Immobilized proteins and use thereof

Abstract

The invention relates to an immobilized protein material comprising a protein that is immobilized on a glass material or organic polymer through affinity tag binding. The glass material may be a porous glass material such as (hybrid) controlled porosity glass. The invention also relates to the use of an immobilized enzyme material as a heterogeneous biocatalyst in chemical synthesis. The invention further relates to a method for the immobilization of affinity tagged proteins on a glass material or organic polymer, and to a method for the purification and isolation of affinity tagged proteins by the immobilization of such proteins on a glass material or organic polymer.

1 . A method for catalyzing an enzyme-catalyzed cascade reaction, comprising
 (a) providing a solid support and two or more enzymes immobilized on said solid support,   wherein the solid support has pores with diameter ranging between about 10 and 300 nm,   wherein the solid support comprises non-swelling carrier material,   wherein the non-swelling carrier material comprises a chelated metal ion, and wherein each of said immobilized enzymes comprises an affinity tag that binds to the chelated metal ion; and   (b) bringing said immobilized enzymes into contact with a continuous flow of at least one substrate, thereby catalyzing the cascade reaction.   
     
     
         2 . The method according to  claim 1 , wherein at least three enzymes are immobilized on said solid support. 
     
     
         3 . The method according to  claim 1 , wherein the non-swelling carrier material is a porous organic polymer. 
     
     
         4 . The method according to  claim 3 , wherein the porous organic polymer is chosen from the group consisting of polyethylene, ultra-high molecular weight polyethylene (UHMWPE), high-density polyethylene (HDPE), polypropylene (PP), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polystyrene, polymethacrylate and poly(methyl methacrylate). 
     
     
         5 . The method according to  claim 3 , wherein the porous organic polymer is a polystyrene. 
     
     
         6 . The method according to  claim 3 , wherein the porous organic polymer is a polymethacrylate. 
     
     
         7 . The method according to  claim 1 , wherein the non-swelling carrier material is controlled porosity glass (CPG) or hybrid CPG. 
     
     
         8 . The method according to  claim 1 , wherein the chelated metal ion is selected from the group consisting of Fe 2+ , Fe 3+ , Co 2+ , Ni 2+ , Cu 2+  and Zn 2+ . 
     
     
         9 . The method according to  claim 8 , wherein the chelated metal ion is Fe 3+ . 
     
     
         10 . The method according to  claim 8 , wherein the chelated metal ion is Co 2+ . 
     
     
         11 . The method according to  claim 1 , wherein the affinity tag is a polyhistidine tag. 
     
     
         12 . The method according to  claim 1 , wherein the substrate is in an organic solvent. 
     
     
         13 . The method according to  claim 1 , wherein the substrate is in aqueous conditions. 
     
     
         14 . The method according to  claim 1 , wherein a substrate for a first enzyme is transformed into a substrate for a second enzyme. 
     
     
         15 . The method according to  claim 1 , wherein a substrate is transformed by a first enzyme and wherein a co-factor for the first enzyme is regenerated by a second enzyme.