关键词: 凝乳酶I3EB99, 全基因合成, 原核表达, 亲和纯化, 结构预测

Abstract: In this study, in order to further understand the structural properties of microbial milk clotting enzyme (MCE), we firstly designed the whole gene sequence of MCE (I3EB99) from Bacillus methanolicus according to the amino acid sequences obtained from the GenBank and the codon preference of Escherichia coli, and then constructed a prokaryotic expression vector. The fusion protein was expressed in E. coli BL21 (DE3), and it was subjected to His-tag specific affinity purification. The three-dimensional spatial structure of the purified MCE was studied by bioinformatics methods. Results showed that the recombinant MCE was expressed at a level 0.7 mg/mL with milk clotting activity (MCA) of (15 870 ± 1.17) SU/g, proteolytic activity (PA) of (263.81 ± 0.94) U/g and MCA/PA ratio of 60.16, which met the requirements for cheese processing. Studies on its structural properties showed that the MCE exhibited hydrophobicity and it contained a transmembrane structure and a signal peptide; the α-helix secondary structure was less abundant than β-sheet in the MCE, which was unstable and easily degraded during the separation and purification process. The MCE was homologous to an unknown protease from B. methanolicus, and its three-dimensional structure had the highest similarity to the template protein 2ra1.1.A in the PDB protein database. By studying the structural characteristics of MCE from B. methanolicus, we have established the theoretical basis for an in-depth understanding of the mechanism and function of the MCE.

Key words: milk clotting enzyme, total gene synthesis, prokaryotic expression, affinity purification, structural properties