Studies On Genetic Marker And Fuse Breeding Of α-ALDC Producing Strains

α-Acetolactate decarboxylase(α-ALDC) which can remove diacetyl from the beer is used widely in beer industry. Protoplasts fusion method had many advantages in breeding techniques. Bacillus subtilis V-20 and 3226-5 producingα-ALDC were kept in our laboratory. Resistane tag had little effect on the enzyme activitiy of-fuses.lt was a easy method of using resistane tag select fuses.So,the protoplast fusion method of Bacillus subtilis V-20 and 3226-5 was carried out to select some strains with higher enzyme activity ofα-ALDCAt the beginning of this test, penicillin was used on 3226-5 and obtained the resistance strain P-67; streptomycin was used on V-20 and obtained the resistance strain S-17. P-67 and S-17 were the two parents of protoplasts. Then, the proper culture time of the parents were determined basing the growth curves. At first, the strains should be activated in complete culture plates and then transferred into the complete fluid culture to shake cultivate under the condition of 37℃,200rpm for 14 hours. At last, 1mL bacterial fluid should be inoculated to 20mL complete culture fluid to shake culture for 3.5-4 hours.The collection of bacteria was treated by lysozyme. In the test, several lysozyme concentrations and time and temprature were selected to treat parents .The curve of protoplast formation rates and regeneration rates showed that P-67 should be treated at lysozyme concentration of 20μg/mL,32°C,for 20min;S-17 should be treated at lysozyme concentration of 15μg/mL,32°C,for 20min. Under above conditions, the formation rate and regeneration rate were better for fusion . The result of fusion rates presents the better fusion condition of parents. The two strains are fused by PEG6000 at concentration of 35%, in water bath of 38°C for 15min. They were tested the enzyme activity ofα-ALDC. Fusants of F-589 which activity ofα-ALDC 157.59 u/g was selected from all the 287 strains and it's activity ofα-ALDC was 37.2% higher than it's parents.At last, rifampicin was used on F-589 and obtained a strain of R-4-250 which activity ofα-ALDC was 194.78 u/g .The activity ofα-ALDC of R-4-250 was 23.6% higher than F-589 and 69.58% higher than the parents.