M. Dolors Benaiges

Rational Nanoconjugation Improves Biocatalytic Performance of Enzymes: Aldol Addition Catalyzed by Immobilized Rhamnulose-1-Phosphate Aldolase

Gold nanoparticles (AuNPs) are attractive materials for the immobilization of enzymes due to several advantages such as high enzyme loading, absence of internal diffusion limitations, and Brownian motion in solution, compared to the conventional immobilization onto porous macroscopic supports. The affinity of AuNPs to different groups present at the protein surface enables direct enzyme binding to the nanoparticle without the need of any coupling agent. Enzyme activity and stability appear to be improved when the biocatalyst is immobilized onto AuNPs. Rhamnulose-1-phosphate aldolase (RhuA) was selected as model enzyme for the immobilization onto AuNPs. The enzyme loading was characterized by four different techniques: surface plasmon resonance (SPR) shift and intensity, dynamic light scattering (DLS), and transmission electron microscopy (TEM). AuNPs-RhuA complexes were further applied as biocatalyst of the aldol addition reaction between dihydroxyacetone phosphate (DHAP) and (S)-Cbz-alaninal during two reaction cycles. In these conditions, an improved reaction yield and selectivity, together with a fourfold activity enhancement were observed, as compared to soluble RhuA.

Influence of water activity and support material on the enzymatic synthesis of a CCK-8 tripeptide fragment

The kinetically controlled condensation of Z-Gly-Trp-OMe and H-Met-OEt catalyzed by α-chymo-trypsin in organic media is reported. The influence of thermodynamic water activity and the support material used to adsorb α-chymotrypsin, on both the product yield and enzymatic activity was investigated. Polyamide based materials were the best support at low water activity rendering the highest reaction rates and yields. The activity of the adsorbed enzyme at low water activities depends on both the accessible surface area and the hydrophobicity of the support. Polyamide had both adequate hydrophilicity and high surface area yielding the best results. Polypropylene based supports were strongly hydrophobic and, although they presented a high surface area, the enzymatic activity was much lower. The solvents used to carry out the synthesis were acetonitrile and ethyl acetate. No significant differences were observed on the performance of the reaction in either solvent. The tripeptide selected is a fragment of the c...

Performance of an immobilized fuculose-1-phosphate aldolase for stereoselective synthesis

A derivative of fuculose-1-phosphate aldolase, immobilized with high loading on glyoxal–agarose gels, has been characterized and evaluated as a biocatalyst for an aldol addition reaction. The reaction of the solid biocatalyst was diffusion-controlled for conversion of its natural substrate. Nevertheless, when catalyzing the synthesis of a biologically active aminopolyol, the lower reaction rate with non-natural substrates led to a process controlled by the intrinsic enzyme kinetics. The resulting biocatalyst has high synthetic specific activity and has been successfully used in batch synthesis reactions with high conversion. In addition, the immobilized aldolase has been employed in fed-batch synthesis, increasing the selectivity of the reaction and obtaining high conversion (88%).

Integrated process for the enzymatic synthesis of the octapeptide PhAcCCK-8

A process for the enzymatic synthesis of PhAcCCK‐8 is presented. The CCK‐8 (CCK26‐33) peptide fragment is the minimum sequence with biological activity of the cholecystokinin hormone. A synthetic convergent strategy has been developed starting from amino acid derivatives as raw materials, employing proteases as biocatalysts for each peptide coupling. The enzymes have been immobilized by deposition onto solid supports in order to be employed in organic media at low water activity. N‐Terminal protecting groups such as PhAc, which can be introduced and removed enzymatically, have been employed. The synthesis process has been set up at preparative level with focus in the integration of reaction and separation steps with an overall yield of 15%.

N-PROTECTION OF AMINO ACID DERIVATIVES CATALYZED BY IMMOBILIZED PENICILLIN G ACYLASE

The introduction of a N-α-phenylacetyl moiety into glycine, methionine and aspartic acid derivatives using immobilized penicillin G acylase (PGA) as catalyst was studied. High synthetic yields (86–97%) were obtained in biphasic systems under thermodynamic control using fully carboxy-protected derivatives of amino acids such as H-Gly-OMe, H-Gly-OBzl, H-Met-OEt, H-Asp(OBut)-OMe, H-Asp(OMe)-OMe and H-Asp(OBzl)-OBzl. Moderate yields (50–62%) were obtained for the N-α protection of β-carboxy free, α-carboxy esters of aspartic acid such as H-Asp-OMe and H-Asp-OBzl in methanol/buffer mixtures under kinetic control. The synthesis of some phenylacetyl derivatives on a preparative scale under the optimum conditions is also described.

Recombinant Candida rugosa lipase 2 from Pichia pastoris: Immobilization and use as biocatalyst in a stereoselective reaction

The characterization of the recombinant Candida rugosa Lip2 (r‐Lip2) isoenzyme obtained from fed‐batch cultures of Pichia pastoris under PAOX promoter was carried out, determining the optimal pH and temperature as well as their catalytic performance in both hydrolysis and synthesis reactions comparing with purified native Lip2 (n‐Lip2) previously determined. The substrate specificity of r‐Lip2 in hydrolysis reactions was determined with a series of triacylglycerols and p‐nitrophenyl esters of variable acyl chain length. r‐Lip2 showed the maximum specificity for both substrates towards medium‐chain esters (C‐8), similar behavior was observed with n‐Lip2. However, significant differences were observed towards unsaturated substrates (triolein) or short‐chain esters. A statistical design applied to study the effect of pH and temperature on lipase stability shown that r‐Lip2, like n‐Lip2, was more sensitive to pH than temperature changes. Nevertheless, the overall stability of soluble r‐Lip2 was lower than soluble n‐Lip2. The stability of r‐lip2 was significantly improved by immobilization onto EP100, an excellent support for lipases with yields around 95% for offered lipolytic activity lower than 600 AU/mL. Finally, immobilized r‐Lip2 was tested in the resolution of ibuprofen in isooctane by means of enantioselective esterification using 1‐butanol as esterifying agent. r‐Lip2 showed a better performance in terms of enantiomeric excess (74%) and enatiomeric factor (96%) than n‐Lip2 (56 and 80%, respectively) for the same conversion (40%). Thus, r‐Lip2 should be considered a good and pure biocatalyst, easy to produce and with a remaining activity of ca. 90% after one reaction cycle when immobilized on EP100.

Papain Immobilization Study in Enzymatic Synthesis of Dipeptide Gly-Phe

Papain catalysed peptide synthesis was followed to determine the optimum conditions for adsorption and covalent binding immobilization methods. The synthesis of the dipeptide Gly-Phe was studied in two different reaction systems: a) For adsorbed papain, in an organic medium containing low water concentration, b) For covalently bound enzyme, in a two-liquid phase system, using trichloroethylene as organic phase. The amount of enzyme offered to the immobilization support and the pH of the immobilization procedure were the main parameters studied.

Exploring substrate specificities of a recombinant Rhizopus oryzae lipase in biodiesel synthesis

The alcoholysis of triolein was used to explore the specific features of a recombinant Rhizopus oryzae lipase (rROL) for biodiesel synthesis. For this purpose, different acylglycerols were compared as substrates in lipase-catalysed transesterification. rROL was shown to exhibit a higher specificity towards 1-monoolein than triolein compared to other R. oryzae lipases, being more than 4-fold more specific; in contrast, rROL did not accept 2-monoolein as substrate, concluding that it is highly 1,3-positional specific. Comparing ethanol and methanol as acyl-acceptors, it was observed that the latter caused more lipase inactivation. Regarding alcohols, it was also demonstrated that acyl migration occurred in moderate alcohol concentrations.

Rice husk ash as a potential carrier for the immobilization of lipases applied in the enzymatic production of biodiesel

Abstract The potential application of rice husk ash (RH26) as support for the immobilization of a recombinant Rhizopus oryzae (rROL) lipase as biocatalyst in the enzymatic biodiesel production using alperujo oil and the comparison with commercial hydrophobic support OD403 (RelOD) has been made. Although the specific activity (UA mg support−1) was around one-half lower in RH26 than in RelOD when they were used as biocatalyst in biodiesel reaction, the normalized initial rate was similar, between 1.6 and 2.4 μmol FAME mL−1 mL−1 UA−1. Thus in terms of biocatalysis performance, rice husk as is an alternative to commercial supports. However, the main problem is the more complex recovery of RH26 for the reutilization compared with commercial ones.

Biodiesel synthesis in a solvent-free system by recombinant Rhizopus oryzae: comparative study between a stirred tank and a packed-bed batch reactor

Abstract A simultaneous synthesis of biodiesel, as fatty acid methyl esters, and monoacylglycerols catalysed by the recombinant Rhizopus oryzae lipase immobilized by adsorption on Relizyme OD/403M is presented. The use of this 1(3)-positional specific lipase prevents the formation of glycerol as a by-product, thus avoiding its drawbacks. The synthesis was carried out in a solvent-free system and it has been studied in two different reactor systems: stirred tank and packed-bed reactor. Stirred tank reactor presented a high-initial reaction rate and achieved a 33.6% yield, which corresponds to a value of 50.4% of the maximum yield that can be achieved with a 1(3)-positional specific lipase. In packed-bed reactor there was a smaller initial reaction rate, but it was achieved a 49.1% yield, which corresponds to a 73.6% of the maximum yield. When a second batch is performed, the yield decreased only 4% when packed-bed reactor is employed whereas a drastic decrease is observed in a stirred tank operation. Therefore, packed-bed reactor showed a best performance and minor damage to the biocatalyst.