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Here, we report a study of the effect of the blocking agent on the properties of the lipase from Thermomyces lanuginosus (TLL) immobilized on a heterofunctional support (Purolite C18-ethylnediamina (EDA)- vinyl sulfone (VS)-TLL-blocking agent) in different reactions. The performance of the biocatalysts was compared to those immobilized on standard hydrophobic support (Purolite C18-TLL) and the commercial one (TLL-IM). The nature of the blocking agent (Cys, Gly and Asp) altered the enzyme features. TLL-IM always gave a comparatively worse performance, with its specificity for the oil being very different to the Purolite biocatalysts. Under optimized conditions, Purolite C18-TLL yielded 97 % of hydrolysis conversion after 4 h using a water/waste cooking soybean oil (WCSO) mass ratio of 4.3, biocatalyst load of 6.5 wt% and a temperature of 44.2 °C (without buffer or emulsification agent). In esterification reactions of the purified free fatty acids (FFAs) obtained from WCSO, the best TLL biocatalysts depended on the utilized alcohol: linear amyl alcohol was preferred by Purolite C18-TLL and Purolite C18-EDA-VS-TLL-Gly, while higher activity was achieved utilizing isoamyl alcohol as nucleophile by Purolite C18-EDA-VS-TLL-Cys, Purolite C18-EDA-VS-TLL-Asp and IM-TLL as catalysts. All the results indicate the influence of the blocking step on the final biocatalyst features.

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We present the synthesis of a cross-linking enzyme aggregate (CLEAS) of a peroxidase from Megathyrsus maximus (Guinea Grass) (GGP). The biocatalyst was produced using 50%v/v ethanol and 0.88%w/v glutaraldehyde for 1 h under stirring. The immobilization yield was 93.74% and the specific activity was 36.75 U mg-1. The biocatalyst surpassed by 61% the free enzyme activity at the optimal pH value (pH 6 for both preparations), becoming this increase in activity almost 10-fold at pH 9. GGP-CLEAS exhibited a higher thermal stability (2-4 folds) and was more stable towards hydrogen peroxide than the free enzyme (2-3 folds). GGP-CLEAS removes over 80% of 0.05 mM indigo carmine at pH 5, in the presence of 0.55 mM H2O2 after 60 min of reaction, a much higher value than when using the free enzyme. The operational stability showed a decrease of enzyme activity (over 60% in 4 cycles), very likely related to suicide inhibition.

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Immobilization of enzymes on aminated supports using the glutaraldehyde chemistry may involve three different interactions, cationic, hydrophobic, and covalent interactions. To try to understand the impact this heterofunctionality, we study the physical adsorption of the beta-galactosidase from Aspergillus niger, on aminated supports (MANAE) and aminated supports with one (MANAE-GLU) or two molecules of glutaraldehyde (MANAE-GLU-GLU). To eliminate the chemical reactivity of the glutaraldehyde, the supports were reduced using sodium borohydride. After enzyme adsorption, the release of the enzyme from the supports using different NaCl concentrations, Triton X100, ionic detergents (SDS and CTAB), or different temperatures (4 °C to 55 °C) was studied. Using MANAE support, at 0.3 M NaCl almost all the immobilized enzyme was released. Using MANAE-GLU, 0.3 M, and 0.6 M NaCl similar results were obtained. However, incubation at 1 M or 2 M NaCl, many enzyme molecules were not released from the support. For the MANAE-GLU-GLU support, none of the tested concentrations of NaCl was sufficient to release all enzyme bound to the support. Only using high temperatures, 0.6 M NaCl, and 1 % CTAB or SDS, could the totality of the proteins be released from the support. The results shown in this paper confirm the heterofunctional character of aminated supports modified with glutaraldehyde.

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The solvent-free esterification of the free fatty acids (FFAs) obtained by the hydrolysis of castor oil (a non-edible vegetable oil) with 2-ethyl-1-hexanol (a branched fatty alcohol) was catalyzed by different free lipases. Eversa Transform 2.0 (ETL) features surpassed most commercial lipases. Some process parameters were optimized by the Taguchi method (L16'). As a result, a conversion over 95% of the FFAs of castor oil into esters with lubricants properties was achieved under optimized reaction conditions (15 wt% of biocatalyst content, 1:4 molar ratio (FFAs/alcohol), 30 °C, 180 rpm, 96 h). The substrates molar ratio had the highest influence on the dependent variable (conversion at 24 h). FFAs/2-ethyl-1-hexanol esters were characterized regarding the physicochemical and tribological properties. Interestingly, the modification of the FFAs with 2-ethyl-1-hexanol by ETL increased the oxidative stability of the FFAs feedstock from 0.18 h to 16.83 h. The biolubricants presented a lower friction coefficient than the reference commercial mineral lubricant (0.052 ± 0.07 against 0.078 ± 0.04). Under these conditions, ETL catalyzed the oligomerization of ricinoleic acid (a hydroxyl fatty acid) into estolides, reaching a conversion of 25.15% of the initial FFAs (for the first time).

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This study investigated the impact of a support matrix and active group on the support to the nutritional properties of orange juice after juice clarification. Pectinase was immobilized on chitosan and aminated silica supports, activated with genipin or glutaraldehyde, and applied for juice clarification. The effects on various juice properties, including reducing sugars, total soluble solids, vitamin C, and phenolic compounds, juice color, and pH, were evaluated. The results revealed that the immobilization on chitosan activated using genipin resulted in the highest biocatalyst activity (1211.21 U·g-1). The juice treatments using the biocatalysts led to turbidity reduction in the juice (up to 90%), with the highest reductions observed in treatments involving immobilized enzyme on chitosan. Importantly, the enzymatic treatments preserved the natural sugar content, total soluble solids, and pH of the juice. Color differences between treated and raw juice samples were especially relevant for those treated using enzymes, with significant differences in L* and b*, showing loss of yellow vivid color. Analysis of phenolic compounds and vitamin C showed no significant alterations after the enzymatic treatment of the raw juice. According to our results, the clarification of orange juice using immobilized enzymes can be a compromise in turbidity reduction and color reduction to maintain juice quality.

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Bromelains are cysteine peptidases with endopeptidase action (a subfamily of papains), obtained from different parts of vegetable belonging to the Bromeliaceae family. They have some intrinsic medical activity, but this review is focused on their application (individually or mixed with other proteases) to produce bioactive peptides. When compared to other proteases, perhaps due to the fact that they are commercialized as an extract containing several proteases, the hydrolysates produced by this enzyme tends to have higher bioactivities than other common proteases. The peptides and the intensity of their final properties depend on the substrate protein and reaction conditions, being the degree of hydrolysis a determining parameter (but not always positive or negative). The produced peptides may have diverse activities such as antioxidant, antitumoral, antihypertensive or antimicrobial ones, among others or they may be utilized to improve the organoleptic properties of foods and feeds. Evolution of the use of this enzyme in this application is proposed to be based on a more intense direct application of Bromeliaceae extract, without the cost associated to enzyme purification, and the use of immobilized biocatalysts of the enzyme by simplifying the enzyme recovery and reuse, and also making the sequential hydrolysis using diverse proteases possible.

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The increasing worries by the inadequate use of energy and the preservation of nature are promoting an increasing interest in the production of biolubricants. After discussing the necessity of producing biolubricants, this review focuses on the production of these interesting molecules through the use of lipases, discussing the different possibilities (esterification of free fatty acids, hydroesterification or transesterification of oils and fats, transesterification of biodiesel with more adequate alcohols, estolides production, modification of fatty acids). The utilization of discarded substrates has special interest due to the double positive ecological impact (e.g., oil distillated, overused oils). Pros and cons of all these possibilities, together with general considerations to optimize the different processes will be outlined. Some possibilities to overcome some of the problems detected in the production of these interesting compounds will be also discussed.

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This review summarizes the most relevant advances in the biological transformation of fatty acids (or derivatives) into hydrocarbons to be used as biofuels (biogasoline, green diesel and jet biofuel). Among the used enzymes, the fatty acid decarboxylase from Jeotgalicoccus sp. ATCC 8456 (OleTJE) stands out as a promising enzyme. OleTJE may be coupled in cascade reactions with metalloenzymes or reductases from the Old Yellow Enzymes (OYE) family to perform the hydrogenation of α-olefins into paraffins. The photodecarboxylase from Chlorella variabilis NC64A (CvFAP) is an example of coupling biocatalysis and photocatalysis to produce alkanes. Besides the (photo)decarboxylation of free fatty acids and/or triacyclglycerols to produce alkanes/alkenes, by enzymes has also been employed. The cyanobacterial aldehyde decarbonylase (cAD) from Nostoc punctiforme is an outstanding example of this kind of enzymes used to produce alkanes. Overall, these kinds of enzymes open up new possibilities to the production of biofuels from renewable sources, even if they have many limitations on the current situation. The possibilities of improving enzymes features via immobilization or coimmobilization, as well as the utilization of whole cells haves been also reviewed.

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In this review we have focused on the preparation of cross-linked enzyme aggregates (CLEAs) from lipases, as these are among the most used enzyme in bioprocesses. This immobilization method is considered very attractive due to preparation simplicity, non-use of supports and the possibility of using crude enzyme extracts. CLEAs provide lipase stabilization under extreme temperature or pH conditions or in the presence of organic solvents, in addition to preventing enzyme leaching in aqueous medium. However, it presents some problems in the preparation and limitations in their use. The problems in preparation refer mainly to the crosslinking step, and may be solved using an aminated feeder. The problems in handling have been tackled designing magnetic-CLEAs or trapping the CLEAs in particles with better mechanical properties, the substrate diffusion problems has been reduced by producing more porous-CLEAs, etc. The enzyme co-immobilization using combi-CLEAs is also a new tendency. Therefore, this review explores the CLEAs methodology aimed at lipase immobilization and its applications.

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In this study, soybean oil deodorizer distillate (SODD), a mixture of free fatty acids and acylglycerides, and isoamyl alcohol were evaluated as substrates in the synthesis of fatty acid isoamyl monoesters catalyzed by Eversa (a liquid formulation of Thermomyces lanuginosus lipase). SODD and the products were characterized by the chemical and physical properties of lubricant base stocks. The optimal conditions to produce isoamyl fatty acid esters were determined by response surface methodology (RSM) using rotational central composite design (RCCD, 23 factorial + 6 axial points + 5 replications at the central point); they were 1 mol of fatty acids (based on the SODD saponifiable index) to 2.5 mol isoamyl alcohol, 45 °C, and 6 wt.% enzymes (enzyme mass/SODD mass). The effect of the water content of the reactional medium was also studied, with two conditions of molecular sieve ratio (molecular sieve mass/SODD mass) selected as 39 wt.% (almost anhydrous reaction medium) and 9 wt.%. Ester yields of around 50 wt.% and 70 wt.% were reached after 50 h reaction, respectively. The reaction products containing 43.7 wt.% and 55.2 wt.% FAIE exhibited viscosity indices of 175 and 163.8, pour points of -6 °C and -9 °C, flash points of 178 and 104 °C, and low oxidative stability, respectively. Their properties (mainly very high viscosity indices) make them suitable to be used as base stocks in lubricant formulation industries.

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This study aimed the enzymatic decyl esters production by hydroesterification, a two-step process consisting of hydrolysis of refined soybean (RSBO) or used soybean cooking (USCO) oils to produce free fatty acids (FFA) and further esterification of purified FFA. Using free lipase from Candida rugosa (CRL), about 98% hydrolyses for both oils have been observed after 180 min of reaction using a CRL loading of 50 U g-1 of reaction mixture, 40 °C, and a mechanical stirring of 1500 rpm. FFA esterification with decanol in solvent-free systems was performed using lipase from Thermomyces lanuginosus (TLL) immobilized by physical adsorption on silica particles extracted from rice husk, an agricultural waste. For such purpose, non-functionalized (SiO2) or functionalized rice husk silica bearing octyl (Octyl-SiO2) or phenyl (Phe-SiO2) groups have been used as immobilization supports. Protein amounts between 22 and 28 mg g-1 of support were observed. When used in the esterification, they enabled a FFA conversion of 81.3-87.6% after 90-300 min of reaction. Lipozyme TL IM, a commercial immobilized TLL, exhibited similar performance compared to TLL-Octyl-SiO2 (FFA conversion ≈90% after 90-120 min of reaction). However, high operational stability after fifteen successive esterification batches was observed only for TLL immobilized on Octyl-SiO2 (activity retention of ≈90% using both FFA sources). The produced decyl esters presented good characteristics as potential biolubricants according to standard methods (ASTM) and thermal analysis.

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Differently modified Lipozyme 435 (L435) (immobilized lipase B from Candida antarctica) preparations were used as biocatalysts in the esterification reaction to synthesize sugar fatty acid esters (SFAEs) from xylose (acyl acceptor) and lauric/palmitic acids (acyl donors) in methyl ethyl ketone (MEK) solvent. The L435 treatment with polyethyleneimine (PEI) (2; 25; and 750 KDa) prevented the enzyme leakage in the crude sugar ester reaction product. The 2 KDa PEI coating of this enzyme preparation produced the highest enzyme stability in MEK, buffer solutions (pHs 5 and 7), and methanol aqueous phosphate buffer at pH 7. Using an excess of the acyl donor (1:5 xylose: fatty acid molar ratio), high xylose conversions (70-84%) were obtained after 24 h-reaction using both, non-modified and PEI (2 KDa) coated L435, but the PEI treated biocatalyst afforded a higher xylose modification degree. After 5 reuse cycles with the L435 coated with PEI 2 KDa, the xylose conversions only decreased 10%, while with the non-treated biocatalyst they decreased by 37%. The formation of SFAEs was confirmed by mass spectrometry, which showed the presence of xylose mono-, di-, and triesters. They exhibited emulsion capacities close to that of a commercial sucrose monolaurate.

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A review on the enzyme ß-galactosidase from Kluyveromyces lactis is presented, from the perspective of its structure and mechanisms of action, the main catalyzed reactions, the key factors influencing its activity, and selectivity, as well as the main techniques used for improving the biocatalyst functionality. Particular attention was given to the discussion of hydrolysis, transglycosylation, and galactosylation reactions, which are commonly mediated by this enzyme. In addition, the products generated from these processes were highlighted. Finally, biocatalyst improvement techniques are also discussed, such as enzyme immobilization and protein engineering. On these topics, the most recent immobilization strategies are presented, emphasizing processes that not only allow the recovery of the biocatalyst but also deliver enzymes that show better resistance to high temperatures, chemicals, and inhibitors. In addition, genetic engineering techniques to improve the catalytic properties of the ß-galactosidases were reported. This review gathers information to allow the development of biocatalysts based on the ß-galactosidase enzyme from K. lactis, aiming to improve existing bioprocesses or develop new ones.

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Lipase from Thermomyces lanuginosus (TLL) has been covalently immobilized on heterofunctional octyl-vinyl agarose. That way, the covalently immobilized enzymes will have identical orientation. Then, it has blocked using hexyl amine (HEX), ethylenediamine (EDA), Gly and Asp. The initial activity/stability of the different biocatalysts was very different, being the most stable the biocatalyst blocked with Gly. These biocatalysts had been utilized to analyze if the enzyme activity could decrease differently along thermal inactivation courses depending on the utilized substrate (that is, if the enzyme specificity was altered during its inactivation using 4 different substrates to determine the activity), and if this can be altered by the nature of the blocking agent and the inactivation conditions (we use pH 5, 7 and 9). Results show great changes in the enzyme specificity during inactivation (e.g., activity versus triacetin was much more quickly lost than versus the other substrates), and how this was modulated by the immobilization protocol and inactivation conditions. The difference in the changes induced by immobilization and inactivation were confirmed by fluorescence studies. That is, the functional and structural analysis of partially inactivated immobilized enzyme showed that their inactivation pathway is strongly depended on the support features and inactivation conditions.

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This work aimed the application of a new biocatalyst for biodiesel production from residual agro-industrial fatty acids. A recombinant Pichia pastoris displaying lipase from Rhizomucor miehei (RML) on the cell surface, using the PIR-1 anchor system, were prepared using glycerol as the carbon source. The biocatalyst, named RML-PIR1 showed optimum temperature of 45 °C (74.0 U/L). The stability tests resulted in t1/2 of 3.49 and 2.15 h at 40 and 45 °C, respectively. RML-PIR1 was applied in esterification reactions using industrial co-products as substrates, palm fatty acid distillate (PFAD) and soybean fatty acid distillate (SFAD). The highest productivity was observed for SFAD after 48 h presenting 79.1% of conversion using only 10% of biocatalyst and free-solvent system. This is about ca. eight times higher than commercial free RML in the same conditions. The stabilizing agents study revealed that the treatment using glutaraldehyde (GA) and poly(ethylene glycol) (PEG) enabled increased stability and reuse of biocatalyst. It was observed by SEM analysis that the treatment modified the cell morphology. RML-PIR1-GA presented 87.9% of the initial activity after 6 reuses, whilst the activity of unmodified RML-PIR decreased by 40% after the first use. These results were superior to those obtained in the literature, making this new biocatalyst promising for biotechnological applications, such as the production of biofuels on a large scale.

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The use of enzymes in industrial processes requires the improvement of their features in many instances. Enzyme immobilization, a requirement to facilitate the recovery and reuse of these water-soluble catalysts, is one of the tools that researchers may utilize to improve many of their properties. This review is focused on how enzyme immobilization may improve enzyme stability. Starting from the stabilization effects that an enzyme may experience by the mere fact of being inside a solid particle, we detail other possibilities to stabilize enzymes: generation of favorable enzyme environments, prevention of enzyme subunit dissociation in multimeric enzymes, generation of more stable enzyme conformations, or enzyme rigidification via multipoint covalent attachment. In this last point, we will discuss the features of an "ideal" immobilization protocol to maximize the intensity of the enzyme-support interactions. The most interesting active groups in the support (glutaraldehyde, epoxide, glyoxyl and vinyl sulfone) will be also presented, discussing their main properties and uses. Some instances in which the number of enzyme-support bonds is not directly related to a higher stabilization will be also presented. Finally, the possibility of coupling site-directed mutagenesis or chemical modification to get a more intense multipoint covalent immobilization will be discussed.

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In this paper, we have performed the Lipozyme 435-catalyzed synthesis of xylose oleate in methyl ethyl ketone (MEK) from xylose and oleic acid. The effects of substrates' molar ratios, reaction temperature, reaction time on esterification rates, and Lipozyme 435 reuse were studied. Results showed that an excess of oleic acid (xylose: oleic acid molar ratio of 1:5) significantly favored the reaction, yielding 98% of xylose conversion and 31% oleic acid conversion after 24 h-reaction (mainly to xylose mono- and dioleate, as confirmed by mass spectrometry). The highest Lipozyme 435 activities occurred between 55 and 70 °C. The predicted Ping Pong Bi Bi kinetic model fitted very well to the experimental data and there was no evidence of inhibitions in the range assessed. The reaction product was purified and presented an emulsion capacity close to that of a commercial sugar ester detergent. Finally, the repeated use of Lipozyme 435 showed a reduction in the reaction yields (by 48 and 19% in the xylose and oleic acid conversions, respectively), after ten 12 h-cycles.

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In this work, the effect of different immobilization procedures on the properties of a lipase obtained from the extremophilic microorganism Serratia sp. USBA-GBX-513, which was isolated from Paramo soils of Los Nevados National Natural Park (Colombia), is reported. Different Shepharose beads were used: octyl-(OC), octyl-glyoxyl-(OC-GLX), cyanogen bromide (BrCN)-, and Q-Sepharose. The performance of the different immobilized extremophile lipase from Serratia (ESL) was compared with that of the lipase B from Candida antarctica (CALB). In all immobilization tests, hyperactivation of ESL was observed. The highest hyperactivation (10.3) was obtained by immobilization on the OC support. Subsequently, the thermal stability at pH 5, 7, and 9 and the stability in the presence of 50% (v/v) acetonitrile, 50% dioxane, and 50% tetrahydrofuran solvents at pH 7 and 40 °C were evaluated. ESL immobilized on octyl-Sepharose was the most stable biocatalyst at 90 °C and pH 9, while the most stable preparation at pH 5 was ESL immobilized on OC-GLX-Sepharose supports. Finally, in the presence of 50% (v/v) tetrahydrofuran (THF) or dioxane at 40 °C, ESL immobilized on OC-Sepharose was the most stable biocatalyst, while the immobilized preparation of ESL on Q-Sepharose was the most stable one in 40% (v/v) acetonitrile.

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In this paper, 3 different biocatalysts of ß-galactosidase from Kluyveromyces lactis have been prepared by immobilization in chitosan activated with glutaraldehyde (Chi_Glu_Gal), glyoxyl agarose (Aga_Gly_Gal) and agarose coated with polyethylenimine (Aga_PEI_Gal). These biocatalysts have been used to catalyze the synthesis of lactulose from lactose and fructose. Aga-PEI-Gal only produces lactulose at 50 °C, and not at 25 or 37 °C, Aga_Gly_Gal was unable to produce lactulose at any of the assayed temperatures while Chi_Glu_Gal produced lactulose at all assayed temperatures, although a lower yield was obtained at 25 or 37 °C. The pre-incubation of this biocatalyst at 50 °C permitted to obtain similar yields at 25 or 37 °C than at 50 °C. The use of milk whey instead of pure lactose and fructose produced an improvement in the yields using Aga_PEI_Gal and a decrease using Chi_Glu_Gal. The operational stability also depends on the reaction medium and of biocatalyst. This study reveals how enzyme immobilization may greatly alter the performance of ß-galactosidase in a kinetically controlled manner, and how medium composition influences this performance due to the kinetic properties of ß-galactosidase.

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The performance of the previously optimized magnetic cross-linked enzyme aggregate of Eversa (Eversa-mCLEA) in the enzymatic synthesis of biolubricants by transesterification of waste cooking oil (WCO) with different alcohols has been evaluated. Eversa-mCLEA showed good activities using these alcohols, reaching a transesterification activity with isoamyl alcohol around 10-fold higher than with methanol. Yields of isoamyl fatty acid ester synthesis were similar using WCO or refined oil, confirming that this biocatalyst could be utilized to transform this residue into a valuable product. The effects of WCO/isoamyl alcohol molar ratio and enzyme load on the synthesis of biolubricant were also investigated. A maximum yield of around 90 wt.% was reached after 72 h of reaction using an enzyme load of 12 esterification units/g oil and a WCO/alcohol molar ratio of 1:6 in a solvent-free system. At the same conditions, the liquid Eversa yielded a maximum ester yield of only 34%. This study demonstrated the great changes in the enzyme properties that can be derived from a proper immobilization system. Moreover, it also shows the potential of WCO as a feedstock for the production of isoamyl fatty acid esters, which are potential candidates as biolubricants.

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