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

The present work studied the synthesis of biolubricants from by-products of soybean oil processing using the lipase from Candida rugosa (CRL) both in free and immobilized forms, as biocatalysts. Soybean fatty acid distillate (SFAD) was used for first time as source of free fatty acids (FFA) to produce biolubricants via their enzymatic esterification with neopentyl glycol (NPG) and trimethylolpropane (TMP) alcohols in a solvent-free medium. The immobilization of CRL was performed via interfacial activation using a commercial hydrophobic support (Accurel) and a home-made core-shell matrix of poly(methyl methacrylate) (PMMA). Conversions of about 80 % were obtained using lyophilized CRL after 30 or 180 min using NPG and TMP, respectively. When CRL-PMMA/PMMA was used to catalyze the synthesis of NPG esters, 90 % conversion was achieved after only 6 h, whilst CRL-Accurel were more active using TMP. The biocatalysts maintained the reaction conversion during eight batches of 24 h. The lubricant properties of the products were also characterized, showing that it can be an environmentally friendly alternative for petrol lubricants

Synthetic oleochemical esters represents a growing alternative for mineral oil-based lubricants. In this sense, the present study shows the results of enzymatic synthesis of neopentyl glycol-bases biolubricants using biodiesel from soybean and castor bean as raw materials. The potential of two commercial immobilized lipases, lipase B from Candida antarctica (Novozyme 435) and lipase from Rhizomucor miehei (Lipozyme RM IM), to produce NPG esters in a solvent-free medium was evaluated. High conversions (>95%) were obtained for both raw materials, with both enzymes, but in a shorter reaction time for soybean biodiesel (24 h) than castor biodiesel (48 h). Both enzymes could be successfully reused for six reactions, Novozyme 435 maintained ≥70% of the initial conversion using the two raw materials and Lipozyme RM IM maintained ≥90% of the first reaction conversion using castor biodiesel. The soybean NPG esters showed good viscosity index, higher than 198, and the castor NPG esters showed good pour point, lower than −20 °C, and good oxidative stability, higher than 30 min. The physico-chemical properties of the biolubricants were dependent on the biodiesel source which directly influence the final application of the products.

Harnessing enzymes which possess several catalytic activities is a topic where intense research has been carried out, mainly coupled with the development of cascade reactions. This review tries to cover the different possibilities to reach this goal: enzymes with promiscuous activities, fusion enzymes, enzymes + metal catalysts (including metal nanoparticles or site-directed attached organometallic catalyst), enzymes bearing non-canonical amino acids + metal catalysts, design of enzymes bearing a second biological but artificial active center (plurizymes) by coupling enzyme modelling and directed mutagenesis and plurizymes that have been site directed modified in both or in just one active center with an irreversible inhibitor attached to an organometallic catalyst. Some examples of cascade reactions catalyzed by the enzymes bearing several catalytic activities are also described. Finally, some foreseen problems of the use of these multi-activity enzymes are described (mainly related to the balance of the catalytic activities, necessary in many instances, or the different operational stabilities of the different catalytic activities). The design of new multi-activity enzymes (e.g., plurizymes or modified plurizymes) seems to be a topic with unarguable interest, as this may link biological and non-biological activities to establish new combo-catalysis routes.

The aim of this study was to understand estolide enzyme polymerization from free ricinoleic acid (FRA) and its lubricant properties, by controlling the reaction time, during the course of a reaction involving a two-step enzymatic hydroesterification from castor oil. Adding water increased the reaction rate, reducing the total acidity to 30% in 24 h. FRA is the major component of castor oil (up to 90%). A GC analysis of the FRA, expressed as g/100 g of sample, was used to determine the FRA profile during estolide polymerization. These data suggested a relationship between the total acidity of the samples and the FRA concentration. The estolide profile and number (EN) were determined by GPC and NMR analyses. A 14-hour reaction time was needed for total consumption of FRA, with an EN of 5.5 and a degree of polymerization of the major estolide of 6/7. The analysis showed that estolides of several sizes [dimers, trimers, tetramers, and pentamers (+)] were produced during the reaction; between 8 and 14 h the EN grew exponentially until reaching a plateau after about 16 h of reaction. From 14 to 24 h, although the estolide-size profile did not change significantly, EN continued to increase with increasing reaction time, until a polymerization degree of 7/8 at 24 h. The increase in EN corresponds to the increase in the viscosity index and oxidative stability, reaching 164 and 43 min, respectively, after 20 h of reaction; as well as to the decrease in pour point and total acidity number, which reached −48 °C and 36.8 mg KOH/g of sample after 20 h of reaction. This is the first description of a kinetic method to control the degree of estolide polymerization, by controlling the reaction time; and of its relationship between the EN and the lubricant properties of these molecules, aiming toward the production of different biolubricants for several applications and desired uses.

Lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) were immobilized on octyl agarose. Then, the biocatalysts were chemically modified using glutaraldehyde, trinitrobenzenesulfonic acid or ethylenediamine and carbodiimide, or physically coated with ionic polymers, such as polyethylenimine (PEI) and dextran sulfate. These produced alterations of the enzyme activities have, in most cases, negative effects with some substrates and positive with other ones (e.g., amination of immobilized TLL increases the activity versus p-nitro phenyl butyrate (p-NPB), reduces the activity with R-methyl mandate by half and maintains the activity with S-isomer). The modification with PEI increased the biocatalyst activity 8-fold versus R-methyl mandelate. Enzyme stability was also modified, usually showing an improvement (e.g., the modification of immobilized TLL with PEI or glutaraldehyde enabled to maintain more than 70% of the initial activity, while the unmodified enzyme maintained less than 50%). The immobilized enzymes were also mineralized by using phosphate metals (Zn2+, Co2+, Cu2+, Ni2+ or Mg2+), and this affected also the enzyme activity, specificity (e.g., immobilized TLL increased its activity after zinc mineralization versus triacetin, while decreased its activity versus all the other assayed substrates) and stability (e.g., the same modification increase the residual stability from almost 0 to more than 60%). Depending on the enzyme, a metal could be positively, neutrally or negatively affected for a specific feature. Finally, we analyzed if the chemical modification could, somehow, tune the effects of the mineralization. Effectively, the same mineralization could have very different effects on the same immobilized enzyme if it was previously submitted to different physicochemical modifications. The same mineralization could present different effects on the enzyme activity, specificity or stability, depending on the previous modification performed on the enzyme, showing that these previous enzyme modifications alter the effects of the mineralization on enzyme features. For example, TLL modified with glutaraldehyde and treated with zinc salts increased its activity using R-methyl mandelate, while almost maintaining its activity versus the other unaltered substrates, whereas the aminated TLL maintained its activity with both methyl mandelate isomers, while it decreased with p-NPB and triacetin. TLL was found to be easier to tune than CALB by the strategies used in this paper. In this way, the combination of chemical or physical modifications of enzymes before their mineralization increases the range of modification of features that the immobilized enzyme can experienced, enabling to enlarge the biocatalyst library.

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.

Lipases A and B from Candida antarctica (CALA, CALB), and those from Candida rugosa (CRL) and Rhizomucor miehei (RML) have been immobilized on octyl agarose beads under 16 different conditions. The activities of the biocatalysts versus different substrates were analyzed, as well as their thermal stabilities at pH 7.0. All CALB and CRL preparations showed very similar properties, except the CRL biocatalysts prepared at pH 9. Under these conditions the free enzyme was partially inactivated. Immobilized CALA showed some significant differences in activity depending on the immobilization conditions when using esters of mandelic acid as substrates (the activities of the different preparations differed by more than a 2-fold factor), while when using the other substrates the differences were minimal. However, immobilized RML enzyme greatly alters its activity depending on the immobilization conditions, reaching differences up to 4–5 fold in both activity and stability. It is remarkable that the effect of one immobilization variable depends on the substrates, and that there are strong interactions between the different immobilization variables. Thus, this immobilization method was very robust (producing almost identical functional biocatalysts independently from the immobilization conditions) using CRL or CALB, while the immobilization conditions must be carefully controlled using RML to have reproducible results.

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., This research was funded by the São Paulo Research Foundation (FAPESP, grant number 2016/10636-8), in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–Brasil (CAPES)–Finance Code 001, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, project numbers 405889/2016-0 and 308212/2017-7), and Ministerio de Ciencia e Innovación-Spanish Government (project number CTQ2017-86170-R).

This paper outlines the immobilization of the recombinant dimeric unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). The enzyme was quite stable (remaining unaltered its activity after 35 h at 47 °C and pH 7.0). Phosphate destabilized the enzyme, while glycerol stabilized it. The enzyme was not immobilized on glyoxyl-agarose supports, while it was immobilized albeit in inactive form on vinyl-sulfone-activated supports. rAaeUPO immobilization on glutaraldehyde pre-activated supports gave almost quantitative immobilization yield and retained some activity, but the biocatalyst was very unstable. Its immobilization via anion exchange on PEI supports also produced good immobilization yields, but the rAaeUPO stability dropped. However, using aminated agarose, the enzyme retained stability and activity. The stability of the immobilized enzyme strongly depended on the immobilization pH, being much less stable when rAaeUPO was adsorbed at pH 9.0 than when it was immobilized at pH 7.0 or pH 5.0 (residual activity was almost 0 for the former and 80% for the other preparations), presenting stability very similar to that of the free enzyme. This is a very clear example of how the immobilization pH greatly affects the final biocatalyst performance.

1 Tabla.-- 17 Figuras, Trypsin, chymotrypsin, penicillin G acylase and ficin extract have been stabilized by immobilization on glyoxyl agarose, adding different aliphatic compounds bearing a primary amine group during the immobilization: ethyl amine, butyl amine, hexyl amine (at concentrations ranging from 0 to 20 mM) and octyl amine (from 0 to 10 mM) to analyze their effects on the immobilized enzyme stability. As expected, the presence of amines reduced the intensity of the enzyme-support multipoint covalent attachment, and therefore the enzyme stability. However, it is clear that this effect is higher using octyl amine for all enzymes (in some cases the enzyme immobilized in the presence of 10 mM octyl amine was almost inactivated while the reference kept over 50 % of the initial activity). This way, it seems that the most important effect of the presence of aminated compounds came from the generation of steric hindrances to the enzyme/support multi-reaction promoted by the ammines that are interacting with the aldehyde groups. In some instances, just 1 mM of aminated compounds is enough to greatly decrease enzyme stability. The results suggested that, if the composition of the enzyme extract is unknown, to eliminate small aminated compounds may be necessary to maximize the enzyme-support reaction., We gratefully recognize the financial support from Ministerio de Ciencia e Innovación-Spanish Government (project number CTQ2017-86170-R). RMS thank to Ministerio de Educacion -Spanish Government for a FPU fellowship, SAB and EHS thank Algerian Ministry of higher education and scientific research for their fellowships. The help and suggestions from Dr. Ángel Berenguer (Departamento de Química Inorgánica, Universidad de Alicante) are gratefully recognized.

Cottonseed meal is a co-product from the cotton industry that is produced in large quantities worldwide. The aim of this work was to add value to this by-product through the production of Rhizomucor miehei lipases and application of the dry fermented solid in esterification reactions between oleic acid and ethanol or methanol and transesterification of a vegetable acid oil. Reactions were carried out in a solvent-free system and conversions higher than 85% were obtained in 4 h in the esterification reactions. The reusability of the biocatalyst was evaluated and the washing of the biocatalyst with solvent (ethanol) was necessary to recover the activity of the lipase, being obtained conversion of 74% in relation to the initial conversion after 4 sucessive reactions. The dried fermented solid was also able to catalyze the production of ethyl and methyl esters by simultaneous transesterification/esterification reactions of macauba acid oil, with production of an ester content of about 76% after 24 h. This is the first time that fermented solids produced in cottonseed meal without any supplementation are used as biocatalyst of esterification and transesterification reactions.

Boron-rich particles with the boron fraction ca.10-20 wt % of controllable shape and size that can be easily prepared via simple ion co-assembly are promising material for tumor treatment by boron neutron capture therapy. Electroneutral, dynamic core-shell polymeric nanoparticles were prepared by co-assembly of cationic PEO-block-PGEA diblock copolymer with sodium closo-dodecaborate, Na2 [B12 H12 ]. This is the first example of polymer nanoparticles based on [B12 H12 ]2- nano-ion pairing. The high [B12 H12 ]2- loading is proven by calorimetry at physiological salt concentration. As a result of rational design, rod-, worm- and sphere-like particles were produced and further tested using human glioblastoma and cervical carcinoma cell lines. Rod-like particles yielded the highest internalization capability in all tested cell lines.

This contribution describes a sustainable, environmentally benign process for the production of biolubricants. The reaction was performed in a solvent-free system using a side stream from palm-oil refining (palm fatty-acid distillate, PFAD) and polyols as substrates for enzymatic catalysis. Biocatalysts consume less energy than chemical processes, and produce esters with good lubricant properties and environmentally friendly characteristics such as high biodegradability and low toxicity. The effects of different parameters (molar ratio, temperature and enzyme concentration) were evaluated during esterification of PFAD with neopentyl glycol (NPG) or trimethylolpropane (TMP) to produce polyol esters. The products obtained through esterification of PFAD with NPG and TMP in the stoichiometric molar ratio at 45 °C, using 4% (w/w) of Candida rugosa lipase, attained a modification of around 90% of hydroxyl groups (OH) at 10 and 8 h, respectively. These conditions were used to reach the maximum OH esterification for PFAD-TMP esters (94%) and PFAD-NPG esters (87%). NMR analysis of the final ester composition showed that the PFAD-TMP and PFAD-NPG esters were composed mostly of triesters and diesters, respectively. The properties of these esters were also characterized, including fusion and crystallization temperatures, viscosity, pour point and oxidative stability. The polyol esters showed promising lubricant properties, such as oxidative stability and viscosity within the acceptable range for many applications. This study demonstrated the potential of using PFAD for the production of environmentally benign polyol esters through non-conventional catalysis.

Three commercial lipases, Lipomod 34MDP (free lipase from Candida rugosa ), Lipozyme RM IM (immobilized lipase from Rhizomucor miehei ) and Novozym 435 (immobilized lipase B from Candida antarctica ) were evaluated as catalysts in the production of biolubricant base oils. This was accomplished via the esterification of free fatty acids obtained from soybean-oil hydrolysis and different polyols (neopentyl glycol, NPG; trimethylolpropane, TMP; and pentaerythritol, PE). Reactions carried out with free C. rugosa lipase showed the highest conversions for the three polyols (97% for NPG, 100% for TMP and 55% for PE) after 24 h, while Novozym 435 and Lipozyme RM IM resulted, respectively, in 65% and 92% for NPG, 15% and 39% for TMP and 1% and 0% for PE. Then, Accurel MP1000 (a microporous polypropylene support, PP) was used to immobilize the C. rugosa lipase; enzyme loading used was 0.3 mg/g. The zymography analysis showed that the protein band of 60 KDa present in the C. rugosa lipase extract was the main protein responsible for the extract activity in the biolubricant synthesis and it was successfully immobilized onto the support. The immobilized lipase (34MDP-PP) showed conversions of 99% for NPG and 92% for TMP, after 24 h of reaction, maintaining the results obtained with the free enzyme. The 34MDP-PP could be reused for six consecutive reaction cycles without a reduction in the final conversion, using NPG and TMP as substrates. Using Lipozyme RM IM, the conversion decreased from 92 to 56% after six consecutive reactions. The soybean esters of NPG and TMP showed good viscosity indexes, higher than 200.

The paper shows the preparation of a new heterofunctional agarose support: amino-vinylsulfone. This has been employed to immobilize the interesting enzyme β-galactosidase from Aspergillus oryzae. The enzyme cannot be immobilized on just vinylsulfone activated support a pH values ranging from 5.0 to 9.0. Neither the enzyme was immobilized using 200 mM of NaCl on amino-vinylsulfone support. However, the enzyme was readily immobilized at moderate ion strength at pH values from 5.0 to 9.0 via ion exchange on amino-vinylsulfone support, and later some covalent enzyme-support bonds could be formed, more rapidly at alkaline pH value. After optimization of immobilization pH, incubation pH and time, and blocking reagent, several immobilized biocatalysts on amino-vinylsulfone support having 50–80% of the initial activity and a stabilization factor of around 8–15 were prepared, depending on the exact immobilization conditions.

The aim of this work was to produce esters with potential application as biodiesel via esterification with short-chain alcohols of free fatty acids (FFA) obtained from soybean and palm oils refining. Esterification reactions were catalyzed by dry lipase immobilized in fermented solids (DFS), obtained by Rhizomucor miehei grown on babassu cake by solid-state fermentation (SSF). A final conversion above 80% was attained using palm fatty acid distillate (PFAD) and soybean fatty acid distillate (SFAD) as raw materials in esterification reactions with ethanol. The biocatalyst could be reused 5 times in repeated 6 h reactions after its washing with 95% (v/v) ethanol or hexane (using SFAD as raw material). Moreover, the properties of the obtained fatty acid ethyl esters (FAEE)-enriched products were characterized. Both products were in accordance with several limits established by biodiesel-related standardized tests from Brazilian specifications including ester content (97.3% for the product obtained from SFAD), water content (180 mg Kg −1 for the product obtained from PFAD), density (867.6 kg (m 3 ) −1 for the product obtained from SFAD), viscosity kinematic (3.14–4.29 mm 2 s −1 ), flash point (

We performed two-step enzymatic biolubricant production: castor oil hydrolysis and esterification of castor oil free fatty acids (COFFA) rich in ricinoleic acid. The hydrolysis step utilized castor seeds as catalyst yielding 93.13 ± 5.9% COFFA in only 1 h. In the esterification step, C. rugosa lipase (CRL) decreased the acidity by over 80% after 96 h, utilizing as polyols neopenthylglicol (NPG), trimethylolpropane (TMP) and pentaerytritol (PE) and showed about 70% acidity decrease without polyols, indicating the production of COFFA polymers (estolides). Nuclear magnetic resonance (NMR) confirmed estolides as the main product, reaching a polymerization degree of 6/7 without polyols, 4 in the presence of PE and ricinoleic acid dimers in the presence of NPG and TMP. Analyses of biolubricant properties showed that the best product was the estolide with a polymerization degree of 6/7: viscosity index of 162, oxidation stability of 51 min and pour point of −42 °C.

A new battery of biocatalyst was evaluated in the synthesis of biodiesel using a mix of used cooking oil and fats and methanol as substrates. Biocatalysts were prepared using five different commercial supports (Lifetech™ ECR1061M (styrene/methacrylic polymer), Lifetech™ ECR8804M (octadecyl methacrylate), Lifetech™ ECR8806M (octadecyl methacylate), Lifetech™ ECR1090M (styrene) and Lifetech™ ECR1030M (DVB/methacrylic polymer)) for the immobilization of four different lipases (from Rhizomucor miehie (RML), from Thermomyces lanuginosus (TLL) and the A and B forms from Candida antarctica, (CALA and CALB)) and of the phospholipase Lecitase Ultra™ (LU), and their performance were compared with that from commercial biocatalysts (when available: TL-IM, RM-IM and Novozyme 435). Results clearly showed that the methyl ester yields and specific activities greatly depend on the support. The addition of organic solvents usually had positive effects on all preparations activities but not on the yields, and the results depend on the solvent used, enzyme and media. In all cases, at least one of the new preparations was better than the commercial one in solvent free conditions, and in general supports having a layer of acyl groups gave better activities (octadecyl) that when the enzyme was directly immobilized on the support surface. TLL immobilized the new supports (except Lifetech™ ECR1061M) seemed to be very promising in solvent free medium, and yields near to 80% could be achieved in direct addition of 3:1 methanol to triglyceride molar ratio. The results show that is not possible to discard one couple enzyme/support using other couple because the strong dependence.

Graphical abstract, Highlights • Lipase B from Candida antarctica immobilized on activated chitosan. • The loading capacity of the new biocatalyst is around 20 mg/g of support. • Biocatalyst was higher than that of the CALB-Octyl (by a 53-fold factor). • Enzymatic esterification reaction in supercritical or near-critical CO2. • Molecular sieves promoted 16.0% increase in enzymatic esterification reaction., The objective of this new paper was to evaluate the enzymatic esterification reaction conducted in supercritical or near-critical CO2, catalyzed by immobilized lipase B from Candida antarctica (CALB). The biocatalyst was prepared through the immobilization of CALB by covalent attachment using chitosan sequentially activated with Glycidol, ethylenediamine (EDA) and glutaraldehyde as support. In order to determine the best operational conditions of the esterification reaction (1: 1 (alcohol–acid); biocatalyst content, 10% (by substrate mass); 45 °C), an experimental design (23) was conducted to evaluate the effects of the following parameters: alcohol to oil molar ratios, reaction time and temperature. The maximum loading of chitosan was 20 mg protein/g support, and the thermal and solvent stability of the new biocatalyst was higher than that of the CALB-GX (by a 26-fold factor), CALB-OC (by a 53-fold factor) and Novozym 435 (by a 3-fold factor). The maximum conversion was 46.9% at a temperature of 29.9 °C, ethanol to oleic acid molar ratio equal to 4.50:1, and a reaction time of 6.5 h. Additionally, the removal of water from the medium, by using molecular sieves, promoted a 16.0% increase in the conversion of oleic acid into ethyl esters.

The synthesis of ethyl butyrate catalyzed by lipases A (CALA) or B (CALB) from Candida antarctica immobilized onto magnetic nanoparticles (MNP), CALA-MNP and CALB-MNP, respectively, is hereby reported. MNPs were prepared by co-precipitation, functionalized with 3-aminopropyltriethoxysilane, activated with glutaraldehyde, and then used as support to immobilize either CALA or CALB (immobilization yield: 100 &plusmn, 1.2% and 57.6 &plusmn, 3.8%, biocatalysts activities: 198.3 &plusmn, 2.7 Up-NPB/g and 52.9 &plusmn, 1.7 Up-NPB/g for CALA-MNP and CALB-MNP, respectively). X-ray diffraction and Raman spectroscopy analysis indicated the production of a magnetic nanomaterial with a diameter of 13.0 nm, whereas Fourier-transform infrared spectroscopy indicated functionalization, activation and enzyme immobilization. To determine the optimum conditions for the synthesis, a four-variable Central Composite Design (CCD) (biocatalyst content, molar ratio, temperature and time) was performed. Under optimized conditions (1:1, 45 &deg, C and 6 h), it was possible to achieve 99.2 &plusmn, 0.3% of conversion for CALA-MNP (10 mg) and 97.5 &plusmn, 0.8% for CALB-MNP (12.5 mg), which retained approximately 80% of their activity after 10 consecutive cycles of esterification. Under ultrasonic irradiation, similar conversions were achieved but at 4 h of incubation, demonstrating the efficiency of ultrasound technology in the enzymatic synthesis of esters.

Amphiphilic triblock terpolymers have attractive applications in the preparation of nanoparticles with controlled morphology. An additional level of morphology control can be provided by reactive blocks, whose interactions with the solvent vary under different stimuli. In this work, we synthesized a triblock terpolymer (poly(acrylic acid)-b-poly(4-hydroxystyrene)-b-poly{1-[4-(1-methyl-1,2-dicarba-closo-dodecaborane-2-yl methyl)-phenyl]ethylene}) (PAA-b-PHS-b-PSC) containing carboranes as pendant groups by reversible addition fragmentation chain transfer (RAFT) polymerization and subsequently studied its behavior in aqueous solution. Once the nanoparticles were formed, the solubility of the second and third blocks was changed via pH and CsF reactions, respectively. The resulting micelles work as an ON/OFF system, using changes in fluorescence intensity at different pH values., The authors acknowledge the financial support from the Czech Science Foundation Grant No. 17-00289Y. The authors would like to thank Prof. Miroslav Štěpánek for his counseling in SAXS fitting and interpretation, Prof. Jiří Vohlídal for his assistance in polymer nomenclature, Dr Jiří Brus for solid state NMR measurements, and Dr Carlos V. Melo for editing the manuscript. The group from ICMAB thanks the Spanish Ministerio de Economía y Competitividad, MINEICO (CTQ2016-75150-R9) and Generalitat de Catalunya (2017/SGR/1720) for financial support.

A recombinant lipase from Candida antarctica, LIPB, was obtained through fermentation using an alternative low-cost medium and immobilized on the distinct mesoporous silica particles. The performance and versatility of the new biocatalysts was evaluated, for the first time, on two biotechnological interest reactions: resolution of racemic myo-inositol derivative and acidolysis reaction of coconut oil. For the racemate, the covalent biocatalyst SBA-15-F-APTES-GA-LIPB exhibited the highest conversions and enantiomeric excess of up to 99 %. It was the first time that covalent biocatalyst was used for resolution of racemic myo-inositol derivative. The most active biocatalyst (SBA-15-F-APTES-GA-LIPB) was used on ten different reaction cycles maintaining its activity, highlighting the operational stability of the developed biocatalyst. Moreover, it was possible to produce food oils enriched with polyunsaturated fatty acids (PUFA), obtaining lipids containing 181.6 mg of EPA and DHA in coconut oil. The preliminary economic analyses also indicate that the enzymatic production of such nutraceuticals and pharmaceutics compounds may be economically feasible, principally exploring low-cost strategies for enzyme production.

Lipases from Candida antarctica (A and B) (CALA and CALB), Candida rugosa (CRL), Thermomyces lanuginosus (TLL) and Rhizomucor miehei (RML), as well as the chimeric phospholipase Lecitase Ultra (LU) were immobilized on octyl agarose or on heterofunctional octyl supports. RML, CRL and TLL were covalently immobilized on octyl agarose beads activated with divinyl sulfone (OCDVS), while the other lipases were immobilized on octyl-glyoxyl beads (OCGLX). The 12 biocatalysts were utilized in the production of esters using tributyrin and 20% (v/v) methanol, ethanol or isopropanol via a kinetically controlled strategy. All preparations produced the desired ester, except RML, TLL and LU for isopropyl butyrate. CALA showed the best performance in these reactions, with maximum yields over 40%. The immobilization on heterofunctional supports usually reduced the activity and even the maximum yields, although some exceptions were relevant (e.g., CALA or CALB in the production of ethyl butyrate). The effect of the nucleophile was also very different using the just physically adsorbed or the covalently immobilized preparations, some instances one preparation has as best substrate an alcohol while the best substrate was other alcohol using the other lipase preparation. Using CALB as model enzyme, we have shown the advantages of the use of the covalent preparation. The increase of the alcohol permitted the increase in maximum ester yields. However, the combined presence of dibutyrin and alcohol prevented the reuse of OC-CALB due to the enzyme desorption, while the covalent preparation could be reused by 6 cycles.

A new simple and extremely versatile zymography method based on transesterification and esterification reactions was developed in this work. The method consists in building a transesterification or esterification reaction medium according to the goal of the research. Since commercial enzymes and crude extracts commonly consist in pools of proteins, this methodology provides a means to determine which protein are responsible for the enzymatic activity desired. The protocol may be potentially used as a high-throughput screening of lipases or esterases that catalyzes the synthesis reaction for biodiesel and biolubricants production.

Different lipases immobilized on octyl agarose or heterofunctional (glyoxyl or divinylsulfone) octyl supports have been compared in the selective and specific production of 1,2-dibutyrin by hydrolysis of tributyrin. The addition of cosolvents improved the solubility of the substrate thus improving the accumulation of 1,2-dibutyrin. The form B of the lipase from Candida antarctica (CALB) was selected considering reaction both enzyme activity and reaction yields produced by this biocatalyst. Using this enzyme, the highest dibutyrin yield using 100 mM of tributyrin (over 80 %) was obtained using a medium containing 50 % of cosolvent. However, the use of higher tributyrin concentrations required increasing the concentration of cosolvent in order to increase the soluble fraction of substrate. Under these conditions, the enzyme is released to the medium if it is just physically adsorbed on the support. For this reason, the octyl-CALB biocatalyst is unsuitable for this reaction. Using octyl-glyoxyl-CALB in the presence of 75 % cosolvent to facilitate substrate solubility, a conversion yield of 70 % of dibutyrin could be achieved using 1 M of tributyrin as substrate. Using 500 mM tributyrin, the yield under these conditions increased to 80 %. The biocatalyst could be reused for several cycles without any detrimental effect on its performance.