Construction Of Engineering Bacterium For Expression Thermostable β-galactosidase And The Optimization Of Culture Condition

The main intention in this work is Construction of Engineering Bacterium for Expression the Thermostableβ-galactosidase and Culture Condition Optimization., Result as follows: 1. pGJ09 and pGJ16,integration vector contakin spo0A integrated loci, were integrated into B.subtilis 1A747 by double and single crossover homologous-recombine , respectly , getting BGJ09-5 and BGJ16-3 with spo0A mutant. Spo0A gene is a earlier factor sporulation. Result is good by PCR and restriction endonuclease digestion. BGJ09-5 dosen`t form spore by SSM medium, and it`s growth speed is inhibited heavily. It is showed that spo0A gene in BGJ09-5 is knockouted , otherwise BGJ16-3 has the spo0A gene still, but it sporulation velocity is slow than 1A747. 2. The pGJ222, β-galactosidase expression vector , was transformated into 1A747 and BGJ16-3 to get BGJ222 and mutant BGJ222-16,and transformated into BGJ09-5 not get mutant strain. However the expression capacity of β-galactosidase is stronger BGJ222 than mutant BGJ222-16. 3. The optimum medium and culture condition was screened out with high ability to accumulate ?-galactosidase for BGJ222 strain as followings:Tryton 1%, Yeast extract 1%, D-Soribitol 1%, Lactose 1%, Na₂HPO₄·12H₂O 1.3%, NaH₂PO₄·2H₂O 1%, and optimal technological parameters as followings: initial pH 6.6, rotationspeed 225r/min,temperature 37℃,inoculum size 2%, liquid medium 30ml in 250ml conical beaker. 4. Adding maltose end concentration 3% after fermentation culture 6 hour which is preferred induction condition. The ?-galactosidase enzyme activity reached 22992.7 miller unit, 27 times higher than LB culture medium . At the same time,this work discuss effect of glucose concentration in fermenting liquor to promoter pglv activity. It is result that restrain of glucose to pglv activity disappears under glucose concentration less than 0.3mg/ml,and that simultaneity ?-galactosidase begin to expression.