| ?-polyglutamic acid(?-PGA),produced by the fermentation of microorganisms,uses the left and right optically active glutamic acid as a monomer to form a homogeneous polypeptide by polymerization at the?-position on the amide bond.Glutamic acid,which can be degraded into single molecules,is non-toxic to the human body and the environment.It is an environment-friendly polymer material.Due to its excellent water-solubility,superior adsorption and biodegradability,it can be used as a water-retaining agent,heavy metal ion adsorbent,flocculant,sustained-release agent,and drug carrier for cosmetics,environmental protection,food,Medicine,agriculture and other fields.At present,there are many researches on ?-PGA in various fields at home and abroad,and the related literature reports are also increasing day by day.However,they are all in the laboratory research stage and cannot achieve the purpose of industrial production.Therefore,in order to achieve the increase of?-PGA production,screening of its production strains,selection of mutagenesis,optimization of fermentation processes and other work are very important.In this thesis,a ?-PGA producing strain(identified as Bacillus amyloliquefaciens)was screened from soybean meal,and its quantitative detection method was studied.The strain was obtained by mutagenesis using ARTP mutagenesis technology.The high-yielding mutant PI142 was used to optimize the fermentation process of the mutant strain PI142.The following results were obtained:(1)Using the characteristics of polyglutamic acid and the charge of the dye,Bacillus licheniformis B146 was used as a tool strain to study the effects of dye type,dye concentration,and coloration time on the separation of ?-PGA-producing bacteria.The method for obtaining ?-PGA producing bacteria by acid red dye plate separation is as follows: acid red is added at a concentration of 0.004%,and the time for coloring observation is 24 hours.The ?-PGA-producing strain B.amyloliquefaciens N1 was obtained by acid red separation plate method combined with morphological characteristics,physiological and biochemical indicators,qualitative analysis of products and16 s r DNA sequence analysis.(2)Quantitative ?-PGA was determined by UV spectrophotometry.The results showed that the maximum absorption wavelength of ?-PGA in the aqueous solvent system was 218 nm,and the linear relationship was good inthe range of 20-200 ?g/m L.The detection and quantification limits were 4.11?g/m L and 13.71 ?g/m L,respectively.UV spectrophotometry is suitable for p H 5.8-9.0,temperature less than 50 °C,the ionic strength of the detection system has no significant effect on the ?-PGA quantification;the accuracy of the method is 0.642%,and the recovery rate is 95.49%.The CTAB method was superior to the UV spectrophotometry in terms of stability,precision,and recovery,but the fermentation broth samples showed no statistically significant difference between them.The UV spectrophotometry can be used instead of the CTAB method for the fermentation broth.The ?-PGA quantification is used to overcome the insufficiency of CTAB in the analysis process of the CTAB method,the harsh reaction time,and the cumbersome operation process.(3)By comparing the dynamic changes of the circle-to-circle ratio of B.amyloliquefaciens N1 and the yield of ?-PGA during acid red separation plate,well plate culture,and shake flask culture,the initial screening time of the mutant strain during the mutagenesis process was determined to be 24 hours.The mutant liquid fermentation rescreening was performed using 24 microplate culture instead of shake flasks.The mutagenesis of B.amyloliquefaciens N1 by ARTP technique was carried out.After 5 rounds of breeding,a mutant PI142 with improved ?-PGA synthesis ability was screened.The yield of ?-PGA reached 0.54 g/L,which was higher than that of the original strain.About 42.11%,subcultured showed good genetic stability.(4)B.amyloliquefaciens PI142 Fermentative ?-PGA is affected by various nutrient conditions and culture conditions.The use of small molecule carbon source by PI142 is better than that of macromolecular polysaccharide carbon source.The use of organic compound nitrogen source is better than that of small molecule inorganic nitrogen source.When the bean cake powder concentration reaches 0.4%,the biosynthesis of ?-PGA basically tends to Constant;when the precursor substance sodium glutamate is added,the biosynthesis of ?-PGA can be greatly increased;in the fermentation conditions,the p H value has a significant effect on the fermentation yield of?-PGA compared with other conditions.On the basis of single-factor tests,using ?-PGA production as a response indicator and combining three factors and three levels of response analysis,the important factors affecting ?-PGA production were optimized,and the optimal fermentation process parameters for B.amyloliquefaciens PI142 were obtained as: Sucrose concentration3.77%,bean cake powder 0.4%,sodium glutamate 2.01%,dipotassium hydrogen phosphate 0.7%,initial p H 8.24,and the average ?-PGA yield reached 3.19 g/L from 0.54g/L,4.9 times before optimization. |
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