Semi-rational engineering of D-allulose 3-epimerase for simultaneously improving the catalytic activity and thermostability based on D-allulose biosensor.

BACKGROUND: D-Allulose is one of the most well-known rare sugars widely used in food, cosmetics, and pharmaceutical industries. The most popular method for D-allulose production is the conversion from D-fructose catalyzed by D-allulose 3-epimerase (DAEase). To address the general problem of low catalytic efficiency and poor thermostability of wild-type DAEase, D-allulose biosensor was adopted in this study to develop a convenient and efficient method for high-throughput screening of DAEase variants. RESULTS: The catalytic activity and thermostability of DAEase from Caballeronia insecticola were simultaneously improved by semi-rational molecular modification. Compared with the wild-type enzyme, DAEaseS37N/F157Y variant exhibited 14.7% improvement in the catalytic activity and the half-time value (t1/2) at 65°C increased from 1.60 to 27.56 h by 17.23-fold. To our delight, the conversion rate of D-allulose was 33.6% from 500-g L-1 D-fructose in 1 h by Bacillus subtilis WB800 whole cells expressing this DAEase variant. Furthermore, the practicability of cell immobilization was evaluated and more than 80% relative activity of the immobilized cells was maintained from the second to seventh cycle. CONCLUSION: All these results indicated that the DAEaseS37N/F157Y variant would be a potential candidate for the industrial production of D-allulose.

Efficient enzymatic synthesis of d-allulose using a novel d-allulose-3-epimerase from Caballeronia insecticola.

BACKGROUND: Rare sugars have become promising 'sugar alternatives' because of their low calories and unique physiological functions. Among the family of rare sugars, d-allulose is one of the sugars attracting interest. Ketose 3-epimerases (KEase), including d-tagatose 3-epimerase (DTEase) and d-allulose 3-epimerase (DAEase), are mainly used for d-allulose production. RESULTS: In this study, a putative xylose isomerase from Caballeronia insecticola was characterized and identified as a novel DAEase. Caballeronia insecticola DAEase displayed prominent enzymatic properties, and 150 g L-1 d-allulose was produced from 500 g L-1 d-fructose in 45 min with a conversion rate of 30% and high productivity of 200 g L-1 h-1 . Furthermore, DAEase was employed in a phosphorylation-dephosphorylation cascade reaction, which significantly increased the conversion rate of d-allulose. Under optimized conditions, the conversion rate of d-allulose was approximately 100% when the concentration of d-fructose was 50 mmol L-1 . CONCLUSION: This research described a very beneficial and facile approach for d-allulose production based on C. insecticola DAEase. © 2022 Society of Chemical Industry.

Identification of a Novel Alditol Oxidase from Thermopolyspora flexuosa with Potential Application in D-Glyceric Acid Production.

Glycerol is a potential sustainable feedstock, and biorefining processes to convert glycerol into value-added chemicals have been developed over the past decade. Alditol oxidase (AldO) is capable of selectively oxidizing the primary hydroxyl groups of alditols such as glycerol. In this study, a new FAD-binding protein from Thermopolyspora flexuosa was expressed and identified as a novel alditol oxidase (AldOT. fle). AldOT. fle displayed the optimal activity at pH 8.0 and 25 °C. AldOT. fle was not metal-dependent, but the activity was completely inhibited by Fe3+. AldOT. fle had a wide substrate specificity and high catalytic efficiency for glycerol. Furthermore, the recombinant AldOT. fle could produce D-glyceric acid from glycerol with a conversion rate ranging from 86.6% (5 mM glycerol) to 20.5% (500 mM glycerol). The recombinant E. coli with AldOT. fle could also produce 23.8 mM D-glyceric acid from 100 mM glycerol. The recombinant AldOT. fle had the potential to produce other aldehyde products by selectively oxidizing the hydroxyl groups of alditols and many other commodity chemicals by redesigning glycerol metabolism.

Electrophoretic deposition of carbon nanotube on reticulated vitreous carbon for hexavalent chromium removal in a biocathode microbial fuel cell.

For Cr(VI)-removal microbial fuel cell (MFC), a more efficient biocathode in MFCs is required to improve the Cr(VI) removal and electricity generation. RVC-CNT electrode was prepared through the electrophoretic deposition of carbon nanotube (CNT) on reticulated vitreous carbon (RVC). The power density of MFC with an RVC-CNT electrode increased to 132.1 ± 2.8 mW m-2, and 80.9% removal of Cr(VI) was achieved within 48 h; compared to only 44.5% removal of Cr(VI) in unmodified RVC. Cyclic voltammetry, energy-dispersive spectrometry and X-ray photoelectron spectrometry showed that the RVC-CNT electrode enhanced the electrical conductivity and the electron transfer rate; and provided more reaction sites for Cr(VI) reduction. This approach provides process simplicity and a thickness control method for fabricating three-dimensional biocathodes to improve the performance of MFCs for Cr(VI) removal.