High-throughput Breeding Of Glutathione-producing Strains And Extracellular Seceretion Characteristics

Glutathione(GSH)is an important non-protein mercapto tripeptide which is widely found in nature and has important physiological activity.It is extensive used in food,medicine,cosmetics and other fields.Currently,the industrial production of GSH is mainly microbial fermentation production.In this paper,a rapid detection method for GSH in fermentation broth was firstly established and optimized to improve the screening efficiency of mutants after mutagenesis.The yeast cells were treated with atmospheric pressure room temperature plasma and chemical mutagen,and the high yield mutant D-17-51 was obtained by combining with ZnCl2 resistance screening.Finally,the optimization of surfactants increased the cell membrane permeability and further improved fermentation yield.The main research contents and results were as follows:(1)Establishment and optimization of GSH rapid detection methods.The strains deposited in the laboratory were activated,and the high-yield strains were initially screened for subsequent experiments.The measurement of GSH was compared with many literatures.According to the laboratory's own conditions,the DTNB method was used to measure with the microplate reader,and the main factors affecting the DTNB method were optimized.It was confirmed that the reaction system for measuring GSH was 0.25 mol/L tris(pH 8.0)0.6 mL,3%formaldehyde 0.1 mL,and 0.1 mmol/L 2-nitrobenzoic acid analysis solution 1.5 mL.The amount of glutathione sample was 0.5 mL and the precision experiment was performed.Finally,after the mutagenesis of 45 mutant strains was fermented in 24-well plates,the content of glutathione in the fermentation broth was detected by spectrophotometer and microplate reader at 412 nm and the fitting analysis was performed to verify the The feasibility of the method,the correlation coefficient of the microplate reader method and the spectrophotometer method is R=0.9503,which indicates that the two detection methods have good consistency.The experimental results show that the microplate reader method can easily and quickly determine the GSH content in the fermentation broth,which lays an experimental foundation for the subsequent high-throughput screening of high-yield GSH strains.(2)Breeding GSH-producing bacteria by compound mutagenesis.The high-yielding strain screened from previous experiments was used as the starting strain D for mutagenesis,and the initial yield was 122 mg/L.The mutant strains were screened by using zinc chloride resistance plate.When the concentration of zinc chloride was 0.6g/L,the colony was relatively large and the ability to produce GSH was strong.After initial screening,the 24-well plate fermentation was used to screen 37 mutant strains.The correlation between the two methods was verified by shaking flask and 24-well plate fermentation.The correlation coefficient R2 between the two methods was 0.97653,which showed that the two methods had good consistency.It was proved that 24-well plate fermentation could be used for mass screening of GSH-producing strains.The method improves the efficiency of screening.According to this method,the high-yield strain D-17-51 was induced by combination of ARTP and EMS.The yield of the strain reached 359.67 mg/L,which was 194.8%higher than that of the original strain.The genetic stability of mutagenic high-yielding strains was tested.After 5 passages,the yield of mutagenic high-yielding strains decreased slightly and was relatively stable.(3)Effect of surfactant on the extracellular secretory GSH characteristics of mutant D-17-51.Surfactants can be used to increase the permeability of cell membranes due to their unique molecular structure-both hydrophilic and lipophilic,similar to the structure of cell membranes.Four kinds of surfactants were investigated.It was found that the anionic surfactant SDS had a positive effect on the extracellular accumulation of GSH,and the extracellular GSH content increased with the increase of SDS.The effects of SDS on cell growth,synthesis of GSH and its intracellular and extracellular distribution were studied.When the concentration of SDS reached a critical concentration of 4 g/L,the cells grew slowly,the ability to synthesize GSH decreased,and the amount of cells present at the end of the fermentation was small.SDS was added at Oh and 8h after fermentation.With the increase of time,the cells grew slowly and did not promote GSH efflux.When SDS was added for 16h and 24h,the cells entered a stable phase,the amount of synthetic GSH increased and a large amount was secreted to the outside of the cell;Addition of SDS for 40h did not have much effect on the synthesis of GSH,but it could promote extracellular secretion of GSH.