Covalent immobilization of oxylipin biosynthetic enzymes on nanoporous rice husk silica for production of cis(+)-12-oxophytodienoic acid

Abstract

Soybean lipoxygenase, recombinant rice allene oxide synthase-1 and rice allene oxide cyclase were covalently immobilized on nanoporous rice husk silica using two types of linkers: glutardialdehyde and polyethylene glycol. The immobilization efficiency achieved using glutardialdehyde-linked rice husk silica was higher than that achieved using polyethylene glycol-linked rice husk silica (50-92% and 25-50%, respectively). Immobilization on both types of matrices significantly decreased the specific activities of the immobilized enzymes. Solid-phase reaction yields of the enzymes were determined relative to the yields observed for the solution-phase reactions. Yields of the solid-phase reactions catalyzed by immobilized soybean lipoxygenase, rice allene oxide synthase-1, and rice allene oxide cyclase ranged from 50% to 230% and were dependent on both the enzymes and linkers used. Production of cis(+)-12-oxophytodienoic acid from α-linolenic acid by consecutive reactions using all three enzymes in a co-immobilization system resulted in 83.6% and 65.1% yields on glutardialdehyde-linked and epichlorohydrin-polyethylene glycol-linked rice husk silica, respectively. Our results suggest that immobilization of biosynthetic enzymes of the octadecanoid pathway on rice husk silica may be an efficient method for the in vitro production of oxylipins. Additionally, enzyme immobilizations on rice husk silica matrices may be more broadly applicable for producing physiologically important compounds in other biosynthetic pathways.

Jasmonates: News on Occurrence, Biosynthesis, Metabolism and Action of an Ancient Group of Signaling Compounds

“…For research purpose, there is permanent demand for OPDA. Now, OPDA can be synthesized by covalently immobilized recombinant rice AOS-1, together with bound soybean LOX and rice AOC, which are able to form OPDA from α–LeA up to more than 80% yield [ 82 ] or by enzymatic multi-step one-pot synthesis using all three enzymes from A. thaliana and α–LeA as substrate [ 83 ].…”

Facile Immobilization of Soybean Lipoxygenase on Nanoporous Rice Husk Silica by Adsorption

“…and a long arm linker (ECH-PEG-linked RHS) were shown to be maintained during ten cycles although there were differences depending on the linker and enzyme type [13]. In this study, the RHS-adsorbed soybean LOX was reused five times and its activity showed gradual decreases according to the cycles of reuse (Figure 4).…”

“…JA biosynthesis, which is involved in the production of unstable intermediates by participating enzymes located in other cellular organelles, has encountered obstacles with in vivo production and regulation. To overcome this inconvenience, enzyme immobilization can effectively mimic the multistep JA biosynthesis by co-immobilization regardless of cellular location [13].…”

Structural Evidence for the Substrate Channeling of Rice Allene Oxide Cyclase in Biologically Analogous Nazarov Reaction

“…As mentioned above, this would protect the 12,13-EOT intermediate, and prevent its premature dissolution, by effectively coupling catalysis in the active sites of OsAOC and OsAOS1. We previously reported a substrate channeling effect, when OsAOC and OsAOS1 were immobilized on nanoporous rice husk silica (Le et al, 2017 ). Data presented here also demonstrate a hyperbolic dependence of OsAOC reaction yield on the molar ratio of OsAOC to OsAOS1, and this is consistent with the proposed substrate channeling (Figure 7A ).…”