Display of Functionally Active PHB Depolymerase on Escherichia Coli Cell Surface

Abstract

The display of PHB depolymerase (PhaZ(RpiT1) ) from R. pickettii T1 on the surface of E. coli JM109 cells is realized using OprI of P. aeruginosa as the anchoring motif. The fusion protein is stably expressed and its surface localization is verified by immunofluorescence microscopy. The displayed PhaZ(RpiT1) retains its cleaving ability for soluble substrates as well as its ability to adsorb to the PHB surface, and also remains catalycically active in the degradation of insoluble polyester materials, in spite of the possible suppression of the enzyme movement on the polymer surface. The results demonstrate that PhaZ(RpiT1) -displaying E. coli shows potential for use as a whole-cell biocatalyst for the production of (R)-3-hydroxybutyrate monomers from insoluble PHB materials.

Display of a sucrose isomerase on the cell surface of Yarrowia lipolytica for synthesis of isomaltulose from sugar cane by-products

“…Yeast surface display is an extension of the enzyme application technique. One of the most attractive features of surface display is that enzyme molecules are simultaneously synthesized and self-immobilized on the microbial cell surface and the living whole-cell biocatalyst is easily produced by microbial cultivation techniques (Chen et al 2012;Hiraishi et al 2012;Ueda and Tanaka 2000). No further work is required to either purify or immobilize the enzymes.…”

Protein Engineering of Enzymes Involved in Bioplastic Metabolism

“…A variety of surface anchoring motifs, including outer membrane proteins, lipoproteins, autotransporters, subunits of surface appendages, and S-layer proteins, have been employed to achieve the display systems [125,127,128]. We used the OprI anchoring motif for the functional display of PhaZ RpiT1 on Escherichia coli cell surface [129]. The displayed enzyme retained its intrinsic characteristics, that is, hydrolytic activity for p-nitrophenyl butyrate (pNPC4) and the ability to adsorb to and degrade PHB, indicating that the engineered E. coli can be used in the form of a whole-cell biocatalyst by overcoming the uptake limitation of such substrates as insoluble PHB.…”

Cell Surface Display of Poly(3-hydroxybutyrate) Depolymerase and its Application

“…Therefore, the development of new useful enzymes for display systems is an important research objective. Recently, a Japanese research group successfully applied cell surface display of PHB depolymerase, however, the application was limited to the degradation of the polyester to produce 3-hydroxybutyrate [ 16 , 17 ]. In this paper, we report the display of the Ralstonia pickettii T1 depolymerase on the surface of E. coli cells using truncated Pseudomonas OprF as an anchoring motif and demonstrate the biocatalytic application of this system to enantioselective hydrolysis of a racemic ester.…”

“…It is known that PHA depolymerase shows enantioselective behavior towards substrate in aqueous solutions, however, the main application of surface displayed depolymerase has been the hydrolysis of polymer or oligomer, composed of monomeric units of 3-hydroxyacid [ 5 , 16 , 17 ]. Here, we have shown for the first time, that cell surface displayed depolymerase can enantioselectively hydrolyze not only polymers or oligomers of PHB, but racemic esters.…”