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

摘要

According to the wild used of cyclodextrin,the cyclodextrin glycosyltransferase (EC 2.4.1.19,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.%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.