Influencing Factors of Protoplast Regeneration of Bacillus subtilis and Pseudomonas fluorescent

Objective:In order to improve the regeneration rate,the effects of different factors on the regeneration of Gram-positive Bacillus subtilis KR strain and Gram-negative Pseudomonas fluorescent B13 strain were conducted.Methods:The experiments were designed for protoplast regeneration of KR and B13 strains as the following factors:lysozyme treatment time,regeneration mode,stabler in culture medium,and the concentration of Ca2+,Mg2+,sodium succinate acid,L-tryptophan,and culture medium placed time.Results:The optimal protoplast regeneration conditions for KR strain were lysozyme treatment 20 min,interlayer,0.6 mol/L sucrose,0.03 mol/L Ca2+,0.02 mol/L Mg2+,0.3 mol/L sodium succinate acid,0.2 mol/L L-tryptophan and culture medium placed 72 h under 37℃.The optimal protoplast regeneration conditions for B13 strain were lysozyme treatment 15 min,interlayer,0.6 mol/L NaCl,0.02 mol/L Ca2+,0.01 mol/L Mg2+,0.3 mol/L sodium succinate acid,0.1 mol/L L-tryptophan and culture medium placed 48 h under 37℃.The highest protoplast regeneration rates of KR strain and B13 strain were 42.7% and 15.3% respectively.Conclusion:The factors which affected Gram-positive bacterium's protoplast regeneration were different from which affected Gram-negative bacterium.