A pretreatment scheme for plasmid extraction contained sugar, high concentration lysozyme and mild lysozyme removal.

ABSTRACT

To address the issue of low efficiency in extracting plasmid DNA (pDNA) from Lactobacillus plantarum by breaking the cell wall, we proposed an effective pretreatment scheme. This study investigated the impacts of lysozyme concentrations and glucose, as well as centrifugal forces during lysozyme removal in the pretreatment system. The efficiency of pDNA extraction was assessed using non-staining method, acridine orange staining method (AO staining) and agarose gel electrophoresis (AGE). Furthermore, the glucose high lysozyme method was compared to the commercial kit method and the lysozyme removal method using L. plantarum PC518, 9L15, JS193 and Staphylococcus aureus USA300. The results indicated that the pDNA extraction concentrations from the four tested strains were increased by 8.9, 7.2, 8.5, and 3.6 times, respectively, compared to the commercial kit method. Furthermore, they increased by 1.9, 1.5, 1.8, and 1.4 times, respectively, compared to the lysozyme removal method. The maximum average concentration of pDNA extraction (from L. plantarum PC518) reached 590.8 ± 31.9 ng/ul. In conclusion, the incorporation of sugar, high concentration lysozyme and mild lysozyme removal proved to be effective enhancements in improving the efficiency of pDNA extraction from L. plantarum. Using the pretreatment scheme, the concentration of pDNA extraction was significantly increased, approaching levels comparable to pDNA extraction from Gram-negative bacteria.