臺灣博碩士論文加值系統

透明顫菌血紅蛋白(Vitreoscilla hemoglobin ; VHB)，在許多文獻證實不僅可以促進細菌的生長，同時還能促進外源蛋白產量。木質醋酸菌所產生的細菌纖維素，具有相當的產業應用價值，但目前礙於產量少，因此在價格上也比其他類似材質相對較高。為了突破細菌纖維素產量的限制，本研究欲利用基因工程的方式，使木質醋酸菌表現透明顫菌血紅蛋白來促使細菌纖維量產;此外，本實驗室已選殖出能產生4-Coumaroyl-CoA ligase (4CL)重組蛋白之大腸桿菌，此蛋白質可產生具有經濟價值的產物白藜蘆醇，因此，欲利用基因工程的方式，在4CL菌株中表現透明顫菌血紅蛋白，促使大腸桿菌表現更多的4CL重組蛋白。研究結果顯示，於木質醋酸菌實驗中，以tac為promoter，將透明顫菌血紅蛋白基因，構築於pBBR122 載體中，經誘導表現後並未發現有透明顫菌血紅蛋白表現，所以改以不同培養基進行木質醋酸菌的培養，試圖找尋最佳生產細菌纖維素的有利條件，結果顯示以GM培養基靜置五天培養後，Factor A synthase (FAS)基因型的木質醋酸菌有最高的纖維素產量，與野生型相比多了13.81% ; 在大腸桿菌實驗中，以T7為promoter，將透明顫菌血紅蛋白基因構築於pGEM®-T Easy Vector中，經PCR與限制酶進行質體檢測，確認質體無誤後，將質體轉型至BL21(DE3)pLysS中進行蛋白質誘導表現，以Urea萃取出重組蛋白經SDS-PAGE、Western blot分析後，證實此重組蛋白為包涵體(inclusion body)，其表現量為1g菌重可產生2.2375mg的重組蛋白。最後參考前人文獻利用HPLC的方法測試4CL對於對香豆酸的轉換率分析，將4CL與對香豆酸反應30分鐘之後能得到較高的轉換率11.29 %。冀望未來能找尋一個可利於表現具有活性透明顫菌血紅蛋白的條件，並應用於木質醋酸菌與大腸桿菌4CL菌株中，促使產量提升並使之價值提高與增加利用性，應用於相關之生技產業造福社會。

Vitreoscilla hemoglobin (VHB) has been shown in many previous studies to promote not only the growth of bacteria but also the production of exogenous proteins. Bacterial cellulose produced by Gluconacetobacter xylinus has considerable industrial application value, but at present, it is relatively low in output. In this study, we try to express hemoglobin both in G. xylinus and E. coli, promote the production of bacterial cellulose and 4CL (4-Coumaroyl-CoA ligase) recombinant protein. In the G. xylinus experiment, the hemoglobin did not express after the induced expression. Therefore, the cultivation of the G. xylinus with different media, trying to find the optimal conditions for producing bacterial cellulose, showed that the Factor A synthase (FAS) genotype of G. xylinus had the highest cellulose yield after culturing for five days in GM medium, which was 13.81 % more than the wild type. In the E.coli experiment, hemoglobin protein was expression after induced, but the recombinant protein was inclusion body, its expression amount was 1 g of bacterial weight to produce 2.2375 mg of VHB recombinant protein. We take the previous studies as a reference, used the HPLC method to test the conversion rate of 4CL protein for coumaric acid. After reacting 4CL with coumaric acid for 30 minutes, have the highest conversion rate of 11.29 %. In the future, we can find the condition to expression activity hemoglobin, and application in the G. xylinus and E. coli 4CL strains, which promotes the increase of production and enhances its value.

目錄
中文摘要 i
英文摘要 iii
目錄 iv
圖目錄 viii
第一章、 前言 1
1-1 細菌纖維素之構造、合成 1
1-2 生產細菌纖維素的產業應用 3
1-3 木質醋酸菌簡介 4
1-4 利用基因轉殖方式增加細菌纖維素量產之相關研究 5
1-5 VHB基因之介紹 6
1-6 4-Coumaroyl-CoA ligase ( 4CL ) 7
1-7 研究目的 7
第二章、 實驗材料 9
2-1 實驗藥品 9
2-2 實驗儀器設備 10
2-3 實驗套組(Kit) 11
2-4 宿主與載體 12
2-5 DNA與蛋白質電泳染劑與Marker 13
2-6 引子使用表 13
2-7 抗體與轉漬膜 14
2-8 SDS-PAGE配方 15
2-9 培養基 16
2-9-1材料 16
2-9-2配置E.coli medium 17
2-9-3配置G. xylinus Medium 17
2-10 HPLC mobile phase 配置 19
第三章、 實驗方法 19
3-1 細菌培養 19
(1) 大腸桿菌培養 19
(2) 木質醋酸菌培養 19
3-2 VHB基因密碼子優化與構築至表現載體 19
3-3 質體DNA萃取 20
3-4 限制內切酶處理 20
3-5 PCR反應 21
3-6 DNA電泳分析 21
3-7 DNA電泳膠萃取 21
3-8 黏合作用 22
3-9 勝任細胞製備與轉型 22
3-9-1 氯化鈣轉型法 22
3-9-2電穿孔轉型法 23
3-10 菌落PCR反應 23
3-11 蛋白質表現 24
3-12 蛋白質萃取、純化 24
3-13 蛋白質定量 25
3-14 蛋白質製備與電泳 25
3-15 西方墨點法 (Western blot) 25
3-16 定序 26
3-17 纖維素測定方法 26
3-18 4CL 酵素轉換率分析 26
3-19 實驗設計 27
第四章、 結果與討論 28
4-1 VHB基因選殖、質體構築、蛋白質表現 28
(1) VHB基因的選殖與優化 28
(2) 質體驗證 29
(3 ) VHB基因之蛋白質表現與純化 29
(4) 構築單質體雙基因(FAS、VHB)之質體 31
(5) 選殖具有pMBFAS與pMBVHB雙質體之木質醋酸菌株 31
4-2 木質醋酸菌之纖維素測定 32
4-3 pGEMVHB質體構築 32
4-4 VHB之蛋白誘導與純化 33
4-5 pGEMT7rbsVHB質體構築 33
4-6 VHB蛋白之誘導與純化 34
4-7 4CL蛋白的轉換率分析 35
4-8 pBRT7rbsVHB質體構築 36
總結論 37
第五章、 實驗結果圖 39
參考文獻 73
附錄 79
 

楊祐俊、洪爭坊、趙雲鵬。(2009)。醋酸菌Gluconacetobacter xylinus轉殖類血紅素。臺中區農業改良場研究彙報。102:1-14。

馬承鑄、顧真榮。(2001)。細菌纖維素生物理化特性和商業用途(綜述)。上海農業學報。17: 93-98。

林明煌。(2017)。利用基因轉殖木質醋酸菌以增加纖維素產量。國立彰化師範大學生物技術研究所碩士論文。

Austin S, Nordström K. (1990). Partition-mediated incompatibility of bacterial plasmids. Cell. 60(3):351-4.

Banzon, J.A. (1990). Coconut as food. Philippine Coconut Research and Development Foundation, Diliman, Quezon City, Philippines. 239.
Blackwell, J. (1982). The macromolecular organization of cellulose and chitin. Cellulose and Other Natural Polymer Systems. 403–428pp.

Bae SO, Sugano Y, Ohi K, Shoda M. (2004). Features of bacterial cellulose synthesis in a mutant generated by disruption of the diguanylate cyclase 1 gene of Acetobacter xylinum BPR 2001. Appl Microbiol Biotechnol. 65(3):315-22.

Camacho-Zaragoza JM, Hernández-Chávez G, Moreno-Avitia F, Ramírez-Iñiguez R, Martínez A, Bolívar F, Gosset G. (2016). Engineering of a microbial coculture of Escherichia coli strains for the biosynthesis of resveratrol. Microb Cell Fact. 15(1):163.



Chen YM, Xu HY, Wang Y, Zhang JF, Wang SM. (2014). Vitreoscilla hemoglobin promotes Salecan production by Agrobacterium sp. ZX09. J Zhejiang Univ-Sci B. 15(11):979–985.

Deng F, Pan Y, Chang F, Fang W, Fang Z, Xiao Y. (2018). Co-expression of β-glucosidase and Vitreoscilla hemoglobin in Escherichia coli. Chinese Journal of Biotechnology. 34(3): 379−388.

Dudman, W. F. (1959). Cellulose production by Acetobacter actigenum in defined medium. J Gen Microbiol. 21:327-37.

Dikshit KL, Webster DA. (1988). Cloning, characterization and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli. Gene. 70(2):377-86.

Hestrin S, Schramm M. (1954). Synthesis of cellulose by Acetobacter xylinum. 2. Preparation of freeze-dried cells capable of polymerizing glucose to cellulose. Biochem J. 58(2):345–352.

Haigler, C. H. (1985). The function and biogenesis of native cellulose. In Cellulose Chemistry and its Applications (T. P. Nevell and S. H. Zeronian eds). Chichester, England: Ellis Horwood, 30-83.

Jozala AF, Pértile RA, dos Santos CA, de Carvalho Santos-Ebinuma V, Seckler MM, Gama FM, Pessoa A Jr. (2015). Bacterial cellulose production by Gluconacetobacter xylinus by employing alternative culture media. Appl Microbiol Biotechnol. 99(3):1181-90.

Kallio PT, Bailey JE. (1996). Intracellular expression of Viteroscilla hemoglobin (VHb) enhances total protein secretion and improves the production of alpha-amylase and neutral protease in Bacillus subtilis. Biotechnol Prog. 12(1):31-39.

Khosravi M, Webster DA, Stark BC. (1990). Presence of the bacterial hemoglobin gene improves alpha-amylase production of a recombinant Escherichia coli strain. Plasmid. 24(3):190-4.


Khosla C, Curtis JE, DeModena J, Rinas U, Bailey JE. (1990). Expression of intracellular hemoglobin improves protein synthesis in oxygen-limited Escherichia coli. Biotechnology. 8(9):849-53.
Khosla C, Bailey JE. (1988). The Vitreoscilla hemoglobin gene: molecular cloning, nucleotide sequence and genetic expression in Escherichia coli. Mol Gen Genet. 214(1):158-61.

Kunkel SA, Pagilla KR, Stark BC. (2015). Engineering of Nitrosomonas europaea to express Vitreoscilla hemoglobin enhances oxygen uptake and conversion of ammonia to nitrite. AMB Express. 5(1):135.

Khosla C, Bailey JE. (1988). Heterologous expression of a bacterial hemoglobin improves the growth properties of recombinant Escherichia coli. Nature. 331(6157):633-5.

Lin J, Zhang X, Song B, Xue W, Su X, Chen X, Dong Z. (2017). Improving cellulase production in submerged fermentation by the expression of a Vitreoscilla hemoglobin in Trichoderma reesei. AMB Express. 7(1):203.

Li HJ, He YL, Zhang DH, Yue TH, Jiang LX, Li N, Xu JW. (2016). Enhancement of ganoderic acid production by constitutively expressing Vitreoscilla hemoglobin gene in Ganoderma lucidum. J Biotechnol. 227:35-40.

Liu T, Yao R, Zhao Y, Xu S, Huang C, Luo J, Kong L. (2017). Cloning, Functional characterization and site-directed mutagenesis of 4-Coumarate: Coenzyme A Ligase (4CL) involved in coumarin biosynthesis in Peucedanum praeruptorum Dunn. Frontiers in plant science. 8:4.

Liu XF, Pei JZ, Du GJ, Yang ZM. (2011). Recent Progress in Renaturation of Inclusion Bodies of Prokaryotically Expressed Snake Venom Proteins. China Biotechnology. 31(03):113-119.

Liu M, Li S, Xie Y, Jia S, Hou Y, Zou Y, Zhong C. (2018). Enhanced bacterial cellulose production by Gluconacetobacter xylinus via expression of Vitreoscilla hemoglobin and oxygen tension regulation. Applied Microbiology and Biotechnology. 102(3):1155–1165.

Muangman P, Opasanon S, Suwanchot S, Thangthed O. (2011). Efficiency of microbial cellulose dressing in partial-thickness burn wounds. J Am Col Certif Wound Spec. 3(1):16-9.
Nordström K, Austin SJ. (1989). Mechanisms that contribute to the stable segregation of plasmids. Annual Review of Genetics. 23:37-69.

Novick RP. (1987). Plasmid incompatibility. Microbiology Reviews. 51(4):381-95.

Okiyama, A. Shirae, H. Kano, H., Yamanaka, S. (1992). Bacterial cellulose I. Two-stage fermentation process for cellulose production by Acetobacter acet. Food-Hydrocolloids. 6(5):471-477.

Okiyama, A., Motoki, M., Yamanaka, S. (1992). Bacterial cellulose II. Processing of the gelatinous cellulose for food materials. Food Hydrocolloids. 6(5):479-487

Ross P, Mayer R, Benziman M. (1991). Cellulose biosynthesis and function in bacteria. Microbio Rev. 55(1):35-58.

Ross P, Mayer R, Weinhouse H, Amikam D, Huggirat Y, Benziman M, de Vroom E, Fidder A, de Paus P, Sliedregt LA. (1990). The cyclic diguanylic acid regulatory system of cellulose synthesis in Acetobacter xylinum. J Biol Chem. 265(31):18933-43.

Singh SM, Panda AK. (2005). Solubilization and refolding of bacterial inclusion body proteins. Journal of Bioscience and Bioengineering. 99(4):303-310.

Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.

Schein, C. H., Noteborn, M. H. M. (1988).Formation of soluble recombinant proteins in Escherichia coli is favored by lower growth temperature. Biotechnology 6:291–294.



Speed MA, Wang DI, King J. (1996). Specific aggregation of partially folded polypeptide chains: The molecular basis of inclusion body composition. Nature Biotechnology. 14(10):1283–1287.

Strandberg L, Enfors SO. (1991). Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli. Appl Environ Microbiol. 57(6):1669–1674.

Sørensen MA, Kurland CG, Pedersen S. (1989). Codon usage determines translation rate in Escherichia coli. Journal of Molecular Biology. 207(2):365-77.

Setyawati MI, Chien LJ, Lee CK. (2007). Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production. Journal of Biotechnology. 132(1):38-43.

Seras-Franzoso J, Peternel S, Cano-Garrido O, Villaverde A, García-Fruitós E. (2015). Bacterial inclusion body purification. Methods Mol Biol. 1258:293-305.

Shafferman A, Helinski DR. (1983). Structural properties of the beta origin of replication of plasmid R6K. J Biol Chem. 258(7):4083-90.

Stark BC, Dikshit KL, Pagilla KR. (2012). The Biochemistry of Vitreoscilla hemoglobin. Comput Struct Biotechnol J. 3(4). e201210002.

Toyosaki, H. Nacitomi, T., Seto, A., Matsuoka, M., Tsuchida, T., Yoshinaga, F. (1995). Screening of Bacterial Cellulose-producing Acetobacter Strains Suitable for Agitated Culture. Bioscience, Biotechnology, and Biochemistry. 59(8):1498-1502.

Velappan N, Sblattero D, Chasteen L, Pavlik P, Bradbury AR. (2007). Plasmid incompatibility: more compatible than previously thought? Protein Engineering, Design and Selection. 20(7):309–313.



Villaverde A, Carrió MM. (2003). Protein aggregation in recombinant bacteria: biological role of inclusion bodies. Biotechnol Lett. 25(17):1385-95.

Webster DA, Hackett DP. (1966). The purification and properties of
cytochrome o from Vitreoscilla. J. Biol. Chem. 241(14):3308-3315.
Wu JM, Fu WC. (2012). Intracellular co-expression of Vitreoscilla hemoglobin enhances cell performance and β-galactosidase production in Pichia pastoris. Journal of Bioscience and Bioengineering. 113(3):332-7.

Wang S, Liu F, Hou Z, Zong G, Zhu X, Ling P. (2014). Enhancement of natamycin production on Streptomyces gilvosporeus by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb). World J Microbiol Biotechnol. 30(4):1369-76.

Zähringer F, Massa C, Schirmer T. (2011). Efficient enzymatic production of the bacterial second messenger c-di-GMP by the diguanylate cyclase YdeH from E. coli. Appl Biochem Biotechnol. 163(1):71-9.

Zhang SP, Zubay G, Goldman E. (1991). Low-usage codons in Escherichia coli, yeast, fruit fly and primates. Gene. 105(1):61-72.

Zhang H, Feng Y, Cui Q, Song X. (2017). Expression of Vitreoscilla hemoglobin enhances production of arachidonic acid and lipids in Mortierella alpine. BMC Biotechnology. 17(1):68.