Itoh Y, Sumi M, Nakamura K, Yamane K
Institute of Biological Sciences, University of Tsukuba, Ibaraki.
J Biochem. 1990 Dec;108(6):954-9. doi: 10.1093/oxfordjournals.jbchem.a123320.
To analyze the processing of extracellular enzymes of Bacillus subtilis, an NH2-terminally extended hybrid alpha-amylase [pTUBE638-alpha-amylase (E24)] was purified from the periplasm of E. coli(pTUBE638) as the substrate for the in vitro processing reaction, in which a 21-amino-acid extra-peptide was added at the NH2-terminus of the mature thermostable alpha-amylase. The extended peptide in pTUBE638-alpha-amylase (E24) was completely processed by the extracellular alkaline protease of B. subtilis alone at pH 7.5 to 10.0. The processing was inhibited by 2 mM PMSF. In contrast, the neutral protease did not process the extended peptide. The processing activity of the purified alkaline protease was fully active in 100 mM phosphate and glycine-NaCl-NaOH buffer while it was partially active in 100 mM Tris-HCl or MOPS buffer. The optimum pH of the activity ranged from 8.0 to 9.0, although the optimum pH of the alkaline protease activity toward casein and Azocoll was 10.5. The NH2-terminal amino acid sequences of the enzymes processed in vitro coincided with those of the mature extracellular thermostable alpha-amylases in the culture medium of B. subtilis (pTUBE638). The appearance of the processing activity of alkaline protease was correlated with the changes of the pH in the culture medium.
为分析枯草芽孢杆菌胞外酶的加工过程，从大肠杆菌（pTUBE638）的周质中纯化出一种氨基末端延伸的杂合α-淀粉酶[pTUBE638-α-淀粉酶（E24）]作为体外加工反应的底物，其中在成熟的耐热α-淀粉酶的氨基末端添加了一个21个氨基酸的额外肽段。pTUBE638-α-淀粉酶（E24）中的延伸肽段在pH 7.5至10.0时仅被枯草芽孢杆菌的胞外碱性蛋白酶完全加工。该加工过程被2 mM苯甲基磺酰氟（PMSF）抑制。相反，中性蛋白酶不加工该延伸肽段。纯化的碱性蛋白酶的加工活性在100 mM磷酸盐和甘氨酸-氯化钠-氢氧化钠缓冲液中完全有活性，而在100 mM Tris-HCl或MOPS缓冲液中部分有活性。尽管碱性蛋白酶对酪蛋白和偶氮酪蛋白的活性的最适pH为10.5，但该活性的最适pH范围为8.0至9.0。体外加工的酶的氨基末端氨基酸序列与枯草芽孢杆菌（pTUBE638）培养基中成熟胞外耐热α-淀粉酶的序列一致。碱性蛋白酶加工活性的出现与培养基中pH的变化相关。
