A protein showing endoglucanase-peptidase activity was prepared from a newly isolated bacterium (ST15c10). We identified ST15c10 as Brevibacillus agri based on electron-microscopic images and its 16S-rDNA sequence (GenBank accession No. HM446043), which exhibits 98.9% sequence identity to B. agri (KZ17)/B. formosus (DSM-9885T)/B. brevis. The enzyme was purified to homogeneity and gave a single peak during high-performance liquid chromatography on a Seralose 6B-150 gel-matrix/C-18 column. MALDI-TOF mass-spectrometry and bioinformatics studies revealed significant similarity to M42-aminopeptidases/endoglucanases of the CelM family. These enzymes are found in all Brevibacillus strains for which the genome sequence is known. ST15c10 grows optimally on carboxymethyl cellulose (CMC)-gelatin (40°C/pH 8-9), and also shows strong growth/carboxymethyl cellulase (CMCase) activity in submerged bagasse fermentation. The purified enzyme also functions as endoglucanase with solid bagasse/rice straw. Its CMCase activity (optimal at pH 5.6 and 60°C/Km = 35.5 µM/Vmax = 1,024U) was visualized by zymography on a CMC-polyacrylamide gel, which provided a strong band of approximately 70 kDa. The purified enzyme also showed strong peptidase (gelatinase) activity (pH 7.2/40°C during zymography on 6-12% gelatin/1% gelatin-PAGE (at approx. 70 kDa). The CMCase activity is inhibited by the metal ions Mn/Cu/Fe/Co (50%), Hg/KMnO4 (100%), and by glucose or lactose (50-75%; all at 1 mM). The observed dose/time-dependent inhibition by Hg ions could be prevented with 2-mercaptoethanol. A comparison of the B. agri endoglucanase-aminopeptidase (ELK43520; 350 aa) with other members of the M42-family revealed the conservation of active-site residues Cys256/Cys260, which were previously identified as metal-binding sites. Regulation of the endoglucanase activity probably occurs via metal binding-triggered changes in the redox state of the enzyme. Studies on this type of enzyme are of high importance for basic scientific and industrial research.

中文翻译：

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来自农业短螺旋杆菌ST15c10的新分离蛋白的羧甲基纤维素酶和明胶酶的双重活性在底物的利用中具有相互调节作用。

从新分离出的细菌（ST15c10）中制备出一种具有内切葡聚糖酶-肽酶活性的蛋白质。基于电子显微镜图像和其16S-rDNA序列（GenBank登录号HM446043），我们将ST15c10鉴定为农杆菌，其与农杆菌（KZ17）/ B的序列同一性为98.9％。孔（DSM-9885T）/ B。布雷维斯。将该酶纯化至均质，并在Seralose 6B-150凝胶基质/ C-18色谱柱上进行高效液相色谱分析时给出一个峰。MALDI-TOF质谱和生物信息学研究表明，其与CelM家族的M42-氨基肽酶/内切葡聚糖酶具有显着相似性。在所有已知基因组序列的短杆菌属菌株中都发现了这些酶。ST15c10在羧甲基纤维素（CMC）-明胶（40°C / pH 8-9）上生长最佳，在淹没的蔗渣发酵中也显示出强大的生长/羧甲基纤维素酶（CMCase）活性。纯化的酶还与固体蔗渣/稻草一起用作内切葡聚糖酶。其CMCase活性（在pH 5.6和60°C / Km = 35.5 µM / Vmax = 1,024U时最佳）在CMC-聚丙烯酰胺凝胶上通过酶谱法显示，其强带约为70 kDa。纯化的酶还具有很强的肽酶（明胶酶）活性（在6-12％明胶/ 1％明胶-PAGE（约70 kDa）上酶切过程中，pH 7.2 / 40°C），金属离子Mn抑制了CMCase活性。 / Cu / Fe / Co（50％），Hg / KMnO4（100％）以及葡萄糖或乳糖（50-75％；均在1 mM下），可以防止观察到的剂量/时间依赖性的Hg离子抑制a。2-巯基乙醇的比较。B. agri内切葡聚糖酶-氨基肽酶的比较（ELK43520；350 aa）与M42家族的其他成员一起揭示了活性位点残基Cys256 / Cys260的保守性，该残基先前被鉴定为金属结合位点。内切葡聚糖酶活性的调节可能通过金属结合触发的酶氧化还原状态变化而发生。对这类酶的研究对于基础科学研究和工业研究具有重要意义。