由Haloferax spp.生產C50類胡蘿蔔素之研究

Abstract

Haloferax spp., an extremely halophilic archaea bacterium, must grow in the environment containing 1.5-4.5 M NaCl. It can produce a group of 50-carbon (C50) carotenoids, mainly bacterioruberin. According to the previous reports, the physiological role of bacterioruberin, the major pigment in halobacteria, was suggested to protect the cells against severely solar irradiation. The aim of this study was to investigate the cultural conditions for the production of C50-carotenoids by Haloferax spp. using a two-stage strategy. At the 1st stage culture, we used ATCC 1176 medium as the basal medium and studied on increasing the production of Haloferax spp. biomass. The 2nd stage culture was simulated as resting cells culture of the 1st stage harvested cells to conduct the bioconversion of C50-carotenoids and studied on the influence of nutrient factors and cultural conditions for C50-carotenoids production at the stage. The results indicated that the optimum conditions for C50-carotenoids production by Haloferax spp. were to use ATCC 1176 medium contained 1% glucose as the initial medium and incubated the cells to the mid-log phase of growth under 37℃, 150 rpm of agitation and 1 vvm of aeration at the 1st stage culture. Successively, the 2nd stage was proceed to the bioconversion of C50-carotenoids by means of incubating the 1st stage harvested cells in the synthetic salts medium (briefly contained at 5% NaCl, 0.1% CH3COONa and 8% MgSO4．7H2O) under 37℃,120 rpm of agitation for 24 hr. The yield of C50-carotenoids was reached 0.604±0.005 Abs/mL broth. After the chromatographic fractionations, the main components of crude pigment extracts were analyzed and identified according to the TLC profiles and the corresponding UV-VIS spectra. The results revealed that crude extracts were mainly composed of three C50-carotenoids (Spot 1-3) and a C45-carotenoid (Spot 4). Spot 1 was the major component of crude extracts (70% around of total intensity) and was identified as the compound of bacterioruberin. Spot 2 and Spot 3 were respectively conducted to monoanhydrobacterioruberin and bisanhydrobacterioruberin. Additionally, Spot 4 was suggested as the compound of 2-isopentenyl-3, 4-dehydrorhodopin (a C45-carotenoid) according to the information of UV-VIS spectrum. Furthermore, Fraction containing Spot 1, Spot 2 or Spot 3 was successfully separated from the crude extracts by silica gel chromatography and was respectively corresponded with 40% EtOAc frc. (bisanhydrobacterioruberin major), EtOAc frc. (monoanhydrobacterioruberin major) and MeOH frc. (bacterioruberin major). Antioxidative activities of the crude extracts and the three fractions were determined using the methods of reducing power analysis and DPPH radical scavenging ability. The results of reducing power analysis were shown in the order of crude extract≒MeOH frc.＞40% EtOAc frc.≒EtOAc frc.≒BHA＞β-carotene＞α-tocopherol. And the results of DPPH radical scavenging ability were shown in the order of crude extract≒MeOH frc.≒BHA＞β-carotene≒40% EtOAc frc.＞EtOAc frc.＞α-tocopherol.