The Research And Application On Bacillus Amyloliquefaciens SWJS22 Producing Glutaminase

Soy sauce has become more and more popular in the world, owing to this, people pay more attention on improving the flavor of soy sauce. L-Glutaminase catalyzes the hydrolysis of L-glutamine to L-glutamic acid and ammonia, and glutamate is a widely claimed flavor-enhancing amino acid, so improving the glutaminase activity is the key to make soy sauce better. During the soy sauce fermentation, high NaCl concentration limited the glutaminase activity of Aspergillus oryzae, considering the similar high NaCl environment of ocean, people put their sights on marine organism. In this research, Bacillus amyloliquefaciens SWJS22 screened from South China Sea’s deep-sea mud, to optimize its seed liquid, solid fermentation medium. Then we applied this bacteria strain and its neo-solid fermentation medium to soy sauce fermentation in order to improve soy sauce quality. Besides, glutaminase made by Bacillus amyloliquefaciens SWJS22 was extracted, separated, purified and characterized. The main contants and results are as follows:Using the amount of biomass index（OD600） as measured, LB medium as a basic medium, Bacillus amyloliquefaciens SWJS22 seed liquid was optimized in terms of nitrogen, carbon, a metal element, salt content. The optimized seed solution（%） is tryptone 1, yeast extract 0.5, sucrose 1, Fe2（SO₄）3·XH₂O 0.01,MnSO₄·H₂O 0.0001,CuSO₄·5H₂O 0.0001. The amount of biomass was increased by 171.09% after optimization. Meanwhile its physiological and biochemical characteristics was deeply investigated. As a result, it entered logarithmic growth phase after 2 hours of fermentation, and it entered the stationary growth phase after 12 hours of fermentation. In addition, the strain is a salt-tolerant bacteria, but not a halophiles.Solid fermentation showed significant advantages over liquid fermentation on enzyme production. Single factor test and orthogonal test, including culture media and the fermentation parameters, were applied to optimize glutaminase production technology; optimal fermentation conditions were: 2%（v/w） seed liquid was inoculated in a culture medium composed of 20 g soy flakes, 5 g wheat flour, 0.02 g SE-1170, water to solid in a ratio of 1.0:0.6（w/w）, and fermented at 37℃ for 48 h. Under this optimized condition, the L-glutaminase activity was increased from 83.10±4.64 U/gds to 2690.02±28.80 U/gds.New soy sauce was fermentated using Bacillus amyloliquefaciens SWJS22 and Aspergillus oryzae as fermentation strain, complexing during the koji making, and mixing the Bacillus amyloliquefaciens SWJS22’s solid-state fermentation with Aspergillus oryzae koji, fermenting new soy sauce. Meanwhile, the effects on different ways of soy sauce koji making for the soy sauce basic physical and chemical indicators were compared. In conclusion, Bacillus amyloliquefaciens SWJS22 can improve the amino acid conversion rate, color index, glutamine acid content and aroma components.The extraction of glutamines was optimized, as a result, the optimal extraction technology method was to add 8 volume of water to the culture and shaking in a vibrator at 37℃ for 1 h. It was shown that the crude enzyme can transform glutamine to glutamic acid. Ethanol precipitation has less damage for protein, and less hybrid protein than ammonium sulfate precipitation, and the process is simpler, safer and with higher multiple purification. After partly purification, some properties of the enzyme were determined, the optimal temperature is 55℃, the stability temperature is 37℃, the optimal pH value is 9, the stability pH value is 7, the enzyme activity remained 80.65 % when the salt concentration was 18%, 10% to 30%（v/v） ethanol concentration can promote glutaminase activity, 0.1 mM and 1 mM Ca²⁺, Li2, Zn²⁺, Fe²⁺, K+, Fe3+, Mg²⁺ can inhibite the activity conversely, 1 mM DTT, NaN3 can make the glutaminase lose its activity completely.