Zhang Yayuan, Chen Jixiang, Wang Yonggang, Li Yanlin, Rui Wenhong, Zhang Jiyi, Luo Dan
School of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou, China.
School of life science and enginerring, Lanzhou University of Technology, Lanzhou, China.
PeerJ. 2020 May 18;8:e9061. doi: 10.7717/peerj.9061. eCollection 2020.
The glycopeptidase GCP and its homologue proteins are conserved and essential for survival of bacteria. The gene (Glycopeptidase homologue) was cloned from strain SF-1. The gene consisted of 1,017 bp, which encodes a 338 amino acid polypeptide. The nucleotide sequence similarity of the gene with that of FDAARGOS 107 was 95%. The gene also showed similarities of 68%, 67% and 50% with those of , and . The gene was expressed in BL21 (DE3) and the recombinant YgjD was purified by Ni affinity chromatography column. The purified YgjD showed a specific 37 kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and exhibited protease activities of 59,000 units/mg, 53,700 units/mg and 8,100 units/mg, respectively, on N-Acetyl-L-tyrosine ethyl ester monohydrate (ATEE), N-Benzoyl-L-tyrosine ethyl ester (BTEE) and N-Benzoyl-DL-arginine-4-nitroanilide hydrochloride (BAPNA) substrates. When the conserved amino acids of His, Glu and His in the YgjD were replaced with alanine, respectively, the protease activities of the mutants were partly decreased. The two conserved His and His of YgjD were mutated and the protein lost the protease activity, which implied that the two amino acid played very important roles in maintaining its protease activity. The addition of the purified YgjD to the culture medium of strain SF-1 can effectively promote the bacteria growth. These results indicated that the protease activities may be involved in the survival of bacteria.
糖肽酶GCP及其同源蛋白对于细菌的存活是保守且必需的。从菌株SF-1中克隆了该基因（糖肽酶同源物）。该基因由1017个碱基对组成，编码一个338个氨基酸的多肽。该基因与FDAARGOS 107的核苷酸序列相似性为95%。该基因与、和的核苷酸序列相似性也分别为68%、67%和50%。该基因在BL21(DE3)中表达，重组YgjD通过镍亲和层析柱纯化。纯化后的YgjD在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上显示出一条特异性的37 kDa条带，并且在N-乙酰-L-酪氨酸乙酯一水合物（ATEE）、N-苯甲酰-L-酪氨酸乙酯（BTEE）和N-苯甲酰-DL-精氨酸-4-硝基苯胺盐酸盐（BAPNA）底物上分别表现出59000单位/毫克、53700单位/毫克和8100单位/毫克的蛋白酶活性。当YgjD中保守的组氨酸、谷氨酸和组氨酸分别被丙氨酸取代时，突变体的蛋白酶活性部分降低。YgjD的两个保守组氨酸发生突变后，该蛋白失去了蛋白酶活性，这表明这两个氨基酸在维持其蛋白酶活性方面起着非常重要的作用。将纯化后的YgjD添加到菌株SF-1的培养基中可以有效促进细菌生长。这些结果表明蛋白酶活性可能与细菌的存活有关。
