Coimmobilization of L-asparaginase and glutamate dehydrogenase onto highly activated supports

In the present research work, production of coimmobilized derivatives of L-asparaginase and glutamate dehydrogenase was attempted. Comparison of immobilization of each enzyme independently with coimmobilization of the two enzymes unfolded important advantages of the latter, namely a decrease in the induction period (time before the maximum reaction rate is virtually achieved) and an increase in the maximum reaction rate. The effectiveness of the independent enzyme derivatives was low; however, it was enhanced by three-fold when the enzymes were coimmobilized onto the same agarose-glutaraldehyde support. Each supporting agarose bead may in fact be viewed as a nano-reactor with in situ reaction and separation (i.e. elimination of the ammonia formed), with the nanoenvironment surrounding each enzyme molecule being essentially devoid of steric hindrance

New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD+-recycling enzyme

<p>Abstract</p> <p>Background</p> <p>The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD⁺pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD⁺. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds.</p> <p>Results</p> <p>The NADH-oxidase (NOX) presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C) under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C). The hyperactivated form presented a high specific activity (37.5 U/mg) at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme). The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols.</p> <p>Conclusion</p> <p>We have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of <it>rac</it>-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations.</p

Disulfide Engineered Lipase to Enhance the Catalytic Activity: A Structure-Based Approach on BTL2

Enhancement, control, and tuning of hydrolytic activity and specificity of lipases are major goals for the industry. Thermoalkaliphilic lipases from the I.5 family, with their native advantages such as high thermostability and tolerance to alkaline pHs, are a target for biotechnological applications. Although several strategies have been applied to increase lipases activity, the enhancement through protein engineering without compromising other capabilities is still elusive. Lipases from the I.5 family suffer a unique and delicate double lid restructuration to transition from a closed and inactive state to their open and enzymatically active conformation. In order to increase the activity of the wild type Geobacillus thermocatenulatus lipase 2 (BTL2) we rationally designed, based on its tridimensional structure, a mutant (ccBTL2) capable of forming a disulfide bond to lock the open state. ccBTL2 was generated replacing A191 and F206 to cysteine residues while both wild type C64 and C295 were mutated to serine. A covalently immobilized ccBTL2 showed a 3.5-fold increment in esterase activity with 0.1% Triton X-100 (2336 IU mg-1) and up to 6.0-fold higher with 0.01% CTAB (778 IU mg-1), both in the presence of oxidizing sulfhydryl agents, when compared to BTL2. The remarkable and industrially desired features of BTL2 such as optimal alkaliphilic pH and high thermal stability were not affected. The designed disulfide bond also conferred reversibility to the enhancement, as the increment on activity observed for ccBTL2 was controlled by redox pretreatments. MD simulations suggested that the most stable conformation for ccBTL2 (with the disulfide bond formed) was, as we predicted, similar to the open and active conformation of this lipase.Financial and logistic support from Colombian Universidad del Valle and COLCIENCIAS (CI 71083-Grant 745-2016-Project 110671250425), Spanish CICYT project BIO-2005-6018576, BFU2017-90030-P, and BFU2011-25326, B. Di G. In addition, thanks to the Spanish MINECO for a FPU fellowship.S

Dextran-coated nanoparticles as immunosensing platforms: Consideration of polyaldehyde density, nanoparticle size and functionality

Magnetic nanoparticles (MNPs) can be used as antibody carriers in a wide range of immunosensing applications. The conjugation chemistry for preparing antibody-MNP bionanohybrids should assure the nanoparticle’s colloidal dispersity, directional conformation and high biofunctionality retention of attached antibodies. In this work, peroxidase (HRP) was selected as model target analyte, and stable antibody-MNP conjugates were prepared using polyaldehyde-dextrans as multivalent linkers, also to prevent nanoparticles agglomeration and steric shielding of non-specific proteins. Under the manipulation of the oxidation variables, MNP-conjugated antibody showed the highest Fab accessibility, of 1.32 μmol analyte per μmol antibody, corresponding to 139 μmol aldehyde per gram of nanocarrier (5 mM NaIO4, 4 h). Demonstrating anti-interference advantage up to 10% serum, colorimetric immunoassay gave a detection limit (LOD) of 300 ng mL− 1 , while electrochemical transduction led to a considerable (680 times) improvement, with a LOD of 0.44 ng mL− 1 . In addition, polyaldehydedextran showed priority over polycarboxylated-dextran as the multivalent antibody crosslinker for MNPs in terms of sensitivity and LOD value, while immunosensors constructed with carboxylated magnetic microbeads (HOOC-MBs) outperformed MNPs-based immunoplatforms. This work sheds light on the importance of surface chemistry (type and density of functional groups) and the dimension (nanosize vs micrometer) of magnetic carriers to conjugate antibodies with better directional orientation and improve the analytical performance of the resulting immunosensors

Copper-catalyzed diastereo- and enantioselective desymmetrization of cyclopropenes: Synthesis of cyclopropylboronates

This document is the accepted manuscript version of a Published Work that appeared in final form in Journal of American Chemical Society 136.45, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see DOI: 10.1021/ja510419zA novel Cu-catalyzed diastereo- and enantioselective desymmetrization of cyclopropenes to afford nonracemic cyclopropylboronates is described. Trapping the cyclopropylcopper intermediate with electrophilic amines allows for the synthesis of cyclopropylaminoboronic esters and demonstrates the potential of the approach for the synthesis of functionalized cyclopropanesWe thank the European Research Council (ERC-337776) and MINECO (CTQ2012-35957) for financial support. M. T. and A. P. thank MICINN for RyC and JdC contract

Novel enzyme-polymer conjugates for biotechnological applications

In the present research, a rapid, simple and efficient chemoselective method for the site-directed incorporation of tailor-made polymers into protein to create biocatalysts with excellent properties for pharmaceutical industrial purpose has been performed. First we focused on the protein engineering of the Geobacillus thermocatenulatus lipase 2 (BTL2) to replace the two cysteines (Cys65, Cys296) in the wild type enzyme (BTL-WT) by two serines. Then, by similar mode, a unique cysteine was introduced in the lid area of the protein. For the site-directed polymer incorporation, a set of different tailor-made thiol-ionic-polymers were synthesized and the protein cysteine was previously activated with 2,2-dithiodipyridine (2-PDS) to allow the disulfide exchange. The protected BTL variants were specifically modified with the different polymers in excellent yields, creating a small library of new biocatalysts. Different and important changes in the catalytic properties, possible caused by structural changes in the lid region, were observed. The different modified biocatalysts were tested in the synthesis of intermediates of antiviral and antitumor drugs, like nucleoside analogues and derivatives of phenylglutaric acid. In the hydrolysis of per-acetylated thymidine, the best biocatalyst was the BTL*-193-DextCOOH , where the activity was increased in 3-fold and the regioselectivity was improved, reaching a yield of 92% of 3’-O-acetyl-thymidine. In the case of the asymmetric hydrolysis of dimethyl phenylglutarate, the best result was found with BTL*-193-DextNH2-6000, where the enzyme activity was increased more than 5-fold and the enantiomeric excess was >99%.Fil: Romero, Oscar Eduardo. Consejo Superior de Investigaciones Cientificas; EspañaFil: Rivero, Cintia Wanda. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Biotecnología Sustentable; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Guisán, José M.. Consejo Superior de Investigaciones Cientificas; EspañaFil: Palomo, José M.. Consejo Superior de Investigaciones Cientificas; Españ

Selective oxidation of glycerol to 1,3-dihydroxyacetone by covalently immobilized glycerol dehydrogenases with higher stability and lower product inhibition

Funding Este trabajo ha contado con el apoyo del Ministerio de Economía e Innovación de España (proyecto CTQ2009-07568 ). Queremos agradecer a IKERBASQUE , Fundación Vasca para la Ciencia por la financiación al doctor Fernando López Gallego. También agradecemos al gobierno de la Comunidad de Madrid por financiar la beca de doctorado de J. Rocha-Martín.Glycerol dehydrogenase (GlyDH) catalyzes the regioselective oxidation of glycerol to yield 1,3-dihydroxyacetone (DHA); an important building block in chemical industry. Three recombinant GlyDHs from Geobacillus stearothermophilus, from Citrobacter braakii and from Cellulomonas sp. were stabilized by covalent immobilization. The highest activity recoveries (40–50%) of the insoluble preparations were obtained by immobilizing these enzymes in presence of polyethylene glycol (PEG). Noteworthy, these immobilized preparations were more stable and less inhibited by DHA than their soluble counterparts. In particular, GlyDH from G. stearothermophilus immobilized on agarose activated with both amine and glyoxyl groups and crosslinked with dextran aldehyde was 3.7-fold less inhibited by DHA than its soluble form and retained 100% of its initial activity after 18 h of incubation at 65 °C and pH 7. This is one of the few examples where the same immobilization protocol has minimized enzyme product inhibition and maximized thermal stability.Ministerio de Economía e Innovación de EspañaComunidad de MadridFundación Vasca para la Ciencia (IKERBASQUE)Depto. de Bioquímica y Biología MolecularFac. de Ciencias BiológicasTRUEpu

Oxidation of phenolic compounds catalyzed by immobilized multi-enzyme systems with integrated hydrogen peroxide production

Suicide inactivation of peroxidases by hydrogen peroxide is the major deterrent to using such biocatalysts in oxidative processes. This has been successfully addressed by the in situ generation of H2O2. In this study, we have developed a novel multi-enzyme biocatalyst that has been immobilized on agarose-type carriers to oxidize phenols using oxygen and formic acid as indirect oxidants. This original system couples the in situ production of H2O2 to the phenol oxidation via an enzymatic cascade that involved three different enzymes (formate dehydrogenase, NADH-oxidase and peroxidase) and two different redox cofactors: nicotinamide and flavin derivatives. The cascade reaction only works when enzymes are immobilized on the solid support since soluble enzymes are dramatically inactivated under the reaction conditions. The highest oxidation efficiency was achieved by combining two different solid biocatalysts: (1) formate dehydrogenase and NADH-oxidase co-immobilized onto agarose beads activated with glyoxyl groups and (2) peroxidase immobilized onto agarose beads as well but activated with boronate groups. Unlike conventional peroxidase-mediated oxidations with exogenous H2O2, this novel system enables the quantitative oxidation of phenol without the addition of H2O2. Furthermore, this novel system can use a broad range of redox cofactors with similar oxidative effectiveness. Therefore, this novel immobilized tri-enzyme system removes important pollutants such as hydroxylated aromatic derivatives (phenol, 4-aminophenol, 2,4-dichloro-phenol or α-naphthol) using formic acid and molecular oxygen as substrates. In addition, this system generates CO2 as waste beyond the oxidized phenols that can be easily separated from the aqueous solution. The sustainability of this system is supported by an E-factor of 1.3 and an atom economy of 43%.Ministerio de Economía e InnovaciónGobierno de EspañaDepto. de Bioquímica y Biología MolecularFac. de Ciencias BiológicasTRUEpu

Coimmobilization and colocalization of a glycosyltransferase and a sucrose synthase greatly improves the recycling of UDP-glucose: Glycosylation of resveratrol 3-O-β-D-glucoside

Funding Este trabajo fue apoyado por el proyecto SuSy del 7PM de la UE (Sacarosa sintasa como mediador rentable de reacciones de glicosilación, C-KBBE/3293). Javier Rocha-Martin agradece la beca Juan de la Cierva (IJCI-2014-19260) financiada por el Ministerio de Economía, Industria y Competitividad de España . El Prof. Tom Desmet y la Dra. Margo Diricks (Centro de Biotecnología y Biocatálisis Industrial, Universidad de Gante, Bélgica) proporcionaron amablemente el plásmido para la expresión de SuSyAc. Se agradece la ayuda y sugerencias del Prof. Bernd Nidetzky y el Dr. Alexander Gutmann (Instituto de Biotecnología e Ingeniería Bioquímica, Universidad de Graz, Austria). El Prof. Nidetzky amablemente proporcionó el plásmido para la expresión de UGT71A15. La producción de enzimas se llevó a cabo en la 'Instalación de Fermentación CBMSO'. Agradecemos al Laboratorio de Espectrometría de Masas (Servicio Interdepartamental de Investigaciones (SIdI), Universidad Autónoma de Madrid) por facilitar las instalaciones y asistencia técnica para el análisis RP-HPLC–ESI-Q-TOF-MS, en particular a la Dra. María Teresa Alonso Pascual. También agradecemos al Centro de Microscopía y Confocal (Instituto de Investigaciones Biomédicas “Alberto Sols”, IIBm-CSIC) su amable ayuda con los estudios CLSM. Se agradece la ayuda y sugerencias de María Romero-Fernández (Facultad de Química, Universidad de Nottingham).Glycosylation is one of the most efficient biocompatible methodologies to enhance the water solubility of natural products, and therefore their bioavailability. The excellent regio- and stereoselectivity of nucleotide sugar-dependent glycosyltransferases enables single-step glycosylations at specific positions of a broad variety of acceptor molecules without the requirement of protection/deprotection steps. However, the need for stoichiometric quantities of high-cost substrates, UDP-sugars, is a limiting factor for its use at an industrial scale. To overcome this challenge, here we report tailor-made coimmobilization and colocalization procedures to assemble a bi-enzymatic cascade composed of a glycosyltransferase and a sucrose synthase for the regioselective 5-O-β-D-glycosylation of piceid with in situ cofactor regeneration. Coimmobilization and colocalization of enzymes was achieved by performing slow immobilization of both enzymes inside the porous support. The colocalization of both enzymes within the porous structure of a solid support promoted an increase in the overall stability of the bi-enzymatic system and improved 50-fold the efficiency of piceid glycosylation compared with the non-colocalized biocatalyst. Finally, piceid conversion to resveratrol 3,5-diglucoside was over 90% after 6 cycles using the optimal biocatalyst and was reused in up to 10 batch reaction cycles accumulating a TTN of 91.7 for the UDP recycling.Unión EuropeaMinisterio de Economía, Industria y Competitividad (MINECO)Depto. de Bioquímica y Biología MolecularFac. de Ciencias BiológicasTRUEpu