Evaluation of the operational conditions of the glucose oxidase and catalase multienzymatic system through enzyme co-immobilization on amino hierarchical porous silica.

Hexaric acids have attracted attention lately because they are platform chemicals for synthesizing pharmaceuticals. In particular, gluconic acid is one of the most studied because it is readily available in nature. In this work, operational conditions like temperature and pH were evaluated for the enzymatic production of gluconic acid. For this purpose, glucose oxidase (GOx) and catalase (CAT) were individually immobilized and co-immobilized using amino-silica as support. The catalytic performance of the enzymes both as separate biocatalysts (GOx or CAT) and as an enzymatic complex (GOx-CAT) was assessed in terms of enzymatic activity and stability at temperatures 45 °C and 50 °C and pH 6 to 8. The results show that CAT is a key enzyme for gluconic acid production as it prevents GOx from being inhibited by H2O2. However, CAT was found to be less stable than GOx. Therefore, different GOx to CAT enzymatic ratios were studied, and a ratio of 1-3 was determined to be the best. The highest glucose conversion conditions were 45 °C and pH 7.0 for 24 h. Regarding the biocatalyst reuse, GOx-CAT retained more than 70% of its activity after 6 reaction cycles. These results contribute to further knowledge and application of oxidases for hexaric acid production and shed greater light on the role of the glucose oxidase/catalase pair in better catalytic performance. Both enzymes were immobilized in one pot, which is relevant for their potential use in industry; an enzyme system was obtained in a single step.

Preparation of a Mesoporous Biosensor for Human Lactate Dehydrogenase for Potential Anticancer Inhibitor Screening.

Cancer is the second leading cause of death worldwide, with a dramatic impact due to the acquired resistance of cancers to used chemotherapeutic drugs and treatments. The enzyme lactate dehydrogenase (LDH-A) is responsible for cancer cell proliferation. Recently the development of selective LDH-A inhibitors as drugs for cancer treatment has been reported to be an efficient strategy aiming to decrease cancer cell proliferation and increase the sensitivity to traditional chemotherapeutics. This study aims to obtain a stable and active biocatalyst that can be utilized for such drug screening purposes. It is conceived by adopting human LDH-A enzyme (hLDH-A) and investigating different immobilization techniques on porous supports to achieve a stable and reproducible biosensor for anticancer drugs. The hLDH-A enzyme is covalently immobilized on mesoporous silica (MCM-41) functionalized with amino and aldehyde groups following two different methods. The mesoporous support is characterized by complementary techniques to evaluate the surface chemistry and the porous structure. Fluorescence microscopy analysis confirms the presence of the enzyme on the support surface. The tested immobilizations achieve yields of ≥80%, and the best retained activity of the enzyme is as high as 24.2%. The optimal pH and temperature of the best immobilized hLDH-A are pH 5 and 45 °C for the reduction of pyruvate into lactate, while those for the free enzyme are pH 8 and 45 °C. The stability test carried out at 45 °C on the immobilized enzyme shows a residual activity close to 40% for an extended time. The inhibition caused by NHI-2 is similar for free and immobilized hLDH-A, 48% and 47%, respectively. These findings are significant for those interested in immobilizing enzymes through covalent attachment on inorganic porous supports and pave the way to develop stable and active biocatalyst-based sensors for drug screenings that are useful to propose drug-based cancer treatments.

Industrial bioelectrochemistry for waste valorization: State of the art and challenges.

Bioelectrochemistry has gained importance in recent years for some of its applications on waste valorization, such as wastewater treatment and carbon dioxide conversion, among others. The aim of this review is to provide an updated overview of the applications of bioelectrochemical systems (BESs) for waste valorization in the industry, identifying current limitations and future perspectives of this technology. BESs are classified according to biorefinery concepts into three different categories: (i) waste to power, (ii) waste to fuel and (iii) waste to chemicals. The main issues related to the scalability of bioelectrochemical systems are discussed, such as electrode construction, the addition of redox mediators and the design parameters of the cells. Among the existing BESs, microbial fuel cells (MFCs) and microbial electrolysis cells (MECs) stand out as the more advanced technologies in terms of implementation and R&D investment. However, there has been little transfer of such achievements to enzymatic electrochemical systems. It is necessary that enzymatic systems learn from the knowledge reached with MFC and MEC to accelerate their development to achieve competitiveness in the short term.

Enzymatic Synthesis of Ascorbyl Palmitate in a Rotating Bed Reactor.

Ascorbyl palmitate, an ascorbic acid ester, is an important amphipathic antioxidant that has several applications in foods, pharmaceuticals, and cosmetics. The enzymatic synthesis of ascorbyl palmitate is very attractive, but few efforts have been made to address its process scale-up and implementation. This study aimed at evaluating the enzymatic synthesis of ascorbyl palmitate in a rotating basket reactor operated in sequential batches. Different commercial immobilized lipases were tested, and the most suitable reaction conditions were established. Among those lipases studied were Amano Lipase PS, Lipozyme® TL IM, Lipozyme® Novo 40086, Lipozyme® RM IM and Lipozyme® 435. Initially, the enzymes were screened based on previously defined synthesis conditions, showing clear differences in behavior. Lipozyme® 435 proved to be the best catalyst, reaching the highest values of initial reaction rate and yield. Therefore, it was selected for the following studies. Among the solvents assayed, 2-methyl-2-butanol and acetone showed the highest yields, but the operational stability of the catalyst was better in 2-methyl-2-butanol. The tests in a basket reactor showed great potential for large-scale application. Yields remained over 80% after four sequential batches, and the basket allowed for easy catalyst recycling. The results obtained in basket reactor are certainly a contribution to the enzymatic synthesis of ascorbyl palmitate as a competitive alternative to chemical synthesis. This may inspire future cost-effectiveness studies of the process to assess its potential as a viable alternative to be implemented.

Simultaneous CO2 reduction and NADH regeneration using formate and glycerol dehydrogenase enzymes co-immobilized on modified natural zeolite.

In this work, the co-immobilization of formate dehydrogenase (FDH) and glycerol dehydrogenase (GlyDH) enzymes is proposed to reduce CO2 into formic acid, an important chemical intermediate. The reduction of carbon dioxide is carried out by FDH to obtain formic acid, simultaneously, the GlyDH regenerated the nicotinamide cofactor in the reduced form (NADH) by the oxidation of glycerol into dihydroxyacetone. Natural zeolite was selected as immobilization support given its good properties and low cost. The natural zeolite was modified with subsequent acid-alkaline attacks to obtain a mesostructurization of the clinoptilolite. The two enzymes were co-immobilized on clinoptilolite, previously hetero-functionalized with amino and glyoxyl groups. The distribution of the enzymes was confirmed by fluorescence microscopy analysis. Furthermore, a great increase in the retained activity for the formate dehydrogenase enzyme was noted, passing from 18% to 89%, when the mesostructured clinoptilolite was used as support. The immobilization yield of formate dehydrogenase and glycerol dehydrogenase is around 100% with all the supports studied. The promising results suggest a possible development of this procedure in enzyme immobilization and biocatalysis. The biocatalysts were characterized to find the optimal pH and temperature. Furthermore, a thermal stability test at 50 °C was carried out on both enzymes, in free and immobilized forms. Finally, it was shown that the biocatalyst is effective in reducing CO2, both by using the cofactor in the reduced form (NADH) or the oxidized form (NAD+), obtaining NADH through the regeneration with glycerol in this latter case.

Covalent Immobilization of Dehydrogenases on Carbon Felt for Reusable Anodes with Effective Electrochemical Cofactor Regeneration.

This study presents the immobilization with aldehyde groups (glyoxyl carbon felt) of alcohol dehydrogenase (ADH) and formate dehydrogenase (FDH) on carbon-felt-based electrodes. The compatibility of the immobilization method with the electrochemical application was studied with the ADH bioelectrode. The electrochemical regeneration process of nicotinamide adenine dinucleotide in its oxidized form (NAD+ ), on a carbon felt surface, has been deeply studied with tests performed at different electrical potentials. By applying a potential of 0.4 V versus Ag/AgCl electrode, a good compromise between NAD+ regeneration and energy consumption was observed. The effectiveness of the regeneration of NAD+ was confirmed by electrochemical oxidation of ethanol catalyzed by ADH in the presence of NADH, which is the no active form of the cofactor for this reaction. Good reusability was observed by using ADH immobilized on glyoxyl functionalized carbon felt with a residual activity higher than 60 % after 3 batches.

ZnO Materials as Effective Anodes for the Photoelectrochemical Regeneration of Enzymatically Active NAD.

This work reports the study of ZnO-based anodes for the photoelectrochemical regeneration of the oxidized form of nicotinamide adenine dinucleotide (NAD+). The latter is the most important coenzyme for dehydrogenases. However, the high costs of NAD+ limit the use of such enzymes at the industrial level. The influence of the ZnO morphologies (flower-like, porous film, and nanowires), showing different surface area and crystallinity, was studied. The detection of diluted solutions (0.1 mM) of the reduced form of the coenzyme (NADH) was accomplished by the flower-like and the porous films, whereas concentrations greater than 20 mM were needed for the detection of NADH with nanowire-shaped ZnO-based electrodes. The photocatalytic activity of ZnO was reduced at increasing concentrations of NAD+ because part of the ultraviolet irradiation was absorbed by the coenzyme, reducing the photons available for the ZnO material. The higher electrochemical surface area of the flower-like film makes it suitable for the regeneration reaction. The illumination of the electrodes led to a significant increase on the NAD+ regeneration with respect to both the electrochemical oxidation in dark and the only photochemical reaction. The tests with formate dehydrogenase demonstrated that 94% of the regenerated NAD+ was enzymatically active.

Synthesis and characterization of ordered mesoporous silicas for the immobilization of formate dehydrogenase (FDH).

This work studied the influence of the pore size and morphology of the mesoporous silica as support for formate dehydrogenase (FDH), the first enzyme of a multi-enzymatic cascade system to produce methanol, which catalyzes the reduction of carbon dioxide to formic acid. Specifically, a set of mesoporous silicas was modified with glyoxyl groups to immobilize covalently the FDH obtained from Candida boidinii. Three types of mesoporous silicas with different textural properties were synthesized and used as supports: i) SBA-15 (DP = 4 nm); ii) MCF with 0.5 wt% mesitylene/pluronic ratio (DP = 20 nm) and iii) MCF with 0.75 wt% mesitylene/pluronic ratio (DP = 25 nm). As a whole, the immobilized FDH on MCF0.75 exhibited higher thermal stability than the free enzyme, with 75% of residual activity after 24 h at 50 °C. FDH/MCF0.5 exhibited the best immobilization yields: 69.4% of the enzyme supplied was covalently bound to the support. Interestingly, the specific activity increased as a function of the pore size of support and then the FDH/MCF0.75 exhibited the highest specific activity (namely, 1.05 IU/gMCF0.75) with an immobilization yield of 52.1%. Furthermore, it was noted that the immobilization yield and the specific activity of the FDH/MCF0.75 varied as a function of the supported enzyme: as the enzyme loading increased the immobilization yield decreased while the specific activity increased. Finally, the reuse test has been carried out, and a residual activity greater than 70% was found after 5 cycles of reaction.

Immobilization strategies of photolyases: Challenges and perspectives for DNA repairing application.

Photolyases are enzymes that repair DNA damage caused by solar radiation. Due to their photorepair potential, photolyases added in topical creams and used in medical treatments has allowed to reverse skin damage and prevent the development of different diseases, including actinic keratosis, premature photoaging and cancer. For this reason, research has been oriented to the study of new photolyases performing in extreme environments, where high doses of UV radiation may be a key factor for these enzymes to have perfected their photorepair potential. Generally, the extracted enzymes are first encapsulated and then added to the topical creams to increase their stability. However, other well consolidated immobilization methods are interesting strategies to be studied that may improve the biocatalyst performance. This review aims to go through the different Antarctic organisms that have exhibited photoreactivation activity, explaining the main mechanisms of photolyase DNA photorepair. The challenges of immobilizing these enzymes on porous and nanostructured supports is also discussed. The comparison of the most reported immobilization methods with respect to the structure of photolyases show that both covalent and ionic immobilization methods produced an increase in their stability. Moreover, the use of nanosized materials as photolyase support would permit the incorporation of the biocatalyst into the target cell, which is a technological requirement that photolyase based biocatalysts must fulfill.

Biocatalysis in the winemaking industry: Challenges and opportunities for immobilized enzymes.

Enzymes are powerful catalysts already being used in a large number of industrial processes. Impressive advantages in enzyme catalysts improvement have occurred in recent years aiming to improve their performance under harsh operation conditions far away from those of their cellular habitat. Production levels of the winemaking industry have experienced a remarkable increase, and technological innovations have been introduced for increasing the efficiency at different process steps or for improving wine quality, which is a key issue in this industry. Enzymes, such as pectinases and proteases, have been traditionally used, and others, such as glycosidases, have been more recently introduced in the modern wine industry, and many dedicated studies refer to the improvement of enzyme performance under winemaking conditions. Within this framework, a thorough review on the role of enzymes in winemaking is presented, with special emphasis on the use of immobilized enzymes as a significant strategy for catalyst improvement within an industry in which enzymes play important roles that are to be reinforced paralleling innovation.

Recovery of humic acids from anaerobic sewage sludge: Extraction, characterization and encapsulation in alginate beads.

Wastewater production is rising all over the world and one of the most difficult problems is the disposal of sewage sludge (SS). It is known that SS contains certain quantities of added-value compounds, such as humic acids (HA) which in turn have beneficial effects on soil quality and plant growth. On the other hand, SS can retain many pollutants, such as heavy metals. The present work aimed to implement an HA alkaline extraction protocol from anaerobic sewage sludge (ASS). Subsequently, the HA were quantified in ASS, in HA extract and in commercial HA, used as a benchmark, which gave results of 12.53%, 26.87% and 77.87% (on dry matter basis), respectively. FESEM and EDX analyses on lyophilized HA extract confirmed that no heavy metals had passed into the extract. Afterwards, in order to allow controlled release of the HA in soils, alginate beads containing the HA extract were created. Finally, a pot experiment in a greenhouse was performed using Chilean lettuce plants (Lactuca sativa L.) treated with alginate-HA extract beads. At the end of the greenhouse experiments, the hypogean dry biomass of the treated plants was significantly higher than for non-treated plants. The relevance of this study relies not only on the exploitation of green chemistry principles, by converting a waste stream into a high-value product, but also on the application of an approach following a circular economy model.

Entrapment of enzyme aggregates in chitosan beads for aroma release in white wines.

Glycosidases are enzymes involved in the cascade reactions leading to the release of aromatic compounds in white wines. However, the use of commercial soluble glycosidases is facing difficulties due to their fast inactivation, poor reaction control, low efficiency of enzyme use, and the presence of catalyst residues in the product. Co-immobilization as cross-linked enzyme aggregates (combi-CLEAs) is a sound alternative allowing the immobilization of enzymes in their own protein matrix, yielding highly stable and active biocatalysts. Notwithstanding, their micrometer sized particles limit their application in industrial processes. To overcome this, combi-CLEAs of ß-D-glucosidase (ßG) and α-L-arabinofuranosidase (ARA) were entrapped in polymeric chitosan beads. The effect of crosslinking reagents and crosslinking time on the specific activity and stability of combi-CLEAs was studied, and the best conditions for the entrapment of the combi-CLEAs in polymeric chitosan beads were determined varying the concentration of the chitosan solution and the pH of the gelation agent solution. The resulting biocatalyst beads (average diameter 1.24 mm), retained full activity after 91 days of incubation under winemaking conditions, having specific activities of 0.91 and 0.88 international units of activity per gram for ßG and ARA, respectively. Such characteristics make them suitable for aroma enhancement in wines.

Synthesis with Immobilized Lipases and Downstream Processing of Ascorbyl Palmitate.

Ascorbyl palmitate is a fatty acid ester endowed with antioxidant properties, used as a food additive and cosmetic ingredient, which is presently produced by chemical synthesis. Ascorbyl palmitate was synthesized from ascorbic acid and palmitic acid with a Pseudomonas stutzeri lipase immobilized on octyl silica, and also with the commercial immobilized lipase Novozym 435. The latter was selected for optimizing the reaction conditions because of its high reactivity and stability in the solvent 2-methyl-2-butanol used as reaction medium. The reaction of the synthesis was studied considering temperature and molar ratio of substrates as variables and synthesis yield as response parameter. The highest yield in the synthesis of ascorbyl palmitate was 81%, obtained at 55 °C and an ascorbic acid to palmitic acid molar ratio of 1:8, both variables having a strong effect on yield. The synthesized ascorbyl palmitate was purified to 94.4%, with a purification yield of 84.2%. The use of generally recognized as safe (GRAS) certified solvents with a polarity suitable for the solubilization of the compounds made the process a viable alternative for the synthesis and downstream processing of ascorbyl palmitate.

Enhanced long-chain fatty alcohol oxidation by immobilization of alcohol dehydrogenase from S. cerevisiae.

This work reports on the oxidation of long-chain aliphatic alcohols catalyzed by a stabilized alcohol dehydrogenase from S. cerevisiae (yeast alcohol dehydrogenase (YADH)). In particular, the oxidation of the fatty alcohol tetracosanol (C24H50O) to yield lignoceric acid (C23H47COOH) was studied. The immobilization of YADH onto glyoxyl agarose supports crosslinked with a polymer (polyethylenimine) produced a highly stable catalyst (60-fold higher than the soluble enzyme at 40 °C). Aliphatic alcohols with different chain lengths (ranging from 2 to 24 carbons) were studied as substrates for YADH. The activity of YADH with aliphatic alcohols with a chain length higher than five carbon atoms is reported for the first time. The activities obtained with the immobilized YADH were all similar in magnitude, even with long-chain fatty alcohols such as docosanol and tetracosanol. As far as the oxidation of tetracosanol is concerned, the best values of reaction rate and substrate conversion were obtained at pH = 8.2 and T = 58 °C. At these conditions, the soluble enzyme inactivated rapidly, precluding its use in batch reaction. However, using the immobilized YADH, up to three sequential reaction batches were performed by recovering the catalyst after each batch. Several applications in the green oleochemical industry, e.g., for making plasticizers, lubricants, detergents, and personal care products, may benefit from having novel and stable biocatalysts able to oxidize long-chain fatty alcohols.

Spin-Coated vs. Electrodeposited Mn Oxide Films as Water Oxidation Catalysts.

Manganese oxides (MnOx), being active, inexpensive and low-toxicity materials, are considered promising water oxidation catalysts (WOCs). This work reports the preparation and the physico-chemical and electrochemical characterization of spin-coated (SC) films of commercial Mn2O3, Mn3O4 and MnO2 powders. Spin coating consists of few preparation steps and employs green chemicals (i.e., ethanol, acetic acid, polyethylene oxide and water). To the best of our knowledge, this is the first time SC has been used for the preparation of stable powder-based WOCs electrodes. For comparison, MnOx films were also prepared by means of electrodeposition (ED) and tested under the same conditions, at neutral pH. Particular interest was given to α-Mn2O3-based films, since Mn (III) species play a crucial role in the electrocatalytic oxidation of water. To this end, MnO2-based SC and ED films were calcined at 500 °C, in order to obtain the desired α-Mn2O3 crystalline phase. Electrochemical impedance spectroscopy (EIS) measurements were performed to study both electrode charge transport properties and electrode-electrolyte charge transfer kinetics. Long-term stability tests and oxygen/hydrogen evolution measurements were also made on the highest-performing samples and their faradaic efficiencies were quantified, with results higher than 95% for the Mn2O3 SC film, finally showing that the SC technique proposed here is a simple and reliable method to study the electrocatalytic behavior of pre-synthesized WOCs powders.