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VEXAS is caused by somatic mutations in UBA1 (UBA1mut) and characterized by heterogenous systemic auto-inflammation and progressive hematologic manifestations, meeting criteria for myelodysplastic syndrome (MDS) and plasma cell dyscrasias. The landscape of myeloid-related gene mutations leading to typical clonal hematopoiesis (CH) in these patients is unknown. Retrospectively, we screened 80 VEXAS patients for CH in their peripheral blood (PB) and correlated findings with clinical outcomes in 77. UBA1mutwere most common at hotspot p.M41 (median variant allele frequency/VAF = 75%). Typical CH mutations co-occurred with UBA1mut in 60% of patients, mostly in DNMT3A and TET2, and were not associated with inflammatory or hematologic manifestations. In prospective single-cell proteogenomic sequencing (scDNA), UBA1mutwas the dominant clone, present mostly in branched clonal trajectories. Based on integrated bulk and scDNA analyses, clonality in VEXAS followed two major patterns: with either typical CH preceding UBA1mutselection in a clone (Pattern 1), or occurring as an UBA1mutsubclone or in independent clones (Pattern 2). VAF in PB differed markedly between DNMT3A and TET2 clones (median VAF of 25% vs 1%). DNMT3A and TET2 clones associated with hierarchies representing patterns 1 and 2, respectively. Overall survival for all patients was 60% at 10 years. Transfusion-dependent anemia, moderate thrombocytopenia, and typical CH mutations, each correlated with poor outcome. In VEXAS, UBA1mut cells are the primary cause of systemic inflammation and marrow failure, being a new molecularly defined somatic entity associated with MDS. VEXAS-associated MDS is distinct from classical MDS in its presentation and clinical course.

Key Points Somatic UBA1 mutations define VEXAS in men with late-onset systemic inflammatory disease and cytopenia.Features summarizing VEXAS include cytopenia, hypercellularity, lack of hematogones, and vacuoles in myeloid and erythroid precursors., VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is caused by somatic mutations in UBA1 and is identified by a genotype-driven method. This condition affects unrelated men with adultonset inflammatory syndromes in association with hematologic manifestations of peripheral cytopenia and bone marrow myeloid dysplasia. Although bone marrow vacuolization restricted to myeloid and erythroid precursors has been identified in patients with VEXAS, the detailed clinical and histopathological features of peripheral blood and bone marrows remain unclear. The current case report describes the characteristic hematologic findings in patients with VEXAS, including macrocytic anemia, thrombocytopenia, marked hypercellular bone marrow with granulocytic hyperplasia, megaloblastic changes in erythroid precursors, and the absence of hematogones in addition to prominent vacuoles in myeloid and erythroid precursor cells. Characterizing the clinical and hematologic features helps to raise awareness and improve diagnosis of this novel, rare, but potentially underrecognized disease. Prompt diagnosis expands the general knowledgeable and understanding of this disease, and optimal management may prevent patients from developing complications related to this refractory inflammatory syndrome and improve the overall clinical outcome.

Objective Somatic mutations in UBA1 cause a newly defined syndrome known as VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic syndrome). More than 50% of patients currently identified as having VEXAS met diagnostic criteria for relapsing polychondritis (RP), but clinical features that characterize VEXAS within a cohort of patients with RP have not been defined. We undertook this study to define the prevalence of somatic mutations in UBA1 in patients with RP and to create an algorithm to identify patients with genetically confirmed VEXAS among those with RP. Methods Exome and targeted sequencing of UBA1 was performed in a prospective observational cohort of patients with RP. Clinical and immunologic characteristics of patients with RP were compared based on the presence or absence of UBA1 mutations. The random forest method was used to derive a clinical algorithm to identify patients with UBA1 mutations. Results Seven of 92 patients with RP (7.6%) had UBA1 mutations (referred to here as VEXAS-RP). Patients with VEXAS-RP were all male, were on average ≥45 years of age at disease onset, and commonly had fever, ear chondritis, skin involvement, deep vein thrombosis, and pulmonary infiltrates. No patient with VEXAS-RP had chondritis of the airways or costochondritis. Mortality was greater in VEXAS-RP than in RP (23% versus 4%; P = 0.029). Elevated acute-phase reactants and hematologic abnormalities (e.g., macrocytic anemia, thrombocytopenia, lymphopenia, multiple myeloma, myelodysplastic syndrome) were prevalent in VEXAS-RP. A decision tree algorithm based on male sex, a mean corpuscular volume >100 fl, and a platelet count

Somatic mutations in UBA1 involving hematopoietic stem and myeloid cells have been reported in patients with the newly defined VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. Here, we report clinical hematologic manifestations and unique bone marrow (BM) features in 16 patients with VEXAS. All patients were male and had a history of severe autoinflammatory and rheumatologic manifestations and a somatic UBA1 mutation (p.Met41). Ten patients had hematologic disorders: myelodysplastic syndrome (MDS; 6 of 16), multiple myeloma (2 of 16), monoclonal gammopathy of undetermined significance (2 of 16), and monoclonal B-cell lymphocytosis (2 of 16), and a few of those patients had 2 co-existing clonal processes. Although macrocytic anemia (100%) and lymphopenia (80%) were prevalent in all patients with VEXAS, thrombocytopenia and neutropenia were more common in patients with progression to MDS. All BMs in VEXAS patients had prominent cytoplasmic vacuoles in myeloid and erythroid precursors. In addition, most BMs were hypercellular with myeloid hyperplasia, erythroid hypoplasia, and varying degrees of dysplasia. All patients diagnosed with MDS were lower risk (low blast count, very good to intermediate cytogenetics) according to standard prognostic scoring with no known progression to leukemia. In addition, 10 of 16 patients had thrombotic events, including venous thromboembolism and arterial stroke. Although VEXAS presents symptomatically as a rheumatologic disease, morbidity and mortality are associated with progression to hematologic disease. Given the increased risk of developing MDS and multiple myeloma, surveillance for disease progression is important.

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

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.

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.

Enzymes are biological catalysts capable of recognizing a substrate and catalyze reactions of hydrolysis and synthesis. The most significant property of enzymes is their high specificity toward their substrates since they are able to recognize and act upon a molecule from a pool of similar compounds.

Green production of biodiesel via enzymatic transesterification necessitates technological improvements for practical implementation. Heterogenization of biocatalysts has long demonstrated its advantages in biocatalytic processes. In this work, a lipase from Thermomyces lanuginosus (TL) has been immobilized on biomimetic silica nanoparticles using two different strategies: in situ entrapment and adsorption/covalent surface immobilization. Variables such as mass of nanobiocatalyst, amount of immobilized enzyme, and activity per gram of silica were studied following a factorial design for the in situ immobilization. The influence of the enzymatic load on the stability and activity of the catalyst was studied for both the surface immobilized and the entrapped lipase. Immobilized preparations were characterized and assayed in the production of fatty acid methyl esters (FAMES). The entrapped nanobiocatalysts were more stable and active than the soluble TL, the commercial immobilized TL and the surface immobilized counterparts. However, surface immobilized lipase reached a maximum yield of 88% in the synthesis of FAMES from canola oil and methanol, surmounting from the yield obtained with the commercial immobilized TL by 10%. Accumulated specific productivity for the entrapped biocatalyst reached 65.6 μmoles FAME/g catalyst/min after repeated batch operation. Biomimetic silica demonstrated its versatility and robustness as a support for TL immobilization in the synthesis of FAMES.

The objective of this paper was to carry out an integral study of the use of hydrophobic chitosan as a low-cost support for immobilizing lipases and their further application in the selective hydrolysis of fish oil. Chitosan functionalized with different alkyl chains (C4, C8, C12) were characterized by FTIR, TGA, SEM, and Rose Bengal adsorption. Lipase B from Candida antarctica (CalB) and lipase from Rhizomucor miehei (RML) were immobilized obtaining a higher expressed activity at a longer alkyl chain length of support. Biocatalyst thermal stability showed that the impact of the alkyl chain length on enzyme stabilization varied according to the lipase source. The biocatalysts were applied in menhaden oil hydrolysis. Total polyunsaturated fatty acids released after 30 h of reaction with lipases immobilized in butyl, octyl and dodecyl-chitosan was 60, 107, and 90 mM for CalB biocatalysts, and 560, 392, and 50 mM for RML biocatalysts, respectively. Selectivity of CalB was not affected by the alkyl chain, while in the case of RML, a higher selectivity to cis-4,7,10,13,16,19-docohexaenoic acid release was obtained with dodecyl-chitosan. In conclusion, the adequate functionalization of chitosan varied according to lipase source, affecting their activity, stability and performance in the hydrolysis of fish oil.

The enzymatic production of lactofructose syrup from lactose coming from surplus whey represents a sound example of circular economy. The production process requires the sequential action of two enzymes, namely β-galactosidase and glucose (xylose) isomerase, which can act independently in different reactors or in a one-pot system. The latter is an interesting option, even more if considering the co-immobilization of the enzymes involved in the biotransformation. This review analyzes the different options to carry out this process, with a focus on the key aspects of the co-immobilization of the enzymes in a carrier-free system, and the operation conditions for such catalysts, which are key issues for developing a successful technology. The production of lactofructose syrup from lactose is an illustrative case study, but the analysis done both on the preparation and use of the biocatalyst can be extrapolated to other co-immobilized multienzyme systems.

One-pot systems are an interesting proposal to carry out multi-enzymatic reactions, though this strategy implies establishing an optimal balance between the activity and operability of the involved enzymes. This is crucial for enzymes with marked differences in their operational stability, such as one-pot production of lactofructose syrup from cheese whey permeate, which involves two enzymes—β-galactosidase (β-gal) and glucose isomerase (GI). The aim of this work was to study the behavior of one-pot sequential batch production of lactofructose syrup considering both enzymes immobilized individually, in order to evaluate and design a strategy of replacement of the catalysts according to their stabilities. To this end, the modelling and simulation of the process was carried out, considering simultaneously the kinetics of both reactions and the kinetics of inactivation of both enzymes. For the latter, it was also considered the modulating effect that sugars present in the medium may have on the stability of β-gal, which is the less stable enzyme. At the simulated reaction conditions of 40 °C, pH 7, and 0.46 (IUGI/IUβ-gal), the results showed that considering the stability of β-gal under non-reactive conditions, meaning in absence of the effect of modulation, it is necessary to carry out four replacements of β-gal for each cycle of use of GI. On the other hand, when considering the modulation caused by the sugars on the β-gal stability, the productivity increases up to 23% in the case of the highest modulation factor studied (η = 0.8). This work shows the feasibility of conducting a one-pot operation with immobilized enzymes of quite different operational stability, and that a proper strategy of biocatalyst replacement increases the productivity of the process.

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.

Reaction conditions were optimized and the donor substrate selected for maximizing the reaction yield and productivity of the enzymatic synthesis of hexyl-β-galactoside with β-galactosidase from Aspergillus oryzae . We independently studied the effect of water content, type of cosolvent, temperature, donor substrate concentration, and leaving group of the donor substrate on the yield and productivity of hexyl-β-galactoside synthesis. Reaction yield was maximum in the medium with 70% water content and acetone as cosolvent, corresponding to a water activity of 0.94. Temperature and donor substrate concentration had very little effect on the yield. The leaving group of the donor substrate was the most relevant variable. Lactose, lactulose, o-nitrophenyl-β- d -galactopyranoside, and propyl- and butyl-β- d -galactoside (the last two are enzymatically synthesized from lactose) were evaluated as donor substrates. Use of propyl- and butyl-β-galactoside as donor substrates allowed us to increase the product yield by 683% and 716% [vs. lactose (0.06 mol/mol)]. Because propyl- and butyl-β-galactosides can be synthesized at low cost from lactose, using them in a two-step process could be much better alternative than a one-step process with lactose; leading to a theoretical global molar yield of 0.41 and 0.37 mol/mol from propyl- and butyl-β-galactosides, respectively.

Three commercial β-galactosidase preparations from Aspergillus oryzae, Bacillus circulans and Kluyveromyces lactis were evaluated in the synthesis of butyl-β-galactoside. The enzyme from A. oryzae performed the best, producing the highest product yield and having the highest operational stability, being selected for further studies. Then, lactose and o -NPG were evaluated as substrates, and temperature and 1-butanol concentration were optimized using response surface methodology. Higher yields (ranging between 0.7 and 0.92 mol/mol) were obtained with o -NPG rather than lactose. However, under optimized conditions, a yield of 0.58 mol/mol was obtained with lactose as substrate, which is interesting because of being much cheaper than o -NPG. Three immobilization strategies were evaluated, the catalyst immobilized in glyoxyl-agarose being selected for producing a yield from lactose of 0.76 mol/mol. Catalyst reuse was evaluated in the synthesis of butyl-β-galactoside in consecutive batch mode during ten cycles of 2 h. Immobilization allowed an increase in the efficiency of catalyst use with respect to the soluble enzyme, the amount of product per unit mass of enzyme protein, being higher from the fifth batch on. Butyl-β-galactoside was easily purified by extraction with acetone and characterized by liquid electrospray ionization mass spectrometry.

The characterization of immobilized enzymes allows the evaluation of the immobilization process itself and also the projection of the immobilized enzyme performance under process operation conditions. Based on such characterization, strategies for support functionalization and enzyme immobilization into the activated support can be selected, determining the best conditions for conducting such steps in view of the intended use of the biocatalyst, establishing a linkage between biocatalyst production and biocatalyst use. The determination of the catalytic potential of the immobilized enzyme under operational conditions is a priceless parameter that takes into account both activity and stability, including the effect of both mass transfer limitations (diffusional restrictions) and intrinsic enzyme inactivation upon the immobilization process.

The characterization of immobilized enzymes allows the evaluation of the immobilization process itself and also the projection of the immobilized enzyme performance under process operation conditions. Based on such characterization, strategies for support functionalization and enzyme immobilization into the activated support can be selected, determining the best conditions for conducting such steps in view of the intended use of the biocatalyst, establishing a linkage between biocatalyst production and biocatalyst use. The determination of the catalytic potential of the immobilized enzyme under operational conditions is a priceless parameter that takes into account both activity and stability, including the effect of both mass transfer limitations (diffusional restrictions) and intrinsic enzyme inactivation upon the immobilization process.

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 β-D-glucosidase (βG) 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 βG and ARA, respectively. Such characteristics make them suitable for aroma enhancement in wines.

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 &deg, 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.

Background:Somatic mutations in ubiquitin activating enzyme 1 (UBA1) cause a newly defined syndrome known as VEXAS. [1] More than fifty percent of patients currently identified with VEXAS meet diagnostic criteria for relapsing polychondritis (RP).Objectives:To determine the prevalence VEXAS within a cohort of patients with RP, to compare their clinical, laboratory, and immunologic features and to develop a clinical algorithm to inform genetic screening for VEXAS among patients with RP.Methods:Exome and targeted sequencing of the UBA1 gene was performed in a prospective observational cohort of patients with RP. Clinical and immunological characteristics of patients with RP were compared based on presence or absence of UBA1 mutations. Random forest was used to derive a clinical algorithm to identify patients with UBA1 mutations. Immune populations were quantified by multipanel flow cytometry. Categorical and continuous variables were compared using the chi square or Kruskal-Wallis test. PResults:Seven of 92 patients with RP (7.6%) were confirmed to have UBA1 mutations (VEXAS-RP). Six additional patients with VEXAS-RP from other cohorts were included for subsequent analyses. Patients with VEXAS-RP were all male, older at disease onset, and commonly had fever, ear chondritis, skin involvement, deep vein thrombosis, and pulmonary infiltrates. Patients with RP as compared with VEXAS-RP had a significantly higher prevalence of airway chondritis, costochondritis and tenosynovitis/arthralgias. (Table). Mortality was significantly greater in VEXAS-RP than RP (27% vs 2% p=0.01). Maximum ESR, CRP, and mean corpuscular volume (MCV) values were significantly greater in VEXAS-RP. Absolute monocyte, lymphocyte, and platelet counts were significantly lower in VEXAS-RP. A decision tree based on male sex, MCV>100 fl and Platelet countTable 1.Clinical Characteristics of patients with RP vs VEXAS-RPAll Patientsn=98RPn=85VEXAS-RPn=13p valueDemographic CharacteristicsRace, White n (%)90 (92)77 (91)13 (100)0.59Sex, Male n (%)26 (27)13 (15)13 (100)Age, Symptom onset, years, Median (IQR)38 (30-47)37 (28-43)56 (54-64)Clinical SymptomsFever n (%)33 (34)20 (24)13 (100)Ear chondritis n (%)61 (62)48 (56)13 (100)0.0015Nose chondritis n (%)83 (85)71 (84)12 (92)0.68Airway chondritis n (%)37 (38)37 (44)0 (0)0.0015Tenosynovitis/arthalgias n (%)83 (85)77 (91)6 (46)0.0005Skin involvement n (%)33 (34)22 (2611 (85)Laboratory ValuesESR, mm/hr, median (IQR)12 (6-22)11 (5-19)66.5 (42-110)CRP, mg/L, median (IQR)2.9 (0.8-9.6)1.9 (0.6-6.3)17.7 (9.6-99.5)Platelet count (k/uL)246(201-299)258 (227-312)145 (100-169)MCV fL93.05 (90-98)92.2 (89-95)105 (102-115)Absolute lymphocyte count1.6 (1.1-2.3)1.78(1.4-2.4)0.92 (0.5-1.2)CT scan abnormalitiesPulmonary infiltrates n (%)16 (16.33)6 (7.06)10 (77)ComplicationsDeath n (%)6 (6)3 (4)3 (23)0.029Unprovoked DVT12 (12)4 (5)8 (62)N number; IQR = interquartile rangeConclusion:Mutations in UBA1 are causal for disease in a subset of patients with RP. These patients are defined by disease onset in the fifth decade of life or later, male sex, ear/nose chondritis and hematologic abnormalities. Early identification is important in VEXAS given the associated high mortality rate.References:[1]Beck DB, Ferrada MA, Sikora KA, Ombrello AK, Collins JC, Pei W, Balanda N, Ross DL, Ospina Cardona D, Wu Z et al: Somatic Mutations in UBA1 and Severe Adult-Onset Autoinflammatory Disease. N Engl J Med 2020, 383(27):2628-2638.Disclosure of Interests:None declared

Synthesis of propyl-β-galactoside catalyzed by Aspergillus oryzae β-galactosidase in soluble form was optimized using response surface methodology (RSM). Temperature and 1-propanol concentration were selected as explanatory variables; yield and productivity were chosen as response variables. Optimal reaction conditions were determined by weighing the responses through a desirability function. Then, synthesis of propyl-β-galactoside was evaluated at the optimal condition previously determined, with immobilized β-galactosidase in glyoxyl-agarose and amino-glyoxyl-agarose, and with cross-linked aggregates (CLAGs). Yields of propyl-β-galactoside obtained with CLAGs, amino-glyoxyl-agarose and glyoxyl-agarose enzyme derivatives were 0.75, 0.81 and 0.87 mol/mol and volumetric productivities were 5.2, 5.6 and 5.9 mM/h, respectively, being significantly higher than the corresponding values obtained with the soluble enzyme: 0.47 mol/mol and 4.4 mM/h. As reaction yield was increased twofold with the glyoxyl-agarose derivative, this catalyst was chosen for evaluating the synthesis of propyl-β-galactoside in repeated batch operations. Then, after ten sequential batches, the efficiency of catalyst use was 115% higher than obtained with the free enzyme. Enzyme immobilization also favored product recovery, allowing catalyst reuse, and avoiding browning reactions. Propyl-β-galactoside was recovery by extraction in 90%v/v acetone with a purity higher than 99% and its synthesis was confirmed by mass spectrometry.

The production of prebiotic galacto-oligosaccharides (GOS) considers the enzymatic synthesis by lactose transgalactosylation and the purification from the reacted mixture (raw GOS). Simultaneous synthesis and purification of GOS (SSP) was conducted in a one-pot operation using β-galactosidase from Aspergillus oryzae and Saccharomyces cerevisiae or Kluyveromyces marxianus cells, allowing the selective removal of unwanted carbohydrates (monosaccharides and lactose). The reaction was carried out in a stirred reactor at 40 °C, pH 4.5 and 200 rpm, using lactose without additional supplements, evaluating the effect of yeast genus ( S . cerevisiae or K . marxianus ), enzyme-substrate ratio (20–400 IU H /g lactose ), initial lactose concentration (20–50% w/w) and substrate mass-biomass ratio (0.05–0.4). The best GOS yield was obtained with S . cerevisiae at 50 IU H /g lactose and 0.25 g cell biomass /g carbohydrates . These conditions were used for scaling-up into a bioreactor (1000 g of 40% w/w lactose) operated under temperature and pH control at an aeration rate of 5 vvm, obtaining a GOS yield of 40% at 24 h of reaction, which is significantly higher than obtained in the conventional system of GOS synthesis. SSP may represent a technological advantage in terms of productivity and yield of GOS production.

The enzyme encapsulation is a very well‐known stabilization pathway. However, there are some challenges in order to avoid the enzyme denaturation under encapsulation conditions. The β‐galactosidase from Bacillus circulans was immobilized through sol‐gel encapsulation route assisted by Triton X‐100 surfactant and sugars. The effects of sugar presence in the immobilization process and the gelation time on the biocatalyst activity/stability were explained taking into account the characteristics of the formed silica matrix and the changes of the enzyme environment. The enzyme was effectively immobilized by this strategy, with high immobilization yield in terms of activity (29%) and expressed activity (47 IU/g). The immobilization through silica sol‐gel in the presence of 1×10−3 M Triton X‐100 and fructose conferred 28.4‐fold higher stability to the enzyme compared with the soluble form. This is an advantage for its use in the synthesis of the galacto‐oligosaccharides at 50ºC. The total lactose conversion to galacto‐oligosaccharides was 26%wt, which is comparable with that reported in the literature. The obtained biocatalyst is useful for the synthesis of galacto‐oligosaccharides and its catalytic behavior is rationalized in this work.

A new bi-enzymatic catalyst has been produced by precipitation and crosslinking (combi-CLEAs) of β-galactosidase and glucose isomerase for catalyzing the cascade reactions of lactose conversion into fructose, producing a lactose-fructose syrup (LFS). Glucose isomerase was chemically aminated to increase its reactive surface groups for favour the crosslinking step. The effect of β-galactosidase to glucose isomerase activity ratio and glutaraldehyde to protein mass ratio in combi-CLEAs production was evaluated. The selected combi-catalyst was successfully used in the production of fructose syrup from lactose in a single reaction vessel. The biocatalyst could be used at least in five sequential batches of LFS production, remaining fully stable after a total of 50 h of reaction, obtaining a product of constant quality. A robust bi-enzymatic catalyst was produced that can be repeatedly used in LFS production, an attractive mild sweetener for the dairy food industry.

Simultaneous synthesis and purification (SSP) of galacto-oligosaccharides (GOS) from lactose was conducted using a combi-biocatalyst formed by crosslinked enzyme aggregates of Aspergillus oryzae β-galactosidase and Saccharomyces cerevisiae cells co-immobilized by entrapment in calcium alginate gel particles. Product yield obtained with the combi-biocatalyst was similar than obtained with the soluble enzyme (23.3%), having a final purity of 25.7%. During the simultaneous process, ethyl-β-galactoside was produced from the ethanol generated as a metabolic product of yeast cells, but ethyl-β-galactoside was considerably decreased at high aeration (4 vvm). The combi-biocatalyst can be recovered and reused but its performance is limited by the reduction of the metabolic capacity of the cells. In this way, a process was developed for the SSP of GOS from lactose, obtaining a comparable product yield and higher specific productivity than in a conventional synthesis.

The work refers to the immobilization of the Alcaligenes sp. lipase for its use in the synthesis of biodiesel from canola oil, which has never been reported before. The enzyme was immobilized on different supports: polyethyleneimine, agarose, glutaraldehyde agarose, octyl agarose, glyoxyl agarose, Sepabeads ® and also by aggregation and crosslinking to produced enzyme aggregates (CLEAs), so to consider different catalyst options for the reaction under study. The lipase biocatalysts were evaluated and compared based on their specific hydrolytic activities, immobilization yields and thermal stabilities under non-reactive and reactive (in the presence of oil and methanol) conditions. The lipase immobilized on Sepabeads ® Ec-BU and CLEAs were selected to catalyze the methanolysis of canola oil. The synthesis of methyl esters from vegetable oil was performed at 40 °C, using oil:methanol molar ratios of 1/3 and 1/4, and also using a six-step addition of methanol to the reaction mixture (oil/methanol molar ratio of 1/6) in order to decrease its inhibitory effect. At oil:methanol molar ratios of 1/3 and 1/4 a maximum conversion yield of 70% was obtained after 30 h of reaction; on the other hand, using the six-step addition of methanol the maximum conversion yield obtained was of 80% in only 10 h of reaction, demonstrating that the inhibitory effect of methanol was significantly reduced. The operational stability of the catalysts was assessed in three sequential batches of 10 h each, obtaining a yield decay of 13% and 3,2% for lipase immobilized on Sepabeads ® Ec-BU and CLEAs respectively, so the latter had a higher operational stability; however, recovery is simpler in the former so both catalyst are competitive for biodiesel production.

α-Chymotrypsin was immobilized in activated agarose support and the stability of the biocatalyst was assessed in three polar organic solvents, namely, ethanol, diglyme, and acetonitrile. Ethanol was the solvent in which the stability of the enzyme was higher and was then selected to perform the synthesis of the kyotorphin derivative benzoyl-tyrosine argininamide, evaluating enzyme reactivation after synthesis. Substrates for reaction were benzoyl tyrosine ethyl ester and argininamide, the reaction being performed under kinetic control. High conversion yield (85%) was obtained and the immobilized enzyme was successfully used in sequential batch reactor operation with enzyme reactivation after three batches.

The main goal of this work was to study different strategies of immobilization of Candida antarctica lipase B (CALB) in order to evaluate changes in the selectivity of this enzyme when R -α-monobenzoate glycerol ( R -α-MBG) is obtained through of asymmetric esterification. CALB was immobilized on sepharose and silica functionalized with octyl groups (monofunctional supports) and undecanol–glyoxyl and octyl-epoxide groups (heterofunctional supports). Our results showed that the enzyme could be immobilized by all carriers, with activity yields ranging from 52% to 83%. CALB immobilized on silica octyl was the most active (367 IU g −1 biocatalyst ) with immobilization yields in terms of protein and expressed activity of 72% and 50% respectively and exhibited a higher half-life in 100% 1,4-dioxane at 50 °C (85,000 h). In contrast, CALB immobilized on heterofunctional silica support was the most selective biocatalyst, reaching an enantiomeric excess of 99% of R -α-MBG in 100% 1,4-dioxane. In terms of configuration the sepharose biocatalysts results in a S -enantiomer, while silica biocatalysts results in a R -enantiomer. The catalysis of asymmetric esterification of glycerol with benzoic acid to obtain R -α-MBG by CALB immobilized in heterofunctional silica is highly selective and, to our knowledge, is the most selective reported to date.

The main goal of this work was to study the stereoselective behavior of immobilized Geobacillus thermocatenulatus lipase (BTL2) in a sequential batch reactor using the partial and asymmetric hydrolysis of dimethyl-3 phenylglutarate (DMFG) as a model reaction. To reach this goal, BTL2 lipase was immobilized on Sepharose and silica supports with cyanogen bromide and octyl groups (monofunctional supports) and undecanol-glyoxyl and octyl-epoxides groups (heterofunctional supports), to determine the effect of the enzyme orientation during the immobilization process on their catalytic properties. In the hydrolysis of DMFG, the biocatalyst obtained with undecanol-glyoxyl Sepharose proved to be the most stereolective with an enantiomeric excess (e.e.) value of 90% in aqueous media. This behavior can be attributed to differences in the orientation of the lipase on the support. In sequential batch reactor operation, the e.e. remained constant in the first two batches; however, from the third batch on the e.e. decreased slightly maybe due to a change in the conformation of the enzyme at the reaction conditions. Finally, the high purity S -methyl-3-phenyl glutarate produced in sequential batch reactor operation shows that the biocatalyst can be reused at least twice without losing stereoselectivity, favoring a reduction in the process cost.

Glycosidases are frequently used in winemaking to liberate glycosidically bound aroma compounds. Since most of the glycosidases used for diglycoside hydrolysis act sequentially, their co-immobilization is an attractive alternative from a technical and economical perspective. The enzymes α- l -arabinosidase (ARA) and β- d -glucosidase (βG) from the preparation Rapidase ® AR2000 were co-immobilized in CLEAs (combi-CLEAs), evaluating the effect of bovine serum albumin (BSA) addition and the concentration of glutaraldehyde (Glu) on enzyme immobilization yield and expressed activity. Combi-CLEAs prepared with a Glu to Rapidase protein mass ratio of 0.053 and BSA to Rapidase protein mass ratios of 1, 0.33, and 0.2 were selected, evaluating their stability at simulated winemaking conditions: 25 °C, pH 3.5, and 10% (v/v) of ethanol. All combi-CLEAs were more stable than the soluble enzymes, the best result being obtained at a BSA to Rapidase protein mass ratio of 0.33. Half-lives of βG and ARA in combi-CLEAs were 43.9 and 54.9 days, respectively, whereas in the case of the soluble enzymes were only 1.3 and 6.2 days, respectively. Immobilization yields were 79.1 and 47.1% in terms of βG and ARA activity, respectively. Combi-CLEAs of glycosidases are technologically relevant robust biocatalysts for their application as aroma enhancers in winemaking.

Enzymes are important industrial catalysts whose use has evolved from simple degradation processes—conducted mostly with hydrolases dissolved in the aqueous reaction medium—to processes of organic synthesis in which different classes of enzymes catalyze more complex reactions in different catalyst formats and reaction media. Extending the use of enzymes from the former low added-value processes to ones in which added-value is considerable is an exciting opportunity for biocatalysis; however, converting these remarkable physiological catalysts into robust process catalysts represents a complex technological challenge. Impressive advances both in enzyme production and enzyme molecular (re)design has opened a whole new field of enzyme use that projects into the fine chemicals and pharmaceutical industries where the outstanding properties of enzymes in terms of molecular precision are highly appreciated. Enzyme processes are compliant with green chemistry principles representing another important asset of biocatalysis. This chapter describes the evolution of enzyme catalysis, its present status and future prospects. Special emphasis is made on its impact on processes of organic synthesis and in non-conventional reaction media. Advances in the field related to the use of enzymes as industrial catalysts as well as the present and future challenges are analyzed in a hopefully unbiased perspective.

Chitosan partially functionalized with aldehyde groups was used for enzyme immobilization, favoring first the enzyme adsorption through its amino groups and then the covalent bonding of the adsorbed catalyst through the aldehyde groups of the support. Using this strategy, immobilized A. oryzae β-galactosidase had a better performance than when only the aldehyde groups were used. The performance was further improved by modifying the support aldehyde group density to 200 μmoles⋅g−1. The biocatalyst under optimized immobilization conditions had 2951 IU⋅g−1 and half-life of 46.3 min at 60 °C, while its agarose counterpart had 2294 IU⋅g−1 and half-life of 59.5 min. Both biocatalysts were applied in galacto-oligosaccharide synthesis. After 10 sequential batches, the cumulative productivity (gGOS⋅h−1ˑgprotein−1) obtained with the chitosan and the agarose biocatalysts were 4.7 and 4.0 times the value when soluble enzyme was used respectively. This methodology had not been reported previously with chitosan, showing the high versatility of this low cost carrier and its high potential for enzyme immobilization.

Chitosan was derivatized by two methodologies to design a robust biocatalyst of immobilized Bacillus circulans β-galactosidase from a low-cost support for its further application in the synthesis of galacto-oligosaccharides (GOS). In the first one, chitosan was derivatized by cross-linking with glutaraldehyde and activated with epichlorohydrin; in the second one, cross-linking and activation were done with epichlorohydrin in a two-step process, favoring first support cross-linking and then support functionalization (C-EPI-EPI). Epoxy groups were hydrolyzed and oxidized, obtaining two supports activated with different aldehyde concentrations (100-250 μmol/g). The expressed activity and stability of the immobilized biocatalysts varied according to the derivatization methodology, showing that both the cross-linking agent and the activation degree are key parameters in the final biocatalyst performance. The best compromise between expressed activity and thermal stability was obtained using C-EPI-EPI with 200 μmol of aldehyde groups per gram of support. The immobilization conditions were optimized, obtaining a biocatalyst with 280 IU/g, immobilization yields in terms of activity and protein of 17.3 ± 0.4 and 61.5 ± 3.9%, respectively, and a high thermal stability, with a half-life of 449 times the value of the soluble enzyme. The biocatalyst was applied to the synthesis of GOS in repeated batch operation without affecting the product composition. Four successive batches were required for obtaining a cumulative specific productivity higher than the one obtained with the soluble enzyme.

Two carbonaceous–siliceous composite materials, produced by hydrothermal and carbonization processes, were evaluated as immobilization support for lipase from Alcaligenes sp. These materials exhibited similar chemical characteristics but their carbon content and porous characteristics were different, which explain the catalytic behavior and stability of the biocatalysts immobilized on them. Higher activity and immobilization selectivity was achieved with the microporous material that had higher carbon content. The lipase immobilized on the mesoporous material had a higher thermal stability at 55 °C, pH 7.0 or at 40 °C in tert-butanol, simulating the reaction conditions required for organic synthesis. Both biocatalysts were tested in the synthesis of palmitoyl ascorbate and they were compared with the commercial biocatalyst QLC. The synthesis conversions with the lipase immobilized in mesoporous materials and with the biocatalyst QLC were similar (50%), but only the former could be reused. These are promising biocatalysts for industrial applications.

Lipase-catalyzed synthesis of sugar esters, as lactulose palmitate, requires harsh conditions, making it necessary to immobilize the enzyme. Therefore, a study was conducted to evaluate the effect of different chemical surfaces of hierarchical meso-macroporous silica in the immobilization of two lipases from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL), which exhibit esterase activity. Porosity and chemical surface of silica supports, before and after functionalization and after immobilization, were characterized by gas adsorption and Fourier transform infrared (FTIR) spectroscopy. PsL and AsL were immobilized in octyl (OS), glyoxyl (GS), and octyl-glyoxyl silica (OGS). Hydrolytic activity, thermal and solvent stability, and sugar ester synthesis were evaluated with those catalysts. The best support in terms of expressed activity was OS in the case of PsL (100 IU g(-1)), while OS and OGS were the best for AsL with quite similar expressed activities (60 and 58 IU g(-1), respectively). At 60 °C in aqueous media the more stable biocatalysts were GS-PsL and OGS-AsL (half-lives of 566 and 248 h, respectively), showing the advantage of a heterofunctional support in the latter case. Lactulose palmitate synthesis was carried out in acetone medium (with 4% of equilibrium moisture) at 40 °C obtaining palmitic acid conversions higher than 20% for all biocatalysts, being the highest of those obtained with OGS-AsL and OS-PsL. Therefore, screening of different chemical surfaces on porous silica used as supports for lipase immobilization allowed obtaining active and stable biocatalyst to be employed in the novel synthesis of lactulose palmitate.

Synthesis of ascorbyl palmitate by enzymatic esterification of palmitic acid and ascorbic acid was conducted in an organic medium with Pseudomonas stutzeri lipase TL immobilized in different supports and its performance was compared with commercial Novozym 435 lipase used as a reference. The enzyme was immobilized in different supports and the best catalyst was selected in terms of immobilization yield and mass specific activity to perform the reactions of synthesis. Synthesis of ascorbyl palmitate was optimized considering temperature, substrate molar ratio and enzyme to limiting substrate mass ratio as variables, and substrate conversion and specific productivity as evaluation parameters. The best reaction conditions for immobilized lipase TL were 55 °C, 1:5 ascorbic to palmitic acid molar ratio, and 1:10 lipase to ascorbic acid mass ratio, obtaining 57 % substrate conversion and a specific productivity of 0.013 [g ascorbic acid/(g enzyme × min)]; the best conditions for Novozym 435 were 70 °C, ascorbic to palmitic acid molar ratio 1:10, and 1:10 lipase to ascorbic acid mass ratio, obtaining 51 % substrate conversion and a specific productivity of 0.016 [g ascorbic acid/(g enzyme × min)].

Hierarchical meso-macroporous silica (average mesopore diameter 20 nm) was synthesized and chemically modified to be used as a support for the immobilization of lipases from Candida antarctica B and Alcaligenes sp. and β-galactosidases from Bacillus circulans and Aspergillus oryzae. Catalytic activities and thermal stabilities of enzymes immobilized by multipoint covalent attachment in silica derivatized with glyoxyl groups were compared with those immobilized in glyoxyl-agarose, assessing biocatalyst performance under non-reactive conditions in aqueous medium. In the case of A. oryzae β-galactosidase and Alcaligenes sp. lipase, an additional step of amination was needed to improve immobilization yield. Specific activities of lipases immobilized in glyoxyl-silica were high (232 and 62 IU per gram, for C. antarctica B and Alcaligenes sp. respectively); thermal stabilities were higher than those immobilized in glyoxyl-agarose. Although in the case of β-galactosidases from B. circulans and A. oryzae, the specific activities (250 and 310 IU per gram, respectively) were lower than the ones obtained with glyoxyl-agarose, expressed activities were similar to values previously reported. Thermal stabilities of both β-galactosidases immobilized in glyoxyl-silica were higher than when glyoxyl-agarose was used as support. Results indicate that hierarchical meso-macroporous silica is a versatile support for the production of robust biocatalysts.

The synthesis of the dipeptide N-benzoyl-l-tyrosine-l-argininamide (BTAA) was conducted under kinetic control with N-benzoyl-l-tyrosine ethyl ester as acyl donor and argininamide as nucleophile using immobilized α-chymotrypsin as catalyst. Using a mathematical procedure, the kinetic constants corresponding to the proposed mechanism of peptide synthesis were determined in three different cosolvent media, namely, ethanol, diglyme and acetonitrile. These constants were used for evaluating the selectivity of glyoxyl-agarose immobilized α-chymotrypsin in the synthesis of BTAA by determining the ratios of synthesis to hydrolysis rates.

Lipoaminoacids, as surfactants, are an excellent option for food industry due to the currently trends in consumption of functional and natural ingredients. Synthesis of lauroyl glycine lipoaminoacid was carried out with a lipase from Pseudomonas stutzeri and a protease from Bacillus subtilis, which were immobilized in octyl-glyoxyl silica and glyoxyl-silica supports, respectively, comparing their catalytic performance. The enzymatic selectivity towards the lipoaminoacid instead of the dipeptide glycylglycine and synthesis yield were evaluated with respect to the characteristics of the immobilized biocatalysts and synthesis conditions. Three solvents were tested as reaction media for evaluating the expressed activity, stability and catalytic behavior during synthesis. Results indicate that both enzymes favor the lauroyl glycine synthesis over the peptide synthesis, but the immobilized protease has the best balance between selectivity and yield: 40% yield for lauroyl glycine and less than 5% for dipeptide after 96 h of synthesis, at 45 °C and acetone as solvent.

Synthesis of cephalexin with immobilized penicillin acylase at high substrates concentration at an acyl donor to nucleophile molar ratio of 3 was comparatively evaluated in aqueous and ethylene glycol media using a statistical model. Variables under study were temperature, pH and enzyme to substrate ratio and their effects were evaluated on cephalexin yield, ratio of initial rates of cephalexin synthesis to phenylglycine methyl ester hydrolysis, volumetric and specific productivity of cephalexin synthesis, that were used as response parameters. Results obtained in both reaction media were modeled using surface of response methodology and optimal operation conditions were determined in terms of an objective function based on the above parameters. At very high substrates concentrations the use of organic co-solvents was not required to attain high yields and actually almost stoichiometric yields were obtained in a fully aqueous media with the advantages of higher productivities than in an organic co-solvent media and compliance with the principles of green chemistry.

The type of enzyme immobilization has been associated with the degree of stabilization/modification of the protein structure, which ultimately determines its activity and stability under different conditions (temperature, solvent and substrate). The thermal stability and the stability in solvated reaction medium of different derivatives of Candida cylindracea lipase (CCL) were evaluated for their hydrolysis activity of p-nitrophenyl butyrate and in obtaining Omega-3 fatty acids EPA and DHA from fish oil triacylglycerols. CCL was immobilized by hydrophobic interactions and by covalent binding. In addition, hydrophobic derivatives were cross linked with functional polymers, such as dextran aldehyde and dextran sulfate, after chemical amination of its surface to prevent lipase desorption. Fish oil hydrolysis reactions were carried out using 35 U of biocatalyst per gram of oil, at 37 °C and with a 1:1 aqueous/organic phase ratio. The aqueous phase was a 40% polyethylene glycol/phosphate buffer solution, pH 7, and the organic phase a 1:4 fish oil/hexane solution. According to its half-life time, the most thermostable derivative corresponds to CCL immobilized in glyoxyl-agarose. Derivatives of CCL immobilized on octyl-agarose (O-CCL) and CCL immobilized on octyl-agarose cross linked with dextran sulfate (O-CCL-NH 2 -DxSO 4 ) showed an activation effect of about 10 folds compared to its initial activity when they were incubated in solvent and co-solvent biphasic reaction media. After 48 h of reaction, O-CCL and O-CCL-NH 2 -DxSO 4 were those derivatives with highest yields of fish oil hydrolysis; its differential rate of hydrolysis detected for polyunsaturated fatty acids make them a valuable tool for EPA/DHA glycerides concentration.