| At present, the nicotine content is a little high in some tobacco planting areas; lots of waste material containing high nicotine was producted during making cigarette; nicotinic pesicides were used in agriculture; all of these brought the economy and environment problems. Agriculture and chemical ways can slove the high nicotine content in some tobacco, but they took lots of time and energy; while the application of microbiology and enzyme provide new channels to solve the above problems.This study was aimed to observe the bacterium Pseudomonas marginalis. ND which was found to have the ability to degrade nicotine. The study introduced the method of strain isolation and identification; optimized the medium and growth condition of strain ND in order to improve the rate of nicotine degradation; as well as purified the nicotine degradation enzyme from strain ND and studied the properties of the enzyme. The main conclusions of the thesis were listed as follow:1. Soil which has been planted by tobacco for years was chosen as the source of bacterium strain isolation, bacterium which can use nicotine as sole carbon and nitrogen source. Strain ND obtained after primary and repeated isolation was able to degrade70.40%nicotine in1mg/ml nicotine liquid medium within2d. Colonies observed were small, milky white, opaque, gibbosity in middle, surface moist, and smooth rim, while the individual morphology was small, short rod, blunt round in two ends, without caspual. The16SrDNA sequence of strain ND had99%homeology Pseudomonas marginalis sequences in GeneBank, thus strain ND was identified as Pseudomonas marginalis.2. Acceptable range conditions for the growth and nicotine degradation of strain ND were observed after several single factorial tests. The optimum condition was temperature20-30℃; pH6.5-8.0; nicotine concentration of the medium0.01%-0.2%. When innoculum concentration was increased from1%to10%, the nicotine degradation rate was increased from27.13%to66.31%. When different carbon sources were added into medium, the synergy was heightened gradually from trisodium citrate, fractose, maltose and glucose, respectively. Similar to it when different nitrogen sources were added, the synergy was heightened gradually from ammonium nitrate, ammonium sulfate, peptone and tryptone. According to the results of single factorial tests, the multi factorial experiment was conducted. After the range analysis and variance analysis results of orthogonal test, the best condition for strain ND to grow and degrade nicotine was pH8.0, inoculum concentration7%, maltose concentration0.1%, peptone concentration0.1%, nicotine concentration0.1%, temperature28℃, rotate speed120r/m. Under this condition, the nicotine degradation rate nicotine was82.40%, higher than70.4%in medium without optimization. Finally, the optimum medium for strain ND to degrade nicotine was Nicotine1.0g; K2HPO4·3H2O13.3g; KH2PO44g; MgSO4·7H2O0.2g; maltose1.0g; peptone1.0g; trace elements solution (MnSO4·7H2O0.4g; CaCl2-2H2O0.2g; FeSO4·7H2O0.2g adding0.1mol/L HCl to get the final volume100ml)0.5ml; Water1000ml; pH8.0. The culture was incubated best at28℃,120r/min.3. After cultured in optimum medium under optimum condition, the strain ND cells were collected and shattered before the degrading nicotine enzyme was isolated. The enzyme was precipitated at the fastest speed in40%-80%saturated ammonium sulfate solution. After dialysis, concentrating and Sephadex chromatography, the single enzyme component degrading nicotine was isolated; with molecular weight was50kDa. Its enzyme activity was increased about160times after purification, from1.76U/ug to293.47U/ug. Through the study of the enzyme properties, it was found that:the enzyme was active in narrow pH range; the optimum pH range for its activity was from6.4to7.0.; the enzyme had the strongest activity at30℃though it can be kept active between15℃and40℃. Its Kmwas3.10×10-5mol/L, Vmax was6.21×10-3g/(L·min). |
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