在本研究中,溶菌酶通过以下多步方法纯化：盐（硫酸铵）沉淀，透析,和超滤。在每个纯化步骤后通过酶活性测量溶菌酶潜能。然而,超滤后，所得材料被认为是额外纯化的。浓缩在超滤离心管中,并将所得的蛋白质/溶菌酶用于确定其对五种细菌菌株的杀菌潜力，包括三个革兰氏阳性(枯草芽孢杆菌168，藤黄微球菌，和蜡状芽孢杆菌）和两个革兰氏阴性（鼠伤寒沙门氏菌和铜绿假单胞菌）菌株。ZOI和MIC/MBC结果表明，溶菌酶对革兰氏阳性菌株的抗菌活性高于革兰氏阴性菌株。将溶菌酶的抗菌活性结果与环丙沙星（抗生素）的抗菌活性进行了比较。为此,本研究应用了两个指标：抗菌指数（AMI）和活性百分比指数（PAI）。研究发现，纯化的溶菌酶对蜡样芽孢杆菌(AMI/PAI;1.01/101)和枯草芽孢杆菌168(AMI/PAI;1.03/103)具有较高的抗菌活性，与本研究中使用的抗生素（环丙沙星）相比。原子力显微镜（AFM）用于确定溶菌酶对细菌细胞的杀菌作用。通过凝胶柱色谱进一步处理纯化的蛋白质并收集洗脱液,酶活性为21.93U/mL,而洗脱液通过天然-PAGE处理。通过这种分析,获得酶活性为40.9U/mL的未变性蛋白。该步骤显示蛋白质(溶菌酶)具有甚至更高的酶潜力。为了确定可能引起杀菌潜力和细胞裂解/酶活性的特定肽（在溶菌酶中），通过SDS-PAGE技术进一步处理分离的蛋白质(溶菌酶)。SDS-PAGE分析显示大小为34kDa的不同条带，24kDa,10kDa,分别。为了确定肽的化学成分，条带（来自SDS-PAGE）被切割，酶消化，脱盐,并通过LC-MS(液相色谱-质谱)分析。LC-MS分析显示纯化的溶菌酶具有以下组成:样品中蛋白质的数量为56,肽的数量为124,PSM的数量(肽谱匹配)为309。其中,与溶菌酶和杀菌活性相关的两种肽被鉴定为:A0A1Q9G213(N-乙酰胞壁酰-L-丙氨酸酰胺酶)和A0A1Q9FRD3(D-丙氨酰-D-丙氨酸羧肽酶)。通过与数据库比较确定相应的蛋白质序列和核酸序列。
In the present study, lysozyme was purified by the following multi-step methodology: salt (ammonium sulfate) precipitation, dialysis, and ultrafiltration. The lysozyme potential was measured by enzymatic activity after each purification step. However, after ultrafiltration, the resulting material was considered extra purified. It was concentrated in an ultrafiltration centrifuge tube, and the resulting protein/lysozyme was used to determine its bactericidal potential against five bacterial strains, including three gram-positive (Bacillus subtilis 168, Micrococcus luteus, and Bacillus cereus) and two gram-negative (Salmonella typhimurium and Pseudomonas aeruginosa) strains. The results of ZOI and MIC/MBC showed that lysozyme had a higher antimicrobial activity against gram-positive than gram-negative bacterial strains. The results of the antibacterial activity of lysozyme were compared with those of ciprofloxacin (antibiotic). For this purpose, two indices were applied in the present study: antimicrobial index (AMI) and percent activity index (PAI). It was found that the purified lysozyme had a higher antibacterial activity against Bacillus cereus (AMI/PAI; 1.01/101) and Bacillus subtilis 168 (AMI/PAI; 1.03/103), compared to the antibiotic (ciprofloxacin) used in this study. Atomic force microscopy (AFM) was used to determine the bactericidal action of the lysozyme on the bacterial cell. The purified protein was further processed by gel column chromatography and the eluate was collected, its enzymatic activity was 21.93 U/mL, while the eluate was processed by native-PAGE. By this analysis, the un-denatured protein with enzymatic activity of 40.9 U/mL was obtained. This step shows that the protein (lysozyme) has an even higher enzymatic potential. To determine the specific peptides (in lysozyme) that may cause the bactericidal potential and cell lytic/enzymatic activity, the isolated protein (lysozyme) was further processed by the SDS-PAGE technique. SDS-PAGE analysis revealed different bands with sizes of 34 kDa, 24 kDa, and 10 kDa, respectively. To determine the chemical composition of the peptides, the bands (from SDS-PAGE) were cut, enzymatically digested, desalted, and analyzed by LC-MS (liquid chromatography-mass spectrometry). LC-MS analysis showed that the purified lysozyme had the following composition: the number of proteins in the sample was 56, the number of peptides was 124, and the number of PSMs (peptide spectrum matches) was 309. Among them, two peptides related to lysozyme and bactericidal activities were identified as: A0A1Q9G213 (N-acetylmuramoyl-L-alanine amidase) and A0A1Q9FRD3 (D-alanyl-D-alanine carboxypeptidase). The corresponding protein sequence and nucleic acid sequence were determined by comparison with the database.
