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Hybrid bioinorganic biocatalysts have received much attention due to their simple synthesis, high efficiency, and structural features that favor enzyme activity and stability. The present work introduces a biomineralization strategy for the formation of hybrid nanocrystals from β-galactosidase. The effects of the immobilization conditions were studied, identifying the important effect of metal ions and pH on the immobilization yield and the recovered activity. For a deeper understanding of the biomineralization process, an in silico study was carried out to identify the ion binding sites at the different conditions. The selected β-galactosidase nanocrystals showed high specific activity (35,000 IU/g biocatalyst) and remarkable thermal stability with a half-life 11 times higher than the soluble enzyme. The nanobiocatalyst was successfully tested for the synthesis of galacto-oligosaccharides, achieving an outstanding performance, showing no signs of diffusional limitations. Thus, a new, simple, biocompatible and inexpensive nanobiocatalyst was produced with high enzyme recovery (82%), exhibiting high specific activity and high stability, with promising industrial applications.

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.

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.

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.

Aroma is a remarkable factor of quality and consumer preference in wine, representing a distinctive feature of the product. Most aromatic compounds in varietals are in the form of glycosidic precursors, which are constituted by a volatile aglycone moiety linked to a glucose residue by an O-glycosidic bond; glucose is often linked to another sugar (arabinose, rhamnose or apiose). The use of soluble β-glycosidases for aroma liberation implies the addition of a precipitating agent to remove it from the product and precludes its reuse after one batch. An attractive option from a technological perspective that will aid in removing such constraints is the use of immobilized glycosidases. Immobilization by aggregation and crosslinking is a simple strategy producing enzyme catalysts of very high specific activity, being an attractive option to conventional immobilization to solid inert supports. The purpose of this work was the evaluation of co-immobilized β-glycosidases crosslinked aggregates produced from the commercial preparation AR2000, which contains the enzymes involved in the release of aromatic terpenes in Muscat wine (α-l-arabinofuranosidase and β-d-glucopyranosidase). To do so, experiments were conducted with co-immobilized crosslinked enzyme aggregates (combi-CLEAs), and with the soluble enzymes, using an experiment without enzyme addition as control. Stability of the enzymes at the conditions of winemaking was assessed and the volatiles composition of wine was determined by SPE-GC-MS. Stability of enzymes in combi-CLEAs was much higher than in soluble form, 80% of the initial activity remaining after 60 days in contact with the wine; at the same conditions, the soluble enzymes had lost 80% of their initial activities after 20 days. Such higher stabilities will allow prolonged use of the enzyme catalyst reducing its impact in the cost of winemaking. Wine treated with combi-CLEAs was the one exhibiting the highest concentration of total terpenes (18% higher than the control) and the highest concentrations of linalool (20% higher), nerol (20% higher) and geraniol (100% higher), which are the most important terpenes in determining Muscat typicity. Co-immobilized enzymes were highly stable at winemaking conditions, so their reutilization is possible and technologically attractive by reducing the impact of enzyme cost on winemaking cost.

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.

7 páginas, 3 figuras, 3 tablas., Organic cosolvents may improve the properties of lipases (e.g., selectivity); however, organic cosolvents also promote the desorption of the enzyme from its hydrophobic supports. In this study, adsorbed lipase molecules were cross-linked with polyfunctional polymers, such as aldehyde-dextrans, to prevent this desorption. The desorption of adsorbed lipases was greatly reduced by optimizing the polymer size, polymer/lipase ratio, and cross-linking time. More than 95% of cross-linked, immobilized Rhizomucor miehei lipase (RML) remained adsorbed on the support after washing with cosolvents or detergents. This new, immobilized RML preparation mediated the hydrolysis of sardine oil in the presence of organic cosolvents. The presence of cosolvents promoted small losses of hydrolytic activity. Interestingly, however, 50% 2-propanol also promoted increased selectivity in the release of eicosapentaenoic acid (EPA) in relation to docosahexaenoic acid (DHA). An EPA/DHA ratio of 4:1 in the absence of 2-propanol was increased to a ratio of 22:1 in the presence of 2-propanol. The new RML derivatives were relatively stable under the selected reaction conditions. Their overall half-life was 100 h, but, in a second inactivation phase (below 60% of remaining activity), it took 600 h to reach 30% of their remaining activity., This work was sponsored by the Spanish Ministry of Science and Innovation (project AGL-2009-07526) and the Comunidad Autonoma de Madrid (Project S0505/PPQ/03449). We gratefully recognize the Spanish Ministry of Science and Innovation for the ‘‘Ramón y Cajal’’ contract for Dr. Fernandez-Lorente and Dr. Betancor.

A mathematical model that describes the heterogeneous reaction–diffusion process involved in a batch reactor with immobilized enzyme is presented. The model is based on equations considering reaction and diffusion components including biocatalyst particle size distribution. The reaction system includes the bulk liquid phase containing the dissolved substrate (and products) and the solid biocatalyst phase represented by spherical porous particles carrying the enzyme. The model developed is illustrated for the case of penicillin G hydrolysis with immobilized penicillin acylase, which is a complex reaction system in which both products of reaction and the substrate itself are inhibitors. Significant differences in batch reactor performance simulation are observed when considering biocatalyst particles of a single radius and particle size distribution. The magnitude of these differences is proportional to the dispersion (standard deviation) considered in that size distribution function.

As long-chain alcohol dehydrogenases are not easily available and seldom reported enzymes, it is worthwhile to appraise the potential of well known dehydrogenases, like horse liver alcohol dehydrogenases (HLAD), for the oxidation of long-chain aliphatic alcohols. Oxidation of docosanol (C-22) and tetracosanol (C-24) is of technological relevance within an industrial platform for the fractionation and upgrading of tall-oil from the Kraft pulping process. Results are presented on the characterization of free and immobilized HLAD with respect to their potential for oxidizing long-chain aliphatic alcohols. Enzyme activity with respect to chain length and pH is presented. Activity for both free and immobilized HLAD increased with pH up to 8.8, but behavior with respect to chain length varied from one biocatalyst to the other. Even though both biocatalysts were less active towards very long-chain aliphatic alcohols, immobilized HLAD had an activity on docosanol and tetracosanol higher than 50% of the value obtained with ethanol, butanol and octanol, which is encouraging and has not been previously reported. Investigation on thermophilic sources and further immobilization strategies are underway to obtain more active and stable catalysts amenable for working at high temperatures which is quite relevant in this case due to the poor solubility of substrates. (C) 2008 Elsevier Inc. All rights reserved.

Galacto-oligosaccharides (GOSs) are properly considered as a prebiotic based on scientific evidence about their physiological effects, having some unique properties among prebiotics that allow them to be used as supplement in formulas for infants where they mimic the physiological effect of human milk oligosaccharides, which are key components in breast milk. GOSs are produced by enzymatic transgalactosylation of lactose with β-galactosidases, so their production technology is tightly connected to the field of enzyme biocatalysis. The chapter thoroughly reviews all aspects of GOS production with whole cells and isolated enzymes, both free in solution and immobilized. Special attention is given to downstream processing of the raw GOS produced to cope with the quality standards imposed by their use as special food components. Cost of downstream operations is a major issue, so advances in GOS purification by different strategies are analyzed. Finally, current applications and perspectives of GOS within the prebiotic sector are reviewed and appraised. Competition with other well-established prebiotics (inulin and fructo-oligosaccharides) and potential prebiotic newcomers is envisaged, so the unique properties of lactose in terms of their physiological effect and functional properties are on the basis of GOS consolidation in an increasingly competitive market.

The presence of organic cosolvents was previously considered necessary to obtain high conversion yields in the synthesis of β-lactam antibiotics with immobilized penicillin acylase, and it is so when working at moderate substrate concentrations. Conversion yields close to stoichiometric and high productivities were recently reported for the synthesis of cephalexin at high substrate concentrations in ethylene glycol medium. Under such conditions, the effect of cosolvent concentration on yield is not significant so we raised the hypothesis that stoichiometric yields and high productivities are attainable at very high substrate concentrations in fully aqueous medium leading to substantial process improvement in terms of costs and environment. To test the hypothesis, the kinetically controlled synthesis of cephalexin with immobilized penicillin acylase was conducted in aqueous medium at substrates concentrations up to and beyond their solubilities at varying temperature, pH, enzyme to substrate and acyl donor to nucleophile ratios. At the best conditions, 99% conversion yield was attained with volumetric productivity of 300 mM/h and specific productivity of 7.8 mmol/h g cat . These values are slightly higher than those previously obtained under optimized conditions in organic medium so that the hypothesis has been confirmed, which opens up the possibility of efficiently produce the antibiotic through an environmentally friendly process.

Different immobilization strategies have been issued to prepare a stable and active biocatalyst of the formate dehydrogenase from Candida boidinii : immobilization on glutaraldehyde, epoxy, amino-epoxy, glyoxyl or treatment of enzymes adsorbed on aminated supports with glutaraldehyde. The best results in terms of stability were achieved using amino-epoxy supports (by a 12-fold factor compared to soluble and BrCN immobilized enzyme) and glyoxyl agarose supports (by a 150-fold factor), although in both cases activity recovery was just over 15%. The use of milder conditions permitted to improve the recovered activity, but decreased in a parallel way the enzyme stability, until being almost identical to the soluble enzyme. Alternatively, ionic adsorption of the enzyme was assayed. Supports coated with PEI permitted to keep full activity of the enzyme, permitted a very strong adsorption and the reutilization of the support after enzyme inactivation. Moreover, all the immobilization techniques prevent enzyme inactivation under strong stirring, necessary to keep the pH value and eliminate the CO 2 formed during the reaction. In this way, a battery of solutions to immobilize this enzyme has been proposed, fulfilling the different requirements that the use of this NADH recycling enzyme may have, depending on the particular reaction.

The kinetically controlled synthesis of cephalexin in ethylene glycol was previously optimized at moderate substrate concentrations obtaining yields close to stoichiometric. A study is now presented on the production of cephalexin at very high substrates concentrations, up to 750 mM acyl donor, with immobilized and cross-linked enzyme aggregates (CLEA) of penicillin acylase. Since conversion yield close to 100% was already obtained, attention was given to productivity under the hypothesis that increasing substrates concentration will produce a substantial increase in productivity without reducing yield. An increase of 29 times in volumetric productivity and 4.5 times in specific productivity was obtained with PGA-450 with respect to the results obtained at moderate substrates concentrations (below 100 mM acyl donor). Volumetric productivity was lower for CLEA than for PGA-450, but specific productivity was almost the same for both. Sequential batch reactor operations were conducted to assess the biocatalyst operational stability and global productivity, considering one half-life as biocatalyst life cycle. Under such criterion, 40.1 and 135.5 g of cephalexin/g of biocatalyst were obtained for PGA-450 and CLEA, respectively. Yields remained close to 100% during the whole cycle. These are very good values which can be improved by optimizing the biocatalyst replacement criterion.

Sugar esters are considered as surfactants due to its amphiphilic balance that can lower the surface tension in oil/water mixtures. Enzymatic syntheses of these compounds are interesting both from economic and environmental considerations. A study was carried out to evaluate the effect of four solvents, temperature, substrate molar ratio, biocatalyst source, and immobilization methodology on the yield and specific productivity of lactulose palmitate monoester synthesis. Lipases from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL), immobilized in porous silica functionalized with octyl groups (adsorption immobilization, OS) and with glyoxyl-octyl groups (both adsorption and covalent immobilization, OGS), were used. The highest lactulose palmitate yields were obtained at 47 °C in acetone, for all biocatalysts, while the best lactulose:palmitic acid molar ratio differed according to the immobilization methodology, being 1:1 for AsL-OGS biocatalyst (20.7 ± 3%) and 1:3 for the others (30-50%).

Immobilization of alcohol dehydrogenase (ADH) from Horse Liver inside porous supports promotes a dramatic stabilization of the enzyme against inactivation by air bubbles in stirred tank reactors. Moreover, immobilization of ADH on glyoxyl-agarose promotes additional stabilization against any distorting agent (pH, temperature, organic solvents, etc.). Stabilization is higher when using highly activated supports, they are able to immobilize both subunits of the enzyme. The best glyoxyl derivatives are much more stable than conventional ADH derivatives (e.g., immobilized on BrCN activated agarose). For example, glyoxyl immobilized ADH preserved full activity after incubation at pH 5.0 for 20h at room temperature and conventional derivatives (as well as the soluble enzyme) preserved less than 50% of activity after incubation under the same conditions. Moreover, glyoxyl derivatives are more than 10 times more stable than BrCN derivatives when incubated in 50% acetone at pH 7.0. Multipoint covalent immobilization, in addition to multisubunit immobilization, seems to play an important stabilizing role against distorting agents. In spite of these interesting stabilization factors, immobilization hardly promotes losses of catalytic activity (keeping values near to 90%). This immobilized preparation is able to keep good activity using dextran-NAD(+). In this way, ADH glyoxyl immobilized preparation seems to be suitable to be used as cofactor-recycling enzyme-system in interesting NAD(+)-mediated oxidation processes, catalyzed by other immobilized dehydrogenases in stirred tank reactors.

Standard CLEAs preparation using commercial preparations of lipases from Alcaligenes sp. (QL) and Candida antarctica (fraction B) (CAL-B) is not fully effective, some leakage of enzyme from the CLEA can be observed, and the SDS-PAGE of that preparations reveals that many enzyme molecules have not cross-linked properly. The co-precipitation of the lipases with poly-ethyleneimine (PEI) or PEI-sulfate dextran (DS) mixtures permitted to get fully physically stable CLEAs, with higher stability in the presence of organic solvents. Very interestingly, the conditions of precipitation and the nature of the polymers permitted to significantly alter the lipases activity, enantio-selectivity and specificity. For example, the QL showed changes in activity and enantio-selectivity in the hydrolysis of (±)-glycidyl butyrate when the derivative was prepared in presence or absence of Triton X-100. Results were further improved if the enzyme was co-precipitated with DS (from around 4 to more than 14). Similar changes in the lipase properties were found using CAL-B.

Lipase QL from Alcaligenes sp. is a quite thermostable enzyme. For example, it retains 75% of catalytic activity after incubation for 100 h at 55 °C and pH 7.0. Nevertheless, an improvement of the enzyme properties was intended via immobilization by covalent attachment to different activated supports and by adsorption on hydrophobic supports (octadecyl-sepabeads). This latter immobilization technique promotes the most interesting improvement of enzyme properties: (a) the enzyme is hyperactivated after immobilization: the immobilized preparation exhibits a 135% of catalytic activity for the hydrolysis of p -nitrophenyl propionate as compared to the soluble enzyme; (b) the thermal stability of the immobilized enzyme is highly improved: the immobilized preparation exhibits a half-life time of 12 h when incubated at 80 °C, pH 8.5 (a 25-fold stabilizing factor regarding to the soluble enzyme); (c) the optimal temperature was increased from 50 °C (soluble enzyme) up to 70 °C (hydrophobic support enzyme immobilized preparations); (d) the enantioselectivity of the enzyme for the hydrolysis of glycidyl butyrate and its dependence on the experimental conditions was significantly altered. Moreover, because the enzyme becomes reversibly but very strongly adsorbed on these highly hydrophobic supports, the lipase may be desorbed after its inactivation and the support may be reused. Very likely, adsorption occurs via interfacial activation of the lipase on the hydrophobic supports at very low ionic strength. On the other hand, all the covalent immobilization protocols used to immobilize the enzyme hardly improved the properties of the lipase.

Formate dehydrogenase (FDH) is a stable enzyme that may be readily inactivated by the interaction with hydrophobic interfaces (e.g., due to strong stirring). This may be avoided by immobilizing the enzyme on a porous support by any technique. Thus, even if the enzyme is going to be used in an ultra-membrane reactor, the immobilization presents some advantages. Immobilization on supports activated with bromocianogen, polyethylenimine, glutaraldehyde, etc., did not promote any stabilization of the enzyme under thermal inactivation. However, the immobilization of FDH on highly activated glyoxyl agarose has permitted increasing the enzyme stability against any distorting agent: pH, T, organic solvent, etc. The time of support-enzyme reaction, the temperature of immobilization, and the activation of the support need to be optimized to get the optimal stability-activity properties. Optimized biocatalyst retained 50% of the offered activity and became 50 times more stable at high temperature and neutral pH. Moreover, the quaternary structure of this dimeric enzyme becomes stabilized by immobilization under optimized conditions. Thus, at acidic pH (conditions where the subunit dissociation is the first step in the enzyme inactivation), the immobilization of both subunits of the enzyme on glyoxyl-agarose has allowed the enzyme to be stabilized by hundreds of times. Moreover, the optimal temperature of the enzyme has been increased (even by 10 degrees C at pH 4.5). Very interestingly, the activity with NAD(+)-dextran was around 60% of that observed with free cofactor.

The encapsulation of crosslinked enzyme aggregates (CLEA) of penicillin G acylase into a very rigid polymeric matrix based on polyvinyl alcohol (LentiKats) has been used successfully to improve the inadequate mechanical properties of CLEA. This encapsulation decreased CLEA activity by only around 40%. As compensation, a significant improvement in the stability of the CLEA in the presence of organic solvents was detected. This could be related to the highly hydrophilic environment inside the LentiKats biocatalysts: Partition experiments showed that the concentration of dioxane inside LentiKats was lower than in the reaction medium. In fact, thermal stability was about the same as in the corresponding CLEA. This permitted great improvement in the reaction rate for thermodynamically controlled synthesis of a model antibiotic (using phenylacetic acid and 7-amino-deacetoxycefalosporanic acid). Even more importantly, yields could be improved by using LentiKats-encapsulated CLEA, very likely by a favorable product/substrate partition. Thus, this very simple technique not only provides an efficient technique for solving the mechanical stability problem associated with CLEA, but also greatly improves the behavior of CLEA in organic media.