The Mutation Breeding And Process Optimization Of High-producing Strain Of Bacteriorhodopsin

Bacteriorhodopsin（BR）can be synthesized on the cell membrane by Halobacterium salinarium.Bacteriorhodopsin has photosensitivity.Under light conditions,a series of molecular configuration changes of Bacteriorhodopsin molecules occur rapidly,then the transformation of the configuration and intermediates is back to the original state,and the optical cycle is completed.It is found that Bacteriorhodopsin is a kind of photosensitive protein,which is widely used in photoelectric converter,signal storage,biological new material and so on.It has a wide range of application prospects.In this paper,Halobacterium salinarium F0 was as a starting strain,then be treated with microwave-ultraviolet-atmospheric pressure plasma composite mutation.At first,it was treated with ultraviolet light.The condition was that 20W UV light exposure 3 minutes and 30 centimetres apart.A mutant of high-yield Bacteriorhodopsin producing UV53 was obtained.Its yield of Bacteriorhodopsin reached 67.65mg/L increased by 35.35%.Comparing with the original strain F0（yield is 49.98mg/L）.Then W75 was obtained after treated with microwave.The condition was that output power 700 w,pulse frequency 2450 hz,irradiation time 110 s,and the yield of Bacteriorhodopsin reached 94.87 mg/L increasd89.81%.Finally AP33 was obtained after treated with ARTP.The condition was that 120W mutation power,high purity helium ventilation 10SLM and 2 mm apart,exposure 200s,and the yield of Bacteriorhodopsin reached 110.42 mg/L increased by 120.92%.This thesis studies the influence of fermentation condition on the Bacteriorhodopsin production of high-yielding strains.First is the single-factor experiment.I choose those factors which affect significantly and optimize the condition of culture by orthogonal test.The condition after optimization is as following:pH 7.0,Rotation rate of shaking table:200 rpm,Liquid Leading:100mL,Temperature:37℃,Inoclum Amount:6%,the intensity of light:33.32μmol/m²s.At the same time this single-factor experiment shows that the NaCl,yeast extract as well as MgSO₄·7H₂O has marked impact on Bacteriorhodopsin producing.I optimized the three constituents through Response Surface Method in order to see their effect on the fermentation of bacteriorhodopsin.I did the multiple regression statistic analysis by SAS and the regression coefficient of the fitted equation is 0.9157.This result shows that this model applies to the theoretical speculation of Bacteriorhodopsin fermentation.The constituents after optimization are:NaCl 191.82 g/L,yeast extract 15.17 g/L,MgSO₄·7H₂O 26.68 g/L.Under this condition of culture medium and fermentation,the output of Bacteriorhodopsin reached 142.16 mg/L,which was29.86%higher than the output before optimization.The sequencing company compared strain AP33 with the Bacteriorhodopsin sequence of the original strain.The sequencing results showed no change.The paper also investigated the effect of adding different kinds of amino acids on the production of Bacteriorhodopsin in the culture medium.The addition of certain amino acids to the fermentation medium,which can promote the growth rate of the bacteria and allow the bacteria to reproduce rapidly.At the same time,the production of Bacteriorhodopsin increase.This paper studied that the effect of light on the mutant strain Halobacterium salinarum AP33.Using a 5 L fermentor amplify experiment.The lighting conditions were set to light intensity 33.32μmol/m²s and no light conditions.Fermentation cultures were used to measure daily microbial biomass and Bacteriorhodopsin production.After analysis,the requirement for light exposure was lower in the first three days.Halomyces salina had lower light requirements from the fourth day of fermentation.Biomass and Bacteriorhodopsin production are significantly different.This experiment provided an important basis for the expansion of the fermentation for Halobacterium salinarium.