Autodisplay of glucose‐6‐phosphate dehydrogenase for redox cofactor regeneration at the cell surface

Abstract

Inherent cofactor regeneration is a pivotal feature of whole cell biocatalysis. For specific biotechnological applications, surface display of enzymes is emerging as a tool to circumvent mass transfer limitations or enzyme stability problems. Even complex reactions can be accomplished applying displayed enzymes. Yet, industrial utilization of the technique is still impeded by lacking cofactor regeneration at the cell surface. Here, we report on the surface display of a glucose-6-phoshate dehydrogenase (G6PDH) via Autodisplay to address this limitation and regenerate NADPH directly at the cell surface. The obtained whole cell biocatalyst demonstrated similar kinetic parameters compared to the purified enzyme, more precisely K values of 0.2 mM for NADP and calculated total turnover numbers of 10 . However, the K for the substrate G6P increased by a factor of 7 to yield 1.5 mM. The whole cell biocatalyst was cheaper to produce, easy to separate from the reaction mixture and reusable in consecutive reaction cycles. Furthermore, lyophilization allowed storage at room temperature. The whole cell biocatalyst displaying G6PDH was applicable for NADPH regeneration in combination with soluble as well as surface displayed enzymes and model reactions in combination with bacterial CYP102A1 and human CYP1A2 were realized. Biotechnol. Bioeng. 2017;114: 1658-1669. © 2017 Wiley Periodicals, Inc.

Biochemical and Kinetic Characterization of the Glucose-6-Phosphate Dehydrogenase from Helicobacter pylori Strain 29CaP

“…To determine the effect of pH on the purified recombinant HpG6PD protein activity, we carried out an activity analysis at various pH values, ranging from 3.0 to 10.0. The curve obtained in this assay (Figure 3A) showed a classical bell-shaped curve, as previously reported for different G6PDs enzymes, in which we observed the highest point of activity (100%) at a pH of 7.5 [34,44,[47][48][49]. Interestingly, the enzyme's activity decreases until it loses activity (0%) at a pH of 10.0.…”

Molecular Cloning and Exploration of the Biochemical and Functional Analysis of Recombinant Glucose-6-Phosphate Dehydrogenase from Gluconoacetobacter diazotrophicus PAL5

“…Furthermore, this result agrees with those previously reported, where the enzyme had a similar pH profile compared with the other previously purified G6PDs. For example, pH values of around 7.5 to 8 were found in G6PDs from Homo sapiens , P. aeruginosa , B. malayi , buffalo liver, camel liver, dog liver, T. crassiceps , T. cruzy , T. maritima , E. coli DH5α, and A. Oryzae [20,21,22,24,25,28,29,30,31,32,33]. However, an optimal pH value of around 9 has also been reported for G6PDs from A. niger and A. midulms [34].…”

NADH Regeneration Promoted by Solar Light Using Gold Nanoparticles/Layered Double Hydroxides as Novel Photocatalytic Nanoplatforms

“…This type of systems are able to solve enzyme stability problems and the mass transfer limitations. [11] The major barrier in surface display technology to industrial application is the low enzyme concentration in the cell wall. [9] Besides the enzymatic route, a broad range of non-enzymatic NAD + reduction methods have been developed to overcome the shortcoming of the traditional regeneration way.…”

“…Promising results were obtained with cell surface‐displayed enzymes . This type of systems are able to solve enzyme stability problems and the mass transfer limitations [11] . The major barrier in surface display technology to industrial application is the low enzyme concentration in the cell wall [9] .…”