Study On The Breeding Of α-ALDC Producing Strain

a-acetolactate decarboxylase（a-ALDC, EC.4.1.1.5） is used widely in beer industry. It can remove diacetyl from the beer, reduce the production cycle and increase the quality of the beer. So radioactive breeding of a-acetolactate decarboxylase producing strain is very important. To obtain a strain with higher production, Bacillus subtilis W195 with a-acetolacetate decarboxylase activity as the initial strain was induced by radioactive breeding. Through optimizing microbial culture conditions and studying extract condition, the enzyme yield was reincreased. The main concent are showed as following aspects: 1. Mutagenic effect of microwave radiationTo research mutagenic effect of the microwave, and definite the best radioactive dosage of microwave, Bacillus subtilis W195 with a-acetolacetate decarboxylase activity was induced by different microwave intensity and different time. The result indicated that, the effect of dying by different microwave irradiation dosage was tremendous. When the strain was induced by the same microwave intensity but different time, the lethality of the strain increased along with the radiation time; When the strain was induced by the same time but different intensity, the lethality of the strain increased along with the intensity. Thermal reaction of microwave was extremely obvious, but it can be obviously reduced by batch treaded. The rate of total mutating increased when the radiactive time of low intensity microwave or medium intensity microwave increasing. The rate of positive mutating rised to a certain degree first, but then dropped. Based on the comparison and analysis, the optimal dosage of microwave irradiation was obtained, that is low intensity microwave of 30-40s. 2. Compound mutation and filtrationTo increase the enzyme yield of a-acetolactate decarboxylase, the initial strain was induced by microwave, ultraviolet radiation, and the genetic stability of the mutants was determined. After Bacillus subtilis W195 was induced by microwave, ultraviolet+microwave, microwave radiation, the mutant CM91 which a-acetolacetate decarboxylase yield was 174% higher than that of the initial strain was obtained, the enzyme yield of CM91 achieves 237.66u/g. The genetic stability trial indicated that the enzyme yield of the strain was quite stablable.3. Preliminary research of fermentation conditionsThrough the preliminary research of cultivation condition of the mutant strain CM91, the culturing conditions such as the optimal carbon and nitrogen source and their optimal concentration and so on, were determined. Through single factor and multi-factorial experiments, the best fermentation medium and the best ferment condition of CM91 was finally determined. The best ferment culture medium:glucose 25g/L, yeast extract 12.5g/L, beef extract 7.5g/L, （NH4）2SO₄8g/L, NaCl 0.4g/L, K2HPO₄ 0.03g/L, Mg²⁺0.030g/L, Mn²⁺ 0.00076g/L, Zn2+0.003g/L; The best ferment condition: inoculum 9%, initial pH9.0, temperature 37℃,220rpm, fermentation time 19h. At last the integration experiment showed the optimization culture was very good, which improved on enzyme activity a lot.