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-  2018 

来源于Bacillus circulans的重组β-CGT酶的分离纯化及其生化性质分析

DOI: 10.3969/j.issn.1673-1689.2018.04.005

Keywords: 环糊精葡萄糖基转移酶 环糊精 枯草杆菌 分离纯化 生化性质
cyclodextrin glycosyltransferase
,cyclodextrin,Bacillus subtilis,purification,characterization

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作者将来源于Bacillus circulans STB01的重组β-CGT酶进行分离纯化,并对其生化性质进行了分析。结果表明,采用Phenyl HP柱疏水层析、Q-HP柱阴离子交换层析两步能很好的对重组β-CGT酶进行纯化,酶的回收率达到45.3%。重组β-CGT酶的表观相对分子质量约为76 500,且相对分子质量呈单分散,说明酶蛋白分子在溶液中是以单聚体形式存在。该酶的最适pH为6.5,且在甘氨酸-氢氧化钠缓冲液中体现出更好的热稳定性;最适温度为60 ℃。该酶的活性不依赖于金属离子,在酶催化反应的整个过程中,其主要产物均为β-环糊精。以玉米淀粉、马铃薯淀粉、木薯淀粉为底物时,该酶环化反应的动力学性质不符合米氏方程;而分别以可溶性淀粉、麦芽糊精(DE 5、15、25)为底物时,其环化反应的动力学性质能用米氏方程很好的进行描述。
According to the wild used of cyclodextrin,the cyclodextrin glycosyltransferase(EC,CGTase) used to product cyclodextrins industrially has become the focus of scientific research nowadays. In this study,the purification and characterization of recombinant β-CGTase from Bacillus circulans STB01 were measured.The results showed that the recombinant β-CGTase could be purified by a combination of Phenyl HP hydrophobic chromatography and Q-HP anion exchange chromatography. The recovery of the enzyme was 45.3%. The apparent molecular weights of the β-CGTase was about 76 500 and presented monodisperse,which indicated that the purified enzyme was a monomer in solution. The optimum cyclization reaction pH of the β-CGTase was 6.5,and showed more thermostable in glycine-NaOH buffer. The optimum cyclization reaction temperature was 60 ℃. The thermostability of β-CGTase increased gradually with increased concentration of the enzyme. The function of β-CGTase did not required the metal cofactor. During the whole cyclization reaction,β-cyclodextrin was the main product. The kinetics of the β-CGTase catalyzed cyclization reaction could not be described by the Michaelis-Menten equation with corn starch,potato starch or cassava starch as the substrate. However,the kinetics of cyclization reaction could be fairly well described by the Michaelis-Menten equation while used soluble starch or maltodextrins(DE 5,15,25) as the substrate,respectively


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