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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 271 records · Page 15
All sourcesNASA NTRSDOE OSTI
DOE OSTI2019

Biotransformation of Isoeugenol into Vanillin Using Immobilized Recombinant Cells Containing Isoeugenol Monooxygenase Active Aggregates

For efficiently enhancing the activity of isoeugenol monooxygenase, a whole cell overproducing active aggregate IEM720-18A was successfully fabricated via the fusion of amphiphilic short peptide 18A (EWLKAFYEKVLEKLKELF) and isoeugenol monooxygenase and then efficiently expressed in E. coli BL21 (DE3). Such resulting bacteria, E. coli BL21 (DE3) harboring pET30a-IEM720-18A was applied in the biotransformation of isoeugenol to vanillin with the optimization of cultivation conditions. Our results revealed that the vanillin concentration reached to the highest level (14.5 mmol/L) under the optimized reaction conditions including 1.5-g cells containing active aggregate of IEM720-18A, 10% (v/v) dimethyl sulfoxide (DMSO), 100 mmol/L isoeugenol, 50 mmol/L glycine-sodium hydroxide buffer (pH 10.5) in 10 mL reaction volume, and 200 rpm at 25 °C for 36 h. Moreover, the active aggregate IEM720-18A was immobilized with 100 mmol/L glutaraldehyde at 4 °C to improve the operational stability. The highest activity could be achieved when the reactions were carried out at 25 °C and the relative activity of the immobilized enzyme maintained over 60% after seven recycles. Our study provides a new approach to the biotransformation of isoeugenol into vanillin.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

A characterization of recombinant Arabidopsis FRIABLE1 (FRB1) reveals robust rhamnogalacturonan-I rhamnosyltransferase activity and critical catalytic residues

Plant cell walls are glycan-rich extracellular matrices that fundamentally impact essential cellular processes, such as growth, adhesion, and cell shape acquisition. Understanding plant cell wall glycans requires the identification and characterization of the biosynthetic enzymes that produce these polymers. Most successful in vitro protein expression studies of plant cell wall glycosyltransferases have relied on insect, fungal/yeast, or human cell expression systems, whereas prokaryotic expression systems have been generally unsuccessful. Here, we show that Arabidopsis FRIABLE1 (FRB1)/rhamnogalacturonan-I rhamnosyltransferase 8 (RRT8) can be produced in Escherichia coli RosettaGami2 cells as N-terminal maltose-binding protein fusion proteins containing C-terminal 6X-His-tags. We also report the catalytic constants of FRB1/RRT8 with apparent K M and K cat values of 226 μM and 33 min -1 for UDP-Rhamnose and 117 μM and 28.7 min -1 for rhamnogalacturonan-I (RG-I), respectively. We examine the catalytic activities of mutated FRB1/RRT8 proteins based on an AlphaFold 3-generated FRB1/RRT8 protein structural model with a virtually docked UDP-Rha donor. Enzymatic characterization of the mutated and wildtype FRB1/RRT8 protein confirmed that mutation of predicted catalytic site amino acid residues resulted in a 20-fold reduction in RRT activity. FRB1 also robustly polymerizes RG-I in combination with RG-I galacturonosyltransferase 1. These results show how a robust E. coli expression system combined with artificial intelligence tools can be used to increase understanding of plant cell wall glycosyltransferase structure and function.

glycosyltransferase↗