Self-Immobilized Putrescine Oxidase Biocatalyst System Engineered with a Metal Binding Peptide

Abstract

Flavin oxidases are valuable biocatalysts for the oxidative synthesis of a wide range of compounds, while at the same time reduce oxygen to hydrogen peroxide. Compared to other redox enzymes, their ability to use molecular oxygen as an electron acceptor offers a relatively simple system that does not require a dissociable coenzyme. As such, they are attractive targets for adaptation as cost-effective biosensor elements. Their functional immobilization on surfaces offers unique opportunities to expand their utilization for a wide range of applications. Genetically engineered peptides have been demonstrated as enablers of the functional assembly of biomolecules at solid material interfaces. Once identified as having a high affinity for the material of interest, these peptides can provide a single step bioassembly process with orientation control, a critical parameter for functional immobilization of the enzymes. In this study, for the first time, we explored the bioassembly of a putrescine oxidase enzyme using a gold binding peptide tag. The enzyme was genetically engineered to incorporate a gold binding peptide with an expectation of an effective display of the peptide tag to interact with the gold surface. In this work, the functional activity and expression were investigated, along with the selectivity of the binding of the peptide-tagged enzyme. The fusion enzyme was characterized using multiple techniques, including protein electrophoresis, enzyme activity, and microscopy and spectroscopic methods, to verify the functional expression of the tagged protein with near-native activity. Binding studies using quartz crystal microbalance (QCM), nanoparticle binding studies, and atomic force microscopy studies were used to address the selectivity of the binding through the peptide tag. Surface binding AFM studies show that the binding was selective for gold. Quartz crystal microbalance studies show a strong increase in the affinity of the peptide-tagged protein over the native enzyme, while activity assays of protein bound to nanoparticles provide evidence that the enzyme retained catalytic activity when immobilized. In addition to showing selectivity, AFM images show significant differences in the height of the molecules when immobilized through the peptide tag compared to immobilization of the native enzyme, indicating differences in orientation of the bound enzyme when attached via the affinity tag. Controlling the orientation of surface-immobilized enzymes would further improve their enzymatic activity and impact diverse applications, including oxidative biocatalysis, biosensors, biochips, and biofuel production.

Probing Selective Self-Assembly of Putrescine Oxidase with Controlled Orientation Using a Genetically Engineered Peptide Tag

“…25 We have previously demonstrated that the AuBP (CPGWALRRSIRRQSYGPC) binds selectively to the Au (111) surface with high affinity. 17,35,46,47 To identify appropriate experimental conditions, the coverage of the PutOx−AuBP was measured as a function of adsorption time at 0.05 μM PutOx−AuBP (Figure 1). Template-stripped gold surfaces were used due to the relatively large terraces of flat, Au (111) grains (see Figure 1b), which would allow protein molecules to be easily distinguished from the surrounding gold surface topography.…”

“…The concentration of the protein solution, 0.05 μM, was optimized based on previous studies. 17 The amount of protein bound as a function of time was determined by placing 100 μL of a 0.05 μM PutOx−AuBP solution onto the TSG surface for different time intervals followed by washing with buffer and pH-adjusted water. The protein adsorption study for calculating surface density was done in duplicate for each data point, and images were collected from five different locations for each surface (for each data point, 10 AFM images were analyzed).…”

“…The protein for analysis was prepared by recombinant overexpression in Escherichia coli (E. coli), as described in detail in our prior publication. 17 In summary, mutagenic PCR was used to insert the AuBP peptide (CPGWALRRSIRRQSYGPC) plus linker (PGGG) sequence at the C-terminus of the coding sequence for the putrescine oxidase from Rhodococcus erythropolis (UniProtID: B0F9F6) contained within the N-terminal MBP fusion expression vector, pTBMalE. Expression of the engineered gold binding putrescine oxidase and the native (wildtype) fusion protein in BL21(DE3) E. coli was performed at 20 °C for 24 h in modified terrific broth.…”

“…39−45 However, the immobilization of flavin oxidases (such as the putrescine oxidase investigated here) has only recently started to be explored by our group. We have recently provided the first demonstration of the ability to change the affinity, coverage, and orientation of the surfacebound putrescine oxidase 17 on gold surfaces through the incorporation of a gold binding peptide, AuBP (sequence: WALRRSIRRQSY). 35,46,47 In this previous work, we expressed two versions of the protein putrescine oxidase (homodimer with ∼100 kDa subunits) 48 from actinomycete Rhodococcus erythropolis with comparable solution phase activity and demonstrated critical differences in their binding to gold surfaces: (1) native putrescine oxidase (PutOx) and (2) a fusion enzyme incorporated with a gold binding peptide sequence (CPGWALRRSIRRQSYGPC) on the N-terminus through a PGGG linker (PutOx−AuBP).…”

“…Another approach that has been developed recently by our groups and others is the use of genetically engineered affinity peptides for the selective, specific immobilization of biomolecules at particular material interfaces. − These peptides have been developed using cell surface display and phage display to have a high affinity for a specific material (e.g., Au, Ag, silica, graphite). − While a number of peptides with high affinity for different materials have been developed and demonstrated, detailed investigation of the immobilization of active enzymes using these peptides is relatively unexplored. ,,,− We and others studied solid binding peptides to control orientation of the proteins and demonstrated self-immobilization of several enzymes, including alkaline phosphatase, glutathione S-transferase, and lactate dehydrogenase on specific materials, − but details including information regarding the correlation of orientation and coverage with activity are still lacking …”