由Haloferax spp.生產C50類胡蘿蔔素之研究

Abstract

Haloferax spp.為一極端嗜鹽古菌，必須生長於含有1.5-4.5 M NaCl之環境中。過去之研究顯示該菌株會產生一類C50類胡蘿蔔素，主要為菌紅素(bacterioruberin)，其作用為保護細胞免於強烈光照的致命傷害。本研究即利用Haloferax spp.進行兩階段生產菌紅素相關之C50類胡蔔素，並探討最適之生產條件。第一階段使用ATCC 1176培養基作為基礎培養基進行菌體的生產，第二階段則是利用第一階段生成之菌體，以靜止細胞培養方式進行C50類胡蔔素之生產。藉由改變第二階段培養之NaCl濃度、鎂離子(Mg2+)添加量、碳源種類、通氣量及培養時間等，探討對C50類胡蘿蔔素產量之影響。結果顯示第一階段使用含1%葡萄糖之ATCC 1176培養基，以37℃、150 rpm、1vvm培養至對數期中期時將培養液轉出離心，得到之菌體利用含5% NaCl、0.1% CH3COONa並添加8% MgSO4．7H2O之全合成培養基，以37℃、120 rpm震盪培養24 hr可得到合理之最高C50類胡蘿蔔素產量(0.604±0.005 Abs/mL broth)。 色素粗萃物之組成分析與鑑定主要利用TLC及UV-VIS光譜分析。結果顯示色素粗萃物中主要可能存在有3種C50類胡蘿蔔素(Spot 1-3)及1種C45類胡蘿蔔素 (Spot 4)。Spot 1為菌紅素約佔了紅色色素之70%，Spot 2為單縮水菌紅素，約佔紅色色素之20%，Spot 3為雙縮水菌紅素，約佔紅色色素之15%。Spot 4為C45類胡蘿蔔(2-isopentenyl-3,4-dehydrorhodopin)。 色素粗萃物以silica gel進行管柱區分，收集40% EtOAc frc.、EtOAc frc.及MeOH frc.三個區分，經TLC分析比對可知40% EtOAc frc.之主要成分對應為Spot 3；EtOAc frc.對應為Spot 2；而MeOH frc.對應為Spot 1。 色素粗萃物及經管柱區分得到之三個區分物分別進行抗氧化能力測定之結果如下：還原能力由強至弱依序為：Crude extract≒MeOH frc.＞40% EtOAc frc.≒EtOAc frc.≒BHA＞β-carotene＞α-tocopherol；DPPH自由基清除能力由強至弱依序為：Crude extract≒MeOH frc.≒BHA＞β-carotene≒40% EtOAc frc.＞EtOAc frc.＞α-tocopherol。

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.