Isolation And Characterization Of Bacteriorhodopsin And Unsaturated Fatty Acids From Halobacterium Halobium

In this study, the culture condition of Halobacterium halobium, the preparation process of Bacteriorhodopsin and the extraction of unsaturated fatty acids were investigated. The purple membrane of archaeon halophile Halobacterium halobium contains bacteriorhodopsin which is a retinal-containing membrane protein that functions as light-driven proton pump packaged in an ordered two-dimensional crystalline array at high concentration. The bacteriorhodopsin has very good adaptability to outside environments, and thus much attention has been focused on the property research of biologic optical material and the utilization of new material.The halophile Halobacterium halobium was a kind of ancient bacteria who lived in very similar high salinity conditions to sea fish and diatoms. In addition, recent research indicated that various fish species and diatoms in oceans could produce unsaturated fatty acids which played an important role in human nutrition. Thus, we assumed that it had the capability to synthesize unsaturated fatty acids, which had not been reported as a fact in literatures yet.This experiment was to study the culture condition of Halobacterium halobium which was used to prepare bacteriorhodopsin. In our study, the influence of various culture periods and culture medium on the cell density were investigated to optimize the ferment condition: Cell fermentation was terminated at 7 days post; The composition of culture medium was Peptonefrom Casein (acid hydrolysate) 10 g·L^-1 , the yeast extract 10 g·L^-1 , MgSO₄·7H₂O 20 g·L^-1 , Sodium citrate 3．0 g·L^-1 , KC1 2．0 g·L^-1 , FeSO₄·7H₂O 0．05 g·L^-1 and NaCl 200．0 g·L^-1 .Halobacterium halobium was cultivated in a composite medium. According to its growth curve, the Halobacterium halobium culture entered its stationary phase when the OD₆₀₀ value exceeded 1．7．After ferment, the broth was centrifuged at 3 000 rpm for 15min to collect the cells. Cell disruption was carried out by suspending each gram of cells in 15 mL of water followed by ultrasonification. The suspension was centrifuged at 8 000 rpm for 10 minutes. The supernatant was to prepare bacteriorhodopsin while the pellet was for the isolation of crude fatty acids. Then the supernatant was centrifuged at 12 000 rpm for 30 minutes. This pellet was utilized to prepare bacteriorhodopsin while the supernatant was used for the isolation of crude fatty acids. The pellet was rinsed by deionized water and centrifuged at 12 000 rpm for 30 minutes until the supernatant turns colourless. Finally cane sugar of 60%, 43%, 35% density grads were used to purify the sample.The supernatant and the pellet preserved during bacteriorhodopsin preparation were used for isolation of crude fatty acids by saponification and extraction. The supernatant was transferred into a 250 mL round bottom flask with pH12 ajusted by KOH, and stirred for 60 minutes at 80℃.The mixture was subject to filtration for removing the insoluble debris after a cool to room temperature. The filtrate was adjusted to pH2．0 with hydrochloric acid and was extracted twice with methanol/chloroform. The organic phase was evaporated to obtain pale yellow crude fatty acids. Crystallization then took place in acetone at-16℃to remove fatty acids in which the carbon chain length was shorter than 13．The sample was obtained after purification and analyzed by gas chromatography. The results demonstrated that Halobacterium halobium could synthesize multiple unsaturated fatty acids, particularly the three important polyunsaturated fatty acids arachidonic acid (1．12%), eicosapentaenoic acid (16．76%), and docosahexaenoic acid (9．38%).The purified protein bacteriorhodopsin, with a molecular weight of 26 kDa, displayed a single band in SDS-PAGE and its absorption spectrum within the range of 400～750nm was consistent with the literature. Halobacterium halobiwn has already been used for decades to prepare bacteriorhodopsin. We found that several important unsaturated fatty acids could be extracted from the bacterial waste, which extends its application scope and might bring additional benefits to humanity.