Studies on a Novel Thermostable Acid Proteinase "Kumamolysin"

在对嗜热微生物中一种对已知特异性抑制剂不敏感的酸性蛋白酶进行筛选的过程中，我们在新芽孢杆菌中发现了一种新的酶，命名为“Kumamolysin”。MN-32。本研究旨在通过蛋白测序和/或克隆来阐明该酶独特的底物专一性和一级结构。实验结果总结如下：(1)枯草杆菌溶血素的独特性质是其底物专一性：枯草杆菌溶血素专一性地降解氧化胰岛素B链上亮氨酸(15)-酪氨酸(16)的多肽键。亚基要求从合成肽底物中揭示，模拟氧化胰岛素B链中的SCILE位点。确定底物的最小长度为六肽(VEALYL)。(2)一级结构：用全自动蛋白质测序仪从胰酶、赖氨酰内肽酶片段中测定了枯草杆菌裂解的部分氨基酸序列(共100个残基)。根据这些结果，设计了用于克隆的DNA探针。杂交后，推测染色体DNA的Sal I片段含有溶血素基因。将此片段插入到pUC18中，得到pK1。对该质粒进行了亚克隆，并对枯草杆菌溶血素基因的部分碱基序列进行了分析。

In the course of our screening of an acid proteinase, insensitive to the known specific inhibitors, in thermophilic microorganism, we found a novel enzyme designated "Kumamolysin" in Bacillus novosp. MN-32. The present project is to elucidate the unique substrate specificity and primary structure of the enzyme from protein sequencing and/or coloning of the kumamolysin gene. The summary of experimental results are as follows.(1) The unique character of kumamolysin is its substrate specificity : kumamolysin specificaly hydrolyzed the peptide bond of Leu (15) -Tyr (16) in oxidized insulin B chain. The subsite requirement was disclosed from synthetic peptide substrate, mimic to the sccile site in oxidized insulin B chain. The minimum length of substrate was determined to be hexapeptide (VEALYL).(2) Primary structure : Partial amino acid sequence (total 100 residues) of kumamolysis was determined from tryptic, lysyl endopeptidase fragements by automated protein sequencer. From these results, DNA probe for cloning was designed. After hybridization, Sal I fragment of chromosomal DNA was deduced to contain kumamolysin gene. Insertion of this fragemnt in pUC18 to yield plasmid pK1. Subcloning of this plasmid was also carried out, and partial base sequence of kumamolysin gene was elucidated.