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研究生:顏文章
研究生(外文):Ven-Chang Yen
論文名稱:芽孢桿菌蛋白質分解酵素生產菌株之開發
論文名稱(外文):Developing of a potential strain for the production of bacillus protease
指導教授:李根永李根永引用關係
指導教授(外文):Ken-Yuon Li
學位類別:碩士
校院名稱:東海大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:1994
畢業學年度:82
語文別:中文
論文頁數:85
中文關鍵詞:芽孢桿菌蛋白質酵素篩選
外文關鍵詞:bacillusproteinenzymeisolate
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由臺灣的土壤中分離出能產生高活性單位的蛋白質分解酵素之芽孢桿菌
THS 2-3,由菌學性質鑑定結果,推論其可能為Bacillus subtilis。以合
成培養基來探討影響此菌株蛋白質分解酵素生成的環境及營養素,得到其
最適的培養溫度為35℃,最初培養基pH值為9.0。最佳的碳水化合物來源,
氮源分別是:1%甘油,2%酪蛋白。且發現無機態氮源Ammonium Sulfate9.5
mM與L- glutamic acid16.0mM的濃度配合下亦可獲致良好的生成量。此
外,0.5% 的 Dipotassium Hydrogenphosphate也可促進總活性的增加。細
胞培養液離心分離除去菌體後,經由Amicon薄膜超過濾濃縮純化後之粗酵
素,再進一步以Sephadex G-200膠體層析後,膠體層析圖中有 兩個波峰
,經由性質測定得知小的波峰為蛋白質分解酵素,較大的波峰為澱粉分解
酵素。蛋白質分解酵素的分子量約為41,000道爾頓。粗酵素的最適作用溫
度與 pH 範圍值為50℃,pH7-9,而在40℃,pH 7之環境下有最佳的穩定性。
當加入鈣離子可明顯的提高酵素之耐熱性。此酵素作用於酪蛋白的Vmax值
為5.05μg/ml /min ,Km值為2.2mg/ml。由酵素之理化性質推論此蛋白質
分解酵素為金屬型中性蛋白質分解酵素。
A bacillus strain ,THS2-3,with high yield of protease was
isolated from the soil of Taiwan .According to the taxonomic
analysis , this strain is belongs to the genus of Bacillus,and
similar to Bacillus subtilis.To study the optimum conditions
for the protease fermentation with a synthetic medium.the
optimum conditions were determined as following:theincubation
temperature ,35℃;medium initial pH,9.0;the best carbon
source,1% glycerol; the best nitrogen source,2% casein.It was
also found that a combined nitrogen source of Ammonium Sulfate
(9.5mM) and L-glutam ate (16.0mM) was adequate for the protease
fermentation.Phosphate (Dipotassium Hydrogenphosphate) at the
concentration of 0.5% in the medium showed a positive effect on
the producing of prote ase.As the fermentation proceeding,the
protease activity was detected at the initiation of log phase,
and then the amount of protease producing was in concert with
the increasing of cell number .Finally,the maximum protease
concentrtion reached 60pu/ml at the stationary phase after 48
hours fermenting.The crude enzyme was recovered from the
fermented broth with centrifugation and was concentrated with a
thin filter(molecular weight cut at 10,000 Daltons),then the
concentrated enzyme suspension was chromatographed with
Sephadex G-200 gel.The chromtogram showed two apparaent peaks,
the small one with protease activity and the large one with
amylase activity.The partial purified protease was used to
study the characteristics of the enzyme.For the enzyme reaction,
the optium pH range is pH 7-9,and the optium temperature is
50℃.The Vmax and Km values was,5.05 μg/ml /min and 2.2 mg/ml
respectively,determined with casein as substrate. metal ions
Ca2+ is able to protect the enzyme at temperature higher than
40℃.The enzyme was inhibited by EDTA ,and could be resumed
partial activity with Zinc ion.The enzyme was considered as a
neutral-metalloprotease.
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