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Noncovalent interactions associated with the enzyme, enzyme-substrate complexes, and transition states have profound influences on varying the biocatalytical activity and enantioselectivity. The recent rediscovery of halogen bonds incorporating in biocatalysis has induced our motivation on introducing halogen substituents into the substrate for improving the enzyme performance. By selecting Candida antarctica lipase B (CALB)-catalyzed kinetic hydrolysis (or butanolysis) of trans-2-(p-halophenyl)cyclopropyl 1,2,4-triazolide in methyl tert-butyl ether (MTBE) as the model reaction system, (R, R)-2-(p-halophenyl)cyclopropane-1-carboxylic acid (or ester) of high-optical purity that acts as the key building block for synthesizing the corresponding (1 R, 2S)-2-(p-halophenyl)cyclopropylamine derivatives was obtainable. The thermodynamic and kinetic analysis was then employed for elucidating effects of the halogen substituent and acyl acceptor on enhancing the noncovalent interactions of the rate-limiting tetrahedral intermediate and hence CALB activity and enantioselectivity. A putative model was moreover proposed for interpreting the possible hydrogen and halogen bonding involving the halogen substituent in turning the noncovalent interactions. [ABSTRACT FROM AUTHOR]

The effect of four kosmotropic salts additives, namely Na2CO3, Na2SO4, Na2HPO4, and NaCl, on the reactivity and selectivity of immobilized Candida antarctica lipase B (CAL-B) and a free lipase of Pseudomonas cepacia (PCL) during the hydrolysis of rac-4-(1-methoxyphenyl) ethyl and rac − 1-phenyl ethyl acetates (1a-2a) was investigated in this study. The study was carried out in two different mediums: non-aqueous conditions and in low water content media. The impact of the anionic counter-ions was examined in four solvents: nonpolar, semipolar, protic, and aprotic, with and without the aforementioned salts. In non-aqueous media, the addition of Na2CO3 significantly enhanced CAL-B hydrolysis rates, resulting in a conversion of 50% for 1a and 44% for 2a, with high enantioselectivities (E > 200). These effects were independent of the solvent hydrophobicity. In contrast, PCL required the presence of external water for effective hydrolysis of both acetates. The addition of salt additives had an adverse impact on PCL activity during the hydrolysis of 1a in hexane, toluene, and TBME solvents. However, when 2M2B was used, the inclusion of salt additives had a notable positive effect on the conversion rates, except for NaCl, which resulted in a lower conversion rate with a value of 15.5%. The presence of Na2CO3 improved the conversion rate during the hydrolysis of 2a in both hexane and TBME. Na2HPO4 further enhanced the conversion rate, reaching 44% in TBME. However, in 2M2B solvent, the addition of Na2HPO4 significantly reduced the enantioselectivity. [ABSTRACT FROM AUTHOR]

Kinetic resolution of racemates with lipases is the preferred method for producing bioactive compounds. One strategy for obtaining highly enantioselective lipases that are stable in the organic media that are often used in these reactions is to improve existing lipases by protein engineering. In this work, we engineered the lipase LipC12, which has good stability in organic media, but only moderate enantioselectivity. Molecular docking with LipC12 identified V261 as a key position influencing, first, enantioselectivity in the transesterification of (RS)-1-phenylethanol and, second, activity in the hydrolysis of p-nitrophenyl octanoate. Variants were then obtained by site-directed mutagenesis, expressed in Escherichia coli, and their performance in these reactions was evaluated. Enzymes immobilized on Immobead 150 were used in the transesterification while free enzyme was used in the hydrolysis reaction. It was not possible to increase the hydrolytic activity and enantioselectivity simultaneously: some variants had increased enantioselectivity but lower hydrolytic activity, and others had increased hydrolytic activity but lower enantioselectivity. The best result for enantioselectivity was obtained for LipC12V261Q, with an increase of the E-value (for (R)-1-phenylethanol) from 46 to 110, however, its hydrolytic activity decreased 4-fold in comparison to LipC12wt. The highest hydrolytic activity was obtained for LipC12V261F, with a value almost 6-fold higher than that of LipC12wt. This variant also had an inverted enantiopreference (i.e. for (S)-1-phenylethanol), but with a very low E-value of only 4. [ABSTRACT FROM AUTHOR]

Layered double hydroxides (LDHs) are brucite-like nanomaterials that have been used to immobilize several enzymes. However, layered hydroxide salts (LHSs), another group of brucite-like nanomaterials, have not yet been used for enzyme immobilization. In this work, we prepared two types of layered hydroxide salts, zinc hydroxide nitrate (ZHN: Zn5(OH)8(NO3)2.2H2O) and zinc hydroxide chloride (ZHC: Zn5(OH)8Cl2.H2O) and used them to immobilize Pseudomonas cepacia lipase (LipPS). The best protein loading for both ZHN and ZHC was 162.5 mg g−1 of LHS, which gave high values of triolein-hydrolyzing activity in organic medium (103 U g−1 for LipPS-ZHN and 105 U g−1 for LipPS-ZHC), immobilization efficiencies above 90% and activity retentions above 170%. In the kinetic resolution of (R,S)-1-phenylethanol, LipPS-ZHN gave better results, with 50% conversion being obtained in 2 h and an ees of 99%. With LipPS-ZHC, the conversion at 2 h was 40% and the ees, was lower, only 73%. For both immobilized materials, the eep was higher than 99% and E was higher than 200. The immobilized materials were stable after 5 cycles of reuse in successive 2-h kinetic resolutions. These results demonstrate that the layered hydroxide salts ZHN and ZHC have good potential as supports for the immobilization of lipases. [ABSTRACT FROM AUTHOR]

The present work deals with the preparation and characterization of Zn2Al layered double hydroxides (LDHs) intercalated with chloride and dodecyl sulfate ions and their use as supports for the immobilization of Pseudomonas cepacia lipase (LipPS). LipPS was immobilized on LDH by adsorption, in situ entrapment and delamination entrapment methods. The performance of free and immobilized LipPS in the transesterification of (R,S)-1-phenylethanol was compared. The immobilized derivatives prepared by adsorption gave the highest conversions (c = 50%), with an enantiomeric excess of the product (eep) greater than 99% and a high enantiomeric ratio (E > 200). For immobilization by in situ entrapment (coprecipitation), a conversion of 31.5% and E > 200 were obtained. For immobilization by delamination entrapment, a conversion of 36% and E= 155 were obtained. Although the best results were obtained by adsorption, these results show that the delamination entrapment method can also be used to prepare bioinorganic-enzyme materials based on LDH. Our work therefore increases the range of methods that can be used to immobilize lipases for use in the production of optically pure secondary alcohols. [ABSTRACT FROM AUTHOR]

Biolubricants are oleochemical esters that are non-toxic to the environment, and they are preferred because of their high-performance activities in various automobile and industrial fields. In this study, chicken tallow has been explored as an alternative to a non-renewable source, which acts as a low-cost feedstock for synthesizing biolubricants in a two-step hydro esterification process catalysed by Candida rugosa lipase (CRL). The enzymatic hydrolysis of chicken tallow is optimized at different time intervals at 40 °C, and the maximum release of 160.2 µmol min−1 free fatty acids (FFAs) is observed at 100 min. The hydrolysed FFAs are characterized by the gas chromatography-mass spectrometry (GC-MS) analysis that has depicted the fatty acid methyl esters (FAMEs) composition of the tallow oil. The recovered FFA is then enzymatically esterified with trimethylolpropane (TMP) to produce TMP esters. Optimization studies are performed to explore the effect of factors such as time, temperature, amount of the enzyme, and TMP: FFA molar ratio for the esterification process. Thus, the optimal conditions for achieving a maximal hydroxyl group conversion of 60% during TMP ester production include incubation time (120 min), temperature (35 °C), lipase concentration (3% w/w), and TMP: FFA molar ratio (1:4). Fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance (NMR) studies confirm the synthesized TMP esters. Moreover, physicochemical properties such as viscosity, cloud point (CP), and calorific value (CV) are found to be 36 mm2/s, 7159.14 cal/g, and 20 °C, respectively. [ABSTRACT FROM AUTHOR]

In this study, a Taguchi experimental design was used for optimzing the immobilization of the lipase Eversa® Transform 2.0 (EVS) onto a hybrid support consisting of chitosan (CHI) and agarose (AGA), with glutaraldehyde (GLU) used as the support activator. The biocatalyst obtained was characterized by X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetry (TGA), Energy Dispersive Spectroscopy (EDS), and Scanning Electron Microscopy (SEM). The optimized reaction conditions (60 min, 5 mM ionic strength, 1% GLU concentration, and 5 mg protein load per g of support) resulted in a highly active biocatalyst (74.39 ± 0.48 U/g) and delivered an immobilization yield of 74.20 ± 0.28%. The biocatalyst produced was observed to lose only 15.3% of its activity after 61 days of storage. The activity was also observed to increase by 96.70% ± 0.76, 27.34% ± 2.34, and 84.35% ± 1.68 in the presence of the organic solvents hexane, cyclohexane, and methanol, respectively. Additionally, the byocatalist showed more pronounced activity at temperatures above 50 °C and was still able to retain approximately 30% of it at 70 °C. These values were found to be higher at alkaline pHs, as the activity of Eversa® 2.0 Transform saw an increase of up to 140% at pH 9. The desorption tests performed did not reveal any enzymatic detachment from the support. The novel biocatalyst also showed promising ester-lubricating properties. Furthermore, the in silico study revealed a binding affinity of −5.1 kcal/mol between oleic acid and the enzyme, suggesting that the combination of the substrate and the lipase was more stable and therefore, suitable for esterification. [ABSTRACT FROM AUTHOR]

AbstractAn enzymatic approach for the synthesis of intermediate alcohol of the drug tegoprazan in 99% ee at 20 °C in hexane has been developed utilizing commercially available immobilized Thermomyces lanuginosus lipase (TL lipase) as a biocatalyst and vinyl acetate as a acyl donor. This method involves the enantioselective esterification of (RS)-5,7-difluorochroman-4-ol with vinyl acetate using TL Lipase. This enzymatic approach provides the (S)-chiral alcohol in 44% yield with 99% enantiomeric excess (ee) and the (R)-acetate in 43% yield and >99% ee. It is a sustainable approach for the synthesis of enantiopure tegoprazan intermediate, contributing to the advancement of green chemistry. The recovered TL lipase was reused in 13 subsequent runs without loss of activity. [ABSTRACT FROM AUTHOR]

Lipases and cellulases have been extensively used in the field of biotechnology for varied purposes. Mainly, the ongoing researches focus on the improvements of kinetic and enzymatic characteristics of enzymes to meet industrial needs. With the discovery of psychrophilic cellulase and lipase sources, a new era has opened for protein research by allowing the discovery of novel functions together with the description of unique cold adaptation mechanisms to harsh environments. The ability of cold-adapted lipase and cellulases to enable chemical reactions at lower temperatures provides a great opportunity to cut the cost of the finished product by lowering energy and purifying expanses. The advances in the cold-adapted lipase and cellulase enzymes are the cumulative efforts of organic chemists, biophysicists, biotechnologists, and process engineers who greatly contribute to a better understanding of cold-adaptation phenomena from different points of view. In this review, we cover the cold-adaptation aspects of cellulase and lipase enzymes from structural points of view by referring to the roles of Gly and Pro residues. Gly and Pro residues accelerate the cold-adaptation of enzymes by altering the conformational changes in the 3 D structure of proteins The list of microorganisms as a source of cold-adapted cellulases and lipases is given by referring to biotechnological applications. After introducing the thermodynamic background of Gly and Pro residues in the phenomena of cold-adaptation, specific examples are given to emphasise how introducing Gly and Pro into 3 D structure of protein molecules adds value in terms of biotechnological application by contributing to cold-adaptation. [ABSTRACT FROM AUTHOR]

Herein, we report the preparation of enantiomerically pure forms of 2,3-dihydrobenzofuran-3-ol (1), chroman-4-ol (2), thiochroman-4-ol (3), 1-(furan-2-yl) ethanol (5) and 1-(thiophen-2-yl) ethanol (6), through a kinetic resolution catalysed by Candida antarctica lipase B/Na2CO3 hydrolysis sequence in organic media. The (R)-furnished alcohols and the (S)-remained acetates are recovered enantiopures (ee >99%, E ≫ 200, Conv = 50%). Those ideal enzymatic kinetic resolution (EKRs) are well incorporated to the Mitsunobu inversion protocol in a one pot procedure to give (S)-heterocyclic acetates (1a–3a) in good to high enantiomeric excess (88%–92% ee). Whilst, the (S)-heteroaromatic acetates (5a and 6a) are given with moderate enantiomeric excess (51%–62% ee). All the (S)-acetates are given in good isolated chemical yields (>80%) allowing to overcome the maximum of 50% yield which could be usually reached in a regular kinetic resolution processes. [ABSTRACT FROM AUTHOR]

Enantiomerically pure secondary alcohols are useful in the synthesis of several natural products and as active pharmaceutical intermediates (API). Due to the high demand for these chiral compounds, much progress has been made in the areas of asymmetric synthesis and catalysis. In this context, biocatalysis together with continuous flow technology can be a valuable tool for more versatile and sustainable methods, with lower cost, greater stereoselectivity and less environmental impact. This work aims to obtain an enantiomerically pure alcohol of industrial interest, (4 Fluorophenyl) (furan-2-yl) methanol (3), by performing a kinetic resolution using immobilized Candida antarctica lipase B (Novozyme 435, N435) under continuous-flow conditions. Initial study was carried out to optimize batch reaction conditions. The best results were obtained using isooctane as solvent, 37.7 mg of N435 and three equivalents of isopropenyl acetate as acyl donor at 60 °C for 24 h. Under these conditions, a conversion of 49% and 91 of enantiomeric ratio was obtained. Optimized batch conditions were translated to the continuous flow reactor leading to the desired product in 30 min of residence time, 47% conversion and an enantiomeric ratio of 61. [ABSTRACT FROM AUTHOR]

Sebacic esters have excellent lubricity, thermal stability, and biodegradability and therefore are widely used as aerospace lubricants, metal working oil, or engine oil. They are mainly produced by chemical synthesis which, however, may cause environmental pollution, its enzymatic synthesis represents a more environmentally friendly alternative. A few reports have described the synthesis of sebacic esters employing immobilised lipases, but these biocatalytic reactions were exclusively carried out in solvent-free systems and thus, could have been limited by slow reaction rates and high reaction temperatures due to poor enzyme dispersion, low substrate solubility, and high viscosity of the reaction mixture. The current study investigated the biosynthesis of dioctyl sebacate in toluene by Novozym 435, a commercial immobilised lipase. The reaction parameters were investigated using the single factor approach and an orthogonal array design. The optimal conditions obtained were as follows: 10 mL toluene, sebacic acid,1 mmol (202.25 mg); molar ratio of sebacic acid to 1-octanol, 1:3; Novozym 435, 0.03 g; 4 Å molecular sieves, 1.5 g; reaction temperature, 40 °C; reaction time, 30 h. A dioctyl sebacate conversion rate of 93% was achieved under these optimal conditions. In particular, the addition of molecular sieves to the reaction mixture markedly improved the product yield. The reaction temperature was low enough to make the operation easy and energy-efficient and therefore, well suited for large-scale production. [ABSTRACT FROM AUTHOR]

Metal-organic frameworks (MOFs) are versatile materials because they have a large internal surface area and tuneable pores, making them suitable for enzyme immobilization. In this study, we prepared a typical microporous Fe-BDC MOF through a thermal treatment to produce additional meso and macropores interconnected to each other, capable of immobilizing the Burkholderia lata LBBIO-BL02 (BLL) lipase by entrapment and physical adsorption. The immobilization efficiency (E) was 90%, and the activity retention (R) was 400% (pNPP hydrolysis). The immobilized lipase (BLL@BDC) also showed excellent activity in the hydrolysis of vegetable oils in aqueous medium, achieving up to 3,200 U g−1 for olive oil, as well as high stability in organic solvents, especially for polar ones, such as iso-propanol (101.5 ± 2.6%), ethanol (103.0 ± 6.0%) and acetone (107.7 ± 8.3%). The results indicate that the multiporous Fe-BDC MOF is a promising support for lipase immobilization and further application in biocatalysis performed in organic media. [ABSTRACT FROM AUTHOR]

Glyceryl monostearate is extensively used as an emulsifier in many industries. Mono acylation of glycerol was carried out by utilizing immobilized Candida antarctica lipase B (Cal B) as a biocatalyst and vinyl stearate as an acyl donor. Different reaction parameters, such as selection of lipases from various sources (like Candida antarctica, Candida rugosa, and Mucor meihei) and their quantity, shaking speed, temperature, substrate concentration, and reusability were studied in detail to achieve excellent conversion. Overall, 98% conversion of glycerol was obtained at a mole ratio of 1:1 of glycerol to vinyl stearate, using 12 mg of immobilized Cal B at 45 °C for 3 h. The mechanism of the given reaction was anticipated based on the results of the Lineweaver-Burk plots. It was found that the reaction followed the Ping-Pong Bi Bi mechanism with inhibition of glycerol. As it was a kinetically controlled synthesis, different kinetic constants were estimated by non-linear regression analysis. The activation energy for Cal B was found to be 10.3 kcal/mol. Further, biocatalyst can be reused up to four catalytic cycles with an average four percent loss of activity. A molecular docking study was done to find out the confirmation of substrates and their binding positions in an enzyme. It was noticed that the reaction proceeds through acyl-enzyme complex formation followed by the transfer of that acyl group to another substrate. [ABSTRACT FROM AUTHOR]

Chemoenzymatic oxidations involving the CAL-B/H2O2 system was developed in a sugar derived Natural Deep Eutectic Solvent (NaDES) composed by a mixture of glucose, fructose and sucrose. Good to excellent conversions of substrates like cyclooctene, limonene, oleic acid and stilbene to their corresponding epoxides, cyclohexanone to its corresponding lactone and 2-phenylacetophenone to its corresponding ester, demonstrate the viability of the sugar NaDES as a reaction medium for epoxidation and Baeyer-Villiger oxidation. [ABSTRACT FROM AUTHOR]

Medicinally active labdane diterpene lactones, andrographolide (1), neoandrographolide (2), dehydroandrographolide (3) and deoxyandrographolide (4) are isolated from medicinal plant, Andrographis paniculata. These diterpenes have many beneficial health effects such as anti-viral, anti-inflammatory, anti-cancer and anti-diabetes. However, to improve bioactivity and water solubility, analogues of labdene diterpenes have been synthesised using synthetic and biotransformation routes. Biocatalytic modification has extensive potential for the preparation of a wide variety of complex, structurally diverse and more potent organic compounds at milder and eco-friendly reaction conditions. Therefore, it is necessary to systematically accumulate the biotransformation reports for isolated lactones. This article reviews the regioselective transformations of bioactive labdane diterpenes isolated from Andrographis paniculata. The whole-cell and pure enzymatic transformations of andrographolide (1) and its derivatives are presented concisely. [ABSTRACT FROM AUTHOR]

Recently, several scientific groups investigated the suitable enzymatic route for synthesis of structured phenolic lipids for cosmetic and nutritive purposes. The feruloylated palm stearin (FPS) was obtained from enzymatic transesterification reactions of palm stearin with ferulic acid in solvent-free media with 1 mmol of glycerol or without it, using non-specific lipases, NovoCor ADL (Candida antarctica lipase A); Novozyme435® (Candida antarctica lipase B), and 1,3-specific lipase Lipozyme TL 100L from Thermomyces lanuginosus. The individual lipases and their combinational systems were examined. ESI+-MS/MS confirmed the formation of 19 feruloylated compounds. FPSs were produced by all lipases and their combinations. The positive synergetic effect occurred for 1,3-specific lipase and unspecified lipases with mass ratio 8:2, respectively. The antioxidant activities of all FPSs were examined. FPS produced by NovoCor ADL lipase in presence of free glycerol showed an antioxidant activity 10.3-times higher. In conclusion, the two low-cost lipases NovoCor ADL and Lipozyme TL 100L and its combi-lipase system could be considered suitable low-cost alternative to the expensive wide use N435 lipase in the solvent free production process of FPS for food and cosmetic applications. [ABSTRACT FROM AUTHOR]

Lipase B from Candida antarctica (CAL-B) was immobilised on epoxy-activated polymer support functionalised with polyethyleneimine (PEI) via preparation of enzyme aggregates by precipitation with polyethylene glycol (PEG 600). This involves adsorption of the aggregates on the functionalised polymer followed by cross-linking with glutaraldehyde. The apparent activity of the immobilised enzyme was found to be 750 TBU/g at optimum pH 7.5 and temperature 30 °C. The immobilisation yield was 24%. The immobilised enzyme was used for desymmetrization of cis-3,5-diacetoxy-1-cyclopentene in aqueous buffer (70% yield, >99% e.e.) and methyl tert-butyl ether (95% yield and > 99% e.e) to obtain (1 R,4S)-4-hydroxycyclopent-2-en-1-yl acetate. The immobilised enzyme could be recycled (5 times) in both aqueous buffer and organic solvents. Further, the expensive polymer support was also recycled (5 times) after the immobilised enzyme lost its activity by washing with 1 N HCl. [ABSTRACT FROM AUTHOR]

The characteristics of a purified lipase from Rhizopus oryzae ZAC3 (RoL-ZAC3) were investigated. RoL-ZAC3, a 15.8 kDa protein, which was optimally active at pH 8 and 55 °C had a half-life of 126 min at 60 °C. The kinetic parameters using p-nitrophenylbutyrate as substrate were 0.19 ± 0.02 mM, 126 ± 5.6 U/ml and 122 s−1 for Km, Vmax and kcat respectively. RoL-ZAC3 showed stability in methanol and isopropanol with Na+ enhancing the activity. p-nitrophenyloleate and castor oil were the best preferred substrates among the p-nitrophenyl esters and vegetable oils tested respectively. About 43% residual activity was observed after incubation for 30 min at 75 °C. Circular dichroism thermal scan showed that the lipase displayed intense negative ellipticities even at high temperature. Perturbation of the tertiary structure with increasing temperature caused the exposure of hydrophobic side chains to the aqueous environment as revealed by tryptophan fluorescence, with a t−Tm of 50 °C. Differential scanning calorimetry analysis showed melting temperature and calorimetric enthalpy of 55.5 °C and 444 kJ/mol respectively. Dynamic light scattering analysis indicated that the lipase was prone to aggregation upon unfolding at high temperature. It can be concluded that RoL-ZAC3 possesses promising potential for numerous biotechnological applications. [ABSTRACT FROM AUTHOR]

Fe3O4 magnetic nanoparticles were prepared by chemical co-precipitation and modified with 3-aminopropyltriethoxysilane (APTES) and CS2 to form Fe3O4-APTES-CS2 magnetic carriers. Magnetically immobilized enzymes are attractive owing to their potential applications in many biological fields. When an alternating magnetic field is applied, the magnetic nanoparticles experience a force in the direction of the field, and the magnetic moments of the magnetic nanoparticles are apt to align in the direction of the field. Thus, magnetic nanoparticles behave like microscopic stirrers. The optimal reaction conditions of the acylation reaction using Fe3O4-APTES-CS2-lipase were 200 mmol/L 1-methyl-3-amphetamine, 100 mg immobilized lipase, 200 μL ethyl acetate, 12.6 × g rotating speed, 40 °C, and 5 days in the shaker. The conversion and enantiomeric excess of R-2a reached a maximum of 36.8% and 98.5%, respectively. 1-Methyl-3-phenylpropylamine was resolved using Fe3O4-APTES-CS2-lipase in an alternating magnetic field. The optimal magnetic field frequency and magnetic field intensity were determined to be 500 Hz and 12 Gs, respectively. The conversion and enantiomeric excess of R-2a reached a maximum of 40.3% and 98.5%, respectively. The results revealed that conversion under a magnetic field was significantly higher than that without a magnetic field. Highly optically active (R)-N-acetyl-1-methyl-3-amphetamine can be obtained via an efficient resolution of racemic 1-methyl-3-phenylpropylamine with immobilized lipase magnetic nanoparticles as a catalyst and ethyl acetate as an acyl donor in organic solvents in an alternating magnetic field. [ABSTRACT FROM AUTHOR]

The study demonstrates the lab-scale production of biodiesel from soybean acid oil (SAO) in a 5 L lab-scale bioreactor using immobilized whole cells as a catalyst. The maximum lipase activity 2.89 ± 0.6 U/mL of immobilized whole cells was achieved at optimum conditions viz; pH (7.1), temperature (31 °C), peptone (1.2% w/v) and immobilized pieces (10) in 100 mL growth medium. The esterification approach was fit to Michaelis–Menten model, based on concentration-time profile with 75 gL−1 Michaelis constant (KM) and 78 gL−1h−1 maximum velocities (Vmax) resulting in a maximum yield of up to 95.4% over 24 h of reaction. A 5% decrease in the catalytic potential of immobilized whole cells from 1st to 5th recycle of immobilized support. The fuel properties of resulted biodiesel were noted to meet the specifications of major international biodiesel standards such as ASTM D6751 to a significant extent. The study reveals the potential use of immobilized whole-cell catalyst and acid oil as an alternative, low-cost bearing and quality feedstock for the generation of biodiesel for a diverse variety of industrial/commercial use. [ABSTRACT FROM AUTHOR]

Two biocatalysts with high lipolytic activity were obtained – freeze-dried supernatant and freeze-dried biomass of Yarrowia lipolytica KKP 379. The biocatalysts were compared with Candida antarctica lipase B in the synthesis of ethyl hydrocinnamate – a flavour compound with a floral-honey aroma. Ester synthesis was completed after 2 h when 3 grams of freeze-dried biomass was used. Using smaller amounts of biomass (1.2 and 0.6 g) also allowed to achieve high ester conversion and results were comparable with those obtained for CALB used as a catalyst. Freeze-dried supernatant showed a weaker lipase activity (22.17 U/g) compared to freeze-dried biomass (46.13 U/g), which resulted in a lower conversion of 3-phenylpropionic acid to its ethyl ester, and after 36 h a conversion peaked at around 70%, then began to decrease and finally the conversion reached 50%. Moreover, antimicrobial and antioxidant properties of the synthesised ester were evaluated. Ethyl hydrocinnamate showed no antibacterial activity and weak antioxidant activity towards DPPH radical. In contrast to bacteria, the obtained compound moderately inhibited the growth of tested yeast species, where inhibition zones ranged from 10 to 16 mm. [ABSTRACT FROM AUTHOR]

Solid-State Fermentation (SSF) defined as a system in which the growth of microorganisms occurs on a moist solid substrate, shows tremendous potential in applications of microbial enzyme production as a low-cost, low-energy option. In this work, the production of lipase from Penicillium aurantiogriseum by using SSF was investigated. For this purpose, the sunflower pulp, an agro-industrial by-product, was utilized as a solid substrate in the SSF method. The optimization and characterization studies for lipase extract were performed by using the p-nitrophenyl palmitate (pNPP) (C16) as substrate. The lipase activity was found as 29.6 ± 0.11 U/mg protein at optimum conditions (50 mM of Tris-HCl buffer, pH 7.0, 40 °C). In thermal stability assay, the catalytic activities of lipase which are kept at 40, 50 and, 60 °C in a water bath for 1 h, were calculated as 100%, 60.48 ± 1.3%, 32.67 ± 0.2%, respectively. Also, the enzyme lost rapidly its activity within 15 min at 70 °C. The kinetic data, Km and Vmax, were determined as 0.17 mM and 2.9 µM/min, respectively. Also, 10 mM of Ba+2 ion had a slight activating effect (15%) showed on lipase. Lipase kept about 50% of its activity at 4 °C at the end of the 30 days. The hydrolytic activities of lipase for commercial olive oil and sunflower oil were found as 3.52 ± 0.1 U/mg and 3.90 ± 0.12 U/mg, respectively. The SSF method had obvious potential for the sustainable and cost-effective production of lipase from P. aurantiogriseum. Also, these results confirmed that the sunflower pulp could be used as an alternative substrate to produce enzymes in SSF conditions. [ABSTRACT FROM AUTHOR]

Biodiesel, fatty acid methyl ester (FAME), is an environmentally friendly alternative fuel produced by transesterification of renewable lipid feedstocks for diesel engines. Methanolysis of waste frying oil (WFO) was catalyzed by Rhizopus stolonifer 1aNRC11 mutant F whole-cell lipase that produced under solid state fermentation (SSF) using wheat bran (WB) and black cumin meal as the main substrate. Cultivation SSF parameters of R. stolonifer mutant were optimized using Plackett-Burman (PB) experimental design and Face-centred Central Composite Design (FCCD), taking FAME yield as a response for whole-cell lipase production ability. PB results showed that cultivation temperature, medium pH and moisture content were the most significant parameters, which were selected for optimization by FCCD. The optimal determined conditions were as follows: WB (10 g), black cumin meal (6%, w/w of WB), fodder yeast (1%, w/w of WB), moisture content (1.5 g water/g solid), inoculum size (3 discs), initial pH (8), and cultivation time (4 day) at 28 °C. With R. stolonifer mutant cultivated in the optimized conditions, the FAME yield reached 82.6% that was close to the predicted yield of 85.7%. The analysis of variance showed that the model was statistically significant (p value of.0001) and could explain 89% of the variability in the response. These results indicated that the optimization conditions increased biodiesel production by about 43% compared to the basal medium (57.60%). The optimization of SSF conditions using low cost components makes the whole-cell lipase production process from the R. stolonifer 1aNRC11 mutant F economically effective for application in biodiesel production from WFO. [ABSTRACT FROM AUTHOR]

Yarrowia lipolytica is a yeast that presents high biotechnological potential due to its ability to produce many metabolites, among them lipases and esterases, which are important industrial biocatalysts. Since Brazil is an agroindustrial country, it generates an enormous diversity of residues or byproducts that can be used as a platform for biomolecules production. This work aims to evaluate lipase and esterase production by Y. lipolytica via solid-state fermentation using soybean bran and soybean bran supplemented with watermelon peels in different contents, and subsequent use of the enzyme extracts for poly(ethylene terephthalate) (PET) hydrolysis. Supplementation of watermelon peels in the lowest content led to an improvement of lipase activity in almost 31%, reaching 75.22 U g−1. Esterase productivity was 1.5-fold higher when 20 wt% of watermelon peels were added to the media culture. Timecourse evaluation of enzymes production showed a maximum lipase activity in 14 h and similar esterase activity in 14 and 20 h of fermentation. Proteases production were also intensified in supplemented samples. Enzymes produced with 5 wt% watermelon peels supplementation led to higher terephthalic acid concentration (up to 42.02 µmol L−1) during PET depolymerization. Results suggest a great potential of enzyme production in low cost fermentative media to act as biocatalysts in PET hydrolysis reactions. [ABSTRACT FROM AUTHOR]

Lipases are one of the most important catalysts for several industries such as detergent, dairy, and textile industry due to their bio-catalytic ability in aqueous and non-aqueous media. Stability to extreme conditions is an important property since it makes enzymes suitable to several industrial processes. In this study, lipase producing soil bacteria were screened and identified with 16S rDNA sequencing. A new hyper-thermophilic lipase named as Bacillus subtilis LP2 isolate was partially purified by ammonium sulphate precipitation with 17.8-fold purification and 583 U/mg specific activity. Maximum activity was exhibited at pH 7 and 80 °C with the substrate tween 80 KM and Vmax values were calculated as 18.3 mM and 680 U/mg with a catalytic efficiency (kcat/KM) of 307 s−1M−1. These results indicate that lipase from Bacillus subtilis LP2 can be a valuable candidate for industrial applications such as organic synthesis and fats and oils industry due to their efficient catalysis in higher temperatures. [ABSTRACT FROM AUTHOR]

The present study aims to exploit microbial potential from colder region to produce lipase enzyme stable at low temperatures. A newly isolated bacterium GBPI_508 from Himalayan environment, was investigated for the production of cold-active lipase emphasizing on its aggregation properties. Plate based assays followed by quantitative production of enzyme was estimated under different culture conditions. Further characterization of partially purified enzyme was done for molecular weight determination and activity and stability under varying conditions of pH, temperature, and in presence of organic solvents, inhibitors, and metal ions. The psychrotolerant bacterium was identified as Pseudomonas palleroniana following 16S rRNA gene sequencing. Maximum lipase production by GBPI_508 was recorded in 7 days at 25 °C utilizing yeast extract as nitrogen source and olive oil as substrate in the lipase production medium. Triton X-100 (1%) in the medium as emulsifier significantly enhanced the lipase production. Lipase produced by bacterium showed aggregation which was confirmed by dynamic light scattering and native PAGE. SDS-PAGE followed by zymogram analysis of partially purified enzyme showed two active bands of ∼50 kDa and ∼54 kDa. Optimum activity of partially purified enzymatic preparation was recorded at 40 °C while the activity remained nearly consistent from pH 7.0 to 12.0, whereas, maximum stability was recorded at pH values 7.0 and 11.0 at 25 °C. Interestingly, lipase in the partially purified fraction retained 60% enzyme activity at 10 °C. Medium chain pNP ester (C10) was the most preferred substrate for the lipase of GBPI_508. The lipase possessed >50% residual activity when incubated with different organic solvents (25% v/v) except toluene and dichloromethane which inhibited the activity below 50%. Partially purified enzyme was also stable in the presence of metal ions and inhibitors. The study suggests applicability of GBPI_508 lipase in low temperature conditions such as cold-active detergent formulations and cold bioremediation. [ABSTRACT FROM AUTHOR]

Immobilization of enzymes from different sources on various supports in designed systems increases enzymes' stability by protecting the active site of it from undesired effect of reaction environment. Also, immobilization decreases the cost of separation and facilities the reuse of the enzymes. Therefore, the design of new immobilization enzyme preparations has been an inevitable area of modern biotechnology. Herein, Rhizomucor miehei lipase (RML) was immobilized on montmorillonite K-10 (MMT-RML) by adsorption and in polyvinyl alcohol (PVA-RML) by entrapment to obtain a more stable and active lipase preparation. The free and immobilized lipase preparations were characterized for p-nitrophenyl palmitate hydrolysis. The apparent Michaelis–Menten (Kmapp) constant was almost the same for the free RML and PVA-RML, whereas the corresponding value was 17.7-fold lower for MMT-RML. PVA-RML and MMT-RML have shown a 1.1 and 23.8 folds higher catalytic efficiency, respectively, than that of the free RML. The half-lives of PVA-RML and MMT-RML were found to be 7.4 and 3.4 times longer than the free RML at 35 °C, respectively. PVA-RML and MMT-RML maintained 65% and 87% of their initial activities after four reuses. These results showed that the catalytic performance of RML has improved significantly by immobilization. [ABSTRACT FROM AUTHOR]

This study evaluated coupled effects of molar ratio of substrates and enzyme loading in a solvent-free system using a simple mathematical approach to obtain high conversions on octyl caprylate synthesis with Novozym 435. When molar ratios of caprylic acid to n-octanol (1:1 and 1:3) were evaluated with enzyme loadings of 1% to 4% (wt/wt acid), an interdependence between the masses of reagents and the enzymes was observed, that was expressed as a mathematical relation. The study of this relation, named as SER, indicated a specific range of reaction conditions that resulted in conversions above 90%. The most suitable condition corresponded to an acid:alcohol molar ratio of 1:1.3 and an enzyme loading of 1.5%, resulting in 94.5% of conversion at 65 °C in 3 hours of reaction. A different reaction system (bottle reactor) was used to evaluate the influence of reagents mixture and heat distribution. The use of a bottle reactor allowed yield improvement that reached 99.3%. At this condition, Novozym 435 was reused, without washing steps, in three subsequent batches keeping high conversion. A possible balance between the shift of chemical equilibrium by stoichiometric excess of reagents and enzymatic inhibition effects by substrates can be expressed mathematically in a convenient way, helping to predict the behaviour of synthesis in different conditions. The mathematical relation proposed, SER, allowed the achievement of 99% of conversion on enzymatic synthesis of octyl caprylate. [ABSTRACT FROM AUTHOR]

Many studies describe the advantages of using hydrophobic particles on lipase immobilisation. However, many of these works neglect the effect of other variables of the supports, such as specific area and porosity, on the biocatalyst performance, and do not evaluate the influence of the hydrophobicity level of the particles on the biocatalysts' activity as a single variable. Thus, the focus of the present work was to evaluate the effect of the hydrophobicity degree of polymeric particles on the biocatalysts' activities, mitigating the influence of other variables. The study was divided into two steps. Firstly, distinct particles, exhibiting different composition and hydrophobicity levels, were used for the immobilization of a commercial lipase B from Candida antarctica (CAL-B). Then, distinct core-shell polymeric particles presenting different functional compounds on the surface were produced, using as comonomers styrene, divinylbenzene, 1-octene, vinylbenzoate and cardanol. Such particles were subsequently used for CAL-B immobilisation and the performance of the biocatalysts was evaluated on hydrolysis (using p-nitrophenyl laurate, as substrate) and esterification (using ethanol and oleic acid, as substrate) reactions. Based on the screening step, it was observed that for non-porous particles the correlation coefficients between the hydrophobicity level of the supports and the biocatalysts performance, for both hydrolysis and esterification reactions, were very low (0.32 and 0.45, respectively). It highlights that there was no significant correlation between these variables and that, probably, the chemical composition of the polymeric chains affects more significantly the biocatalyst performance. Then, analysing the subsequent stage, it was observed that small changes in the surface composition of the core-shell particles result in significant changes on the textural properties of the supports (specific area ranging from 1.2 m2.g−1 to 18.3 m2.g−1; and contact angles ranging from 71° (hydrophilic particles) to 92° (hydrophobic supports) when polymer films were put in contact with water). Such particles were also employed on CAL-B immobilization and it was noticed that higher correlation coefficients were achieved for hydrolysis (ρ = 0.53) and esterification (ρ = 0.74) reactions. Therefore, it is shown that the hydrophobicity degree of such supports starts to affect more effectively the biocatalysts performance when other textural features of the supports become more significant, such as specific area and porosity. [ABSTRACT FROM AUTHOR]

A cosmetic ester, cetyl oleate was synthesized using microwave irradiated system. The esterification reaction was carried using Candida antarctica lipase B in a solvent-free media. The influence of various reaction parameters was studied, and the efficiency of Fermase CALBTM10000 was compared with other enzymes. Equilibrium conversion of 97.5% was obtained within 20 min at 60 °C temperature, 1:2 oleic acid to cetyl alcohol molar ratio and 4% w/w dose of lipase. A comparative study showed that microwave irradiation is a much more efficient method than ultrasound irradiation and conventional heating. Fermase CALBTM10000 was reusable over 6 enzymatic cycles as its stability improved under microwave system. Physicochemical parameters of cetyl oleate were tested in order to analyze its suitability for further cosmetic use. [ABSTRACT FROM AUTHOR]

Chiral 1-phenylethanol and its ester derivative are important chiral chemicals in diverse industries and the preparation of those optically pure enantiomers is of great importance. One bacterium, Bacillus sp. DL-2, whose extracellular proteases could efficiently asymmetrically hydrolyse (±)-1-phenylethyl acetate, was isolated from the deep sea of the Western Pacific. After the immobilization of extracellular proteases and the optimization of enzymatic reactions, (R)-1-phenylethanol was prepared with the enantiomeric excess (e.e.) being 97% and the yield being 41%, respectively. The optimal resolution reaction condition for the preparation of (R)-1-phenylethanol using immobilized extracellular proteases was found to be 5-mM (±)-1-phenylethyl acetate, 360 mg/mL immobilized extracellular proteases, pH 6.5, and 20 °C for 2 h. (S)-1-phenylethyl acetate was generated through enzymatic kinetic resolution with the e.e. being as high as 99% and the yield being 71%, respectively. The optimal resolution reaction condition for the preparation of (S)-1-phenylethyl acetate was found to be 2.5-mM (±)-1-phenylethyl acetate, 440 mg/mL immobilized extracellular proteases, pH 7.5, and 35 °C for 10 h. Our report is the first report about the kinetic resolution of (±)-1-phenylethyl acetate using proteases and the enantio-preference of the proteases was found to be the same as those of most other esterases/lipases. Also notably, the optical purity of (S)-1-phenylethyl acetate generated through kinetic resolution using the proteases of Bacillus sp. DL-2 was the highest report so far. Proteases from deep-sea Bacillus sp. DL-2 are new contributions to the biocatalyst library for the preparation of valuable chiral alcohols and chiral esters through kinetic resolution. [ABSTRACT FROM AUTHOR]

In this study, Rhizomucor miehei lipase (RML) was immobilized on the hexagonally-ordered nanoporous aluminium oxide membranes (RML-Al2O3-NP) by adsorption and as protein-coated microcrystals (RML-PCMCs) by simultaneously precipitating RML on micron-sized potassium sulfate crystals (K2SO4) in pre-chilled acetone. The hydrolytic activities of immobilized lipase preparations were investigated in terms of p-nitrophenyl palmitate hydrolysis and their esterification activities were examined for the synthesis of some aroma esters such as butyl acetate, isoamyl acetate, hexyl acetate, heptyl acetate, and geranyl acetate. The immobilization yields were 33.8 and 25.1%, respectively for RML immobilized on Al2O3-NP membranes and potassium sulfate crystals. The catalytic efficiency ratios of RML-Al2O3-NP and RML-PCMCs were 2.3- and 3.9-fold higher than that of the free lipase, respectively in terms of hydrolytic activity. The free lipase was stabilized as 4.1- and 10.5-fold, respectively at 40 and 50 °C when immobilized on Al2O3-NP. The corresponding stabilization factors were 4.6- and 12.8-fold higher for RML-PCMCs. RML-Al2O3-NP and RML-PCMCs maintained 84 and 86% of their initial hydrolytic activities, respectively after 10 reuses. Of the synthesized aroma esters, the highest yield was obtained for the geranyl acetate. After 4 h reaction time, no geraniol was detected in the preparative-scale (196 g/L) synthesis of geranyl acetate for both the immobilized lipases when the initial geraniol amount, vinyl acetate amount, RML-PCMCs amount, and reaction temperature values were 1 mmol, 3 mmol, 100 mg (or 300 mg RML-Al2O3-NP), and 50 °C, respectively. These results show that the immobilization of R. miehei lipase by adsorption on nanoporous aluminium oxide and as protein-coated microcrystals leads to the obtention of highly stable, catalytically more active, and reusable lipase preparations. [ABSTRACT FROM AUTHOR]

The extracellular lipase produced by Acinetobacter sp. K5b4 was purified to homogeneity using ultrafiltration (cutoff 30 KDa) followed by gel filtration chromatography on Sephadex G-50. The enzyme was purified to homogeneity with an apparent molecular mass of 133 KDa by SDS-PAGE. This purification resulted on 10.24 fold with 18.3% recovery. The Km and Vmax of purified enzyme when using pNPL hydrolysis were 4.0 mM and 73.53 nmol/ml/min, respectively. The pure enzyme was greatly stimulated in the presence of 20, 40 and 60% (v/v) methanol, DMSO and acetone whereas, ethanol, acetonitrile and propanol decreased the enzyme activity. Maximum enzyme activity was achieved at pH 7.0 and incubation temperature of 27 °C. The enzyme was stable within a pH range of 6.5 to 7 at 27 °C for 1 h. The enzyme activity was enhanced up to 36% by KCl, BaCl2, MgCl2 and CaCl2 while obviously inhibited (10-20%) by CoCl2, ZnCl2, MnCl2 and CuCl2. No inhibitory effects were observed with 1.0 and 5.0 mM of 2-mercaptoethanol and EDTA. Similarly, SDS at 1.0 mM does not affect the enzyme activity while high reduction (80%) was observed at 5.0 mM SDS concentration. The enzyme was active against p-nitrophenyl esters of C8, C12 and C16 with highest preference to the medium carbon chain p-nitrophenyl caprylate (C8). The fact that the enzyme displays distinct stability in the presence of methanol, DMSO and acetone suggests that this lipase is suitable as biocatalyst in organic synthesis where such hydrophilic organic solvents are used as a reaction media. [ABSTRACT FROM AUTHOR]

The combination of Deep-eutectic-solvents (DES) with water as "co-solvent" enables a low-viscous reaction medium that keeps its "non-conventional" nature and thus enables synthetic lyophilization reactions (e.g. esterification) catalyzed by hydrolases. Substrates with different polarity may be employed. This paper shows how the enzyme immobilization with cross-linking aggregates (CLEA) leads to highly stable and active immobilized catalysts in different DES. As a remarkable case, when choline chloride-glycerol DES is used, CLEA derivatives of Candida antarctica lipase B (CLEA-CALB) are stable for at least 14 days without any loss of activity. The immobilized biocatalysts are applied in non-viscous DES-water blends (8% v/v) to catalyze the esterification of benzoic acid and glycerol to furnish glyceryl monobenzoate (α-MBG) in productivities of ∼35 g α-MBG L−1d−1. Compared to other commercial immobilized CALB, the CLEA-CALB derivatives rendered more product (higher conversions by 30%). Moreover, CLEA derivatives were successfully reused for six times without any loss of activity. Given the ease of immobilization (CLEA), their excellent performance in DES and the low viscosity of the DES-water blends, the reported approach may be useful for many synthetic procedures and even for continuous processes with largely optimized outcomes. [ABSTRACT FROM AUTHOR]

Free Candida antarctica lipase B (Lipozyme, CALB L®) was used to produce fatty acid ethyl esters (FAEE) from refined soybean oil in solvent-free media using the conventional (CHS) and microwave (MHS) heating systems. Statistical analyses (95% confidence level) for both reaction products, FAEE and free fatty acids (FFA), were performed. An increase in ethanol:oil molar ratio decreased the catalytic performance of CALB L (p < .05). The best conditions using the microwave radiation were a molar ratio of ethanol:oil of 3:1, a water content of 20.3 wt.% and an enzyme loading of 3 wt.% and this resulted in a total ester content of 64.7% in 15 min, while the same condition using the conventional heating gave only 21.4%. Moreover, the reaction equilibrium was reached 16 times faster with microwave than with conventional heating. High ethanol:oil molar ratios had a negative effect on FAEE synthesis with both CHS and MHS, probably due to the partial inactivation of the enzymes. MHS improved the reaction performance of CALB L, but other process parameters will have to be optimized to enhance the resulting FAEE yields. The recovery and reuse of CALB L using a MHS was demonstrated. Hence, the use of microwave radiation under the conditions applied in this study was not detrimental to the catalytic performance of CALB L for at least one reuse. [ABSTRACT FROM AUTHOR]

Fungal lipases occupy a place of prominence among biocatalysts owing to their novel, multifold applications and resistance to high temperature and other operational conditions. In the present study, Aspergillus fumigatus isolated from oil-contaminated soil produced good amount of lipase activity with galactose (1%) as carbon source and peptone (0.1%) as nitrogen source after 72 h of incubation in the production medium at 45 °C and pH 10.0. The isolated enzyme was found to give its optimum reaction temperature at 40 °C and pH 9.0 with the substrate used as p-nitrophenyl benzoate. The activity of lipase was inhibited by the presence of metal ions. A 6.68-fold increase for lipase production was obtained by one variable at a time. Based on the findings of present study, lipase of A. fumigatus is a potential lipase and a candidate for industrial applications such as bioremediation, detergent, leather and pharmaceutical industries. [ABSTRACT FROM AUTHOR]

The palm oil industry generates large amounts of lignocellulosic co-products. Palm kernel cake (PKC) and palm pressed fibre (PPF) have nutritional limitations as ingredients in animal feed, and are therefore little used. Solid-state fermentation (SSF) is one alternative treatment to improve the nutritional value of these co-products and to increase their possible use, through the production of enzymes such as lipases and xylanases. These enzymes can reduce the contents of undesirable compounds, such as lipids, and degrade some components of the fibres to improve the digestibility of these co-products. The fungi Aspergillus niger, Aspergillus oryzae and Aspergillus awamori were able to grow in PKC/PPF (40/60 w/w) culture medium by SSF, and to produce xylanase and lipase. A. niger showed the highest lipase activity (20.7 U g−1) at 72 h. A. awamori higher xylanase activity than the other fungi at all culture periods, reaching a maximum activity of 134.2 U g−1 at 72 h. The unfermented co-products contained 7.49% lipids and 7.38% non-fibrous carbohydrates (NFC). Lipase produced by these fungi during SSF reduced the lipid content by 36%, 40% and 45% for A. oryzae, A. awamori and A. niger, respectively. The production of xylanases by SSF probably increased the NFC contents by up to 64%. Fermented solids with A. oryzae and A. awamori had the highest levels of NFC, 20.3% and 13.94%, respectively, which improved the nutritional value of these co-products. [ABSTRACT FROM AUTHOR]

In this contribution, a new chromatographic method was applied to study the hydrolysis of non-polar lipids, i.e. triacylglicerols (TAG), diacylglycerols (DAG) and monoacylglycerols (MAG), when crude sunflower lecithin is treated with Lecitase® Ultra, an enzymatic preparation with phospholipase A1 (PLA1) activity. Results not only proved the enzyme lipase activity toward non-polar lipids in selected reaction conditions (aqueous system, T = 50 °C, pH = 5) but also suggested the occurrence of acyl-migration phenomenon observed by other authors in similar systems. Results showed that 1 h of reaction was enough to decrease the content of TAG in 54%, while DAG and MAG concentration increased from 0.4 to 3.5 and from 1.9 to 6.5 g/100 g of crude lecithin, respectively. Along the reaction, different contents of glycerides could be achieved, obtaining products with different composition which, in combination with the presence of phospholipids (PL) and/or lysophospholipids (LPL), could present specific emulsifying/stabilizing properties with a wide range of applications in food and pharmaceutical industry. [ABSTRACT FROM AUTHOR]

Cinnamyl acetate as an important fragrance ingredient could be synthesized by lipase-catalysed transesterification in organic systems, but enzyme proteins tended to denature and inactivate for no water lubrication. To improve the non-aqueous stability of lipases, absorbent cotton was taken as an alternative “water” phase to stabilize enzyme proteins. In a mass ratio of 1:1, Pseudomonas fluorescens lipase was immobilized on cotton fibres by physical absorption in a column glass bottle, forming a facile cotton-lipase bioreactor in which the transesterification between cinnamyl alcohol and vinyl acetate processed efficiently. From the molar conversions after reaction for 2 h at 37 °C and 160 rpm, the ability of cotton-lipase to transform substrate was more than 5-folds of native lipase. And even in static state and at 4 °C, the conversion of reaction catalysed by cotton-PFL had 11-fold increase relative to native lipase after 8 h. Recycles showed that the cotton-lipase had an extra-long half-life of activity (t1/2 = 693 h) and a negligible decay rate in the ability to transform substrate (Dr = 0.08% h−1). All these showed that this lipase had been effectively activated and stabilized by cotton fibres for the numerous hydroxyl groups and fluffy structure. [ABSTRACT FROM AUTHOR]

The presence of two different additives during non-covalent immobilization of lipase was studied. Lipase was immobilized via hydrophobic interactions on an amorphous silica with large pore size bearing octyl groups on the surface. Polyethyleneglycol (PEG) with different molecular weights (MW: 1500, 3000 and 10,000) were added to the suspension during enzyme immobilization, in an enzyme to PEG molar ratio of 1:10, and also 1:20 in the case of PEG1500. The activity after 15 d increased from 10% (absence of PEG) to values close to 40% in samples with PEG except the catalyst immobilized in the presence of 1:10 PEG1500, which kept fully active after 15 d incubation in toluene at 70 °C. The presence of water during storage of immobilized enzymes leads to significant activity loss. Saturated solutions of salts controlling the water activity of the systems were used to reduce in a controlled fashion the moisture of the systems: CaCl2 (aw= 0.037), MgCl2 (aw= 0.328), Mg(NO3)2 (aw= 0.529), Na2PO4.12H2O (aw= 0.74) and KCl (aw= 0.84). The immobilized lipase was suspended in saturated solutions of these salts, and then filtered and incubated in desiccators in the presence of the corresponding saturated salt solutions. Catalysts suspended and incubated in KCl or only suspended in phosphate kept some 20% activity after 33 d incubation whereas the maximal stability was achieved when the catalyst was suspended in phosphate and kept in a desiccator without salt solution. This catalyst kept around 50% activity after 33 d incubation. An inversely proportional relationship can be established between the stability achieved by the enzyme and the water content of the system. [ABSTRACT FROM AUTHOR]

The enzymatic transesterification of docosahexaenoic acid (DHA) ethyl ester with glycerol was carried out by using several immobilized lipases in a solvent-free system. This reaction involves the initial formation of sn-2 docosahexaenyl monoacylglycerol. This DHA derivative is highly relevant for improving the bioavailability of DHA and it has received increasing interest in the field of nutrition. Three commercial lipases, from Rhizomucor miehei (RML), Alcaligenes sp. (AQ) and Candida antarctica-fraction B (CALB) were immobilized by interfacial adsorption on a commercial hydrophobic support (a methacrylate resin containing octadecyl groups, Sepabeads C-18) and tested for glycerolysis of DHA ethyl ester. In certain cases (e.g. immobilized CALB), the transesterification reaction continues to the formation of triacylglycerol (80%) by using a very high excess of DHA ethyl ester ((115 mmols versus 1.24 mmols of glycerol and high temperatures (50 °C). However, the same biocatalyst working at lower temperatures, 37 °C, synthetizes a 90% of sn-2 monoacylglycerol even in the presence of that a high excess of DHA ethyl ester. Interestingly, immobilized RML derivative synthesizes a 98% of sn-2 monoacylglyceride (2-MG) in 15 min at 37 °C with a 4% of immobilized biocatalyst. These high activity and regioselectivity under very mild reaction conditions are very interesting for the thermal oxidative stability of the omega-3 fatty acid as well as for the thermal stability of the biocatalyst. Using Normal Phase HPLC-ELSD and accurate commercial markers, the formation of the 2-MG was confirmed. [ABSTRACT FROM AUTHOR]

The wide use of carbohydrate-based compounds is the pharmaceutical, cosmetic, detergent and food industry has led to the development of efficient synthetic procedures to overcome many drawbacks of conventional synthetic methodologies such as protection/activation/deprotection steps. In this context, due to their high chemo-, regio- and stereoselectivity, enzymes offer very effective and sustainable possibilities, and thus, they are increasingly used in carbohydrate field. In addition, the combination of biocatalysis and the use of green solvents is becoming a real alternative, as many solvents provide interactions with enzymes improving their catalytic behaviour, and they both directly contribute to increase the processes sustainability. This review will provide recent examples of the enzymatic preparation of carbohydrates and glycoconjugates using a combination of hydrolases (lipase and glycosidases) and green solvents. [ABSTRACT FROM AUTHOR]

A lipase-catalysed two-step butanolytic desymmetrization process for the preparation of pyrazolidyl butyl (S)-2-methylmalonate from 2-methylmalonic dipyrazolide was developed. The best reaction condition of using lipase PS-D in anhydrousn-hexane at 55 °C was first selected, leading to high yield and enantiomeric excess for the remained (S)-enantiomer. The kinetic analysis by considering the competitive inhibition from butanol was then carried out for obtaining the stereoselectivity ofE1 = 11.2 for the first desymmetrization and enantiomeric ratio ofE3E2−1=11.8 for the subsequent kinetic resolution. The thermodynamic analysis furthermore revealed that the enzyme stereodiscrimination was enthalpy-driven for the desymmetrization step, but changed as entropy-driven for the kinetic resolution step. [ABSTRACT FROM PUBLISHER]

To overcome the high energy-consuming process of microalgae drying, a two-step lipase catalysis technique for the preparation of biodiesel from microalgae lipid ofChlorella spp. was developed. In the first step, free fatty acids (FAAs) and triacylglycerols (TAGs) are released after cell disruption and extracted, while the TAGs were hydrolysed by free lipase in aqueous phase. In the second step, FAAs were esterified with ethanol in the catalysis of free suspended lipase. The maximum rate of hydrolysis and esterification was 93.6% and 91.3%, respectively. The effects of reaction parameters, such as reaction time, enzyme amount, water content and molar ratio of lipid to ethanol on hydrolysis or esterification, were investigated. The results indicated that two-step reaction process (hydrolyse esterify) for biodiesel production were feasible. [ABSTRACT FROM AUTHOR]

This work describes the immobilization of porcine pancreatic lipase (PPL), obtained from crude extract, on silica coated with octyl groups (OS) by interfacial adsorption, a simple and low-cost immobilization protocol. The biocatalyst PPL-OS was employed to the enzymatic preparation of fatty acid esters ofd-xylose, a product used especially in the field of cosmetics and pharmacy, especially dermatology, improving the functionality of epidermal cells. The yields of the immobilization in terms of enzymatic activity and protein concentration (98% and 75%, respectively) suggested that PPL present in the crude extract was selectively immobilized on the octyl-silica support, which allowed the hyperactivation of the biocatalyst (recovered activity, 144%), a phenomenon that may be attributed to the interfacial activation of the enzyme on hydrophobic surfaces. The biocatalyst PPL-OS showed to be very robust in organic medium and at high temperature, which is an extremely important characteristic to produce sugar fatty acid esters from the industrial point of view. The syntheses of xylose fatty esters (oleate, caprylate and butyrate) yielded conversions around 70% after short reaction period (2 h) at 60 °C in tert-butyl alcohol. The biocatalyst, even after incubation at 60 °C for 24 h, could be reused in four esterification cycles of 2-h reaction at 60 °C, maintaining 100% of its catalytic activity. [ABSTRACT FROM AUTHOR]

Lipase is an important enzyme which can catalyse the hydrolysis of lipids and has several applications and industrial potentials. In addition, different types of lipases are used as efficient catalysts in the enantioselective esterification and/or hydrolysis reactions and produce products in high yields and enantio excess as well. However, immobilization of lipases on the surface of a heterogeneous substrate is necessary to improve its specific catalytic activities as it can be isolated from the reaction mixture easily. Mesoporous silica materials are the best option for this aim due to their high specific surface area, ordered structure, and large pore volume. Hence, in this article, the role of SBA-15 and the modified SBA-15 mesoporous materials as support for different lipases and their catalytic activities are reviewed. [ABSTRACT FROM AUTHOR]

This paper describes the fish oil hydrolysis performed to obtain Omega-3 fatty acids usingCandida rugosalipase (CRL) immobilized and stabilized on different supports. The enzyme was successfully immobilized, presenting higher thermal stability than the free enzyme. Besides, the cationic derivatives were more stable than the others derivatives and free enzyme in methanol, propanol and cyclohexane. Reactions of fish oil hydrolysis were carried out in organic aqueous medium using 10 U of biocatalyst per gram of oil, at 37 °C. After 96 h, the CRL immobilized on cyanogen bromide agarose rendered the lower fish oil hydrolysis, producing 218 μM of Omega-3, which was 1.1-fold more than the hydrolysis catalyzed by free enzyme, while the ionic derivatives rendered the highest fish oil hydrolysis producing 582 and 577 μM of Omega-3 using the carboxymethyl and sulfopropyl derivatives, respectively. The carboxymethyl and the sulfopropyl derivatives resulted in a 2.9-fold increase in the hydrolysis of fish oil, making these derivatives attractive for industrial applications. [ABSTRACT FROM PUBLISHER]

Superparamagnetic Fe3O4hollow sub-microspheres (FHSM) with strong response to an external magnet were prepared via a solvothermal method, followed by acid etching. Lipase fromCandidasp. 99–125 was directly immobilized onto the amino-functional FHSM by simple adsorption, without glutaraldehyde linkage. The immobilized lipase was used to catalyze the esterification/transesterification of waste cooking oil with methanol to produce fatty acid methyl ester (FAME), a major source of biodiesel. FAME yield exceeded 93.4% over a wide range of temperatures from 10 to 40 °C. Notably, stability was clearly improved at the lower temperatures, in particular, giving a FAME yield of 89.6% after eight cycles of use at 10 °C. [ABSTRACT FROM PUBLISHER]

The objective of presented study was to maximize yields of 2-methylbutyl esters, derived by transesterification reactions mediated by sn-1,3-specific lipases, through engineering of reaction medium. Effects of water and diethylamine (DEA) concentrations on the efficiency of plant oils 2-methylbutanolysis, catalyzed by either mycelium-boundMucor circinelloideslipase (powder) or commercial immobilized lipase Lipozyme TL IM, were determined. Water content monitoring in reaction mixtures enabled to optimize the initial water content in terms of preventing the dehydration of enzyme’s microenvironment and increasing 2-methylbutyl esters yields. These yields were found to be increased by addition of either suitable amounts of water (0.5–1.5%) or diethylamine (10–30 mM) to the mixture of substrates. The presented results suggest that at low concentrations, diethylamine molecules contribute to retaining water in the microenvironment of enzyme that gives rise to increased transesterification yields and significantly reduced amounts of residual mono- and 1,2-diacyl-glycerols. [ABSTRACT FROM PUBLISHER]