以一株不需添加麩胺酸之新篩選菌株生產γ－聚麩胺酸及產物特性之研究

High-Level Production and Characterization of Poly-γ-Glutamic Acid by a Newly Isolated Glutamate-Independent Strain

γ－聚麩胺酸（γ-PGA）是一種極有應用潛力的微生物聚合物，可用在食品、化妝品、醫藥材料、農業和環境保護等領域。但以麩胺酸為原料所生產之γ－聚麩胺酸成本高，因而限制其大量之應用。篩選能使用較便宜的原料取代麩胺酸來生產γ-PGA之菌株是克服此限制的重要關鍵。本論文篩選出一株具有高耐鹽性的γ-PGA生產菌株Bacillus subtilis S01，該菌株為不需添加麩胺酸之菌種且具有提高γ-PGA產量之潛力，在不存在L－麩胺酸的情況下會產生γ-PGA。當B. subtilis S01於不含麩胺酸之ME-T培養基中培養至第6天，γ-PGA達最大產量（20.4g／L）。胺基酸分析發現該物質僅由單一胺基酸（麩胺酸）組成；膠體滲透層析（GPC）分析顯示其平均分子量（Mn）為4.2 x 10^6 Da；^1H-NMR及^（13）C-NMR之光譜顯示純化後之γ-PGA純度相當高（＞97％）；光學異構物之組成分析顯示D－麩胺酸／L－麩胺酸比例為97／3，且比例與添加之Mn^（2+）濃度無關。另外結果亦顯示，細胞內之麩胺酸消旋酶參與L-和D-麩胺酸的轉化。另外，B. subtilis S01係一株耐鹽性極高之菌株，在NaCl濃度高達5％時仍能生長與生產γ-PGA，但γ-PGA的分子量隨著鹽濃度的增加而降低，當NaCl鹽濃度為0.05％和5％時，經6天培養時間所產生γ-PGA之分子量分別為3.94×10^6及0.39×10^6Da，降低了約10倍。除會使用檸檬酸及甘油外，研究結果亦顯示B. subtilis S01亦可使用葡萄糖與木糖生產γ-PGA，因此應有利用農產廢棄物之水解液為碳源之潛力。本研究已成功篩選出一株具有高耐鹽性、不需添加麩胺酸且具有高γ-PGA生產力之特殊優良菌株，此應對降低γ-PGA的生產成本與促進γ-PGA工業化生產與應用有很大幫助。

γ-Poly-glutamic acid (γ-PGA) is a microbial polymer with promise for use in various fields, including food, cosmetics, agriculture, medicine, and environmental protection. However, using glutamic acid as a raw material to produce γ-PGA is costly, thus limiting its applicability. One approach to overcoming this limitation is to screen bacterial strains to identify a more efficient producer with lower production costs. This study investigated a strain with a high salt tolerance (Bacillus subtilis S01) that produces γ-PGA. Because this strain does not require glutamic acid, it has potential use to increase the production of γ-PGA. When B. subtilis S01 was cultivated in ME-T medium, a maximum yield of γ-PGA (20.4g / L) was obtained on the sixth day of cultivation. The quality of the produced γ-PGA was comparable to that of an authentic sample obtained from conventional fermentation. An amino acid analysis revealed that the substance consisted of only a single amino acid (glutamic acid). A gel permeation chromatography analysis showed that its average molecular weight (Mn) was 4.2 × 10^6 Da. ^1H-NMR and ^(13)C-NMR spectra revealed a high purity level (＞97%). A composition analysis of the optical isomers reveled that the ratio of D-glutamic acid to L-glutamic acid was 97:3, and the ratio was independent of the added Mn^(2+) concentration. In addition, the results revealed that intracellular glutamate racemase was the enzyme involved in the conversion of L- and D-glutamic acid. B. subtilis S01 has extremely high salt tolerance and can continue to grow and produce γ-PGA with a NaCl concentration of as high as 5%, although the molecular weight of the γ-PGA produced decreases as the salt concentration increases. Because this strain can effectively use glucose and xylose to produce γ- PGA, it has potential to use the hydrolysate in agricultural waste as a carbon source. In this study, a promising strain with high salt tolerance, glutamic acid independence, and high γ-PGA productivity was identified that can greatly reduce the production cost of γ-PGA and increase its industrial production and application.