨基酸序列与来自双翅目昆虫的胰蛋白酶或凝乳胰蛋白酶具有高度的同源性。
A trypsin-like protease, P-1-1, was purified from the culture supernatant of the fungus Cordyeeps militaris by (NH4)(2),SO4 precipitation, chromatography on DEAE Bio-Gel Agarose, TSKgel CM-5PW, and gel-filtration with HiLoad 26/60 Superdex 75 pg, and its properties were examined. Purified P-1-1 showed a single band by SDS-PAGE and was estimated to have a molecular mass of 23,405 by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The optimum pH of the enzyme was between 8.5 and 12.0. It was inhibited strongly by leupeptin and diisopropyl fluorophosphate (DFP), and definitely did by N-alpha-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), phenylmethanesulfonyl fluoride (PMSF) and chymostatin. The carbonyl group sides of Arg and Lys were confirmed as the sites of cleavage by the enzyme toward cecropin B. These results indicate that P-1-1 is a trypsin-type serine protease. The N-terminal amino acid sequence of P-1-1 showed a high homology with those of trypsins or chymotrypsin derived from Diptera insects.