Construction of a fusion enzyme system by gene splicing as a new molecular recognition element for a sequence biosensor

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	Construction of a fusion enzyme system by gene splicing as a new molecular recognition element for a sequence biosensor
	Zhou, YF ; Zhang, XE ; Liu, H ; Zhang, ZP ; Zhang, CG ; Cass, AEG
刊名	BIOCONJUGATE CHEMISTRY
	2001
卷号	12 期号:6 页码:924-931
ISSN号	1043-1802
通讯作者	Zhang, XE, Chinese Acad Sci, Wuhan Inst Virol, Wuhan 430071, Peoples R China
中文摘要	A bifunctional fusion enzyme system constructed by gene splicing is proposed as a new model to develop sequence biosensors, taking maltose biosensor as an example. The cDNA fragment of Aspergillus niger glucoamylase (E.C 3.2.1.3, GA) was fused to the 3 end of Aspergillus niger glucose oxidase (E.C 1.1.3.4, GOD) gene with the insertion of a flexible linker peptide [-(Ser-Gly)(5)-] coding sequence. The fusion gene was cloned into the vector pPIC9 and expressed in Pichia pastoris GS115 under the control of the AOX1 promoter. It was found that, a bifunctional hybrid protein with a molecular weight of 430 kDa was secreted after induction with methanol. The fusion enzyme GOD-(Ser-Gly)(5)-GA (GLG) was purified using Q Sepharose Fast Flow ion-exchang chromatography. Kinetic analysis demonstrated that GLG retained the typical kinetic properties of both GA and GOD. After being immobilized on an aminosilanized glass slide through covalent bonding by glutaraldehyde, GLG showed much higher sequential catalytic efficiency than the mixture of separately expressed GA and GOD (GA/GOD). Maltose biosensors were fabricated with GLG and GA/GOD, respectively. The performance characteristics of the maltose biosensor with respect to reproducibility, signal level, and linearity were effectively improved by using the fusion enzyme. Our findings offer a basis for the development of other sequence biosensors.
学科主题	Biochemical Research Methods; Biochemistry & Molecular Biology; Chemistry, Multidisciplinary; Chemistry, Organic
收录类别	SCI
语种	英语
WOS记录号	WOS:000172451200014
公开日期	2011-09-23
内容类型	期刊论文
源URL	[http://210.72.129.5/handle/321005/55513]
专题	沈阳应用生态研究所_沈阳应用生态研究所_期刊论文
推荐引用方式 GB/T 7714	Zhou, YF,Zhang, XE,Liu, H,et al. Construction of a fusion enzyme system by gene splicing as a new molecular recognition element for a sequence biosensor[J]. BIOCONJUGATE CHEMISTRY,2001,12(6):924-931.
APA	Zhou, YF,Zhang, XE,Liu, H,Zhang, ZP,Zhang, CG,&Cass, AEG.(2001).Construction of a fusion enzyme system by gene splicing as a new molecular recognition element for a sequence biosensor.BIOCONJUGATE CHEMISTRY,12(6),924-931.
MLA	Zhou, YF,et al."Construction of a fusion enzyme system by gene splicing as a new molecular recognition element for a sequence biosensor".BIOCONJUGATE CHEMISTRY 12.6(2001):924-931.