Screening Of Cyclodextrin Glucosyltransferase Producing Strains From Endophytic Of Siraitia Grosvenorii And Study On Enzymatic Modification Of Siraitia Grosvenorii Bitter Glycoside

In actual planting,due to the weather and plant nutrient consumption at the end of each season,many of the fruits are still immature.These glycosides in the immature fruits have not yet been converted into high glucosides.So these immature fruits become the so-called bitter fruit.In the production process,these bitter fruits are generally abandoned,causing a great waste.But both of them have the same basic skeleton.Glucosyltransferase plays an important role in the synthesis of the glucosyltransferase,which adds glucose groups one by one into bitterin for the conversion of bitter low glycosides to sweet high glycosides.The mutualism of Endophytic and Corsvenor Momordica Fruit may creat gene exchange for endophytes have the potential to select glycosyltransferase strain.Endophytic bacteria screened from Momordica grosvenori production of cyclodextrin glucosyltransferase the enzyme(CGTase)strains,and to identify the strain,fermentation conditions of the strain,preparation of fermentation,isolated and purified.The main achievements are as follows:1.solation of endophytic bacteria from Fructus grosvenoris75% ethanol and 3% sodium hypochlorite were used to sterilize the roots,stems and fruits of Siraitia grosvenorii,and the disinfection time was increased or decreased continuously.According to the disinfection's effect,different sterilizing time was chosen for different parts of the plant.Then the disinfected parts were inoculated into three kinds of culture medium: potato glucose Agar medium,beef extract peptone medium and Gaoshi 1 medium to isolate the root and stem of Luohou fruit.The endophytic bacteria in various parts of fruit were isolated from the root of the fruit.20 strains of fungi,9 strains of bacteria,2 strains of actinomycetes,23 strains of fungi,10 strains of bacteria and 4 strains of actinomycetes were isolated from the roots,and 15 strains of fungi,10 strains of bacteria,and 10 strains of actinomycetes were isolated from the fruit.2.Screening of CGTase producing strains and identification of ND-6Eleven strains of CGTase producing bacteria were screened by phenolphthalein-methyl orange plate method and iodine-starch plate method from endophytic bacteria of Siraitia grosvenori.The strains with the highest CGTase activity were obtained by enzyme rescreening.The identification of the strain ND-6 morphology showed that the colonies were round,crumpled in the middle,and the edges were smooth and slightly yellowish.Under the light microscope,the strain was rod-shaped,its Gram stain was positive and spores located in the middle of the bacteria.In order to further determine the species of strain ND-6,Amplification of PCR from genomic DNA of ND-6 by 16 s r DNA universal primers of bacteria A band of 1445 bp was sequenced by PCR amplification product,and the obtained sequence was searched by NCBI.The results showed that the sequence of 16 s r DNA of ND-6 and Bacillus amyloliquefaciensus,Bacillus sp.,Velezensis was 99%.Therefore,ND-6 belonged to Bacillus in phylogenetic taxonomy.The results of morphological observation and molecular biological sequencing were used to determine the ND-6.The strain is Bacillus sp.ND-6,named Bacillus sp.ND-6.3.Study on fermentation conditions of CGTase producing strainThe effects of carbon source,nitrogen source,fermentation temperature and p H value on enzyme production were studied by single factor experiment.The orthogonal experiment was designed to optimize the enzyme production conditions according to the results of single factor experiment.The results showed that the enzyme activity of the strain was affected by carbon source,nitrogen source,fermentation temperature and p H value,and the initial p H value had a significant effect on the enzyme production of the strain.The optimum enzyme production medium of the strain was peptone 10 g,cyclodextrin 10 g,yeast extract 5 g,K2HPO4 0.2 g,Mg SO4·7H2O 0.2 g,Na2CO3 0.2 g,water capacity to 1 L,the initial p H value was 8,the fermentation conditions were as follows: inoculation amount was 4,liquid content was 70 m L(250 m L conical flask),fermentation temperature was 40 ? and 160 r/min for 72 h.The highest CGTase activity of the strain was 9753 U / m L,which was 1.43 times higher than that before optimization.4.Isolation,purification and Enzymatic Properties of CGTaseAccording to the optimized fermentation conditions,the strain ND-6 was fermented in preparation stage,and the fermentation broth was centrifuged to obtain the crude enzyme liquid.After ammonium sulfate salting out and Sephadex G-200 gel filtration chromatography,the crude enzyme solution was purified 5.48 times,and the yield was a band displayed by 34.3%.PAGE gel electrophoresis.The effect of p H,temperature and metal ions on enzyme activity was investigated.The results showed that the optimum reaction temperature was 60 ?,and the enzyme reaction was stable at 30~50 ?.The optimum p H value is 6.0,which is stable in the range of p H 5.0~7.0.Ca2+ can promote the enzyme.The activity of the enzyme was inhibited by Mn2+,Fe2+,Li+,Cu 2+.5.Preliminary study on the effect of CGTase on the conversion of bitter glycosides from Siraitia grosvenoriThe crude extract of Siraitia grosvenori was obtained by crushing it,adding 8 times of water,soaking it at 60 ?for 15 min with an ultrasonic cleaning machine of 80 k Hz and 350 W,and then adsorbing resin by macroporous adsorption.The purified bitter glycosides of Siraitia grosvenori were obtained by ion exchange resin and silica gel column.When the amount of starch was 3 times higher than that of the bitter glycoside of Siraitia grosvenorii,the CGTase 50 ?shock reaction 12 h TLC and HPLC were used to identify the highly polar substances.The sensory evaluation group also agreed after tasting that the bitter taste of Siraitia grosvenori was improved.