The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I. The transaminases were developed by extensive protein engineering followed by optimization of the identified motif. The resulting enzymes exhibited up to 8,900-fold higher activity than the starting scaffold and are highly stereoselective (up to >99.9% enantiomeric excess) in the asymmetric synthesis of a set of chiral amines bearing bulky substituents. These enzymes should therefore be suitable for use in the synthesis of a wide array of potential intermediates for pharmaceuticals. We also show that the motif can be engineered into other protein scaffolds with sequence identities as low as 70%, and as such should have a broad impact in the field of biocatalytic synthesis and enzyme engineering.
Biocatalytic alkylations are important reactions to obtain chemo-, regio-and stereoselectively alkylated compounds. This can be achieved using S-adenosyl-l-methionine (SAM)-dependent methyltransferases and SAM analogs. It was recently shown that a halide methyltransferase (HMT) from Chloracidobacterium thermophilum can synthesize SAM from SAH and methyl iodide. We developed an iodide-based assay for the directed evolution of an HMT from Arabidopsis thaliana and used it to identify a V140T variant that can also accept ethyl-, propyl-, and allyl iodide to produce the corresponding SAM analogs (90, 50, and 70 % conversion of 15 mg SAH). The V140T AtHMT was used in one-pot cascades with O-methyltransferases (IeOMT or COMT) to achieve the regioselective ethylation of luteolin and allylation of 3,4dihydroxybenzaldehyde. While a cascade for the propylation of 3,4-dihydroxybenzaldehyde gave low conversion, the propyl-SAH intermediate could be confirmed by NMR spectroscopy.
Flavonoids are a large group of plant secondary metabolites with a variety of biological properties and are therefore of interest to many scientists, as they can lead to industrially interesting intermediates. The anaerobic gut bacterium Eubacterium ramulus can catabolize flavonoids, but until now, the pathway has not been experimentally confirmed. In the present work, a chalcone isomerase (CHI) and an enoate reductase (ERED) could be identified through whole genome sequencing and gene motif search. These two enzymes were successfully cloned and expressed in Escherichia coli in their active form, even under aerobic conditions. The catabolic pathway of E. ramulus was confirmed by biotransformations of flavanones into dihydrochalcones. The engineered E. coli strain that expresses both enzymes was used for the conversion of several flavanones, underlining the applicability of this biocatalytic cascade reaction.
We examine the immobilization of lipase B from Candida antarctica on functionalized multi‐wall carbon nanotubes (MWCNTs) through physical adsorption. MWCNTs functionalized with carboxyl‐, amine‐ and ester‐ terminal groups on their surface are used as immobilization carriers. Dispersion of the nanotubes and the immobilization procedure take place in aqueous and low‐water media. High enzyme loadings are attained, up to 25% of the weight of the carbon nanotubes. These novel biomaterials are characterized though FT‐IR and Raman spectroscopy. The MWCNT–lipase bioconjugates exhibit high catalytic activity and increased storage and operational stability. The biomaterials retain more than 55% of their initial activity after 6 months at 4 °C, while they retain approximately 25% of their initial activity after 30 d of incubation in hexane at 60 °C. The catalytic behaviour of the immobilized enzyme depends on the terminal group of the carbon nanotubes, the concentration of the enzyme and the immobilization method employed.
Development of effective nanobiocatalytic systems through the immobilization of hydrolases on functionalized carbon-based nanomaterials Pavlidis, Ioannis V.; Vorhaben, Torge; Tsoufis, Theodoros; Rudolf, Petra; Bornscheuer, Uwe T.; Gournis, Dimitrios; Stamatis, Haralambos Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
a b s t r a c tIn this study we report the use of functionalized carbon-based nanomaterials, such as amine-functionalized graphene oxide (GO) and multi-walled carbon nanotubes (CNTs), as effective immobilization supports for various lipases and esterases of industrial interest. Structural and biochemical characterization have revealed that the curvature of the nanomaterial affect the immobilization yield, the catalytic behavior and the secondary structure of enzymes. Infrared spectroscopy study indicates that the catalytic behavior of the immobilized enzymes is correlated with their a-helical content. Hydrolases exhibit higher esterification activity (up to 20-fold) when immobilized on CNTs compared to GO. The covalently immobilized enzymes exhibited comparable or even higher activity compared to the physically adsorbed ones, while they presented higher operational stability. The enhanced catalytic behavior observed for most of the hydrolases covalently immobilized on amine-functionalized CNTs indicate that these functionalized nanomaterials are suitable for the development of efficient nanobiocatalytic systems.
Nanomaterials as solid supports can improve the efficiency of immobilized enzymes by reducing diffusional limitation as well as by increasing the surface area per mass unit and therefore improving enzyme loading. In this work, -glucosidase from almonds was immobilized on two smectite nanoclays. The resulting hybrid biocatalysts were characterized by a combination of powder X-ray diffraction, X-ray photoelectron spectroscopy, thermogravimetric analysis, differential thermal analysis, and infrared spectroscopy. Biochemical studies showed an improved thermostability of the immobilized enzyme as well as enhanced performance at higher temperatures and in a wider pH range.
The immobilization of lipase B from Candida antarctica on smectite group nanoclays (Laponite, SWy-2 and Kunipia), as well as on their organically modified derivatives, was investigated. A combination of techniques, namely X-ray diffraction, thermal analysis, X-ray photoelectron and FT-IR spectroscopy, was used for characterization of the novel immobilized biocatalyst. Structural and biochemical characterization have revealed that the hydrophobic microenvironment created by the organo-modified clays induces minor changes on the secondary structure of the enzyme, resulting in enhanced catalytic behaviour in hydrophobic media. The immobilized lipase on such modified nanoclays can be effectively applied for the indirect epoxidation of alpha-pinene using hydrogen peroxide as substrate. The amount of alpha-pinene epoxide produced in a single-step biocatalytic process is up to 3-fold higher than that of free enzyme or enzyme immobilized in non-modified clays. Moreover, lipase immobilized in modified clays retains up to 90% of its initial activity, even after 48h of incubation in the presence of oxidant, and up to 60% after four reaction cycles, while other forms of the enzyme retain less than 10%.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.