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1.
Cross-linked enzyme aggregates (CLEAs), protein coated microcrystals (PCMCs), cross-linked protein coated microcrystals (CLPCMCs) of Candida antarctica lipase B (CALB) were used for esterification of glycerol with palmitic acid in acetone under low water condition. With CLEAs, 81% monoglyceride (MG) along with 4.5% diglyceride (DG) were produced at 1% (v/v) water content in 24 h. The water content in the medium was managed by stepwise addition of the molecular sieves at appropriate time intervals. With PCMCs (potassium sulfate as a core material), 82% monoglyceride along with 4.0% diglyceride were obtained, with 0.5% water (v/v) added initially to anhydrous acetone with molecular sieves present in the reaction medium. With CLPCMC (prepared by cross-linking with 200 mM glutaraldehyde), 87% monoglyceride and 3.3% diglyceride were produced in 24 h in presence of 1% (v/v) water (added initially) and with appropriate amount of molecular sieves added in the reaction medium. The results offer a comparative study on the performance of three high activity preparations of CALB for preparation of monopalmitin with ≤10% of the diglyceride content. 相似文献
2.
《Journal of Molecular Catalysis .B, Enzymatic》2011,69(3-4):230-239
The Talaromyces thermophilus lipase (TTL) was immobilized by different methods namely adsorption, ionic binding and covalent coupling, using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the most suitable support material preserving the catalytic activity almost intact and offering maximum immobilization capacity (76% and 91%, respectively). The chitosan-immobilized lipase could be reputably used for ten cycles with more than 80% of its initial hydrolytic activity. Shift in the optimal temperature from 50 to 60 °C and in the pH from 9.5 to 10, were observed for the immobilized lipase when compared to the free enzyme.The catalytic esterification of oleic acid with 1-butanol has been carried out using hexane as organic solvent. A high performance synthesis of 1-butyl oleate was obtained (95% of conversion yield) at 60 °C with a molar ratio of 1:1 oleic acid to butanol and using 100 U (0.2 g) of immobilized lipase. The esterification product is analysed by GC/MS to confirm the conversion percentage calculated by titration. 相似文献
3.
《Journal of Molecular Catalysis .B, Enzymatic》2011,72(3-4):85-89
Cross-linked enzyme aggregates (CLEA®s) were prepared from Candida rugosa lipase (CrL) using glutaraldehyde as the cross-linker. The optimum conditions of the immobilization process were determined (precipitant: ethanol, crosslinker concentration: 25 mM, enzyme concentration: 50 mg/ml, crosslinking time: 45 min.). CLEAs were shown to have several advantages compared to the free enzyme. They were more stable at 50 °C and 60 °C and had good reusability; retaining 40% of their initial activity after 15 recycles in aqueous media and remaining constant at that level thereafter, suggesting some initial leaching in water. The CLEAs catalyzed esterification reactions in cyclohexane, affording higher conversions than with the free enzyme, especially when longer fatty acids and alcohols were used as substrates. 相似文献
4.
Esterification degree of fructose laurate exerted by Candida antarctica lipase B in organic solvents
Sugar esters of fatty acids have many applications as biocompatible and biodegradable emulsifiers, which are determined by their degrees of esterification (DE). Direct esterification of fructose with lauric acid in organic media used commercial immobilized Candida antarctica lipase B (CALB) was investigated for DE. Significant difference of DE was observed between 2-methyl-2-butanol (2M2B) and methyl ethyl ketone (MEK), as di-ester/mono-ester molar ratio of 1.05:1 in 2M2B and 2.79:1 in MEK. Fourier transform infrared (FTIR) spectra showed that the secondary structure of the enzyme binding mono-ester presented distinct difference in 2M2B and MEK. Contents of β-turn and antiparallel β-sheet of CALB in 2M2B were 26.9% and 16.2%, respectively, but 19.1% and 13.2% in MEK. To understand the relationship between the conformational changes and differences of DE, mono-ester and fatty acid were directly employed for synthesis of di-ester. The maximum initial velocity of di-ester synthesis in MEK was 0.59 mmol g (enzyme)−1 h−1, which was 2.19-fold as greater as that in 2M2B, indicating that CALB conformation in MEK was preferred for the synthesis of di-ester. These results demonstrated that the conformation of CALB binding mono-ester affected by organic solvents essentially determined DE. 相似文献
5.
Cross-linked enzyme aggregates (CLEAs) of lipase from Thermomyces lanuginosa (TLL) were synthesized using (NH4)2SO4 as precipitant and glutaraldehyde as cross-linking agent. CLEAs were assayed for their hydrolytic activity in a reaction performed in an emulsioned medium. The effects of the amount of precipitant, cross-linker, and different additives such as protein cofeeder, oleic acid, n-heptane, sodium dodecyl sulfate (SDS), polyethylenglicol (PEG) and ethylendiamine were studied at selected ratios with respect to TLL mass. Traditional non-layered CLEAs of TLL showed recovered activities between 3 and 31% when compared with native lipase. Novel TLL layered CLEAs consisting of a protein cofeeder core and successive layers of target lipase showed an important increase in their retained activity. The highest recovered activity was found for the one-layered non-additivated CLEAs of TLL which showed a recovered activity of 75%. 相似文献
6.
Enhanced esterification activity through interfacial activation and cross‐linked immobilization mechanism of Rhizopus oryzae lipase in a nonaqueous medium
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Funda Kartal 《Biotechnology progress》2016,32(4):899-904
Interfacial activation via surfactant (Tween 80, Triton X‐100) treatment was conducted to improve the esterification activity of Rhizopus oryzae lipase that had undergone immobilization through cross‐linked enzyme aggregates (CLEA®) technique. Surfactant pretreated immobilized enzymes exhibited better esterification activity compared to free and non‐pretreated immobilized enzyme (Control CLEAs) since higher conversion rates were obtained within shorter times. The superiority of surfactant pretreated CLEAs, especially Tween 80 pretreated CLEAs (T 80 PT CLEAs), were clearly pronounced when longer alcohols were used as substrates. Conversion values exceeded 90% for octyl octanoate, oleyl octanoate and oleyl oleate synthesis with T 80 PT CLEAs whereas Control CLEAs and free enzyme showed no activity. Maximum conversions were achieved in the case equal molars of the substrates or in the case excess of the alcohol to acid in cyclohexane. In solvent free medium containing equal molars of substrates the conversion rates were 85% and 87% with T 80 PT CLEAs respectively for octyl octanoate and oleyl oleate within 2 hours. T 80 PT CLEAs showed 59% of its original activity after 7 consecutive usage for oleyl oleate synthesis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:899–904, 2016 相似文献
7.
《Process Biochemistry》2014,49(1):90-94
FastPrep cross-linked enzyme aggregates of N-acetylneuraminate aldolase from Staphylococcus carnosus (ScNAL-FpCLEAs) were prepared in order to improve the synthesis of 2-keto-3-deoxy-d-glycero-galactononulosonic acid (KDN), an important building block for therapeutic glycolipids and a possible marker for human prostate cancer. ScNAL-FpCLEAs showed improved thermostability compared with the free enzyme, doubling its half-life at 60 °C. When the effect of substrate ratio (pyruvate:d-mannose) and temperature on the yield of KDN was studied at its optimum pH (pH 7.0), 90% conversion in only 8 h was reached in the presence of 0.6 M d-mannose and 1.2 M pyruvate at 37 °C. This is the highest conversion described to date for enzymatic KDN synthesis. In addition, ScNAL-FpCLEAs exhibited enhanced catalytic activity and stability and could be recycled 10 times with no loss of activity. These results suggest the biotechnological potential of using FastPrepCLEAs to obtain valuable biocatalysts. 相似文献
8.
Lorena Wilson Andrés Illanes Lorena Soler María José Henríquez 《Process Biochemistry》2009,44(3):322-326
Cross-linked enzyme aggregates (CLEAs) are novel type biocatalysts well suited to catalyze reactions of organic synthesis. Penicillin acylase is a versatile enzyme that can both hydrolyze and synthesize β-lactam antibiotics. CLEAs and CLEAs covered with polyionic polymers (polyethyleneimine and dextran sulfate at two different enzyme to polymer ratios) were prepared at varying cross-linking agent to enzyme ratio: 0.15 and 0.25. Results are presented on the effect of such variables on immobilization yield, specific activity, stability and performance of penicillin acylase CLEAs in the kinetically controlled synthesis of cephalexin. The cross-linking agent to enzyme ratio had no significant effect on the specific activity of the CLEAs, but affected immobilization yield, stability in ethylene glycol medium and conversion yield and productivity in the synthesis of cephalexin, being always higher at the lower cross-linking agent to enzyme ratio. Best results were obtained with CLEAs at 0.15 glutaraldehyde to enzyme protein ratio: specific activity of hydrolysis and synthesis was 708 and 325 UI/gCLEA respectively, conversion yield was 87%, specific productivity was 5.4 mmol cephalexin/(gCLEA·h) and 90% of the enzyme remained active after 170 h at operating conditions. 相似文献
9.
We successfully developed an efficient and promising bioprocess for 1,3-diolein synthesis by performing Novozym 435-mediated esterification of oleic acid with monoolein in this work. Under the optimized conditions (60 °C, molar ratio of oleic acid to monoolein 1.2:1), a 1,3-diolein yield of 93.7% could be achieved, and the yield of 1,2-diolien was low (2.6%). The high yield of 1,3-diolein and the optimum reaction time were improved remarkably compared with the results of our previous study, which involved the enzymatic esterification of oleic acid with glycerol. An additional advantage of the new process is the fact that 90% original activity of the enzyme was maintained after being used for 100 reactions. The present work could be seen as a useful enzyme-catalyzed process for the industrial production of 1,3-diacylglycerol. 相似文献
10.
Three new synthetic routes were critically evaluated for the lipase-catalyzed production of 1,3-oleoyl-2 docosahexaenoylglycerol (ODO) in relatively large-scale (approximately 200 g). First, the production of 1,3-diolein by the reaction of glycerol and oleic acid followed by incorporation of docosahexaenoic (DHA) ethyl ester at the sn-2 position was studied. 1,3-Diolein was produced in 68.3% and 84.6% yield when stoichiometric amounts of the substrates were reacted at 25 °C for 8 h in the presence of 10% Novozym 435 and Lipozyme RM IM, respectively. Further increase in reaction temperature and time led to decrease in the 1,3-diolein yield. However, only a 9.4% yield of triacylglycerol was obtained in the subsequent reaction step when the 1,3-diolein was reacted with DHA ethyl ester in the presence of Novozym 435. Secondly, the feasibility of direct acidolysis was studied. Acidolysis of single cell oil (SCO) in excess oleic acid using Novozym 435 as the catalyst occurred twice as fast in solvent (tert-butanol) compared to a solvent-free system, and 63% oleic acid was incorporated into SCO. However, the regio-isomeric purity of the product was poor. Finally, the ethanolysis of SCO to produce DHA-enriched 2-monoacylglycerol followed by esterification with oleic acid or ethyl oleate was investigated. ODO was obtained in 50.9% regio-purity by Lipozyme RM IM-catalyzed esterification. The latter method was the most feasible for preparing ODO in large-scale. This synthetic route could be adapted for related triacylglycerols containing highly polyunsaturated when their productions in large-scale and high regio-purity are required. 相似文献
11.
Enzymes are usually immobilized on solid supports or solubilized when they are to be used in organic solvents with poor enzyme solubility. We have reported previously on a novel immobilization method for subtilisin Carlsberg on fumed silica with results that reached some of the best previously reported catalytic activities in hexane for this enzyme. Here we extend our method to Candida antarctica Lipase B (CALB) as an attractive target due to many potential applications of this enzyme in solvents. Our CALB/fumed silica preparations approached the catalytic activity of commercial Novozym 435 for a model esterification in hexane at 90 wt.% fumed silica (relative to the mass of the preparation). An intriguing observation was that the catalytic activity at first increases as more fumed silica was made available to the enzyme but then decreased precipitously when fumed silica exceeded 90 wt.%. This was not the case for s. Carlsberg where the catalytic activity leveled off at high relative amounts of fumed silica. We determined adsorption kinetics, performed variations of the pre-immobilization aqueous pH, determined the stability, and applied fluorescence microscopy to the preparations. A comparison with recent concepts by Gross et al. may point towards a rationale for an optimum intermediate surface coverage for some enzymes on solid supports. 相似文献
12.
《Journal of Molecular Catalysis .B, Enzymatic》2011,68(3-4):214-218
In this study porcine pancreatic lipase (PPL) was covalently immobilized on cross-linked polyvinyl alcohol (PVA) in organic media in the presence of fatty acid additives in order to improve its immobilized activity. The effects of fatty acid additions to the immobilization media were investigated choosing tributyrin hydrolysis in water and ester synthesis by immobilized PPL in n-hexane. Various fatty acids which are also the substrates of lipases in esterification reactions were used as active site protecting agents during the immobilization process in an organic solvent. The obtained results showed that covalent immobilization carried out in the presence of fatty acids as protective ligands improved the hydrolytic and esterification activity of immobilized enzyme. A remarkable increase in activity of the immobilized PPL was obtained when octanoic acid was used as an additive and the hydrolytic activity was increased from 5.2 to 19.2 μmol min−1 mg−1 as compared to the non-additive immobilization method. With the increase of hydrolytic activity of immobilized lipase in the presence of octanoic acid, in an analogous manner, the rate of esterification for the synthesis of butyl octanoate was also increased from 7.3 to 26.3 μmol min−1 g−1 immobilized protein using controlled thermodynamic water activities with saturated salt solutions. In addition, the immobilized PPL activity was maintained at levels representing 63% of its original activity value after 5 repeated uses. The proposed method could be adopted for a wide variety of other enzymes which have highly soluble substrates in organic solvent such as other lipases and esterases. 相似文献
13.
《Journal of Molecular Catalysis .B, Enzymatic》2011,71(3-4):138-143
A recombinant esterase (EC 3.1.1.1) cloned from Bacillus subtilis 0554 (BSE) was carrier-freely immobilized with the method of cross-linked enzyme aggregates. The conditions for preparing the cross-linked aggregates of BSE (CLA-BSE) were optimized, including the type and concentration of precipitants, and the concentration of cross-linker, and a simple and efficient procedure for preparing CLA-BSE was developed, consisting of a precipitation step with 0.5 g mL−1 (NH4)2SO4 and a cross-linking step with 60 mM glutaraldehyde for a period of 3 h as the cross-linking time. As a result, about 70% of the initial free BSE activity was incorporated into the CLA-BSE. The thermal stabilities of the immobilized enzyme at 30 °C and 50 °C were >360 and 14 times those of free BSE, respectively. More importantly, the operational stability of CLA-BSE was also considerably improved. In the kinetic resolution of dl-menthyl acetate to produce l-menthol with CLA-BSE gave eep > 94% at conversion of >40% and the CLA-BSE could be reused for 10 times with only about 8% reduction in activity. Therefore, the new biocatalyst immobilized through the methodology of CLEAs could significantly decrease the manufacturing cost of l-menthol and would be more beneficial for its practical applications. 相似文献
14.
The enzymatic conversion of lignocellulosic biomass into biofuels has been identified as an excellent strategy to generate clean energy. However, the current process is cost-intensive as an effective immobilization approach to reuse the enzyme(s) has been a major challenge. The present study introduces the concept and application of novel magnetic cross-linked enzyme aggregates (mag-CLEAs). Both mag-CLEAs and calcium-mag-CLEAs (Ca-mag-CLEAs) exhibited a 1.35 fold higher xylanase activity compared to the free enzyme and retained more than 80.0% and 90.0% activity, respectively, after 136 h of incubation at 50 °C, compared to 50% activity retained by CLEAs. A 7.4 and 9.0 fold higher sugar release from lime-pretreated and NH4OH pre-treated sugar bagasse, respectively, was achieved with Ca-mag-CLEAs compared to the free enzymes. The present study promotes the successful application of mag-CLEAs and Ca-mag-CLEAs as carrier free immobilized enzymes for the effective hydrolysis of lignocellulolytic biomass and associated biofuel feedstocks. 相似文献
15.
Lipase from Thermomyces lanuginosus (TLL) was immobilized on mesoporous hydrophobic poly-methacrylate (PMA) particles via physical adsorption (interfacial activation of the enzyme on the support). The influence of initial protein loading (5–200 mg/g of support) on the catalytic properties of the biocatalysts was determined in the hydrolysis of olive oil emulsion and synthesis of isoamyl oleate (biolubricant) by esterification reaction. Maximum adsorbed protein loading and hydrolytic activity were respectively ≈100 mg/g and ≈650 IU/g using protein loading of 150 mg/g of support. The adsorption process followed the Langmuir isotherm model (R2 = 0.9743). Maximum ester conversion around 85% was reached after 30 min of reaction under continuous agitation (200 rpm) using 2500 mM of each reactant in a solvent-free system, 45 °C, 20% m/v of the biocatalyst prepared using 100 mg of protein/g of support. Apparent thermodynamic parameters of the esterification reaction were also determined. Under optimal experimental conditions, reusability tests of the biocatalyst (TLL-PMA) after thirty successive cycles of reaction were performed. TLL-PMA fully retained its initial activity up to twenty two cycles of reaction, followed by a slight decrease around 8.6%. The nature of the product (isoamyl oleate) was confirmed by attenuated total reflection Fourier transform infrared (ATR-FTIR), proton (1H NMR) and carbon (13C NMR) nuclear magnetic resonance spectroscopy analyses. 相似文献
16.
Candida rugosa lipases (CRLs) immobilized by physical adsorption, cross-linking and covalent binding methods on a MSU-H type mesoporous silica previously modified organically by different strategies, respectively, were examined as biocatalysts for esterification of conjugated linoleic acid (CLA) and ethanol in nonaqueous medium. MSU-H silica was modified by nonionic surfactant of triblock copolymer Pluronic P123, amino-functionalization and glutaraldehyde-grafting and confirmed by FT-IR analysis. Interaction mechanisms of CRLs and supports involve covalent and non-covalent interactions including electrostatic repulsion and hydrophobic interaction at pH 7. The immobilized CRLs containing surfactant were prepared by cross-linking via entrapping CRL aggregates inside the pores of silicas. The surfactant located inside the silicas could interfacially activate the immobilized CRLs and favored catalytic esterification. The biocatalyst containing 38 wt.% of surfactant afforded 1111.1 U/mg of specific activity about eight times higher than soluble CRL, and maximal 56.7% of total CLA esterification with 96.5% of 9c, 11t-CLA isomer esterification degree. The immobilized-CRL with 64.5 mg/g of loading amount of protein exhibited maximal hydrolytic activity of 2945.3 U/g-support for grafting glutaraldehyde. This derivative showed a high level of esterification activity and operational stability and remained 43.2–46.9% of total esterification for 32 h consecutive four runs. 相似文献
17.
Ahmed Eid Fazary Suryadi Ismadji Yi-Hsu Ju 《International journal of biological macromolecules》2009,44(3):240-248
Thermal stabilities of a native freeze dried Aspergillus awamori feruloyl esterase (FAE-II) enzyme and a cross-linked feruloyl esterase aggregate (CLEAs) at 25–85 °C were evaluated and discussed. Effects of some metal ions and some chemicals on the activity of both native freeze dried FAE-II enzyme and CLEAs were examined and explained. Differential scanning calorimetry, thermogravimetry, and derived thermogravimetry, were used to observe and explain the thermal denaturation processes. Structural analyses were made for native FAE-II and CLEAs using FT-IR and SEM techniques to investigate whether the cross-linking had any effect on the powder structure of native FAE-II enzyme. 相似文献
18.
álvaro Cruz-Izquierdo Enrique A. Picó Carmen López Juan L. Serra María J. Llama 《PloS one》2014,9(12)
Enzyme-catalyzed production of biodiesel is the object of extensive research due to the global shortage of fossil fuels and increased environmental concerns. Herein we report the preparation and main characteristics of a novel biocatalyst consisting of Cross-Linked Enzyme Aggregates (CLEAs) of Candida antarctica lipase B (CALB) which are covalently bound to magnetic nanoparticles, and tackle its use for the synthesis of biodiesel from non-edible vegetable and waste frying oils. For this purpose, insolubilized CALB was covalently cross-linked to magnetic nanoparticles of magnetite which the surface was functionalized with –NH2 groups. The resulting biocatalyst combines the relevant catalytic properties of CLEAs (as great stability and feasibility for their reutilization) and the magnetic character, and thus the final product (mCLEAs) are superparamagnetic particles of a robust catalyst which is more stable than the free enzyme, easily recoverable from the reaction medium and reusable for new catalytic cycles. We have studied the main properties of this biocatalyst and we have assessed its utility to catalyze transesterification reactions to obtain biodiesel from non-edible vegetable oils including unrefined soybean, jatropha and cameline, as well as waste frying oil. Using 1% mCLEAs (w/w of oil) conversions near 80% were routinely obtained at 30°C after 24 h of reaction, this value rising to 92% after 72 h. Moreover, the magnetic biocatalyst can be easily recovered from the reaction mixture and reused for at least ten consecutive cycles of 24 h without apparent loss of activity. The obtained results suggest that mCLEAs prepared from CALB can become a powerful biocatalyst for application at industrial scale with better performance than those currently available. 相似文献
19.
A low-cost lipase preparation is required for enzymatic biodiesel synthesis. One possibility is to produce the lipase in solid-state fermentation (SSF) and then add the fermented solids (FS) directly to the reaction medium for biodiesel synthesis. In the current work, we scaled up the production of FS containing the lipases of Rhizopus microsporus. Initial experiments in flasks led to a low-cost medium containing wheat bran and sugarcane bagasse (50:50 w/w, dry basis), supplemented only with urea. We used this medium to scale-up production of FS, from 10 g in a laboratory column bioreactor to 15 kg in a pilot packed-bed bioreactor. This is the largest scale yet reported for lipase production in SSF. During scale-up, the hydrolytic activity of the FS decreased 57%: from 265 U g−1 at 18 h in the laboratory bioreactor to 113 U g−1 at 20 h in the pilot bioreactor. However, the esterification activity decreased by only 14%: from 12.1 U g−1 to 10.4 U g−1. When the FS produced in the laboratory and pilot bioreactors were dried and added directly to a solvent-free reaction medium to catalyze the esterification of oleic acid with ethanol, both gave the same ester content, 69% in 48 h. 相似文献
20.
ZnO nanowires were successfully introduced into a macroporous SiO2 by in situ hydrothermal growth in 3D pores. The obtained composites were characterized by SEM and XRD, and used as supports to immobilize Candida antarctica lipase B (CALB) through adsorption. The high specific surface area (233 m2/g) and strong electrostatic interaction resulted that the average loading amount of the composite supports (196.8 mg/g) was 3–4 times of that of macroporous SiO2 and approximate to that of a silica-based mesoporous material. Both adsorption capacity and the activity of the CALB immobilized on the composite supports almost kept unchanged as the samples were soaked in buffer solution for 48 h. The chiral resolution of 2-octanol was catalyzed by immobilized CALB. A maximum molar conversion of 49.1% was achieved with 99% enantiomeric excess of (R)-2-octanol acetate under the optimal condition: a reaction using 1.0 mol/L (R,S)-2-octanol, 2.0 mol/L vinyl acetate and 4.0 wt.% water content at 60 °C for 8 h. After fifteen recycles the immobilized lipase could retain 96.9% of relative activity and 93.8% of relative enantioselectivity. 相似文献