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| Title: | 改良酵母菌種及培養基組成以增進γ-GC(γ-Glutamylcysteine)產量之研究 |
| Other Titles: | Studies of γ-Glutamylcysteine production by improving yeast strains and medium compositions |
| Authors: | 林欣儀
Lin, Xin-Yi |
| Contributors: | 黃進發;楊芳鏘
Hwang, Chin-Fa ,;Yang, Fan-Chiang
東海大學化學工程與材料工程學系 |
| Keywords: | 麩胱?;?;酵母菌
GSH;γ-GC;yeast |
| Date: | 2005 |
| Issue Date: | 2011-05-19T07:57:17Z (UTC)
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| Abstract: | 本研究主要目的為改良酵母菌BCRC 21727，藉由篩選MG (MethylGlyoxal)之敏感性變異株，提高酵母菌體胞內麩胺半胱胺酸(γ-glutamylcysteine ;γ-GC )之生產能力，並分析其特性與培養基組成之影響等。 在實驗項目方面首先以EMS進行酵母菌BCRC 21727突變，篩選γ-GC產能較高之菌株，並比較原始菌株與突變菌株之GSH I及GSH II酵素活性，H2O2、不同培養基組成、與胺基酸之添加等對菌體及γ-GC產能之影響，最後於5L發酵槽進行初步批式與饋料批式發酵試驗，並與三角瓶進行比較。 實驗結果顯示，以EMS進行酵母菌BCRC 21727突變，篩得MG敏感性變異株中，以編號SY-35679其胞內γ-GC含量顯著提高達4.77 ± 0.17 mg/g DCW約為原始菌株（0.59 ± 0.06 mg/g DCW）之8倍，γ-GC/GSH比值為0.53約為原始菌株（0.09 mg/g DCW）之6倍。此γ-GC高產量變異株SY-35679之酵素GSH I比活性提高，而對酵素GSH II則沒有明顯影響。另一方面，突變株單位菌體對H2O2之抗性有提升之現象。 在培養基方面，突變菌株SY-35679，在代號為 TM的培養基中，γ-GC含量為6.85 ± 0.36 mg/g DCW約為YPD培養基的1.4倍。在不同組成份濃度之試驗中，當葡萄糖濃度為20 g/L時結果較好，菌體內GSH含量為8.95 ± 0.33 mg/g DCW，γ-GC含量為4.77 ± 0.16 mg/g DCW。而氮源Peptone濃度在6 g/L時，各項分析指標顯示較高。於YPD培養基中添加適量L-Cysteine有助於細胞生長及增加胞內含硫物質，在12 hr添加5 mM L-Cysteine，可獲得較高之GSH與γ-GC含量。 以三角搖瓶或5L發酵槽初步培養比較，結果顯示突變菌株SY-35679胞內GSH與γ-GC含量均明顯較原始菌株高。而批式發酵槽所生產的菌體胞內γ-GC及GSH含量均較以三角搖瓶培養者高。然而，在5L發酵槽以非連續饋料批式發酵方式培養結果末期菌體量顯著增加，但是胞內γ-GC與GSH含量明顯降低，此結果顯示不同培養環境或條件會顯著影響菌體生長及菌體胞內含硫物質之生產。
The purpose of this study is to improve the γ-glutamylcysteine（γ-GC） production of a yeast strain BCRC 21727 by mutating and screening the MethylGlyoxal-sensitive mutants. Besides, we also analysis the mutant’s characteristics and effects of media on the cell growth and the intracellularγ-GC content. In experimental, first, the parent yeast strain, BCRC 21727, was mutated by EMS in order to isolate higherγ-GC-producing strains. Then, this isolated mutant with improved γ-GC production was compared with the parent strain, BCRC 21727, for their GSH I and GSH II enzyme activities, as well as, the effects of H2O2, medium compositions, or amino acid additions on the cell growth and the γ- GC productivity. Finally, we also do a preliminary test by batch or discontinuous fed-batch fermentation in 5L fermentors in order to provide data information for comparing with those obtained from the shake flasks. The results show that one significantly high γ-GC-producing mutant, SY-35679, was isolated from the MG-sensitive mutants derived from yeast BCRC 21727 through the EMS treatment. The intracellular γ- GC content（4.77 ± 0.17 mg/g DCW）in strain SY-35679 is around eight times compared to that (0.59 ± 0.06mg/g DCW) of the parent strain, meanwhile, the γ-GC/GSH ratio (0.53) of the mutant is about six times higher than that of the parent strain (0.09). Besides, the GSH I specific activity of strain SY-35679 was higher than the parent strain, however, no significant increase in GSH II activity was observed. On the other hand, cell growth against hydrogen peroxide was slightly inhibited, however, the anti-H2O2 ability in mutant was better than the parent strain based on evaluating the equal amount of cell mass. In medium studies, the γ- GC content of strain SY-35679 in TM medium was 6.85 ±0.36 mg/g DCW, which was 1.4 time of that in YPD medium. From the different concentration of media used, 20 g/L of glucose can reach better results with 8.95 ±0.33 mg/g DCW of GSH and 4.77 ±0.16 mg/g DCW of γ- GC. In the meantime, nitrogen source with 6g/L of peptone could reach the best results. On the other hand, higher amounts of GSH andγ- GC content were obtained with adding 5 mM L-Cysteine to the YPD medium at 12hr. Comparison of the γ- GC fermentation in either shake flasks or 5L fermentors, the results showed that intracellular γ- GC and GSH contents in mutant SY-35679 were significantly higher than those in the parent strain. Besides, higher amounts and content of γ- GC were obtained at 30 h batch fermentation in a 5L fermentor when comparing with those from shake flasks. However, different profiles of GSH orγ- GC production were observed in a discontinuous fed-batch fermentation. These results indicate that incubation situations significantly affect the production of sulfur-containing compounds. |
| Appears in Collections: | [化學工程與材料工程學系所] 碩博士論文
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