Selection Of Theanine-Producing Strain

Theanine is a special amino acid in tea plants, Besides its important biological and biochemical roles, theanine is good for health from the pharmaceutical and physiological points of view. Therefore,Theanine not only can be used as an additive in food and health care products, but also has potential applications in pharmaceutical industry. Industrial production of theanine technology is relatively weak, so it has become a limit to further promote the theanine application of the most important factor. Theanine production of microbial transformation was considered the most suitable method for theanine industrial production, Microbial transformation method primarily used two enzymes to produce theanine(γ-glutamyltranspeptidase, glutamine synthetase). The method of usingγ-glutamyltranspeptidase to produce theanine is fully developed. Currently , i n Japan, theanine commercial production country, The use of theγ-glutamyltranspeptidase mainly from modified by genetic engineering of E. coli. Domesticγ-glutamyltranspeptidase-producing theanine research focused on the use of E. coli bacteria genetically engineered to express. The use ofγ-glutamyltranspeptidase in industrial production strains , Bacillus licheniformis to produce theanine has not yet been publicly reported.The main purpose of this study is screening microbial strains which can produceγ-glutamyltranspeptidase to synthesize theanine to breed a theanine high-yielding strain by it mutation selection, and optimization its fermentation conditions . It can provide theoretical basis on industrial production of theanine using microbial fermentation in future.The main result as follow:1. This paper has stuied two commonly used methods,including National standard colorimetric ninhydrin and paper chromatography.The method of GB/T8314 can not rule out other amino acid in samples which interfere with the determination . Paper chromatography can separate theanine and other amino acids . It has more structured and more clear spot and better separation effect.. Using the spot of theanine to do quantitative determination after extraction, Theanine concentration has the linear relationship between theoptical density during 0.0016～0.0040mg/ml. Linear regression equation is y = 59.125x + 0.0 032,R2 = 0.9936. The Rf identification of theanine has proved that this method is stable and reliable. So we can establishe paper chromatography as theanine detection using large-scale screening of strains.2. In seven types ofγ-glutamyltranspeptidase-producing bacteria.such as Bacillus licheniformis, Bacillus subtilis, Bacillus stearothermophilus, Agrobacterium radiobacter, Agrobacterium tumefaciens, Xanthomonas campestris, Brevibacterium ammoniagenes we have screened Bacillus licheniformis as synthesis of theanine strain, by using enzyme activity determination and paper chromatography as detectation theanine production in transformation liquid.3. Using Mutagenic dose as fatality rate for 99.0% and exposure time for 60s to mutagenesis Bacillus licheniformis, selecting 200 Single colonies from diluted separation plate for preliminary screening, selecting 50 strains for re-screening, and getting 10 strains for shake flask, then we can get a strain Bacillus licheniformis C12 which theanine yield is 18.57g/L .ComPared with the yield of the starting strain, the theanine yield has improved 66.19%.4. The study of the optimization fermentation conditions about Bacillus licheniformis C12 such as:seed age 18h,start culture medium30ml, seed volume10%, initial pH 7.2, culture temperature 30℃。Adding 0.4%theanine ,0.04%PLPand 0.8% tween-80 to medium can profit fermentation.