Efficient Synthesis of l-Asparagine by an Immobilized Three-Enzyme Cascade Reaction System DOI
Ran Wang, Wei Song, Huimin Xu

et al.

ACS Sustainable Chemistry & Engineering, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 15, 2025

l-Asparagine (l-Asn) is an important amino acid with broad applications in food, medicine, fine chemicals, and environmental protection. However, its industrial production limited by the high cost of raw materials low catalytic efficiency enzymes. In this study, a three-enzyme cascade pathway (FDN) was designed to produce l-Asn, using fumaric as material. Within pathway, EcAsnA identified rate-limiting enzyme subsequently engineered product rescue strategy reduce inhibition opening closed gate. The optimal mutant, L109 K/K58R, exhibited 6.61-fold reduction 4.24-fold improvement efficiency. This mutant then integrated into Escherichia coli along other two enzymes construct recombinant strain E. 17. Using diatomite-glutaraldehyde cross-linking immobilized 17 biocatalyst, 267.74 g l-Asn (with 5.35 g·L–1·h–1 STY, > 99% ee) produced across 50 batch feedings 1 L reaction volume. purity exceeded after isolation purification. study demonstrates effective integration design, engineering, cell immobilization, providing promising approach for synthesizing high-value products from cost-effective substrates.

Language: Английский

Optimized Substrate Positioning Enables Switches in the C–H Cleavage Site and Reaction Outcome in the Hydroxylation–Epoxidation Sequence Catalyzed by Hyoscyamine 6β-Hydroxylase DOI
Eliott S. Wenger, Ryan J. Martinie, Richiro Ushimaru

et al.

Journal of the American Chemical Society, Journal Year: 2024, Volume and Issue: 146(35), P. 24271 - 24287

Published: Aug. 22, 2024

Hyoscyamine 6β-hydroxylase (H6H) is an iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase that produces the prolifically administered antinausea drug, scopolamine. After its namesake hydroxylation reaction, H6H then couples newly installed C6 oxygen to C7 produce drug's epoxide functionality. Oxoiron(IV) (ferryl) intermediates initiate both reactions by cleaving C–H bonds, but it remains unclear how enzyme switches target site promotes (C6)O–C7 coupling in preference second step. In one possible epoxidation mechanism, would─analogously mechanisms proposed for Fe/2OG halogenases and, our more recent study, N-acetylnorloline synthase (LolO)─coordinate as alkoxide C7–H-cleaving ferryl intermediate enable alkoxyl ensuing radical. Here, we provide structural kinetic evidence does not employ substrate coordination or repositioning step instead exploits distinct spatial dependencies of competitive cleavage (C6 vs C7) C–O-coupling (oxygen rebound cyclization) steps promote two-step sequence. Structural comparisons ferryl-mimicking vanadyl complexes wild-type a variant preferentially 7-hydroxylates epoxidizing 6β-hydroxyhyoscyamine suggest modest (∼10°) shift Fe–O–H(C7) approach angle sufficient change outcome. The 7-hydroxylation:epoxidation partition ratios proteins increase than 5-fold 2H2O, reflecting epoxidation-specific requirement alcohol O–H bond, which, unlike LolO oxacyclization, accomplished iron advance cleavage.

Language: Английский

Citations

5
Efficient Synthesis of l-Asparagine by an Immobilized Three-Enzyme Cascade Reaction System DOI
Ran Wang, Wei Song, Huimin Xu

et al.

ACS Sustainable Chemistry & Engineering, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 15, 2025

l-Asparagine (l-Asn) is an important amino acid with broad applications in food, medicine, fine chemicals, and environmental protection. However, its industrial production limited by the high cost of raw materials low catalytic efficiency enzymes. In this study, a three-enzyme cascade pathway (FDN) was designed to produce l-Asn, using fumaric as material. Within pathway, EcAsnA identified rate-limiting enzyme subsequently engineered product rescue strategy reduce inhibition opening closed gate. The optimal mutant, L109 K/K58R, exhibited 6.61-fold reduction 4.24-fold improvement efficiency. This mutant then integrated into Escherichia coli along other two enzymes construct recombinant strain E. 17. Using diatomite-glutaraldehyde cross-linking immobilized 17 biocatalyst, 267.74 g l-Asn (with 5.35 g·L–1·h–1 STY, > 99% ee) produced across 50 batch feedings 1 L reaction volume. purity exceeded after isolation purification. study demonstrates effective integration design, engineering, cell immobilization, providing promising approach for synthesizing high-value products from cost-effective substrates.

Language: Английский

Citations

0