製備固定化生物觸媒應用於生產細菌纖維素

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Title:	製備固定化生物觸媒應用於生產細菌纖維素 Preparation of Immobilized Biocatalyst For Bacteria Cellulose Production
Authors:	王星堯 Wang, Sine-Yao
Contributors:	化學工程與材料工程學系奈米材料碩士班
Keywords:	β-半乳糖苷酶固定化乳清木質醋酸菌 β-galactosidase Immobilized Whey Gluconacetobacter xylinus
Date:	2012-07
Issue Date:	2013-11-07 13:27:06 (UTC+8)
Abstract:	本研究先以五種不同化學試劑進行酵母菌細胞之通透，再以四種凝膠進行固定化，測其最佳反應條件、穩定性、使用性等，並用於填充床反應器中水解乳清和培養木質醋酸菌生產細菌纖維素。結果顯示以1.94×10-3％ SDS /3.80 ％ chloroform通透酵母菌細胞會有最佳的活性值 7530 U/mg-DCW，再以5% acrylamide進行固定化，於33℃、pH 7 的條件下反應25 min可達最大活性值614 U/mg-DCW，且在使用10次後，還能保有60%的活性。應用於填充床反應器中水解乳清時，在流速24 ml/hr、反應溫度33℃、包埋有600 mg-DCW酵母菌細胞時，水解效率能達到83.8%，再以此固定化觸媒與木質醋酸菌進行共培養其細菌纖維素產量可達1.22 g/l。而以細菌纖維素 (Bacterial cellulose; BC) 固定化酵母菌則是以40 % isopropanol通透後有最大的活性值352 U/mg-DCW，於33℃、pH 6.5的條件下反應30 min 其活性可達最大513 U/mg-DCW，在重複使用8次後，能保有60%的活性。應用於填充床反應器中水解乳清時在24 ml/hr和33℃下，可達79.4%之水解效率。以此固定化觸媒與木質醋酸菌進行共培養其細菌纖維素產量可達1.06 g/l。 β-galactosidase (β-gal) is one of the most important enzymes used in fermentation engineering, which catalyses the hydrolysis of whey for applications of microbial fermentation. Recombinant Pichia pastoris (P. pastoris) cells harboring β-gal were treated with different solvents for cell permeabilization and then were immobilized by various gels. Different parameters (gel concentration, bead size, temperature, pH and reaction time) were studied to maximize the lactose hydrolysis in whey. Maximal β-gal activity (614 U/mg-DCW) was obtained by using yeast cells treated with 1.94×10-3％ SDS /3.80 ％ chloroform and then immobilized in 5% (w/v) acrylamide gel with a bead size of 2 mm reacted for 25 min at 33℃、pH 7.0. The activity of the prepared biocatalyst retained about 60% even after 10 times used. The hydrolysis efficiency of packed-bed reactor using the prepared biocatalyst can reach 83.8% under the temperature of 33℃, speed of 24 ml/hr, biocatalyst amount of 600 mg-DCW. The biocatalyst was used to co-culture with Gluconacetobacter xylinus (G. xylinus) using whey as a carbon-source. The BC yield was 1.22 g/l under this simultaneously hydrolysis and fermentation. Besides, we used the BC pellicles as a new immobilization material for recombinant P. pastoris entrapment. After cell immobilization, 40% isopropanol was used to permeate yeast cells. The highest β-gal activity (513 U/mg DCW) was achieved under the gel size of 5 mm reacted for 30 min at 33℃、pH 6.5. After 8 times used, the prepared biocatalyst retained about 60% of β-gal activity. The hydrolysis efficiency of packed-bed reactor using the novel biocatalyst can reach 79.4%. The biocatalyst was also used to co-incubate with G. xylinus using whey based medium and the BC production was 1.06 g/l.
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