Javier Rocha-Martin

Coating of Soluble and Immobilized Enzymes with Ionic Polymers: Full Stabilization of the Quaternary Structure of Multimeric Enzymes

This paper shows a simple and effective way to avoid the dissociation of multimeric enzymes by coating their surface with a large cationic polymer (e.g., polyethylenimine (PEI)) by ionic exchange. As model enzymes, glutamate dehydrogenase (GDH) from Thermus thermophilus and formate dehydrogenase (FDH) from Pseudomonas sp. were used. Both enzymes are very unstable at acidic pH values due to the rapid dissociation of their subunits (half-life of diluted preparations is few minutes at pH 4 and 25 degrees C). GDH and FDH were incubated in the presence of PEI yielding an enzyme-PEI composite with full activity. To stabilize the enzyme-polymer composite, a treatment with glutaraldehyde was required. These enzyme-PEI composites can be crosslinked with glutaraldehyde by immobilizing previously the composite onto a weak cationic exchanger. The soluble GDH-PEI composite was much more stable than unmodified GDH at pH 4 and 30 degrees C (retaining over 90% activity after 24 h incubation) with no effect of the GDH concentration in the inactivation course. The composite could be very strongly, but reversibly, adsorbed on cationic exchangers. Similarly, FDH could be treated with PEI and glutaraldehyde after adsorption on cationic exchangers, This permitted a stabilized FDH preparation. In this way, the coating of the enzymes surfaces with PEI is used as a simple and efficient strategy to prevent enzyme dissociation of multimeric enzymes. These composites can be used as a soluble catalyst or reversibly immobilized onto a cationic exchanger (e.g., CM-agarose).

Improvement of enzyme properties with a two-step immobilizaton process on novel heterofunctional supports.

Novel heterofunctional glyoxyl-agarose supports were prepared. These supports contain a high concentration of groups (such as quaternary ammonium groups, carboxyl groups, and metal chelates) that are capable of adsorbing proteins, physically or chemically, at neutral pH as well as a high concentration of glyoxyl groups that are unable to immobilize covalently proteins at neutral pH. By using these supports, a two-step immobilization protocol was developed. In the first step, enzymes were adsorbed at pH 7.0 through adsorption of surface regions, which are complementary to the adsorbing groups on the support, and in the second step, the immobilized derivatives were incubated under alkaline conditions to promote an intramolecular multipoint covalent attachment between the glyoxyl groups on the support and the amino groups on the enzyme surface. These new derivatives were compared with those obtained on a monofunctional glyoxyl support at pH 10, in which the region with the greatest number of lysine residues participates in the first immobilization step. In some cases, multipoint immobilization on heterofunctional supports was much more efficient than what was achieved on the monofunctional support. For example, derivatives of tannase from Lactobacillus plantarum on an amino-glyoxyl heterofunctional support were 20-fold more stable than the best derivative on a monofunctional glyoxyl support. Derivatives of lipase from Geobacillus thermocatenulatus (BTL2) on the amino-glyoxyl supports were two times more active and four times more enantioselective than the corresponding monofunctional glyoxyl support derivative.

Rational Co-Immobilization of Bi-Enzyme Cascades on Porous Supports and their Applications in Bio-Redox Reactions with In Situ Recycling of Soluble Cofactors

In bio‐redox cascade reactions that are immobilized on porous supports, mass‐transfer limitations may impede the effective concentration of the cofactor around the corresponding dehydrogenases. This main drawback has been addressed by the co‐immobilization of both the main and recycling dehydrogenases. Herein, we report tailor‐made co‐immobilization procedures to assemble three different bio‐redox orthogonal cascades in vitro (two selective reductions and one selective oxidation) with in situ cofactor‐regeneration. However, the co‐immobilization itself does not guarantee the success of the biotransformation because the same co‐immobilization chemistry may not be suitable for the two enzymes that are involved in the bio‐redox cascade. Therefore, our co‐immobilization system was optimized for each bi‐enzymatic cascade. In all cases, the optimized co‐immobilization procedure was more efficient in the biocatalytic cascade than if the two dehydrogenases were immobilized on two different carriers. In one specific case (one thermophilic cascade), the co‐immobilization of an optimal ratio of main/recycling dehydrogenases (1:5) on the same carrier resulted in a biocatalyst that was able to recycle NADH up to 9000 times per equivalent of substrate in 1 hour at 55 °C. Moreover, uniform distributions of both dehydrogenases across the porous surface also enhanced the recycling efficiency of the cofactor 1.5‐fold versus cascades in which the enzymes were not uniformly distributed across the same porous surface, presumably because of vicinal cooperation effects. Hence, this system for the co‐immobilization of bi‐enzymatic systems may be extended to other biocatalytic cascades, thereby opening a window for the optimization of other multi‐enzyme biotransformations in which cofactor‐recycling is necessary.

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%.

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

BackgroundThe 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.ResultsThe 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.ConclusionWe have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of rac-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations.

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

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 18h 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.

Stabilization of enzymes by multipoint covalent immobilization on supports activated with glyoxyl groups

Stabilization of enzymes via immobilization techniques is a valuable approach in order to convert a necessary protocol (immobilization) into a very interesting tool to improve key enzyme properties (stabilization). Multipoint covalent attachment of each immobilized enzyme molecule may promote a very interesting stabilizing effect. The relative distances among all enzyme residues involved in immobilization has to remain unaltered during any conformational change induced by any distorting agent. Amino groups are very interesting nucleophiles placed on protein surfaces. The immobilization of enzyme through the region having the highest amount of amino groups (Lys residues) is key for a successful stabilization. Glyoxyl groups are small aliphatic aldehydes that form very unstable Schiffs bases with amino groups and they do not seem to be useful for enzyme immobilization at neutral pH. However, under alkaline conditions, glyoxyl supports are able to immobilize enzymes via a first multipoint covalent immobilization through the region having the highest amount of Lysine groups. Activation of supports with a high surface density of glyoxyl groups and the performance of very intense enzyme-support multipoint covalent attachments are here described.

Synthesis of propyl gallate by transesterification of tannic acid in aqueous media catalysed by immobilised derivatives of tannase from Lactobacillus plantarum

Immobilised derivatives of tannase from Lactobacillus plantarum were able to catalyse the transesterification of tannic acid by using moderate concentrations of 1-propanol in aqueous media. Transesterification of tannic acid was very similar to transesterification of methyl gallate. The synthetic yield depended on the pH and concentration of 1-propanol, although it did not vary much when using 30% or 50% 1-propanol. Synthetic yields of 45% were obtained with 30% of 1-propanol at pH 5.0. The product was chromatographically pure, and the reaction by-product was 55% pure gallic acid. On the other hand, immobilised tannase was fairly stable under optimal reaction conditions.

Additives enhancing enzymatic hydrolysis of lignocellulosic biomass

Linked to the development of cellulolytic enzyme cocktails from Myceliophthora thermophila, we studied the effect of different additives on the enzymatic hydrolysis yield. The hydrolysis of pretreated corn stover (PCS), sugar cane straw (PSCS) and microcrystalline cellulose (Avicel) was performed under industrial conditions using high solid loadings, limited mixing, and low enzyme dosages. The addition of polyethylene glycol (PEG4000) allowed to increase the glucose yields by 10%, 7.5%, and 32%, respectively in the three materials. PEG4000 did not have significant effect on the stability of the main individual enzymes but increased beta-glucosidase and endoglucanase activity by 20% and 60% respectively. Moreover, the presence of PEG4000 accelerated cellulase-catalyzed hydrolysis reducing up to 25% the liquefaction time. However, a preliminary economical assessment concludes that even with these improvements, a lower contribution of PEG4000 to the 2G bioethanol production costs would be needed to reach commercial feasibility.

Protein hydrolysis by immobilized and stabilized trypsin

The preparation of novel immobilized and stabilized derivatives of trypsin is reported here. The new derivatives preserved 80% of the initial catalytic activity toward synthetic substrates [benzoyl‐arginine p‐nitroanilide (BAPNA)] and were 50,000‐fold more thermally stable than the diluted soluble enzyme in the absence of autolysis. Trypsin was immobilized on highly activated glyoxyl‐Sepharose following a two‐step immobilization strategy: (a) first, a multipoint covalent immobilization at pH 8.5 that only involves low pKa amino groups (e.g., those derived from the activation of trypsin from trypsinogen) is performed and (b) next, an additional alkaline incubation at pH 10 is performed to favor an intense, additional multipoint immobilization between the high concentration of proximate aldehyde groups on the support surface and the high pKa amino groups at the enzyme surface region that participated in the first immobilization step. Interestingly, the new, highly stable trypsin derivatives were also much more active in the proteolysis of high molecular weight proteins when compared with a nonstabilized derivative prepared on CNBr‐activated Sepharose. In fact, all the proteins contained a cheese whey extract had been completely proteolyzed after 6 h at pH 9 and 50°C, as determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Under these experimental conditions, the immobilized biocatalysts preserve more than 90% of their initial activity after 20 days. Analysis of the three‐dimensional (3D) structure of the best immobilized trypsin derivative showed a surface region containing two amino terminal groups and five lysine (Lys) residues that may be responsible for this novel and interesting immobilization and stabilization. Moreover, this region is relatively far from the active site of the enzyme, which could explain the good results obtained for the hydrolysis of high‐molecular weight proteins.