- 學位論文
嗜高溫菌的分離及其聚木醣分解酵素
Isolation and Characterization of Xylanase in Thermo-tolerant Microbes.
摘要
在堆肥製造過程中，由於發酵熱會累積而使堆肥溫度逐漸升高，抑制中溫細菌的生長，產生的高溫亦有消除病源菌及加速反應速率的效果；當堆積後溫度上升，堆肥中的微生物相會轉為以嗜高溫菌為主。嗜高溫菌生長快速及不容易受污染等有利於發酵的特點，在堆肥穩定化過程中扮演重要角色。 嗜高溫微生物能在高溫環境下生長，其所產生相關酵素之熱安定性極佳，因此，利用嗜高溫菌生產酵素作為生物轉化觸媒有其經濟價值。本研究由臺中市環保局自製廚餘堆肥與昶怡堆肥場之堆肥挑選出45株疑似具有聚木醣酶活性的菌，然後再接種自聚木醣酶篩選培養基，分析水解酵素圈直徑及菌落直徑，培養四天水解酵素圈直徑(mm)/菌落直徑(mm)比值在3.0以上有5株，比值在1.0及3.0間有9株，其餘29株比值平均在1.0以下或無活性。。14株當中有2株是細菌，其餘則皆為放線菌。在培養過程中，分離株T307、T501、T603、T604、T605及A02等6株放線菌活性較佳，另外再以本研究室先前自宏金堆肥場中所篩選出的高溫放線菌Streptomyces thermonitrificans NTU-88為對照菌種。6個放線菌分離株70℃聚木醣分解酵素的活性均比S. thermonitrificans NTU-88還高。分離株T501、T603與T605聚木醣分解酵素的最適反應pH為6；分離株T307、T604、A02與S. thermonitrificans NTU-88則為pH 7。分離株T307與T604在80℃加熱150分鐘後聚木醣分解酵素的活性仍分別有54 %與24 %的活性；各個分離株之粗酵素液均對燕麥聚木醣均展現高的活性，對纖維素、澱粉、水楊素的活性則相對較低。添加HgCl2後，分離株T307與T604的活性大幅降低，而添加EDTA與其他微量金屬則對酵素活性影響不大。經上述一系列生化特性分析並與對照菌株S. thermonitrificans NTU-88比較後，篩選出分離株T307與T604為聚木醣酶較佳的菌種。
並列摘要
During the preparation of compost, the ferment heat will accumulate, make the waste temperature go up gradually, and repress mesophilic bacteria grow. The generate heat will also accelerate the reaction velocity. After the compost temperature rise, the microbial group of compost will transfer to be thermo-tolerant primarily. Thermo-tolerant microbes can grow fast and be not polluted during composting. Because thermotolerant microbes can grow in the high temperature, the enzymes from them are stable than other microbes under the high temperature environment and therefore their economic values as the biocatalysts are accessed. This research at first chooses 45 isolates from the food waste of Taichung Environmental Protection Bureau and the compost of Chian-I compost plant, they are transfered to new xylanase isolation culture medium again, and then their hydrolitic enzyme diameter as well as the colony diameter is analyzed after four days incubation. The ratio of the fourth day hydrolitic enzyme circle diameter (mm)/the fourth day colony diameter (mm) above 3.0 has 5, between 1.0 and 3.0 has 9, other 29 do not have any activity or below 1.0. Two isolates are bacteria and others are actininomycetes. In the incubation process, isolate T307, T501, T603, T604, T605 and A02 are easier to grow, so these actinomycetes choosed, and then their characteristics are analyzed. Streptomyces thermonitrificans NTU-88 is the reference bacterium. All isolates have optimal xylanase reaction temperature at 70℃ and was higher than S. thermonitrificans NTU-88. Isolates T501, T603 and T605 have optimal enzyme activity of pH 6. Isolates T307, T604, A02 and S. thermonitrificans NTU-88 have optimal pH 7. Isolates T307 and T604 are better than other isolates in thermal stability of xylanase. After 150 minutes incubation at 80℃, isolates T307 and T604 xylanase activity still have 54 % and 24 %. If HgCl2 is added to isolates T307 and T604 crude enzyme solution, their xylanase activity is both repressed. EDTA and other metal ions such as Ca2+, Na+, Mg2+, Zn2+, Cu2+, Co2+, Mn2+ didn’t affect isolates T307 and T604 xylanase activity.