【摘要】：糖苷類化合物(Glycoside compounds)是一類廣泛分布于自然界中的功能性分子,其在細胞識別、生長調(diào)控、信號傳遞、結(jié)構(gòu)支持等多個方面起著重要的作用。由糖苷類化合物衍生而來的糖綴合物(Glycoconjugates),由于其高表面活性、抗菌性、高水溶性、高生物相容性等獨特的理化性質(zhì),近年來受到了廣泛的研究與關(guān)注。糖苷水解酶(Glycoside hydrolases,EC 3.2.1)是一類特異性水解糖苷鍵的水解酶類,其既可水解糖苷類化合物中糖基之間的糖苷鍵,又可以水解糖綴合物中糖基與非糖基之間的糖苷鍵。根據(jù)人們對其水解機理的研究發(fā)現(xiàn),絕大多數(shù)糖苷水解酶通過控制底物與水的比例可以行使轉(zhuǎn)糖苷活力。較之糖基轉(zhuǎn)移酶而言,使用糖苷水解酶從糖苷類化合物出發(fā)合成新型的糖綴合物,成本更為低廉,更具有產(chǎn)業(yè)化價值。這其中,源自嗜熱微生物的嗜熱糖苷酶,由于其具有很高的熱穩(wěn)定性、有機溶劑耐受性,并且在高溫下具有較高的反應(yīng)活性,符合工業(yè)生產(chǎn)上的需求,與耐受性差、易失活的常溫糖苷酶相比,更具有產(chǎn)業(yè)化優(yōu)勢。特別是在合成糖綴合物的過程中,高反應(yīng)溫度能夠大大提升反應(yīng)速率,降低體系粘度,避免微生物污染,因此使用嗜熱糖苷酶合成糖綴合物有著十分顯著的優(yōu)勢。分離自日本熱泉中的嗜熱厭氧菌Dictyoglomus thermophilum H-6-12能夠在70℃的條件下利用木質(zhì)纖維正常生長。其基因組中包含大量的糖苷水解酶基因,其中,基因序列ACI18764.1被認為編碼GH3家族的β-葡萄糖苷酶。本課題通過Blast分析,確認該基因為GH3家族的β-葡萄糖苷酶,并通過PCR成功獲得了該基因的體外擴增。通過分子生物學(xué)方法,我們將該基因轉(zhuǎn)入大腸桿菌表達載體p ET-28a中,成功構(gòu)建了表達工程菌,實現(xiàn)了該β-葡萄糖苷酶的異源可溶表達。利用p ET-28a提供的組氨酸標簽,我們使用Ni-NTA層析柱通過金屬離子親和層析,獲得了較純的的蛋白。對重組β-葡萄糖苷酶Dt0262酶學(xué)性質(zhì)的表征表明,該酶對人工合成底物p NPGlc與天然底物纖維二糖均具有水解效果。對p NPGlc的最適水解條件為80℃,p H 6.0;對纖維二糖的最適水解條件為75℃,p H 5.5。動力學(xué)常數(shù)測定結(jié)果表明,重組β-葡萄糖苷酶Dt0262對p NPGlc的Km與kcat分別為0.23m M與0.64s-1,對纖維二糖的Km與kcat分別為3.57m M與0.88s-1,根據(jù)kcat/Km的大小可知,該酶更偏好于水解p NPGlc。對該酶的穩(wěn)定性研究表明,重組β-葡萄糖苷酶Dt0262在70℃下相對穩(wěn)定,t1/2可達到92h,而在80℃與90℃下,其t1/2分別縮短至20.3h與21.8min。此外,該酶還表現(xiàn)出明顯的熱激活效果,在70℃與80℃下孵育0.5h后,酶活分別變?yōu)樵瓉淼?.32倍與1.46倍。p H穩(wěn)定性研究表明,該酶能夠在較為寬泛的p H條件下保持穩(wěn)定。金屬離子依賴性研究顯示,重組β-葡萄糖苷酶Dt0262為非金屬離子依賴性酶。通過對該酶的表征我們發(fā)現(xiàn),Dt0262確為嗜熱β-葡萄糖苷酶,具有應(yīng)用于糖綴合物合成方面的潛力。
[Abstract]:Glycosides (Glycoside compounds) are a class of functional molecules widely distributed in nature. They play an important role in cell recognition, growth regulation, signal transmission, structural support and so on. The glycoconjugate (Glycoconjugates), derived from glycoside compounds has attracted extensive attention in recent years because of its unique physicochemical properties such as high surface activity, antimicrobial activity, high water solubility and high biocompatibility. Glycoside hydrolase (Glycoside hydrolases,EC 3.2.1) is a kind of hydrolytic enzymes that can hydrolyze glycoside bonds between glycosyl and non-glycosylated glycosides in glycosides. According to the study of hydrolysis mechanism, most glycoside hydrolases can exercise transglycoside activity by controlling the ratio of substrate to water. Compared with glycosyltransferase, the use of glycoside hydrolase to synthesize new glycosylated conjugates from glycosides is cheaper and more valuable for industrialization. Among them, thermophilic glycosidase from thermophilic microbes, because of its high thermal stability, organic solvent tolerance, and high reaction activity at high temperature, meet the requirements of industrial production, and poor tolerance, The inactivated glucosidase at room temperature has the advantage of industrialization. Especially in the process of synthesis of glycoconjugates, high reaction temperature can greatly increase the reaction rate, reduce the viscosity of the system, avoid microbial contamination, so the use of thermophilic glycosidase to synthesize the conjugate has a very significant advantage. The thermophilic anaerobes Dictyoglomus thermophilum H-6-12 isolated from hot springs in Japan can grow normally using wood fibers at 70 鈩，

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