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標題: BurkholderiacepaciaCC-Al74分泌溶磷物質之分離及鑑定
Separation and Identification of the P-solubilizing Exudates by Burkholderia cepacia CC-Al74
作者: 林大發
Lin, Ta-Fa
Contributors: 楊秋忠
Chiu-Chung Toung
國立中興大學
關鍵字: Burkholderia cepacia;Phosphate solubilizing bacteria;Organic acid;P-solubilizing metabolite.
Burkholderia cepacia;溶磷微生物;有機酸;溶磷物質
日期: 2005
Issue Date: 2012-08-31 09:14:55 (UTC+8)
Publisher: 土壤環境科學系
摘要: 溶鈣磷細菌具有將難溶性磷酸三鈣轉變成為水溶性磷化合物的能力。多年來的研究顯示微生物所分泌的低分子量有機酸(分子量小於500 Da)是造成溶磷現象的主要原因,但對於分子量大於500 Da的溶磷物質則尚無人加以探討。因此本實驗的目的在於分離及鑑定溶鈣磷細菌分泌物中分子量大於500 Da之溶磷物質。本研究首先由本研究室保存的40株溶鈣磷菌中,篩選出生長速度快、溶磷能力高及溶磷現象穩定的實驗菌株Burkholderia cepacia CC-Al74,以透析膜區隔出該菌株分泌物中分子量大於500 Da的溶磷物質,經過有機溶劑萃取及再結晶處理後,使用高效液相層析儀(HPLC)分離並回收樣品中的溶磷物質,最後使用串聯質譜(ESI-MS/MS)及質譜分析軟體推測化學結構。本研究結果顯示挑選溶鈣磷細菌Burkholderia cepacia CC-Al74菌株的溶磷能力中約55.3 %是由分泌物分子量大於500 Da之物質所貢獻。CC-Al74菌株分泌物中分子量大於500 Da之濾液樣品經過萃取及再結晶處理後可以去除樣品的干擾物質,且證實分析物為極性物質。經HPLC分析的結果顯示使用Mightysil NH2 column及Aminex HPX-87H column配合折射率偵測器(RI detector)可以有效分離及純化樣品,回收的樣品經過溶磷能力分析後,證實樣品含有溶磷物質。HPLC管柱分離回收的溶磷物質經過串聯質譜(ESI-MS/MS)、質譜分析軟體「HighChem Mass Frontier」、化學分子描繪軟體「ISIS/Draw」及3D 分子模擬軟體「DS ViewerPro」分析後,本研究提出分子量為620 Da的溶磷物質之推測結構(proposed structure),命名為「Burkholderic acid」,該推測結構中含有四個羧酸基(-COOH),具有酸化及鉗合的功能,可以分解培養基中的難溶性磷酸三鈣,符合有機酸溶磷學說。本研究首次確認溶鈣磷細菌分泌物中具有分子量大於500 Da的溶磷物質,這是在微生物溶磷機制上的新發現,實驗結果將有助有機酸溶磷學說更趨完整。
Phosphate solubilizing bacteria (PSB) are being successfully applied as bio inoculants to increase phosphate uptake by plant and as an agent to remove excess phosphate from agriculture/aquatic systems. Many soil bacteria have proven efficient in enhancing plant growth and yield by solubilizing the inorganic phosphate present in the soil. Burkholderia cepacia CC-Al74, a bacterial strain isolated from the soil having a high ability for solubilizing tricalcium phosphate (TCP) was used in the present study as to understand the complex mechanism involved in the phosphate solubilization. A novel method was developed which, enables the collected filtrates to precisely assess extent of TCP solubilization by the strain CC-Al74. From the results of this study it was demonstrated that 55.3% of P-solubilization was contributed by the high-molecular weight metabolite of strain CC-Al74. The separation, purification and characterization of P-solubilizing metabolite from the strain CC-Al74 culture medium was performed using Mightysil NH2 and Aminex HPX-87H columns by high-performance liquid chromatography (HPLC). The proposed structure of P-solubilizing metabolite was analyzed by electrospray ionization (ESI) mass spectrometry and Mass Frontier software. The results showed that the molecular weight of P-solubilizing metabolite (M.W. > 500 Da) released by strain CC-Al74 was 620 Da. In the proposed structure of P-solubilizing metabolite (M.W. 620 Da) four carboxylic acid groups (-COOH) were evidenced. Therefore, by the process of acidification and chelation of P-solubilizing metabolite (M.W. 620 Da) TCP in culture medium was dissolved. The proposed structure of P-solubilizing metabolite (M.W. 620 Da) was named Burkholderic acid. In conclusion this study confirms that the high-molecular-weight (M.W. 620 Da) P-solubilizing metabolite is released by PSB, which was the major factor responsible for dissolution of TCP. This study also will give further scope for understanding and derive precise, but less understood, mechanism of P-solubilization by PSB.
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