ABSTRACT
A rapid in situ immobilization process was developed based on conventional separation technique of immobilized metal affinity chromatography (IMAC) and was studied in the case of D-amino acid oxidase (DAAO) with binding-enhancing Heli-tag (His-Arg-Asn-Tyr-Gly-Gly-Cys-Cys-Gly). A recombinant Escherichia coli strain JM105 (Δase)/pGEMK-R-DAAO-Heli was successfully constructed to synthesize chimeric protein DAAO-Heli. Without additional purification procedure, the tagged enzyme DAAO-Heli could be directly immobilized to EP-IDA-Ni(2+) support with purity of 90 % and DAAO activity of over 70 U/g support. Experimental results showed that the immobilized DAAO-Heli was 73 times more thermally stable than free enzyme. Besides, it remained 67 % of initial activity after 100 cycles of batch catalysis and its operational stability was improved 36 times than that of the previously IMAC-immobilized DAAO-His. Furthermore, the epoxy (EP) support could be easily recovered and repeatedly used with simple steps, which could reduce the immobilization costs significantly.
Subject(s)
Chromatography, Affinity/methods , D-Amino-Acid Oxidase/chemistry , D-Amino-Acid Oxidase/isolation & purification , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Saccharomycetales/enzymology , D-Amino-Acid Oxidase/biosynthesis , D-Amino-Acid Oxidase/genetics , Enzymes, Immobilized/biosynthesis , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Fungal Proteins/biosynthesis , Fungal Proteins/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Saccharomycetales/geneticsABSTRACT
Two categories of expression systems with different promoters and substitutive ribosome binding site region (RBS) were constructed in order to improve the soluble expression of d-amino acid oxidase (DAAO) basing on the hypothesis that the optimal promoter and suitable RBS would provide the recombinant expression system with better matched expression rate, which served as key factor to the heterogenous synthesis of soluble protein. The results showed that with rational promoter recombination and delicate RBS substitution, significant increase of DAAO activity (20-fold) was obtained for strain JM105/pGEMKT-Tac-R-DAAO over the previously constructed strain BL21(DE3)/pET-DAAO. Furthermore, similar optimization strategy proved feasible in the active expression of other enzymes such as glutaryl-7-aminocephalosporanic acid acylase (GCA) and N-Carbamyl-D-amino acid amidohydrolase (D-Case).
