Jasilionis Andrius, Kuisiene Nomeda
Department of Microbiology and Biotechnology, Faculty of Natural Sciences, Vilnius University, LT-03101 Vilnius, Lithuania.
J Microbiol Biotechnol. 2015 Jul;25(7):1070-83. doi: 10.4014/jmb.1412.12049.
A gene (GT-SM3B) encoding a thermostable secreted oligoendopeptidase (GT-SM3B) was cloned from the thermophile Geobacillus thermoleovorans DSM 15325. GT-SM3B is 1,857 bp in length and encodes a single-domain protein of 618 amino acids with a 23-residue signal peptide having a calculated mass of 67.7 kDa after signal cleavage. The deduced amino acid sequence of GT-SM3B contains a conservative zinc metallopeptidase motif (His(400)-Glu(401)-X-XHis (404)). The described oligopeptidase belongs to the M3B subfamily of metallopeptidases and displays the highest amino acid sequence identity (40.3%) to the oligopeptidase PepFBa from mesophilic Bacillus amyloliquefaciens 23-7A among the characterized oligopeptidases. Secretory production of GT-SM3B was used, exploiting successful oligopeptidase signal peptide recognition by Escherichia coli BL21 (DE3). The recombinant enzyme was purified from the culture fluid. Homodimerization of GT-SM3B was determined by SDS-PAGE. Both the homodimer and monomer were catalytically active within a pH range of 5.0-8.0, at pH 7.3 and 40°C, showing the Km, Vmax, and kcat values for carbobenzoxy-Gly-Pro-Gly-Gly-Pro-Ala-OH peptidolysis to be 2.17 ± 0.04 × 10(-6) M, 2.65 ± 0.03 × 10(-3) micrometer/min, and 5.99 ± 0.07 s(-1), respectively. Peptidase remained stable at a broad pH range of 5.0-8.0. GT-SM3B was thermoactive, demonstrating 84% and 64% of maximum activity at 50°C and 60°C, respectively. The recombinant oligopeptidase is one of the most thermostable M3B peptidase, retaining 71% residual activity after incubation at 60°C for 1 h. GT-SM3B was shown to hydrolyze a collagenous peptide mixture derived from various types of collagen, but less preferentially than synthetic hexapeptide. This study is the first report on an extracellular thermostable metallo-oligopeptidase.
从嗜热栖热地芽孢杆菌DSM 15325中克隆出一个编码耐热性分泌型寡肽酶（GT-SM3B）的基因（GT-SM3B）。GT-SM3B长度为1857 bp，编码一个618个氨基酸的单结构域蛋白，带有一个23个残基的信号肽，信号肽切割后计算分子量为67.7 kDa。GT-SM3B推导的氨基酸序列包含一个保守的锌金属肽酶基序（His(400)-Glu(401)-X-XHis(404)）。所述的寡肽酶属于金属肽酶的M3B亚家族，在已鉴定的寡肽酶中，与嗜温解淀粉芽孢杆菌23-7A的寡肽酶PepFBa的氨基酸序列同一性最高（40.3%）。利用大肠杆菌BL21(DE3)对寡肽酶信号肽的成功识别，实现了GT-SM3B的分泌表达。从培养液中纯化出重组酶。通过SDS-PAGE确定了GT-SM3B的同二聚化。同二聚体和单体在pH 5.0 - 8.0范围内均具有催化活性，在pH 7.3和40°C下，对苄氧羰基-Gly-Pro-Gly-Gly-Pro-Ala-OH肽解反应的Km、Vmax和kcat值分别为2.17±0.04×10(-6) M、2.65±0.03×10(-3) μmol/min和5.99±0.07 s(-1)。肽酶在5.0 - 8.0的宽pH范围内保持稳定。GT-SM3B具有热活性，在50°C和60°C时分别显示出最大活性的84%和64%。该重组寡肽酶是最耐热的M3B肽酶之一，在60°C孵育1小时后仍保留71%的残余活性。GT-SM3B显示能水解源自各种类型胶原蛋白的胶原肽混合物，但比合成六肽的水解偏好性低。本研究是关于细胞外耐热性金属寡肽酶的首次报道。
