Breeding, Identification Of Marine Lysozyme-Producing Strain And Optimization Of Fermentation Conditions

The lysozyme from marine microorganism has unique advantages compared with those from various animals, plants and other microorganisms. Because of its promising application prospect, breeding, identification of one marine Bacillus producing lysozyme and optimization of fermentation conditions were investigated as a basis of further study and application on lysozyme from marine microorganism.Preparation and regeneration of strain S-12-86 producing lysozyme were studied. The results showed that optimal conditions of the protoplast preparation and regeneration were as follows: incubation time 18h, proportion of the strain to lysozyme 1:1, concentrations of lysozyme 1.0mg/ml, and enzymolysis 30min at 35℃. Under the optimal conditions the rates of protoplast preparation and regeneration were 97.6% and 23.6%, respectively. When the protoplasts of strain S-12-86 were irradiated 120s by a 30w UV lamp from 80cm distance , the optimal condition of protoplasts mutagenesis was achieved. After irradiation, a genetically stable mutant strain YJ007 was obtained by primary and secondary screens and genetic stability analysis, lysozyme activity for the new strain was 46.8% higher than that for the original strain S-12-86.The lysozyme-producing strain YJ007 was identified as a member of the genus Bacillus based on phenotypic and phylogenetic analysis. 16S rDNA gene sequence has been registered in GenBank (number EU016215), which analysis of YJ007 showed that similarity of sequence is 99.68% with Bacillus licheniformis DSM13^T (X68416), so YJ007 was identified as B. licheniformis..The fermentation conditions of strain YJ007 were investigated by unifactor experiment. The results indicated that medium ingredients were corn flour, peptone, beef extract, NaCl, and MgSO₄, and the fermentation conditions were incubation temperature 30℃, initial pH7.5, inoculum size 6%, rotationl speed 200 r/min, inoculum time 18-20h, and fermentation time 20-22h. Quadratic regression orthogonal design was applied to optimize the composition of medium used on shaking flask fermentation. The optimized medium ingredients were corn flour 0.200%, peptone 0.773%, beef extract 0.132%, NaCl 0.320%, and MgSO₄0.759×10^-5 mol/L.