Search Results (12)

In this study, trypsin from the porcine pancreas was immobilized on a heterofunctional support prepared by activating chitosan (Chit) hydrogel with glutaraldehyde (GA), then functionalizing it with glycine (Chit–GA–Gly). The catalytic performance of the immobilized trypsin in the hydrolysis reactions was compared with the catalytic performance of the immobilized enzyme on glutaraldehyde-activated chitosan (Chit–GA) and chitosan hydrogel (Chit). The maximum concentration of immobilized protein on Chit–GA–Gly was approximately 16 mg·g⁻¹ at pH 9.0 (5 mmol·L⁻¹ buffer sodium carbonate) at 25 °C from an offered protein loading of 20 mg·g⁻¹. This biocatalyst exhibited maximum specific activity (SA) of 33.1 ± 0.2 nmol·min⁻¹·mg⁻¹ for benzoyl-DL-arginine-p-nitroanilide (BAPNA) hydrolysis, twice as high as the enzyme immobilized on the classic Chit–GA support (SA values ranging between 6.7 ± 0.1 nmol·min⁻¹·mg⁻¹ and 8.1 ± 0.1 nmol·min⁻¹·mg⁻¹). The Elovich kinetic model was used to describe the adsorption process using low (3 mg·g⁻¹) and high (20 mg·g⁻¹) initial protein loadings. The optimum temperature for BAPNA hydrolysis catalyzed by the immobilized trypsin (60 °C) was 10 °C higher than that of its soluble form. Additionally, the immobilized enzyme was 16 to 20 times more stable than its soluble form at 50–55 °C. Thermodynamic studies were conducted to elucidate the kinetics of the thermal inactivation process of soluble and immobilized forms. Complete hydrolysis of bovine serum albumin (BSA) at 37 °C was achieved after 2 h using a soluble enzyme, while for its immobilized form, the hydrolysis yield was 47%. Reuse tests revealed that this biocatalyst retained 37% of its original activity after 10 successive hydrolysis batches. Based on these results, this support could be used as an interesting alternative for producing heterogeneous biocatalysts with high catalytic activity and thermal stability when producing protein hydrolysates. Full article

Agarose-vinyl sulfone (VS) beads have proven to be a good support to immobilize several enzymes. However, some enzymes are hardly immobilized on it. This is the case of penicillin G acylase (PGA) from Escherichia coli, which is immobilized very slowly on this support (less than 10% in 24 h). This enzyme is also not significantly adsorbed in aminated MANAE-agarose beads, an anionic exchanger. In this study, MANAE-agarose beads were modified with divinyl sulfone (DVS) to produce MANAE-vinyl sulfone (VS) agarose beads. When PGA was immobilized on this support, the enzyme was fully immobilized in less than 1.5 h. PGA cannot be released from the support by incubation at high ionic strength, suggesting that the enzyme was rapidly immobilized in a covalent fashion. Considering that the amount of reactive VS groups was only marginally increased, the results indicated some cooperative effect between the anion exchange on the amine groups of the support, probably as the first step of the process, and the covalent attachment of the previously adsorbed PGA molecules. The covalent reaction of the previously adsorbed enzyme molecules proceeds much more efficiently than that of the free enzyme, due to the proximity of the reactive groups of the support and the enzyme. Finally, the steps of immobilization, incubation, and blocking with different agents were studied to determine the effects on final activity/stability. The stability of PGA immobilized on this new catalyst was improved with respect to the VS-agarose prepared at low ionic strength. Full article

Lipase from Thermomyces lanuginosus (TLL) has been immobilized on a methacrylate macroporous resin coated with octadecyl groups (Purolite Lifetech^®® ECR8806F). This immobilization protocol gave a biocatalyst with significantly higher stability than that obtained using octyl agarose. To further improve the biocatalyst features, we tried to covalently immobilize the enzyme using this support. For this purpose, the support was activated with divinyl sulfone. The results showed that at least 1/3 of the immobilized enzyme molecules were not covalently immobilized. To solve the problem, we produced an aminated support and then activated it with divinyl sulfone. This permitted the full covalent immobilization of the previously immobilized TLL. The use of different blocking agents as the reaction endpoint (using ethylenediamine, Asp, Gly, and Cys) greatly altered the biocatalyst functional features (activity, specificity, or stability). For example, the blocking with ethylenediamine increased the ratio of the activity versus R- and S-methyl mandelate by a three-fold factor. The blocking with Cys produced the most stable biocatalyst, maintaining close to 90% of the activity under conditions where the just adsorbed enzyme maintained less than 55%. That way, this strategy to modify the support has permitted obtaining an enzyme interfacially activated versus the octadecyl layer and, later, covalently immobilized by reaction with the vinyl sulfone groups. Full article

Lipase B from Candida antarctica was immobilized on heterofunctional support octyl agarose activated with vinyl sulfone to prevent enzyme release under drastic conditions. Covalent attachment was established, but the blocking step using hexylamine, ethylenediamine or the amino acids glycine (Gly) and aspartic acid (Asp) altered the results. The activities were lower than those observed using the octyl biocatalyst, except when using ethylenediamine as blocking reagent and p-nitrophenol butyrate (pNPB) as substrate. The enzyme stability increased using these new biocatalysts at pH 7 and 9 using all blocking agents (much more significantly at pH 9), while it decreased at pH 5 except when using Gly as blocking agent. The stress inactivation of the biocatalysts decreased the enzyme activity versus three different substrates (pNPB, S-methyl mandelate and triacetin) in a relatively similar fashion. The tryptophane (Trp) fluorescence spectra were different for the biocatalysts, suggesting different enzyme conformations. However, the fluorescence spectra changes during the inactivation were not too different except for the biocatalyst blocked with Asp, suggesting that, except for this biocatalyst, the inactivation pathways may not be so different. Full article

Penicillin G acylase (PGA) from Escherichia coli was immobilized on vinyl sulfone (VS) agarose. The immobilization of the enzyme failed at all pH values using 50 mM of buffer, while the progressive increase of ionic strength permitted its rapid immobilization under all studied pH values. This suggests that the moderate hydrophobicity of VS groups is enough to transform the VS-agarose in a heterofunctional support, that is, a support bearing hydrophobic features (able to adsorb the proteins) and chemical reactivity (able to give covalent bonds). Once PGA was immobilized on this support, the PGA immobilization on VS-agarose was optimized with the purpose of obtaining a stable and active biocatalyst, optimizing the immobilization, incubation and blocking steps characteristics of this immobilization protocol. Optimal conditions were immobilization in 1 M of sodium sulfate at pH 7.0, incubation at pH 10.0 for 3 h in the presence of glycerol and phenyl acetic acid, and final blocking with glycine or ethanolamine. This produced biocatalysts with stabilities similar to that of the glyoxyl-PGA (the most stable biocatalyst of this enzyme described in literature), although presenting just over 55% of the initially offered enzyme activity versus the 80% that is recovered using the glyoxyl-PGA. This heterofuncionality of agarose VS beads opens new possibilities for enzyme immobilization on this support. Full article

Naringin and limonin are the two main bitter compounds of citrus products such as grapefruit juice. The aim of this investigation was to evaluate the reduction in both bitter components simultaneously using a combined biochemical and physical approach. The proposed strategy was based on the use of heterofunctional supports with glyoxyl groups that allow for the covalent immobilization of naringinase, which hydrolyses naringin and alkyl groups that allow for the adsorption of limonin. The supports were butyl-glyoxyl agarose (BGA) and octyl-glyoxyl agarose (OGA), which were characterized in terms of aldehyde group quantification and FTIR analysis. The optimal pH and temperature of free and immobilized enzymes were assessed. The maximum enzyme loading capacity of supports was analyzed. Debittering of grapefruit juice was evaluated using soluble enzyme, enzyme-free supports, and immobilized catalysts. Enzyme immobilized in BGA reduced naringin and limonin concentrations by 54 and 100%, respectively, while the use of catalyst immobilized in OGA allowed a reduction of 74 and 76%, respectively, obtaining a final concentration of both bitter components under their detection threshold. The use of OGA biocatalyst presented better results than when soluble enzyme or enzyme-free support was utilized. Biocatalyst was successfully applied in juice debittering in five repeated batches. Full article

Rare ginsenoside Rh2 exhibits diverse pharmacological effects. UDP-glycosyltransferase (UGT) catalyzed glycosylation of protopanaxadiol (PPD) has been of growing interest in recent years. UDP-glycosyltransferase Bs-YjiC coupling sucrose synthase in one-pot reaction was successfully applied to ginsenoside biosynthesis with UDP-glucose regeneration from sucrose and UDP, which formed a green and sustainable approach. In this study, the his-tagged UDP-glycosyltransferase Bs-YjiC mutant M315F and sucrose synthase AtSuSy were co-immobilized on heterofunctional supports. The affinity adsorption significantly improved the capacity of specific binding of the two recombinant enzymes, and the dual enzyme covalently cross-linked by the acetaldehyde groups significantly promoted the binding stability of the immobilized bienzyme, allowing higher substrate concentration by easing substrate inhibition for the coupled reaction. The dual enzyme amount used for ginsenoside Rh2 biosynthesis is Bs-YjiC-M315F: AtSuSy = 18 mU/mL: 25.2 mU/mL, a yield of 79.2% was achieved. The coimmobilized M315F/AtSuSy had good operational stability of repetitive usage for 10 cycles, and the yield of ginsenoside Rh2 was kept between 77.6% and 81.3%. The high titer of the ginsenoside Rh2 cumulatively reached up to 16.6 mM (10.3 g/L) using fed-batch technology, and the final yield was 83.2%. This study has established a green and sustainable approach for the production of ginsenoside Rh2 in a high level of titer, which provides promising candidates for natural drug research and development. Full article

Functional properties of each enzyme strictly depend on immobilization protocol used for linking enzyme and carrier. Different strategies were applied to prepare the immobilized derivatives of Rhizomucor miehei lipase (RML) and chemically aminated RML (NH₂-RML). Both RML and NH₂-RML forms were covalently immobilized on glyoxyl sepharose (Gx-RML and Gx-NH₂-RML), glyoxyl sepharose dithiothreitol (Gx-DTT-RML and Gx-DTT-NH₂-RML), activated sepharose with cyanogen bromide (CNBr-RML and CNBr-NH₂-RML) and heterofunctional epoxy support partially modified with iminodiacetic acid (epoxy-IDA-RML and epoxy-IDA-NH₂-RML). Immobilization varied from 11% up to 88% yields producing specific activities ranging from 0.5 up to 1.9 UI/mg. Great improvement in thermal stability for Gx-DTT-NH₂-RML and epoxy-IDA-NH₂-RML derivatives was obtained by retaining 49% and 37% of their initial activities at 70 °C, respectively. The regioselectivity of each derivative was also examined in hydrolysis of fish oil at three different conditions. All the derivatives were selective between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA. The highest selectivity (32.9 folds) was observed for epoxy-IDA-NH₂-RML derivative in the hydrolysis reaction performed at pH 5 and 4 °C. Recyclability study showed good capability of the immobilized biocatalysts to be used repeatedly, retaining 50–91% of their initial activities after five cycles of the reaction. Full article

Immobilization is an exciting alternative to improve the stability of enzymatic processes. However, part of the applied covalent strategies for immobilization uses specific conditions, generally alkaline pH, where some enzymes are not stable. Here, a new generation of heterofunctional supports with application at neutral pH conditions was proposed. New supports were developed with different bifunctional groups (i.e., hydrophobic or carboxylic/metal) capable of adsorbing biocatalysts at different regions (hydrophobic or histidine richest place), together with a glutaraldehyde group that promotes an irreversible immobilization at neutral conditions. To verify these supports, a multi-protein model system (E. coli extract) and four enzymes (Candida rugosa lipase, metagenomic lipase, β-galactosidase and β-glucosidase) were used. The immobilization mechanism was tested and indicated that moderate ionic strength should be applied to avoid possible unspecific adsorption. The use of different supports allowed the immobilization of most of the proteins contained in a crude protein extract. In addition, different supports yielded catalysts of the tested enzymes with different catalytic properties. At neutral pH, the new supports were able to adsorb and covalently immobilize the four enzymes tested with different recovered activity values. Notably, the use of these supports proved to be an efficient alternative tool for enzyme immobilization at neutral pH. Full article

Lipase from Candida rugosa (CRL) was stabilized at alkaline pH to overcome the inactivation problem and was immobilized for the first time by multipoint covalent attachment on different aldehyde-activated matrices. PEG was used as a stabilizing agent on the activity of CRL. At these conditions, CRL maintained 50% activity at pH 10 after 17 h incubation in the presence of 40% (w/v) of PEG, whereas the enzyme without additive was instantaneously inactive after incubation at pH 10. Thus, this enzyme was covalently immobilized at alkaline pH on three aldehyde-activated supports: aldehyde-activated Sepharose, aldehyde-activated Lewatit105 and heterofunctional aldehyde-activated EDA-Sepharose in high overall yields. Heterogeneous stable CRL catalysts at high temperature and solvent were obtained. The aldehyde-activated Sepharose-CRL preparation maintained 70% activity at 50 °C or 30% (v/v) acetonitrile after 22 h and exhibited high regioselectivity in the deprotection process of per-O-acetylated thymidine, producing the 3′-OH-5′-OAc-thymidine in 91% yield at pH 5. Full article

Two different heterofunctional octyl-amino supports have been prepared using ethylenediamine and hexylendiamine (OCEDA and OCHDA) and utilized to immobilize five lipases (lipases A (CALA) and B (CALB) from Candida antarctica, lipases from Thermomyces lanuginosus (TLL), from Rhizomucor miehei (RML) and from Candida rugosa (CRL) and the phospholipase Lecitase Ultra (LU). Using pH 5 and 50 mM sodium acetate, the immobilizations proceeded via interfacial activation on the octyl layer, after some ionic bridges were established. These supports did not release enzyme when incubated at Triton X-100 concentrations that released all enzyme molecules from the octyl support. The octyl support produced significant enzyme hyperactivation, except for CALB. However, the activities of the immobilized enzymes were usually slightly higher using the new supports than the octyl ones. Thermal and solvent stabilities of LU and TLL were significantly improved compared to the OC counterparts, while in the other enzymes the stability decreased in most cases (depending on the pH value). As a general rule, OCEDA had lower negative effects on the stability of the immobilized enzymes than OCHDA and while in solvent inactivation the enzyme molecules remained attached to the support using the new supports and were released using monofunctional octyl supports, in thermal inactivations this only occurred in certain cases. Full article