The Preliminary Study Of Lytic Action Of Nisin And PlyG For The Killing Of Bacillus

Bacillus, the largest feature of which is the generation of spore. With multi-storey of structure, the spores of Bacillus have a stronger resistance to radiation, chemical disinfectants and other physical and chemical factors. As the spores have strong resistance to outside environment, Bacillus are widely distributed, in soil, water, air and animal intestine, etc., and are closely related to human beings. Such as Bacillus anthracis can cause people and livestock in anthrax; Wax-like Bacillus cause food poisoning, etc. Because of the strong resilience and resistance, Bacillus spores can not be killed by general approaches. The spores will turn into a lot of bacillus and breeding in the conditions for germination, and cause food corruption or lead to outbreak of diseases. The researchers in the field of preventive medicine take more focus on finding effective method of killing the Bacillus spores. Traditionally, we use the high-temperature sterilization method (121℃, 30 min) to eliminate total spores, but there is a lot of inconvenience to use this method in practical application. In addition to physical methods, some chemical disinfectants are used to kill spores, such as: chlorine disinfectants, ozone, methyl, double-stranded quaternary, ethylene oxide and so on, but these types of chemical disinfectants will cause harm to the human body or contaminate the environment to some extent.In this study, we selected Nisin and PlyG as lytic agents for Bacillus, compared the lytic action of the two agents for the rapid killing of alternative strains of Bacillus anthracis RSVF1, and laid the foundation for their application in the future. This study consists of two parts. First part is the expression of PlyG in Escherichia coli and its identification. Anthraxγ-phage was used as a template, the primer was designed according to GenBank sequence DQ289556. The gene of Bacillus anthracis phage lyase PlyG was cloned into the pET-22b(+) plasmid,the expression plasmid was verified by DNA sequenceing, and it turned out that the amino acid sequence encoded by the plasmid was the same as expected. The construct was transformed into E.coli BL21(DE3).The hosts were induced by IPTG to express PlyG protein, then gathered, treated with ultrasonic wave, and centrifuged to obtain its supernatant.The supernatant was purified by use of the SP-XL ion chromatography twice. Results showed protein PlyG was expressed efficiently in E.coli, and the amout of soluble PlyG took 40% of the total protein by SDS-PAGE analisis.The purity of PlyG was achieved up to 95% by two steps and the capability purified per 1L media was up to 20mg. N-terminal amino acid sequencing results M-E-I-Q-K, was consistent with the sequence reported. Second, the preliminary study of the lytic action of Nisin and PlyG for the rapid killing of alternative strains of Bacillus anthracis RSVF1. The LD50 to RSVF1 of Nisin and PlyG was measurede with OD₆₅₀, and the LD₅₀ of Nisin is 309μg/mL, while PlyG is 672μg/mL. For exploring the lytic action of Nisin and PlyG for killing spores of Bacillus, we used RSVF1 to generate a lot of spores(the content of spore≥99%). L-Ala (100mmol/L) was used as a satisfactory germinant of spores and the calculation of Bacillus germination rate with L-Ala was 97.80 %. Then we used flat-plate counting to detect the killing rate of PlyG and Nisin on the germination of RSVF1 spores .The experimental results showed that with the germinant of L-Ala(100mmol/L), the killing rate of Nisin for the RSVF1 spores was up to 99.37%, while the killing rate of PlyG increased to 99.12 %.In conclusion, our work compared PlyG with Nisin in the lytic action for the killing of Bacillus and their spores, which will serve as a base for their development and application as a new type of disinfectant in the future.