Screening Of α-ALDC-higher-Producing Strains By Inactivated Protoplast Fusion

a -Acetolactate decarboxylase( a -ALDC) which can remove diacetyl from the beer is used widely in beer industry. Inactivated protoplasts fusion method has more advantages in breeding techniques and also it has been used effectively in parents without any heritable labels. Bacillus subtilis V-20 and 3226-5 producing a -ALDC are kept in our laboratory. The inactivated protoplast fusion method of Bacillus subtilis V-20 and 3226-5 is carried out to select some strains with higher enzyme activity of a -ALDC.At the beginning of this test, the proper culture conditions of the parents are determined and the growth curve of two different culture solution (complete culture fluid and fermented broth ) and two different volume of the complete culture fluid(20mL and 40mL) are selected. At first, the strains should be activated in complete culture plates and then transferred into the complete culture fluid to shake cultivate under the condition of 37?,200rpm for 14 hours. At last, ImL bacterial fluid should be inoculated to 20mL complete culture fluid to shake culture for 3.5-4 hours. The collection of bacteria is treated by lysozyme. In the test, several lysozyme concentrations are selected to treat parents in 32? The curve of protoplast formation rates and regeneration rates show that parents should be treated at lysozyme concentration of 50 P g/mL, for 20min. Under above conditions, the formation rate and regeneration rate are better for fusion of Bacillus subtilis V-20 and 3226-5. Before fusion, the strain V-20 is inactivated by ultraviolet and the strain 3226-5 is inactivated by boiling water bath. The result of fusion rates presents the better fusion condition of parents. The two strains are fused by PEG6000 at concentration of 32%, in water bath of 38癈 for 15min. After the fusants are transferred for ten generation, they are tested the enzyme activity of a -ALDC. Three fusants are got and their enzyme activitiesare higher than their parents. At the end of the test, a simple comparison of DNA quantity between fusants and parents shows these strains are fusants.