Purification and biochemical analysis of WprA, a 52-kDa serine protease secreted by B-subtilis as an active complex with its 23-kDa propeptide
Purification and biochemical analysis of WprA, a 52-kDa serine protease secreted by B-subtilis as an active complex with its 23-kDa propeptide
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DOI：
10.1016/s0167-4838(98)00110-1
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发表时间：
1998-07-28
期刊：
影响因子：
--
通讯作者：
Schmidt, B
中科院分区：
文献类型：
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作者：
Babé, LM;Schmidt, B
革兰氏阳性细菌枯草芽孢杆菌产生大量的蛋白酶，分泌到细胞外环境，随着菌株的产生，缺乏更突出的蛋白酶，次要的就变得可以检测到。我们从蛋白酶缺陷菌WB600中分离到一种52 kDa的分泌型蛋白酶。它由wprA基因编码，它包含一个信号序列，一个46 kDa的前肽，进一步加工到23 kDa，以及52 kDa的成熟蛋白酶。此前在野生型菌株的细胞壁制剂中检测到了52 kDa和23 kDa的多肽。我们从培养上清液中共纯化了这些蛋白，并确认了与膜结合物种相同的N末端和相对分子质量。WprA蛋白酶域与枯草杆菌菌素A有28.5%的同源性，与其他枯草杆菌菌素一样，表现出广泛的底物特异性。WprA和枯草杆菌菌素A具有相似的pH曲线，对于底物中Met、Gln或Lys残基位于P1的底物，在pH 7.5附近显示出最佳活性。在底物P1Asp的作用下，WprA在pH 5和pH 6对枯草杆菌素A的活性出现另一个高峰。某些细菌蛋白水解酶与底物和抑制剂相互作用时的pi-i依赖性可能与生物学相关。(C)1998 Elsevier Science B.V.保留所有权利。
The Gram-positive bacterium Bacillus subtilis produces numerous proteases that are secreted to the extracellular milieu, and as strains are generated which lack the more prominent proteases, minor ones become detectable. We have isolated a 52-kDa secreted protease from the protease-deficient strain WB600. It is encoded by the wprA gene which encompasses a signal sequence, a 46-kDa propeptide further processed to 23 kDa, and the 52-kDa mature protease. The 52-kDa and 23-kDa polypeptides were previously detected in cell-wall preparations of a wild-type strain. We have co-purified these proteins from culture supernatant, and confirmed the same N-termini and molecular weights as the membrane-bound species. The WprA protease domain has 28.5% identity to subtilisin A, and like other subtilisins, it displays a broad substrate specificity. WprA and subtilisin A have similar pH profiles, showing optimal activity near pH 7.5 for substrates with Met, Gln, or Lys residues at P1. Using a substrate with Asp at P1, another peak of activity was observed for WprA at pH 5 and at pH 6 for subtilisin A. The pi-I dependence of some bacterial proteases in their interaction with substrates and inhibitors may be biologically relevant. (C) 1998 Elsevier Science B.V. All rights reserved.