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

A bifunctional fusion enzyme system constructed by gene splicing is propose d as a new model to develop sequence biosensors, taking maltose biosensor a s 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-Gl y)(5)-] coding sequence. The fusion gene was cloned into the vector pPIC9 a nd expressed in Pichia pastoris GS115 under the control of the AOX1 promote r. It was found that, a bifunctional hybrid protein with a molecular weight of 430 kDa was secreted after induction with methanol. The fusion enzyme G OD-(Ser-Gly)(5)-GA (GLG) was purified using Q Sepharose Fast Flow ion-excha ng chromatography. Kinetic analysis demonstrated that GLG retained the typi cal kinetic properties of both GA and GOD. After being immobilized on an am inosilanized glass slide through covalent bonding by glutaraldehyde, GLG sh owed much higher sequential catalytic efficiency than the mixture of separa tely expressed GA and GOD (GA/GOD). Maltose biosensors were fabricated with GLG and GA/GOD, respectively. The performance characteristics of the malto se biosensor with respect to reproducibility, signal level, and linearity w ere effectively improved by using the fusion enzyme. Our findings offer a b asis for the development of other sequence biosensors.