| Canavanine was a natural nonprotein alkaline amino acid with an isoelectric point8.2, and it is easily soluble in water. Canavanine exists in nonprotein alkaline L-alpha-amino acids of some beans and has certain toxic, tumor and metabolic resistance. The extraction methods have two kinds:one is chemical synthesis and the other is the biological extraction method, the latter is easier and simpler. L-canavanine is regarded as a potential biological pesticide; it is a nitrogen storage compound, so it is used as a kind of fertilizer. The most important is that canavanine has lower toxicity and no contamination to natural environment, so it has high research value. At present, extracting canavanine from the plant tissue is reported only by Dongfang Chen in domestic. She found that L-canavanine and2,4-dinitrofluorobenzene in borax buffer had special color reaction. At present, L-canavanine and2,4-dinitrofluorobenzene in borax buffer reaction is the simplest and most rapid method. But we found the sensitivity is not high in the process of testing canavanine with the existing of many kinds of amino acid, and she has not performed spectral scans on the reaction of all reagents and the measurement of the stability of the complex compound. In addition, there are few reports in domestic about how to extract canavanine from the organization of canavalia ensiformis. This paper mainly took the fresh weight of canavanine, contents of canavanine and urea as complete index, performed experiment on multiple factors and multi level, established suspension-cultured system, and induced with chemical factors and physical factors, to improve the canavanine extractive method. The results are as follow:(1) Study on canavanine extraction method and the optimum condition of2,4-dinitrofluorobenzene and canavanine reaction in pH9.5borax acid buffer solution, and optimizing measurement of canavanine. Using spectrophotometry determines the absorbance of2,4-dinitrofluorobenzene and canavanine complex compounds in571nm. The results show that the effect of water extraction of canavanine is the best. When the DNFB-acetonitrile with the dosage of30μL, the complex compounds of canavanine and DNFB reaction in571nm have the maximum absorption value. The complex compounds’ OD value increased after the stable value in30min. The corresponding linear regression equation is y=0.0083x-0.052, and the correlation coefficient is R2=0.9986.(2) In the process of organizing suspension culture, the callus’growth curve was roughly "S" type; the growth period was18days, divided into delay period, index period and stable period. At the beginning, the cells adapt to the new environment and the callus’weight increase slowly. In the cultivation of the12th to18th days, the cells divide and grow rapidly, and in the18th to21st days the cells’weight increase slowly because of the accumulation of harmful substances in the culture medium and the consumption of nutrient. The content of canavanine was first a slight decline and then rise, and the last drop, similar to "S" shape.(3) The cells growth curve of Semen Canavaliae suspension culture and the influence of different sucrose,6-BA, NAA to the cells growth callus and content of L-canavanine and urea in the process of cultivation were mainly studied. The results showed that:30g/L sucrose was best fit for cell growth with organization up to maximum9.02g,20g/L sucrose was the most beneficial to of L-canavanine synthesis, up to maximum at18th day, cell fresh weight and urea content reach maximum at18th day with6-BA for4.Omg/L,content of L-canavanine reach maximum with6-BA for2.Omg/L at18th day;1.Omg/L NAA was best fit for cell growth and the production of L-canavanine.(4) According to the time of growth medium into production training, the contents of L-canavanine and urea were most at12th day by using two step culture methods. With jasmonic acid methyl ester and two hydrogen jasmine ketone acid methyl ester inducting to tissue of Semen Canavaliae suspension culture, L-canavanine yield was the highest when jasmonic acid methyl ester for40μmol/L and two hydrogen jasmine ketone acid methyl este for20μmol/L.(5) Ultrasonic and illumination were used in the process of Semen Canavaliae suspension culture, the results showed that:the contents of L-canavanine and urea were most at9th day by60Hz ultrasonic, the contents of L-canavanine and urea were more in the culture with12hours of illumination than0and24hours of illumination.(6) Water extraction was best for extraction of L-canavanine with the largest amount; L-canavanine purified by732cation exchange resin with ammonia for elution agents. The results showed that the optimal concentration of ammonia water was3mol/L; the ninhydrin experiment showed that Method the basic amino acids could be isolated by cation exchange resin. L-canavanine was basic amino acids, with isoelectric point8.2. |
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