Screening Of High ?-polyglutamic Acid Producing Strains And Studies On The Fermentation Condition And Stress Resistance

?-Polyglutamic acid is a kind of natural macromolecule peptide that consists of glutamic acid units linked by amide linkages between a-amino and ?-carboxylic acid groups.As green and new environmentally-friendly material,?-polyglutamic acid has been used in many fields and attracted more and more attentions because of its excellent performance including excellent water absorption,cohesiveness and biodegradability.Besides,the research on ?-polyglutamic acid is more active.At present,the main preparation method of ?-polyglutamic acid is microbial fermentation,the production condition is mild and easy to control,the synthesis process is short and easy to expand production.However,problems such as microbial synthesis,low strain yield,complex metabolic pathway and vague synthesis mechanism restricte the study of ?-polyglutamic acid.Therefore,on the one hand,we actively look for and screen wild strains that produce ?-polyglutamic acid efficiently and optimize their culture medium and fermentation conditions.On the other hand,we study the synthesis mechanism and pathway of ?-polyglutamic acid,understanding?-polyglutamic acid from a molecular biology perspective.In this paper,we studied from the select of high ?-polyglutamic acid production strains,optimization of feimentation conditions and stress resistance,in order to provide guidance for industrial production and application.1.Screening from a variety of high-amino acid fermented foods,200 strains of strains were screened by the smooth and viscous surface of?-polyglutamic acid,and the yield of ?-polyglutamic acid was measured by CTAB method.Sieve to obtain a high yield of ?-polyglutamic acid with an initial yield of 7.67 g/L,named ZJS18.It was positive bacteria determined by Gram stained,the spore staining,initially identified as Bacillus.Then it was identified by physiological and biochemical identification and molecular biology.Through 16SrDNA sequence amplification and comparison and phylogenetic tree,it was concluded that ZJS18 was Bacillus subtilis.2.The ?-polyglutamic acid-producing strain was optimized by the optimization of various culture medium and culture conditions to improve the yield of ?-polyglutamic acid.The effects of carbon monoxide,nitrogen source,glutamic acid precursor,metal ion,culture temperature,shake flask speed and pH value on the yield of ?-polyglutamic acid were studied by Plackett-Burman test designed to select the three factors,namely,sucrose,yeast powder and sodium glutamate,which had a great influence on the synthesis of ?-polyglutamic acid.The response surface was optimized,and the center point of the response surface was determined by the steepest climbing experiment.The two factors were optimized by Box-Behnken test design and response surface method.The results showed that the optimum fermentation conditions were as follows:sucrose 64.40 g/L,Yeast powder 7.10 g/L,sodium glutamate 57.96 g/L,sodium chloride 30 g/L,magnesium sulfate 0.3 g/L,dipotassium hydrogen phosphate 2 g/L,initial pH 7.5,inoculum 5%40 mL/250 mL,temperature 37?,shaking speed 200 r/min,fermentation time 36 h.Under the above conditions,the yield of y-polyglutamic acid produced by Bacillus subtilis ZJS18 was 13.20 g/L.The optimum fermentation conditions for the production of ?-polyglutamic acid after the optimized Bacillus subtilis were 1.88 times higher than those before the optimization.The results provided a good basis for the production and application of ?-polyglutamic acid.3.?-Polyglutamic acid is an extracellular polymer that has the ability to resist adverse environmental conditions.Three types of environmental impacts including heat shock,pH shock and osmotic shock were used to explore its stress resistance.The results showed that the yield of?-polyglutamic acid of strain ZJS18 significantly increased under the impact of heat and osmotic shocks.Next,through the heat shock and osmotic shock to explore the synthesis pathway of endogenous glutamic acid.Five different catalytic enzymes in different stages of synthetic pathway were chosen including glutamate dehydrogenase,isocitrate dehydrogenase,a-ketoglutarate dehydrogenase,L-glutamate synthase and glucose hexaphosphate dehydrogenase.By measuring the activities of five enzymes under different impacts and finding out how they affectithe synthesis of ?-polyglutamic acid,the synthesis pathway of ?-polyglutamic acid was deduced.And a new method to increase the yield of?-polyglutamic acid was established.