極端嗜熱古生菌kodakarensis KOD1之β-醣苷酶分子特性及酶化葡萄糖之生物感應器於生化燃料製品的應用

Molecular characterization of β-glycosidase from hyperthermophilic archaeon Thermococcus kodakarensis KOD1 and fabrication of an enzymatic glucose biosensor for applications in biofuel production

Boopathi(Boopathi Subramani)

指導教授 ： 華國媛

國立臺北科技大學/工程學院/能源與光電材料外籍生專班研究所/博士(2014年)

https://doi.org/10.6841/NTUT.2014.00291

摘要

本論文的研究主要是運用超嗜熱古菌 Thermococcus kodakarensis KOD1中的極耐熱的β-糖苷酶(Tkβgly)和葡萄糖氧化酶，來設計發展可用於葡萄糖檢測纖維素糖化時的生物傳感器。Tkβgly是一個具多重功能的水解酶，屬於CAZy資料庫糖苷水解酶家族1。我們所研究的Tkβgly，其最適溫度和pH值分別為100 ?C和pH4.5。 Tkβgly具有兩個典型的的半胱氨酸殘基：Cys88、Cys376，並參與雙硫鍵的形成。關於Tkβgly的兩個Cys殘基點突變研究證明Cys88是參與形成分子間的雙硫鍵，其缺失影響Tkβgly的熱穩定性和熱活性。另一個半胱氨酸Cys376的突變,並未有任何影響。 Tkβgly的結構模型顯示，該水解酶的活性點是存在一個TIM桶形成的通道內。這種酵素會以兩種穀氨酸鹽殘基Glu207和Glu399，作為酸/鹼催化劑和親核試劑，來進行水解反應。Tkβgly的活性位點由兩個獨特的胺基酸：谷氨酰胺和天冬氨酸(Q77和D206)。這兩個胺基酸在古細菌的βgly中保有高度相同度。另外Arg和Asn兩個胺基酸也在其它超嗜熱β-糖苷酶的活性區塊經常出現。我們將Q77和D206的突變，研究結果表示這兩個胺基酸可能在催化過程中參與基質結合。Q77R突變使得酵素失去催化活性，而D206N則改變親和力、葡糖苷酶及甘露糖苷酶的催化翻轉速率。從動力學的研究，D206N突變沒有影響岩藻糖苷酶活性。高催化流動率的β-葡萄糖苷酶活性藉由D206N使得它在纖維素降解，可用來生產乙醇。 在木質纖維素降解過程中，β-葡糖苷酶活性是最重要的，因為它的產物葡萄糖會抑制此作用。若要檢測β-葡萄糖苷酶水解纖維雙糖所產生的葡萄糖水平，則可藉由我們設計的氧化鋅-石墨烯碳納米管混合矩陣，與葡萄糖氧化酶所延伸出的生物感測器來檢測。結果顯示我們所製備的生物感測器可以用來檢測從木質纖維素水解生成的葡萄糖。未來，本研究將致力於如何將嗜熱β-糖苷酶運於在木質纖維所產生的生物燃料流程上，及開發這些酵素其他的工業應用。

關鍵字

β-糖苷酶嗜熱kodakarensis KOD1 ； 熱穩定性 ； 熱活動 ； 熱力學 ； 生物傳感器 ； 葡萄糖氧化酶

並列摘要

This thesis works reports the biochemical and molecular characterization of an extremely thermostable β-glycosidase (Tkβgly) from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 and fabrication of Glucose Oxidase based biosensor for glucose detection in cellulose saccharification. Tkβgly is a multi–substrate hydrolyzing enzyme belongs to glycoside hydrolase family 1 in CAZy database. The optimum temperature and pH for Tkβgly activity was 100 ?C and pH 4.5 respectively. Tkβgly has two conserved cysteine residues at Cys88 and Cys376 and involvement in disulfide bond formation. Site-directed mutagenesis studies of two Cys residues proved that Cys88 is involved in forming intermolecular disulfide bond and its deletion affects the thermostability and thermoactivity of Tkβgly. The other cysteine mutant Cys376 did not have any effect on the enzyme. The structural model of Tkβgly revealed that the active site of the enzyme is present inside a tunnel formed by TIM barrel. The enzyme follows retaining mechanism of hydrolysis with two glutamate residues Glu207 and Glu399 as acid/base catalyst and nucleophile respectively.
The active site of Tkβgly consists of two unique residues, Gln and Asp (Q77 and D206) which are highly conserved among the two genus in archaea, Pyrococcus and Thermococcus. These residues are represented by Arg and Asn in all the other hyperthermophilic β-glycosidases. Site-directed mutagenesis studies of Q77 and D206 revealed that both the residues might be involved in substrate binding during catalysis. Q77R mutant inactivated the catalytic activity of the enzyme whereas the D206N only altered the affinity and catalytic turn-over rate for glucosidase and mannosidase activities. D206N mutant did not affect the kinetic parameters for fucosidase activity. The high-catalytic turnover rate for β-glucosidase activity by D206N makes it an useful enzyme in cellulose degradation to produce ethanol. β-glucosidase activity is the most important of all the cellulases in lignocellulose degradation as it gets inhibited by its product, glucose. To monitor the level of glucose produced from the cellobiose hydrolysis by β-glycosidase, we have developed a GOx based electrochemical biosensor by synthesizing ZnO-Graphene-Carbon nanotube hybrid matrix. The results showed that the as-prepared biosensor can be used to detect the glucose from the lignocellulose hydrolysis. This study will be highly useful for the industrial applications of hyperthermophilic β-glycosidase in biofuel production from lignocellulosic biomass.

並列關鍵字

β-glycosidase ； Thermococcus kodakarensis KOD1 ； thermostability ； thermoactivity ； thermodynamics ； biosensor ； glucose oxidase

參考文獻

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