产酶溶杆菌L-43的筛选及其抑菌物质的初步鉴定

Screening of Lysobacter enzymogenes L-43 and Preliminary Identification of the Antibacterial Substances

摘要:

采用透明圈法和琼脂扩散法筛选具有抑菌活性的菌株，通过形态观察、理化分析及16S rDNA序列比对鉴定菌株，采用胞外酶活测定、蛋白酶水解、硫酸铵沉淀与膜分析、稳定性研究鉴定菌株所产抑菌物质。结果表明，从农田土壤中获得一株抑菌活性较好且没有溶血性的产酶溶杆菌（Lysobacter enzymogenes）L-43。该菌所产抑菌物质抑菌谱较广，对G+细菌具有良好的抑制作用，抑菌圈直径大于10 mm。对抑菌物质进行分析，其不具有溶菌酶活性、不含过氧化氢；对蛋白酶E、蛋白酶K、胃蛋白酶、胰蛋白酶敏感，抑菌活性回收率为73.43%~79.00%；经过膜分析，透析袋截留分子量为3 500 u和10 000 u时，抑菌活性回收率分别为93.43%和59.82%，初步判定抑菌物质主要是分子量在3 500~10 000 u之间的多肽类物质；抑菌物质具有良好的热、酸碱、金属离子、化学试剂和储存稳定性，121 ℃处理30 min活性回收率为85.66%，pH值2~10时活性回收率高于80%，进一步确定抑菌物质为多肽。综上，L. enzymogenes L-43所产抑菌物质为抑菌谱广且稳定性好的多肽。该研究为抗菌菌株及其代谢物的开发奠定了基础。

Abstract:

A bacterial strain exhibiting antimicrobial activity was identified in farmland soil and screened using the transparent circle and agar diffusion methods. The strain was identified through morphological observation, physical and chemical analysis, and 16S rDNA sequence alignment, and the antimicrobial substances produced by the strain were identified using extracellular enzyme activity assay, protease hydrolysis, ammonium sulfate precipitation, membrane analysis, and stability analysis. The results showed that the identified strain was Lysobacter enzymogenes L-43, which demonstrated good antibacterial activity and no hemolysis. The antibacterial substances produced by L. enzymogenes L-43 had a broad antimicrobial spectrum and maintained a good inhibitory effect on Gram-positive bacteria. The diameter of the inhibition zone exceeded 10 mm. The antibacterial substances had no lysozyme activity and did not contain hydrogen peroxide. They were sensitive to protease E, protease K, pepsin, and trypsin, and the antibacterial activity recovery rate was 73.43%~79.00%. After membrane analysis, when the molecular weight cut-off of the dialysis bag was 3 500 u and 10 000 u, the antibacterial activity recovery rates were 93.43% and 59.82%, respectively. It was preliminarily determined that the antibacterial substances were mainly polypeptides with molecular weights between 3 500~10 000 u. The substances showed good thermal, acid-base, metal ion, chemical reagent, and storage stability, and the antibacterial activity recovery rate was 85.66% after treatment at 121 ℃ for 30 min. The antibacterial recovery rate was higher than 80% at pH 2~10, further confirming that the antibacterial substance was a polypeptide. In conclusion, the antibacterial substances produced by L. enzymogenes L-43 are stable peptides with a broad antibacterial spectrum. The findings of this study provide a foundation for the development of antibacterial strains and their metabolites.