The Fermentation Process Of Halobacterium Salinarum Producing Bacteriorhodopsin(BR)

Bactriorhodopsin is a kind of nano pigment photosynthetic protein,which is the unique protein in purple membrane of Halobacterium salinarium.The bacteriorhodopsin protein is widely studied,which can be used in medical,optical and materials.Bactriorhodopsin has broad development prospect and important research value.The growth environment of Halobacterium salinarium is high content of salt.The formation of purple film is under the condition of illumination.In this paper,the fermentation conditions,the extraction technology and the properties of bacteriorhodopsin were studied.The growth of Halobacterium salinarium and the content of bacteriorhodopsin were influenced by some factors.The factors were illumination time,nitrogen source,carbon source,sodium chloride,metal ion,temperature,speed,initial pH,inoculation size and liquid volume,which were optimized by single factor experiments.The results of single factor experiments showed that carbon source had no significant effect on the growth of Halobacterium salinarium and the synthesis of bacteriorhodopsin,while nitrogen source could promote the growth of Halobacterium salinarium.Preliminary experimental results were that illumination time were 7days,Nitrogen source were yeast extract powder and acid hydrolyzed casein,sodium chloride was200 g/L,metal ions were Fe2+ and Mn2+,culture temperature was 37°C,speed was 200 r/min,initial pH was 7.2,inoculation was 6% and liquid volume was 100 m L.The content of bacteriorhodopsin of Halobacterium salinarium were influenced dramatically by magnesium sulfate Heptahydrate,potassium chloride and yeast extract powder through the Plackett-Burman experiments.Culture conditions were optimized through the steepest ascent experiment and response analysis.Results showed that yeast extract powder,magnesium sulfate heptahydrate and potassium chloride all had significant influence on the content of bacteriorhodopsin of Halobacterium salinarium.The optimal culture conditions were yeast extract powder at 15.156g/L,magnesium sulfate heptahydrate at 26.718 g/L,and potassium chloride at 3.186 g/L,the maximum predicted value of the content of bacteriorhodopsin was at 62.03 mg/L.After the checking experiment,the maximum predicted value was consistent with mean value of verification test and the content of bacteriorhodopsin increased 24.1% at optimized condition.The optimal conditions were determined as follows: yeast extract was15.156 g/L,magnesium sulphate was 26.718 g/L,potassium chloride was 3.186 g/L,,acid hydrolysis casein was 7.5 g/L,citric acid three sodium was 5 g/L,pH was 7.2,liquid volume was 100 m L,and inoculation was 6%.In this paper,the technological conditions of bacteriorhodopsin of H.salinarium CGMCC 1.2368 in 5 L fermentor were studied,which provided the basis for the further application of the strain.The effects of temperature,pH,rotation speed and aeration rate on the production of bacteriorhodopsin of H.salinarium CGMCC 1.2368 were studied by single factor experiment in the fermentation process of 5 L fermentor.The results showed that the optimal fermentation conditions were temperature at 37 ?,agitation speed at 250 r/min,ventilation volume at 1.3 vvm,initial pH at 7.2.pH was not regulated in the process of fermentation.Based on the results,the feeding process of corn pulp with different flow rate was studied,and the optimal feed flow rate was determined to be 0.2 m L/min.Under these conditions,the maximum biomass was 4.531,the maximum BR yield was 90.86 mg/L,which was about respectively43.8%?36% greater than those obtained with initial fermentation conditions.In this paper,single factor experiment and orthogonal experiment were used to investigate the effects of ultrasonic power,ultrasonic time and the amount of DNase on the extraction of bacteriorhodopsin.The results showed that the optimal extraction conditions were ultrasonic power at 120 W,the ultrasonic time at about 40 minutes,and the addition of DNase at about 60 mg/L,and the optimized extraction rate was up to 65.89 mg/L.The absorption spectrum of BR was determined by UV-visible Spectrophotometry.The functional groups of BR were analyzed by fourier transform infrared spectroscopy.The nanometer protein was determined by atomic force microscopy.The molecular weight of BR was determined by SDS-polyacrylamide gel electrophoresis.The types and contents of amino acids of BR were analyzed by Amino acid analyzer.The secondary structure of BR was analyzed by Circular Dichroism Chiroptical Spectrometer.