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  • 學位論文

利用最適化條件之共培養增進木黴菌葡萄糖苷酶產量之研究

Improved β-glucosidase Production of Trichoderma spp. by Co-cultivation in Optimal Conditions

指導教授 : 羅朝村
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摘要


纖維素酶的用途廣範,尤其應用於木質纖維素分解成發酵性單醣,再將糖轉化後得到生質酒精之使用是為最普遍的方法之一。如眾所知,木黴菌可以產生大量的內切葡萄糖酶和外切葡萄糖酶,但是對於主要可分解成葡萄糖的β-葡萄糖苷酶,則被認為產量不多。因此本研究選用了兩株台灣本地分離出之木黴菌菌株,分別為PT Musa S24-1和PTNC WA S50-3作為試驗。本研究主題在於找出最適纖維素酶量產之條件,特別是針對於β-葡萄糖苷酶產量之研究。即以測試各種物理條件 (溫度、pH值和培養時間)及不同化學物質(碳源和氮源)來做觀察。結果顯示,在各種物理及化學測試條件中,以碳源中含1%微晶纖維素;氮源為0.1%蛋白胨、1.4g/l硫酸銨和0.3g/l尿素混合培養;培養時間則以144小時為最佳。在此一組合之培養條件下,PT Musa S24-1和PTNC WA S50-3可產生β-葡萄糖苷酶最多,分別為0.65 IU/ml 和 0.89 IU/ml;而最適pH則分別為5.5和5;最適溫度為28oC及24oC。在兩株木黴菌混合共培養之條件中,以先加PTNC WA S50-3培養兩天後再加入PT Musa S24-1所測得的β-葡萄糖苷酶產量為最高,酵素活性達0.99 IU/ml;以上述培養條件(先培養PTNC WA S50-3兩天後再加入PT Musa S24-1)之共培養粗萃酵素水解稻稈實驗中,發現在 iv 48小時內所水解之效果最好,葡萄糖產率可達1.23g/l;爾後,水解效果開始逐漸趨於平緩。由此結果發現兩株木黴菌之共培養液可以有效的分泌出 較多具有水解纖維素之酵素。

並列摘要


There was an increasing demand for cellulases in the market for various applications, among which the bioconversion of lignocellulosic biomass for ethanol production was the major one. Trichoderma spp. was well known that could produce the endoglucanase and exoglucanase components of cellulolytic enzyme complex in large quantities. However, the insufficient amount of β-glucosidase inhibited hydrolysis process to produce glucose. Two local Trichoderma strains, T. asperellum strain PT Musa S24-1 and T. atroviride strain PTNC WA S50-3, showing considerable cellulase production was selected as the potential agents for bioethanol production in this study. The current study aimed at optimal conditions of cellulase production, especially β-glucosidase, from our local Trichoderma strains. Optimization of β-glucosidase production was done by using various physical (temperature, pH, and incubation time) and chemical parameters (carbon sources and nitrogen sources) which could influence the enzyme activity. The results indicated that the maximum β-glucosidase production of T. asperellum strain PT Musa S24-1 and T. atroviride strain PTNC WA S50-3, was 0.65 IU/ml and 0.89 IU/ml, respectively, when 1% Avicel was used as the carbon source after 6 days incubation. These strains showed the highest enzyme production with the nitrogen combination includes peptone (0.1%), (NH4)2SO4 (1.4g/l) and urea (0.3g/l). T. asperellum strain PT Musa S24-1 showed an optimum pH of 5.5 whereas T. atroviride strain PTNC-WA S50-3 showed an optimum pH of 5 for β-glucosidase production. Optimum ii incubation temperatures of T. asperellum strain PT Musa S24-1 and T. atroviride strain PTNC WA S50-3 for the enzyme production were 24°C and 28°C, respectively. The mixed culture of both strains in the optimal conditions above resulted in a higher level of β-glucosidase (0.99 IU/ml) with T. atroviride strain PTNC WA S50-3 inoculation delayed for 48 hours after inoculated T. asperellum strain PT Musa S24-1. To test the effectiveness of the enzyme produced by mixed culture, a crude enzyme broth was used for hydrolyzing rice straw. The hydrolysis processes was the most effective within 48 hours with the glucose concentration reached 1.23 g/l and then hydrolysis rate decrease quickly. The results indicated that the co-cultivation of Trichoderma spp. is an effective approach to produce a cellulolytic enzyme system to hydrolyze lignocellulosic materials.

參考文獻


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