Breeding Of Alkaline Pectinases Producing Strain By Protoplast Fusion

Alkaline pectinase is one of important industrial enzymes and is of great significance in the current biotechnological area with wide applications in textile processing, degumming of plant bast fibers, poultry, and paper making.Two halophilic and alkaliphilic strains screened out from the soda lake in the Hulunbeir area of Inner Mongolia were studied for alkaline pectinases activity. The alkaline pectinases yield of Alkalibacterium sp. F26 is higher than Bacillus sp. F26, but as far as the rate of growth is concerned, Alkalibacterium sp. F26 is slower than Bacillus sp. F26. In this research, some new strains were gotten by using protoplast fusion technique in higher productivity and quicker growing and the characteristics of strain were improved.The chloramphenicol resistance mutant Alkalibacterium sp. F26-52 were gotten by dealing with NTG and the enzyme productivity increased by 13.6%. After treatment with UV, the streptomycin resistance mutant Bacillus sp. F26-37 were gotten and the enzyme activity was raised to 115.2%. And the markers were constant. These two mutants were fused as parent strains through protoplast fusion. After 10 generations on the selective culture medium, 3 constant fusants with higher enzyme activity were screened. Two of them could produce more alkaline pectinases and growfaster than Alkalibacterium sp. F26-52. At the same time ,these 3 fusants were compared with parent strains in their physiological and biochemical characteristics.Among these fusants, the fusant 11# was highest in the alkaline pectinases yield. The medium components and fermentation conditions for the fusant 11# were optimized. The operating factors, including the carbon and nitrogen sources, inorganic salt, surfactants, initial pH, fermentation temperature and time were investigated. The optimized fermentation medium by single factors and response surface analysis experiments was: glucose 10 g/L, peptone 5g/L, NaNO₃ 1.7 g/L, NaCl 65 g/L, MgSO₄·7H₂O 0.2 g/L, K₂HPO₄·3H₂O 1 g/L, NaCO₃ 10 g/L and tween 80 10 g/L. The corresponding fermentation conditions were 25mL liquid medium with initial pH 10.8 in a flask of 250mL at culture temperature 36℃ for 22h. Pectinase activity reached 1308U /mL under the optimized medium and condition, which was 28.9% higher than that of the original strain. Alkalibacterium sp. F26 and 115.8% higher than that of the original strain Bacillus sp. F26.