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Biocatalytic production of lactose ester catalysed by mica-based immobilised lipase
Abstract
Enzyme immobilisation technology is an effective means to improve sugar ester production through the employment of biocatalysts. In the present study, immobilisation of Candida rugosa (CRL) lipase onto amino-activated mica is performed via covalent bonding (namely Amino-CRL) and the cross-linking of lipases into nano-reactors through physical adsorption (namely NER-CRL). Free and immobilised lipases were tested for their esterification activities. Specific activities for Amino-CRL and NER-CRL increased by 2.4 and 2.6-fold, respectively, upon immobilisation. Extending this work, immobilised lipases have novel capabilities in the synthesis of sugar esters. The optimised conditions for sugar fatty acid ester syntheses are 48 h at 2:1 of molar ratio of lactose sugar to capric acid at 55 °C. Furthermore, a high operational stability with half-lives of over 13 and 10 runs was achieved for NER-CRL and Amino-CRL, respectively, indicating the efficiency of the immobilisation process.
... The temperature range generally oscillates between 40 °C and 60 °C, a characteristic interval of the immobilized form, which usually guarantees better conditions because it is more stable than the free form. Some of these 6′-lactose esters were synthesized employing green solvents such as acetone [8,9]. All of the reported lipases primarily catalyzed esterification of lactose at the C6hydroxyl position (Table 1). ...
... The temperature range generally oscillates between 40 • C and 60 • C, a characteristic interval of the immobilized form, which usually guarantees better conditions because it is more stable than the free form. Some of these 6 -lactose esters were synthesized employing green solvents such as acetone [8,9]. out regioselective acylation reactions. ...
... The temperature range generally oscillates between 40 °C and 60 °C, a characteristic interval of the immobilized form, which usually guarantees better conditions because it is more stable than the free form. Some of these 6′-lactose esters were synthesized employing green solvents such as acetone [8,9]. The biggest drawback of the lipase is its high price. ...
Glycolipid surfactants are biocompatible and biodegradable compounds characterized by potential applications in various sectors including pharmaceuticals, cosmetics, agriculture, and food production. A specific overview regarding synthetic methodologies and properties of 6′-lactose-based surfactants is presented herein, particularly all the synthetic approaches to this class of lactose esters, such as enzymatic and traditional organic syntheses. Moreover, detailed descriptions of physicochemical data and biocompatibility properties of these molecules, that is, surface tension, critical micelle concentration, emulsifying ability, foaming, particle size distribution, biocompatibility, and safety, are described. Biological applications with a focus on permeability enhancing, antimicrobial activity, and antibiofilm properties of 6′-lactose-based esters are also reported.
... Other enzymes and lipases from other microorganisms were also tested in the biocatalytic esterification of mono-and disaccharides. The immobilized lipase of Candida rugosa was tested in the biocatalytic synthesis of lactose caprate esters [47], fructose oleates [8] and glucose fatty acids esters from coconut oil [48]. In the first case, the fatty acid conversions were about 20 % greater when increasing temperature from 45 C to 55 C. ...
... With a very high molar proportion of fatty acid to saccharide when compared to the other studies, the authors suggested that increasing the proportion of fatty acid could negatively affect the lipase conformation due to an acidification of the reaction medium. Similarly, [47] study of the effect of sugar to acyl donor molar ratio in the synthesis of lactose caprate esters catalysed by the lipase of Candida rugosa. Different molar ratios of lactose to capric acid (3:1, 2:1, 1:1, 1:2 and 1:3) were tested. ...
... Different carbohydrates were studied in the production of lactose, trehalose, ribose and mannose esters catalysed by free and immobilised lipases of Candida rugosa [47]. The lipase-catalysed acylation of sugars was found to be regioselective. ...
Glycolipids, carbohydrate fatty esters or sugar esters are amphiphilic molecules containing hydrophilic groups bonded to hydrophobic parent structures. Recently, glycolipids have shown their antimicrobial and antitumor capacities. Their surface activity properties have applications in the food, pharmaceutical and cosmetic industries. Sugar esters’ building blocks can be obtained from natural resources and/or be transformed by biochemical pathways for uses as surfactants. Biosurfactants are non-ionic, nontoxic, biodegradable, tasteless, and odourless. The biocatalysis of these molecules involves sustainable, green, and safer methods. The advantages of producing biosurfactants from enzymatic catalysis are the energy economy, high selectivity, production of natural products, reduction of the use of fossil-based solvents and chloride compounds. This review presents the most recent studies concerning the evaluation of the impact of the main parameters and their levels influencing the enzymatic synthesis of glycolipids. Various enzyme catalysed synthetic methods were described. The parameters studied were temperature, reaction time, solvent system, type of biocatalyst, substrates molar ratio proportion and the nature of substrates. This review discusses the influence of different biocatalysts in the conversions of glycolipids; The reactivity from mono to polysaccharides and their interaction with fatty acids of different carbon chain lengths in the presence of specific enzymes; The effect of the solvent polarity, the use of multiple solvents, ionic liquids, supercritical CO2, and solvent-free media in sugar ester conversions; And the optimization of temperature and reaction time in different enzymatic systems.
... Other minerals reported as the enzyme carriers are halloysite (Zhai et al. 2010) and mica (Zaidan et al. 2012). Mica is a multi-element mineral of a complex chemical composition that includes mainly aluminium, silicon, calcium, sodium and potassium, and in smaller amounts, lithium, magnesium, iron and manganese. ...
... These give rise to the interactions sufficiently strong for the enzyme-carrier binding (adsorption) to occur (Kosaka et al. 2007;Gustafsson et al. 2012;Wu et al. 2012). When such groups are absent, the carrier is subjected to a chemical modification (Cho et al. 2012;Mendes et al. 2012;Zaidan et al. 2012). ...
... SBA-15 or MSU-H (Yu and Fang 2013), vesicular silica (Wu et al. 2012), fumed silica (Kramer et al. 2010), silanized silica (Zheng et al. 2012), silica sol-gel film (Lee et al. 2010) and commercial silica-based products Celite (Brem et al. 2011) and Celite 545 (Kumar and Kanwar 2011;see Fig. 5). Other inorganic carriers used for the adsorption of lipases are zirconia nanoparticles , mica (Zaidan et al. 2012), and magnetic carriers (Liu et al. 2012a, b;Xie and Wang 2012). An interesting immobilization of lipases was performed in sol-gel derived silica using the multi-walled carbon nanotubes as additives to protect the inactivation of the enzymes during the sol-gel process and to enhance their stability. ...
Endowed with unparalleled high catalytic activity and selectivity, enzymes offer enormous potential as catalysts in practical applications. These applications, however, are seriously hampered by enzymes’ low thermal and chemical stabilities. One way to improve these stabilities is the enzyme immobilization. Among various tested methods of this process that make use of different enzyme-carrier interactions, immobilization by adsorption on solid carriers has appeared most common. According to these findings, in this review we present a comparative analysis of the literature reports on the recent trends in the immobilization of the enzymes by adsorption. This thorough study was prepared in order to provide a deeper understanding of the process. Both carriers, carrier modifiers and procedures developed for effective adsorption of the enzymes are discussed. The review may thus be helpful in choosing the right adsorption scheme for a given enzyme to achieve the improvement of its stability and activity for a specific application.
... It is able to esterify lactose in a direct reaction with fatty acids [35,36] or in a transesterification reaction with organic acids esters [26,37,38] and their halogenated derivatives [25,26,39]. Lipases from other microorganisms have been reported to reach comparable yields with similar substrates to the latter lipase and these include enzymes from Candida rugosa [40], Mucor miehei [13,37,41], Pseudomonas cepacia [37,38,42], Rhizomucor miehei [38,[43][44][45], Thermomyces lanuginose [12,16,37,38]. All of the reported lipases primarily catalyze esterification at the non-reducing end of lactose on the C6 0 hydroxyl position. ...
... The enzymatic reactions described above were performed in potentially hazardous and non-environmentally safe solvents such as pyridine [35,47], N,Ndimethylformamide [45,46], dimethyl sulfoxide [16], acetonitrile [26], 2-methyl-2-butanol [12,37,42], butanone [37,38], tetrahydrofuran [25], toluene [13,39], etc. However, some carbohydrate esters, including those of lactose, were synthesized enzymatically employing far greener and environment friendly technologies, such as ionic liquids [48], supercritical fluids, in particularly supercritical carbon dioxide (scCO 2 ) [8,49,50], ethanol Reaction time 2 days [8,36], acetone [38,40], and even pure water [51,52]. For example, a unique mode of esterification of carbohydrates was reported for acetyloesterase from Trichoderma reesei [52]. ...
... It is able to esterify lactose in a direct reaction with fatty acids [35,36] or in a transesterification reaction with organic acids esters [26,37,38] and their halogenated derivatives [25,26,39]. Lipases from other microorganisms have been reported to reach comparable yields with similar substrates to the latter lipase and these include enzymes from Candida rugosa [40], Mucor miehei [13,37,41], Pseudomonas cepacia [37,38,42], Rhizomucor miehei [38,[43][44][45], Thermomyces lanuginose [12,16,37,38]. All of the reported lipases primarily catalyze esterification at the non-reducing end of lactose on the C6 0 hydroxyl position. ...
... The enzymatic reactions described above were performed in potentially hazardous and non-environmentally safe solvents such as pyridine [35,47], N,Ndimethylformamide [45,46], dimethyl sulfoxide [16], acetonitrile [26], 2-methyl-2-butanol [12,37,42], butanone [37,38], tetrahydrofuran [25], toluene [13,39], etc. However, some carbohydrate esters, including those of lactose, were synthesized enzymatically employing far greener and environment friendly technologies, such as ionic liquids [48], supercritical fluids, in particularly supercritical carbon dioxide (scCO 2 ) [8,49,50], ethanol Reaction time 2 days [8,36], acetone [38,40], and even pure water [51,52]. For example, a unique mode of esterification of carbohydrates was reported for acetyloesterase from Trichoderma reesei [52]. ...
Biodegradable nonionic sugar esters-based surfactants have been gaining more and more attention in recent years due to their chemical plasticity that enables the various applications of these molecules. In this review, various synthesis methods and biotechnological implications of lactose esters (LEs) uses are considered. Several chemical and enzymatic approaches are described for the synthesis of LEs, together with their applications, i.e. function in detergents formulation and as additives that not only stabilize food products but also protect food from undesired microbial contamination. Further, this article discusses medical applications of LEs in cancer treatment, especially their uses as biosensors, halogenated anticancer drugs, and photosensitizing agents for photodynamic therapy of cancer and photodynamic inactivation of microorganisms.
... In most cases, denaturation can occur when there is a change in the microenvironment that surrounds these biocatalyst particles, which results in a change in their protein conformation and loss of activity (Faber 1997). Therefore, the structural conformation, choice of support and properties of the binding sites of each immobilised lipase (Zaidan et al. 2012) as well as their possible inactivation by H 2 O 2 must be overcome. ...
... their conformation and also moved freely in the reaction media. The properties of support materials are responsible for the stability of the enzyme Zaidan et al. 2012). The activity of immobilised enzymes like Novozym 435 would be retained since immobilisation improves its conformational rigidity and enhanced the catalytic pocket availability towards the enzyme-acyl complex binding (Guti é rrez- Ayesta et al. 2007). ...
Here, we describe an improved enzyme-facilitated epoxidation of 1-nonene using a conventional water bath shaker at ambient temperature. Enzymes were used to produce peroxy acids instantly from hydrogen peroxide (H2O2) and various perhydrolysis substrates. The peroxy acid generated was then utilised directly for in-situ oxidation of 1-nonene to 1-nonene oxide. Various parameters affecting the reaction were studied such as the nature of the peroxy acids, organic solvents, enzyme sources and enzyme concentrations. The highest conversion rate was achieved using phenylacetic acid as an oxygen carrier. 1-Nonene was converted most efficiently with 95% of the maximum yield by Novozym 435, an immobilised Candida antarctica lipase B, using dichloromethane as the reaction media. A minimum amount (16 mg, 1.4% w/w) of Novozym 435 was needed to maintain catalytic activity (160.0 Ug−1). In addition, a simple and rapid Gas chromatography mass spectroscopy selective ion monitoring (GC-MS SIM) method was developed using a HP-5ms column for determining 1-nonene oxide. The method was found to be linear in the range of 29.9 to 298.8 mg/L with R2 = 0.9981.
... Table 2 shows the kinetic parameters for the model (Equation (1)) fitted to the long-term experimental data. They were of the same order of magnitude as those found by Zaidan et al. [69], who synthesized lactose fatty esters from capric acid in the presence of free lipase and lipase immobilized on a new support of mica clay. Moreover, it can be expected from the Km values that commercial immobilized CALB (Lipozyme 435) has a poor affinity by both xylose and oleic acid, as the values are over 1 M for oleic acid and over 0.75 M for xylose. ...
In this paper, we have performed the Lipozyme 435-catalyzed synthesis of xylose oleate in methyl ethyl ketone (MEK) from xylose and oleic acid. The effects of substrates’ molar ratios, reaction temperature, reaction time on esterification rates, and Lipozyme 435 reuse were studied. Results showed that an excess of oleic acid (xylose: oleic acid molar ratio of 1:5) significantly favored the reaction, yielding 98% of xylose conversion and 31% oleic acid conversion after 24 h-reaction (mainly to xylose mono- and dioleate, as confirmed by mass spectrometry). The highest Lipozyme 435 activities occurred between 55 and 70 °C. The predicted Ping Pong Bi Bi kinetic model fitted very well to the experimental data and there was no evidence of inhibitions in the range assessed. The reaction product was purified and presented an emulsion capacity close to that of a commercial sugar ester detergent. Finally, the repeated use of Lipozyme 435 showed a reduction in the reaction yields (by 48 and 19% in the xylose and oleic acid conversions, respectively), after ten 12 h-cycles.
... Free enzymes have poor stability, which are easily inactivated and difficult to remove. Currently, immobilized enzymes and whole cell catalysis are often used for organic synthesis experiments (Duetz et al., 2001;Zaidan et al., 2012). Various studies demonstrated that esterified flavonoids usually reach enhanced physiological activity and higher bioavailability compared to the original flavonoid compound (Kontogianni et al., 2001). ...
Neohesperidin (NH) is a natural flavonoid glycoside compound with considerable physiological and pharmacological activities. However, its bioavailability is limited due to poor solubility, and few studies have so far attempted improve the solubility and bioavailability of NH. In this study, we structurally modified NH using an immobilized lipase to improve lipophilicity and therefore expand its applicability in lipophilic media as well as enhance its bioavailability in vivo. In addition, we aimed investigated the pro-apoptoptotic activity of this new compound (propionyl neohesperidin ester, PNHE) in MCF-7 breast cancer cells using a variety of cellular assays, including the MTT (3-(4, 5-dimethyl- 2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide assay, assessment of intracellular reactive oxygen species (ROS) levels, and flow cytometry. We successfully synthesized PNHE using immobilized lipases, and the esterification of NH was confirmed by Fourier transform-infrared spectroscopy (FT-IR). Compared to NH, HNPE showed higher anti-proliferative and pro-apoptotic in MCF-7 breast cancer cells, which may be explained by its increased lipophilicity compared to neohesperidin, benefiting to the action of NH on the cancer cell wall. The IC50 of PNHE for inducing apoptosis of MCF-7 cells was 185.52 μg/mL. PNHE increased both the proportion of cells in Sub-G1 phase and the cellular ROS content, indicating a certain therapeutic effect of HNPE on breast cancer.
... %) was appreciably higher over free CRL (28.61 ± 0.24 U/mg). The data corroborated earlier findings of covalently immobilized lipases exhibiting higher activity over their free counterparts [17,18,57]. Also, the improved activity of CRL-ALG/NC/ MMT over free CRL was symptomatic of the rigidification of CRL structure from a very intense multipoint covalent binding (ether bond) to the surface of ALG/NC/MMT. ...
Biomass from oil palm frond leaves (OPFL) is an excellent reservoir of lignocellulosic material which full potential remains untapped. This study aimed to statistically optimize the covalent immobilization of Candida rugosa lipase (CRL) onto a ternary support comprised of OPFL derived nanocellulose (NC) and montmorillonite (MMT) in alginate (ALG) (CRL-ALG/NC/MMT). The coarser topology and the presence of characteristic spherical globules in the field emission scanning electron micrographs and atomic force micrographs, respectively, supported the existence of CRL on ALG/NC/MMT. In addition, amide peaks at 3478 and 1640 cm-1 in the fourier transform infrared spectra affirmed that CRL was covalently bonded to ALG/NC/MMT. The optimized Taguchi Design-assisted immobilization of CRL onto ALG/NC/MMT (7 h of immobilization, 35℃, pH 5, 7 mg/mL protein loading) gave a production yield of 92.89 % of ethyl levulinate (EL), as proven by gas chromatography-mass spectrometric ([M] +m/z 144, C7H12O3), FTIR and nuclear magnetic resonance (CAS-539-88-8) data. A higher optimal reaction temperature (50℃) and the reusability of CRL-ALG/NC/MMT for up to 9 esterification cycles substantiated the appreciable structural rigidification of the biocatalyst by ALG/NC/MMT, which improved the catalytic activity and thermal stability of the lipase.
... Another reported enzyme also often used is the lipase from C. rugosa. After immobilization, this lipase reached over 70% conversion rate when applied to the synthesis of lactone caprate in acetone after a 48 h reaction time [74]. ...
Glycolipids are promising nonionic biosurfactants with a wide range of application. In contrast to the traditional surfactants from fossil origin, they are not only exhibiting excellent surfactant and emulsifying properties but are in addition biodegradable and non-toxic to the environment. Glycolipids can be produced by microbial fermentation, by chemical or enzymatical synthesis using renewable resources. In this review, strengths and weaknesses of each production method are compared and enzymatic synthesis is highlighted as the method of choice for the tailor-made synthesis of a wide range of novel glycolipids. As for enzymatical and chemical synthesis solvents with low water activity are needed to favor condensation instead of the hydrolysis reaction, organic solvents or ionic liquids are commonly used. However, the unusual deep eutectic solvent system might be a green alternative worth to consider. In terms of enzymatic catalysis, lipases are well known in literature and already widely used for the production of sugar-acyl-esters. However, β-glucosidases can be alternatively used for glycolipid synthesis which is far less studied. The abilities of both enzymes for glycolipid synthesis are therefore compared and discussed.
... Typical wave number for the ester (C=O) were in the ranges of 1716-1751 cm -1 (Coates, 2000;van den Broek & Boeriu, 2013). This was also confirmed by Boruczkowska et al. (2012), Zaidan et al. (2012) & Syamsul et al., (2010 that the wave number of the ester were 1715 cm -1 , 1723 cm -1 , and 1732 cm -1 , respectively. ...
... Typical wave number for the ester (C=O) were in the ranges of 1716-1751 cm -1 (Coates, 2000;van den Broek & Boeriu, 2013). This was also confirmed by Boruczkowska et al. (2012), Zaidan et al. (2012) & Syamsul et al., (2010 that the wave number of the ester were 1715 cm -1 , 1723 cm -1 , and 1732 cm -1 , respectively. ...
... Surface modification of the silica support also occurs via the addition of specific functional groups and is a prerequisite for the immobilization of an enzyme by covalent binding to a solid surface [11,17]. Other surface modifications using e.g., silanized agents based on silanizing with organosilanes, occur by reaction between the reactive groups of alkoxy (methoxy, ethoxy) alkoxysilanes present in mono-, bi-, or tri-functional groups and the silanol on the support surface [22,23]. In addition, bifunctional agents or spacer arms (epichlorohydrin, glutaraldehyde, glyoxal, formaldehyde, carbodiimide, ethylenediamine, glycidol, carbonyldiimidazole, and others) are also used for the surface modification of silica, their function being to promote a strong attachment between the support (silanized or otherwise) and the immobilized enzyme. ...
Treated silica xerogel with protic ionic liquid (PIL) and bifunctional agents (glutaraldehyde and epichlorohydrin) is a novel support strategy used in the effective immobilization of lipase from Burkholderia cepacia (LBC) by covalent binding. As biocatalysts with the highest activity recovery yields, LBC immobilized by covalent binding with epichlorohydrin without (203%) and with PIL (250%), was assessed by the following the hydrolysis reaction of olive oil and characterized biochemically (Michaelis–Menten constant, optimum pH and temperature, and operational stability). Further, the potential transesterification activity for three substrates: sunflower, soybean, and colza oils, was also determined, achieving a conversion of ethyl esters between 70 and 98%. The supports and the immobilized lipase systems were characterized using Fourier transform infrared spectra (FTIR), scanning electron microscopy (SEM), elemental analysis, and thermogravimetric (TG) analysis.
... However, high conversion for syntheses of lactose fatty acid esters has been described. Zaidan et al. [65] reported conversions above 70% in the acylation of lactose with capric acid (molar ratio of 2:1) at 55 • C for 48 h, using acetone as solvent and lipase from Candida rugosa immobilized on mica with amino groups. Neta et al. [63] also reported conversions above 80% in the synthesis of lactose oleate (40 • C, 72 h reaction, stoichiometric ratio, initial reagent concentration of 83.3 mM) catalyzed by lipase B from Candida antarctica immobilized on chitosan and acrylic resin (commercial Novozym 435). ...
Short-chain alkyl esters and sugar esters are widely used in the food, pharmaceutical and cosmetic industries due to their flavor and emulsifying characteristics, respectively. Both compounds can be synthesized via biocatalysis using lipases. This work aims to compare the performance of commercial lipases covalently attached to dry acrylic beads functionalized with oxirane groups (lipases from Candida antarctica type B—IMMCALB-T2-350, Pseudomonas fluorescens—IMMAPF-T2-150, and Thermomyces lanuginosus—IMMTLL-T2-150) and a home-made biocatalyst (lipase from Pseudomonas fluorescens adsorbed onto silica coated with octyl groups, named PFL-octyl-silica) in the syntheses of short- and long-chain carboxylic acid esters. Esters with flavor properties were synthetized by esterification of acetic and butyl acids with several alcohols (e.g., ethanol, 1-butanol, 1-hexanol, and isoamyl alcohol), and sugar esters were synthetized by esterification of oleic and lauric acids with fructose and lactose. All biocatalysts showed similar performance in the syntheses of short-chain alkyl esters, with conversions ranging from 88.9 to 98.4%. However, in the syntheses of sugar esters the performance of PFL-octyl-silica was almost always lower than the commercial IMMCALB-T2-350, whose conversion was up to 96% in the synthesis of fructose oleate. Both biocatalysts showed high operational stability in organic media, thus having great potential for biotransformations.
... However, the free lipase is not favoured in industrial sectors because it is fragile and prone to denaturation under extreme conditions of pH, temperature and organic solvents. These drawbacks could be overcome by immobilization on solid supports, which can facilitate enhancing of catalytic stability, selectivity and reusability of the lipases (Zaidan et al., 2012). Based on this, the development of novel immobilized lipase to synthesize OPO-rich TAGs is essential. ...
In this study, Candida lipolytica lipase (CLL) was immobilized on magnetic multi-walled carbon nanotubes (mMWCNTs) via hydrophobic and cation-exchange interaction. The resultant immobilized CLL showed much better thermal stability, biocatalyst activity and easier recycling than did the free form. A method for efficient enzymatic acidolysis of tripalmitin (PPP) with oleic acid (OA), to produce OPO-rich TAGs, was developed, using the immobilized CLL as the biocatalyst. Under optimized conditions (2% water, 20 mg/ml of enzyme, 1:6 PPP/OA, 50 oC, 2 h), the content of OPO in the final product reached 46.5%. CLL@mMWCNTs had a better activity and manipulative stability than had commercial lipases. More importantly, the feasibility of CLL@mMWCNTs was also validated in the practical production of OPO-rich TAGs, using lard and restructured palm oil as the raw material. These results suggest that CLL@mMWCNTs is a promising biocatalyst for the OPO-rich TAGs production and will be helpful for the infant formula industry.
... Enzymatic synthesis have the advantages of higher selectivity, relatively lower temperatures (lower than 70°C), lower solvent toxicity, and easier separation of products compared to traditional chemical synthesis. Enzyme that have been used for synthesis of esters are subtilisin from Bacillus amyloliquefaciens (Rich, Bedell, & Dordick, 1995), lipase from Candida antarctica (Pedersen, Wimmer, Emmersen, Degn, & Pedersen, 2002), Candida rugosa (Zaidan et al., 2012) Mucor miehei (Degn, Pedersen, & Zimmermann, 1999), Humicola lanuginose (Ferrer, Cruces, Bernabe, Ballesteros, & Plou, 1999), Thermomyces lanuginosus (Tsavas et al., 2002), and alkaline protease from Streptomyces spp. (Kitagawa, Tokiwa, Fan, Raku, & Tokiwa, 2000). ...
The synthesis of glucose esters with palmitic acid, lauric acid and hexanoic acid using lipase enzyme was studied and their emulsion functionality in oil-in-water system were compared. Reactions at 3:1 M ratio of fatty acids-to-glucose had the highest conversion percentages (over 90% for each of the fatty acid). Initial conversion rate increased as substrate solubility increased. Ester bond formation was confirmed by nuclear magnetic resonance technique that the chemical shifts of glucose H-6 and α-carbon protons of fatty acids in the ester molecules shifted to the higher fields. Contact angle of water on esters’ pelleted surface increased as the hydrophobicity increased. Glucose esters’ and commercial sucrose esters’ functionality as emulsifiers were compared. Glucose esters delayed, but did not prevent coalescence, because the oil droplets diameter doubled during 7 days. Sucrose esters prevented coalescence during 7 days since the droplets diameter did not have significant change.
... Typical wave number for the ester (C=O) were in the ranges of 1716-1751 cm -1 (Coates, 2000;van den Broek & Boeriu, 2013). This was also confirmed by Boruczkowska et al. (2012), Zaidan et al. (2012) & Syamsul et al., (2010 that the wave number of the ester were 1715 cm -1 , 1723 cm -1 , and 1732 cm -1 , respectively. ...
Enzymatic synthesis of fructose oleic ester (FOE) in stirred tank reactor using the immobilized lipase may cause a physical damage of support matrix, which causes a leaching of lipase from matrix. The objective of this research was to evaluate the best condition of FOE synthesis in fluidized bed reactor (FBR) using the immobilized lipase on modified hydrophobic matrix. Surface of Amberlite IRA 96 was modified with 2-phenylpropionaldehyde to obtain hydrophobic surface. Candida rugosa lipase was immobilized on the modified matrix. Subsequently, it was used for FOE synthesis in FBR system. Factors, such as reaction time, substrate flow rate, amount of water adsorbent, and substrate molar ratio were evaluated based on the conversion of oleic acid into FOE. Results from FTIR analysis showed that modified matrix had a peak at a specific wavelength ∼1674 cm-1. It indicated that a group of imine (-C=N-) occurred after surface modification. The highest adsorption of lipase was obtained after adsorption for 45min (13.28±0.57mg lipase/g matrix), which was equivalent to 52.95±1.67%. The lipase activity was 73.66±5.35 U/g matrix. The best FOE synthesis condition was obtained at flow rate of 0.4mL/min, molecular sieve of 12%, substrate molar ratio (fructose: oleic acid) 1:4 for 48h. FOE was confirmed by TLC and FT-IR analysis, which they had Rf 0.72 and specific wave number of ∼1712 cm-1, respectively. FOE had emulsion capacity, emulsion stability, droplet size, and HLB value of 92.83±0.46%; 85.65±0.92%; 7.86±0.93μm; 11-16, respectively. Emulsion type of FOE was oil in water emulsion.
... The percentage of immobilized enzyme was calculated as follows (Eq. (1)) [27]. ...
A biosensor for measuring the antidiabetic potential of medicinal plants was developed by covalent immobilization of α-glucosidase (AG) enzyme onto amine-functionalized multi-walled carbon nanotubes (MWCNTs-NH2). The immobilized enzyme was entrapped in freeze-thawed polyvinyl alcohol (PVA) together with p-nitrophenyl-α-D-glucopyranoside (PNPG) on the screen-printed carbon electrode at low pH to prevent the premature reaction between PNPG and AG enzyme. The enzymatic reaction within the biosensor is inhibited by bioactive compounds in the medicinal plant extracts. The capability of medicinal plants to inhibit the AG enzyme on the electrode correlates to the potential of the medicinal plants to inhibit the production of glucose from the carbohydrate in the human body. Thus, the inhibition indicates the antidiabetic potential of the medicinal plants. The performance of the biosensor was evaluated to measure the antidiabetic potential of three medicinal plants such as Tebengau (Ehretis laevis), Cemumar (Micromelum pubescens), and Kedondong (Spondias dulcis) and acarbose (commercial antidiabetic drug) via cyclic voltammetry, amperometry, and spectrophotometry. The cyclic voltammetry (CV) response for the inhibition of the AG enzyme activity by Tebengau plant extracts showed a linear relation in the range from 0.423-8.29 μA, and the inhibition detection limit was 0.253 μA. The biosensor exhibited good sensitivity (0.422 μA/mg Tebengau plant extracts) and rapid response (22 s). The biosensor retains approximately 82.16 % of its initial activity even after 30 days of storage at 4 °C.
... When produced by enzymatic process, more mild conditions are required minimizing side reactions when compared to the chemical process. However, one of the major drawbacks of this process is the low solubility of the starting material leading in some cases to low productivity due to long reaction times [8][9][10][11][12][13][14][15][16]. An example of attempt to overcome the solubility problem is the use of sugar boronates as starting materials. ...
Chemical methods for the synthesis of sugar esters require the use of high temperatures in the presence of alkaline catalysts resulting in undesirable by-products and low regiosselectivity. Sugar-based fatty acid esters are non-ionic, biodegradable, odorless, non-irritating, non-toxic surfactants widely used pharmaceuticals, cosmetics, detergents, and food industry. Here in we report our results on enzymatic esterification of protected glucose (glucose ketal) with several fatty acids using the immobilized lipase from Rhizomucor miehei under batch and continuous flow conditions, to afford new glucose derivatives with potential surfactant properties. The results presented here shows that high conversions can be obtained in short reaction time.
Practical
... These issues can be addressed through the immobilization of the lipase on water insoluble carriers allowing the development of continuous processes, an easier separation of products, the reuse of the catalyst and, in some cases, the enhancement of lipase stability and its catalytic activity in non-aqueous media [2,3]. Enzymes can be immobilized by several techniques, such as entrapment [4], cross-linking [5], covalent bonding [6] and physical adsorption [7]. In the latter, the nature of the support is very important in determining the efficiency of the immobilization, as it can influence the conformation of the enzyme during adsorption [3]. ...
Lipases (triacylglycerol ester hydrolases) are enzymes used in several industrial applications. Enzymes immobilization can be used to address key issues limiting widespread application at industrial level. Immobilization efficiency is related to the ability to preserve the native conformation of the enzyme. MAPLE (Matrix Assisted Pulsed Laser Evaporation) technique, a laser deposition procedure for treating organic/polymeric/biomaterials, was applied for the deposition of lipase enzyme in an ice matrix, using near infrared laser radiation. Microscopy analysis showed that the deposition occurred in micrometric and submicrometric clusters with a wide size distribution. AFM imaging showed that inter-cluster regions are uniformly covered with smaller aggregates of nanometric size. Fourier transform infrared spectroscopy was used for both recognizing the deposited material and analyzing its secondary structure. Results showed that the protein underwent reversible self-association during the deposition process. Actually, preliminary tests of MAPLE deposited lipase used for soybean oil transesterification with isopropyl alcohol followed by gas chromatography–mass spectrometry gave results consistent with undamaged deposition of lipase.
... When produced by enzymatic process, more mild conditions are required minimizing side reactions when compared to the chemical process. However, one of the major drawbacks of this process is the low solubility of the starting material leading in some cases to low productivity due to long reaction times [8][9][10][11][12][13][14][15][16]. An example of attempt to overcome the solubility problem is the use of sugar boronates as starting materials. ...
Chemical methods for the synthesis of sugar esters require the use of high temperatures in the presence of alkaline catalysts resulting in undesirable by-products and low regiosselectivity. Sugar-based fatty acid esters are non-ionic, biodegradable, odorless, non-irritating, non-toxic surfactants widely used pharmaceuticals, cosmetics, detergents, and food industry. Here in we report our results on enzymatic esterification of protected glucose (glucose ketal) with several fatty acids using the immobilized lipase from Rhizomucor miehei under batch and continuous flow conditions, to afford new glucose derivatives with potential surfactant properties. The results presented here shows that high conversions can be obtained in short reaction time.
Practical application: The research of new non-toxic, environmentally friendly and biodegradable surfactants to replace chemicals has increased along with the development of green chemistry. Nonionic biosurfactants, such as sugar fatty acid esters, may be obtained by enzymatic synthesis, a simple manufacturing method for application in several areas, including food, cosmetic, detergent, and pharmaceutical industry. In this work we have described the uncommon esterification of a steric hindered secondary hydroxyl group on glucose ketal leading to new glucose derivatives with potential application as surfactants.
... Enzymes can be immobilized by several techniques, classified in four categories: physical adsorption onto an insoluble carrier [17], entrapment within a matrix [20], cross-linking [21] and covalent bonding [22] to an insoluble support. ...
Matrix Assisted Pulsed Laser Evaporation (MAPLE) is a thin film deposition technique derived from Pulsed Laser Deposition (PLD) for deposition of delicate (polymers, complex biological molecules, etc.) materials in undamaged form. The main difference of MAPLE technique with respect to PLD is the target: it is a frozen solution or suspension of the (guest) molecules to be deposited in a volatile substance (matrix). Since laser beam energy is mainly absorbed by the matrix, damages to the delicate guest molecules are avoided, or at least reduced. Lipase, an enzyme catalyzing reactions borne by triglycerides, has been used in biosensors for detection of β-hydroxyacid esters and triglycerides in blood serum. Enzymes immobilization on a substrate is therefore required. In this paper we show that it is possible, using MAPLE technique, to deposit lipase on a substrate, as shown by AFM observation, preserving its conformational structure, as shown by FTIR analysis.
... One of the most common approaches is immobilization http://dx.doi.org/10.1016/j.bej.2014.12.008 1369-703X/© 2014 Elsevier B.V. All rights reserved. of enzymes on a suitable carrier, addition of which, however, further increases the manufacturing cost [23,24]. The resulting high cost of enzyme production makes this method prohibitive for most commercial applications [25]. ...
... The amount of enzyme in the supernatant at each washing was calculated until no enzyme was leached. The percentage of enzyme immobilization was investigated as follows (Equation 1) [13] Enzyme immobilization (%) = (Amount of enzyme introduced / m) 100 % ...
α-glucosidase (AG) enzyme was immobilized onto amine functionalized multi-walled carbon nanotubes (MWCNTs-NH2) by treatment with cross linkers, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) via amide bonding. The immobilized AG was characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscope (SEM). The performance of the electrode was evaluated by Spectrophotometric method and cyclic voltammetric (CV) method using p-nitrophenyl-α-D-glucopyranoside (PNPG) as a substrate. The immobilized AG enzyme retains about 84.16% activity of its initial response even after 30 days when stored at 4°C.
... The SF at 55°C and pH 7.0 is higher for lipase QL immobilized in C6 than in Tx6 support. The thermal stability of these biocatalysts was also evaluated at 40°C in an organic system (100% t-BuOH with 4% of equilibrium moisture), trying to simulate a reaction medium appropriate for organic syntheses, for example, for sugar ester production [23,37]. The highest SF (21.4) is achieved in this case with lipase QL immobilized in C6 support ( Table 3). ...
Two carbonaceous–siliceous composite materials, produced by hydrothermal and carbon-ization processes, were evaluated as immobilization support for lipase from Alcaligenes sp. These materials exhibited similar chemical characteristics but their carbon content and porous characteristics were different, which explain the catalytic behavior and stabil-ity of the biocatalysts immobilized on them. Higher activity and immobilization selectivity was achieved with the microporous material that had higher carbon content. The lipase immobilized on the mesoporous material had a higher thermal stability at 55 °C, pH 7.0 or at 40 °C in tert-butanol, simulating the reaction conditions required for organic synthe-sis. Both biocatalysts were tested in the synthesis of palmitoyl ascorbate and they were compared with the commercial biocatalyst QLC. The synthesis conversions with the lipase immobilized in mesoporous materials and with the biocatalyst QLC were similar (50%), but only the former could be reused. These are promising biocatalysts for industrial applications.
Glycolipids are biocompatible and biodegradable amphiphilic compounds characterized by a great scientific interest for their potential applications in various technological areas, including pharmaceuticals, cosmetics, agriculture, and food production. This report summarizes the available synthetic methodologies, physicochemical properties, and biological activity of sugar fatty acid ester surfactants, with a particular focus on 6-O-glucose, 6-O-mannose, 6-O-sucrose, and 6′-O-lactose ones. In detail, the synthetic approaches to this class of compounds, such as enzymatic lipase-catalyzed and traditional chemical (e.g., acyl chloride, Steglich, Mitsunobu) esterifications, are reported. Moreover, aspects related to the surface activity of these amphiphiles, such as their ability to decrease surface tension, critical micelle concentration, and emulsifying and foaming ability, are described. Biological applications with a focus on the permeability-enhancing effect across the skin or mucosa, antimicrobial and antifungal activities, as well as antibiofilm properties, are also presented. The information reported here on sugar-based ester surfactants is helpful to broaden the interest and the possible innovative applications of this class of amphiphiles in different technological fields in the future.
Fermentation technology has been widely used in biotechnology sectors for achieving large demand of industrially relevant biomolecules. Industries are facing major challenges for exploring novel method for increasing productivity of biomolecules at a very low cost. Nanodrug delivery systems have been used for target‐specific therapy due to their unique ability to increase solubility and bioavailability. Nanomaterial with excellent physiochemical nature and antimicrobial potential is used for crop protection, bioremediation, and food processing. But scientists are facing a major challenge for using low‐cost processing methods to create biocompatible and nontoxic nanodelivery systems. Nowadays, the integration of microbial fermentation process with nanotechnology are preferred for developing biocompatible, safe, and nontoxic nanostructures. In this book chapter, the role of microbial fermentation process for production of significant biomolecules has been highlighted. The integrated approach of this fermentation technology with nanotechnology has been subsequently discussed for improving productivity of industrially significant biomolecules.
Human milk fat (HMF) is a natural complex lipid possessing a unique fatty acids composition and distribution. In order to mimic HMF, human milk fat substitutes (HMFSs) have been produced via the enzymatic or chemical modifications of natural lipids. HMFSs have attracted increasing attention as functional lipids owing to their beneficial effects on infant
’s growth and development. It is very difficult to prepare HMFSs that will exactly match HMF in all lipid compositions. However, it is possible to produce HMFSs by enzymatic reactions that closely resemble HMF. There are two approaches for HMFSs synthesis. The first approach is performed in one step by acidolysis of one triacylglycerol
with free fatty acids or interesterification between two triacylglycerols or between one triacylglycerol
and fatty acids methyl or ethyl esters. The second approach can be accomplished in two steps through hydrolysis and re-esterification with the acyl groups to be incorporated.KeywordsMilk fatTriacylglycerolAcidolysisInteresterificationHydrolysisEsterification
The biosynthesis of sugar esters, molecules with biosurfactant properties, can occur through the esterification of sugars with fatty acids by enzymatic catalysis. An alternative to reduce the impact of raw materials on the final cost of biosurfactant production and reuse industrial waste is to use residues from vegetable oil industries as source of FFA (Free Fatty Acid, such as oleic acid) and lignocellulosic residues of 2G ethanol as source of sugar (xylose). In this scenario, the present work aimed to model the production process of biosurfactants via heterogeneous biocatalysis by lipase, using oleic acid and xylose. Product separation and purification was performed using a sequence of precipitations (by adding ethanol, water and methyl ethyl ketone). Simulation was performed using the equation-oriented software EMSO (Environment for Modeling, Simulation and Optimization), which is CAPE-OPEN compliant. The percentage of biosurfactants in the product was around 86%, with recovery of 88% in the purification. Regarding the study of energy expenditure, it was observed a value of -604.1 kW of heat associated with cooling and a value of 137.6 kW associated with heating. Developed mathematical models successfully described the process. The initial economic analysis of the process indicates a minimum biosurfactant selling price of US$72.37/kg.
1,3-dioleolyl-2-palmitate (OPO) is an important component of the human milk fat. Its unique fatty acid composition and distribution play an important role in proper infant growth and development. Owing to this, it has been attracting researchers and manufacturers to synthesize and commercialize OPO as an important human milk fat substitute added to infant formulas. In this review, the role of OPO in human milk, the benefits of OPO (sn-2 palmitate)-supplemented infant formulas over the conventional infant formulas on infant growth, and lipase-catalyzed synthesis of OPO are discussed. Over the last 20 years of research on the benefits of OPO (sn2 palmitate)-supplemented infant formulas are summarized. Similarly, studies carried out on lipase catalyzed production of OPO for the last 21 years (1999–2019) are also done focusing on the raw materials, sn1,3-regiospecific lipases, immobilization materials, and solvents used in the laboratory-scale experiments. In addition, OPO-based products currently in the market and future research trends are briefly covered in this review.
Practical applications
This work focuses on lipase-catalyzed synthesis of 1,3-dioleoyl-2-palmitoylglycerol (the most abundant triacyl glycerol in human milk fat) and its benefits to infants when it is added in infant formulas. Over the last 20 years of published research from the literature are summarized and future research trends for efficient OPO synthesis are also covered. This will provide current and future researchers on the field with the necessary background information on OPO synthesis and design their research plans accordingly for cost-effective production of OPO and OPO-supplemented infant formulas.
This article presents a study of the interfacial properties of oil-in-water emulsions containing sugar esters and polysaccharides. Sucrose fatty acid esters were synthesized using immobilized Candida antarctica lipase B. A yield of 53.4% was obtained using 2-methyl-2-butanol and 1:3 molar ratio of sucrose:stearic acid. Equilibrium surface tension was 45 mN/m and low critical micellar concentration (CMC) value was obtained (ca. 10 mg/mL), characteristic of non-ionic surfactant. The interfacial properties of mixtures of sucrose esters and polysaccharides, at the oil-water interface were determined using a pendant drop tensiometer. Addition of polysaccharides increased the interfacial tension. Studies of interfacial viscoelasticity showed that the films were predominantly elastic. The presence of polysaccharides in emulsions resulted in flocculated droplets. All the emulsions presented great stability along 28 days with no creaming formation.
Continuous production of the sugar ester (SEs) of a medium-chain fatty acid was conducted in a column reactor packed with a strongly basic anion-exchange resin and its production behavior was compared with that of a conventional long-chain fatty acid SE. Using the resin catalyst under mild conditions (60 °C and atmospheric pressure) facilitated continuous production of the medium-chain fatty acid SE in almost 60% yield, even with a short residence time of 8 min. The productivity per unit time and total output before resin deactivation were determined as scale-up parameters. Both values were five times higher for medium-chain fatty acid SE production compared with long-chain fatty acid SE production. Based on these values, the required amount of the resin can be estimated according to the desired production volume. Furthermore, the resin catalyst activity decreased gradually during continuous production, but repeated resin use was achieved with periodic regeneration.
Microbial lipase from Burkholderia cepacia was immobilized by covalent bond and physical adsorption on SBA-15 mesoporous support and its catalytic efficiency was measured in the formation of fatty acids ethyl esters from palm kernel oil. The immobilized biocatalyst and its support were characterized by X-ray Diffraction, Fourier Transform Infrared Spectroscopy, N2 Adsorption–desorption, and thermal analysis TG/DTG. The immobilization of the enzyme onto the support maintained the typical structure of the SBA-15. From the TG measurements, it can be deduced that about 25% of lipase was immobilized in SBA-15 via covalent bond and 15% was incorporated via physical adsorption. Both immobilized lipase preparations were used to perform the transesterification reactions using different oil: ethanol molar ratios and time. The biocatalyst prepared by physical adsorption promoted better results, measured by the apparent yield (> 90%) and conversion (98.9%) in all reactions, which were also confirmed by viscosity and density data. The regeneration of SBA-15 support was found to be feasible and demonstrated efficiency for 5 times, reducing the associated costs of the process.
Graphic Abstract
Free enzymes employed as biological catalysts have many advantages such as low reaction time, involving low energy and less waste output when compared to conventional chemical catalysts. However, commercial utilization of free enzymes is often hampered by the lack of operational stability, high cost, and non‐reusability. Immobilization of enzyme is an option to overcome these obstacles. Immobilized enzyme expresses stable performance in organic solvents even in adverse pH, which makes the biomolecules reusable and prosperous as a biological catalyst. Biological catalysis with immobilized enzymes is found to be an alternative method instead of chemical catalysis for chemical reactions in the foreseeable future. Sources of lipase, techniques in immobilization and cross‐linkers are dealt with in this paper. Lipases are used to catalyze transesterification in the production of biodiesel. Commercial use of free enzymes is impracticable, but immobilization is able to overcome many of the disadvantages. As such, immobilized enzymes provide a reasonable alternative to chemical catalysts. This first part of a two‐part paper discusses the lipases and the immobilization techniques.
Carriers used in the immobilization process play a major role in enhancing the properties of the biocatalyst. Polymers are used as organic carriers for enzyme immobilization to improve the thermal, chemical, and operational stability of the enzyme. A variety of carriers are used in the immobilization of enzymes, e.g., mica, silica, zeolites, hydrotalcites, activated carbon, gold and magnetic nanoparticles. Silica‐based carriers offer suitable matrices for enzyme immobilization to manufacture industrial products. Immobilization on nanoparticles has become attractive in biocatalytic applications due to the combination of physical, chemical, catalytic, electronic, and optical properties. Gold nanoparticles also have received attention in the preparation of biocatalysts due to the above‐mentioned reasons. Magnetic nanoparticles are used as a carrier since they can be isolated by an external magnetic field, which is of special interest in the synthesis of organic products such as biodiesel. Different carrier materials involved in the immobilization of enzymes are discussed in this paper. In enzyme immobilization, the carrier plays a major role since it influences the properties of the catalyst. It also has to offer specific characteristicts to establish stable interactions between the enzyme and the carrier. Different carrier materials currently in use in the industry are reviewed in this second part of the lipase review, among others, polymers, silica‐based carriers, magnetic nanoparticles are discussed.
Lactose fatty acid esters are high value-added derivatives of lactose and represent a class of biodegradable, nonionic, low molecular weight surfactants (emulsifiers) that have considerable potential in the food, cosmetic, and pharmaceutical industries. Certain lactose esters have also garnered attention for their biological activities. In this work, we detail syntheses of a homologous series of 6ʹ-O-acyllactose esters of varying alkyl chain length (from 6 carbons to 18 carbons) and report on their activities as surfactants as well as their anti-microbial and cytotoxic properties. The structure-property profiles established in this work reveal that while the medium-chain esters display excellent emulsifying properties and moderate antimicrobial activities, their longer-chain congeners exhibited the highest cytotoxicities. As such, we have established that certain 6ʹ-O-acyllactose esters are superior to their sucrose-derived and commercially exploited counterparts. These results will serve as a useful guide for the development of lactose esters as, inter alia, emulsifiers in the food industry.
Surfactants are an essential part of detergent or emulsifier formulations and find applications in many cleaning, cosmetic and pharmaceutical products. For many reasons, the trend today is going towards the use of biosurfactants. Among them glycolipids are a highly interesting group of substances and have gained considerable interest. Being biocompatible and biodegradable, they can be obtained either by chemical synthesis, microbial fermentation processes, by enzymatic synthesis or by mixed chemical and biotechnological methods fully originating from renewable resources. Recently, there is much interest in having tailor tailor-made glycolipid surfactants for various applications, but nevertheless the spectrum of substances available is still restricted and their use in industry is still limited, e.g.for example due to high prices and unrecognizedunrecognised structure-function relationships. Here we present a review on existing strategies and methods for the enzymatic synthesis of glycolipid surfactants considering their advantages and still exiting limitations.
To increase the functionality and broaden the potential application of neokestose, neokestose laurate was bio-synthesized using Candida antarctica lipase B as biocatalyst, for which a mixture of 20% DMSO in 2-methyl-2-butanol (v/v) was chosen as reaction medium. The optimum conditions for biosynthesis were as follows: molar ratio of vinyl laurate to neokestose at 12, temperature at 50 °C, molecular sieves at 100 g/L, and enzyme loading at 10 g/L. Under the optimal conditions, the conversion rate was achieved over 80%. The synthesized chemical 6’-O-lauroylneokestose confirmed by nuclear magnetic resonance (NMR) exhibited good emulsification with critical micelle concentration (CMC) of 352 μM, and broad antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus, Listeria monocytogenes, Streptococcus mutans, Bacillus subtilis and Bacillus cereus. Conclusively, 6’-O-lauroylneokestose was evidenced to be a dual-functional agent with emulsification and antibacterial activity, showing promising application potential in food industry.
In this study, nanocellulose (NC) was successfully extracted from oil palm frond leaves (OPFL) using a combination of bleaching, alkaline treatment and acid hydrolysis. X-ray diffractogram revealed the extracted NC was crystalline with a crystallinity index of 70.2%. This indicates its suitability as nano-fillers for preparing the chitosan/nanocellulose (CS-NC) supports to immobilize Candida rugosa lipase (CRL) to produce the CRL/CS-NC biocatalysts. FTIR, FESEM and TGA characterizations of the CRL/CS-NC confirm the CRLs were successfully conjugated to the CS-NC supports. The air-dried CS-NC supports gave satisfactory immobilization of the CRLs (5.2 mg/g) with the resultant CRL/CS-NCs catalysed conversions of ≥80% of butyl butyrate within 6 h. Time course reaction profile revealed that 76.3% butyl butyrate conversion was achieved at 4 h immobilization time using 3 mg/mL of CRL/CS-NCs. NMR analyses on the purified butyl butyrate confirmed that the ester was successfully synthesized.
An inexpensive carrier of mica clay finds the potential use as an enzyme
matrix for the lipase immobilization. Despite its low cost, industrial applications of
lipase require efficient methods to immobilize the enzyme, to yield a biocatalyst with
high-lipase productivity and stability rather than a free lipase. Objectives: This paper
suggests an immobilization of lipase from Candida rugosa (CRL) onto a mica-based
carrier through different glutaraldehyde activation approaches of immobilization and
evaluation of their use in biocatalytic system. Results: Immobilized lipase preparations
of enzyme-aggregate-coating (EAC-CRL) and nanoscale-enzyme-reactor (NER-CRL)
had demonstrated of high-protein loadings of 8.46 and 8.65 mg/g carrier and specific
esterification activities of 91.0 and 91.34 U/mg, respectively. Furthermore, both
immobilized lipases were successfully performed in the synthesis of fatty acid sugar
ester (FASEs) with optimized reaction parameters studied (time course, fatty acid chain
length and organic solvent) in comparison to free lipase. Conclusion: Efficiency of the
immobilization process has enhanced the biocatalyst potential in numerous
biotechnological applications.
The lipase catalysed synthesis of xylose caproate ester was performed by condensation of xylose, an aldopentose and caproic acid in organic solvents. A dual-solvent system containing DMSO and acetone (1:10 v/v) was used to determine the optimal conditions for the reaction. Different reaction parameters (solvent system, reaction time, substrate molar ratio and the amount of enzyme loaded) were studied. The highest conversion rate (64%) was obtained within 24 hours with the optimal conditions of 16% (w/v) Novozym 435 and a molar ratio of xylose to caproic acid of 1:4.
Matrix Assisted Pulsed Laser Evaporation (MAPLE) is a thin film deposition technique which uses a pulsed laser beam impinging, inside a high vacuum chamber, on a frozen target containing the guest molecules in a volatile matrix to induce fast “evaporation” of the matrix, and ejection of the guest molecules. Lipase, an enzyme acting as a catalyst in hydrolysis of lipids, is widely used in biosensors for detection of triglycerides in blood serum. A key action to this purpose is lipase immobilization on a substrate. In a recent paper, we have shown that MAPLE technique is able to deposit lipase on a substrate in an active form. Here we show that addition to the guest/matrix target of a small amount of m-DOPA (3-(3,4-dihydroxyphenyl)-2-methyl-l-alanine) in order to improve adhesion and protect lipase secondary structure, also allows the lowering the laser pulse energy required for matrix evaporation and therefore the risk of damaging the enzyme.
In this work it was evaluated for the first time, the ester synthesis catalyzed by the phospholipase Lecitase-Ultra immobilized styrene-divinylbenzene beads (MCI-Lecitase), comparing the mechanical stirring and the ultrasonic energy. It was studied the specificity of the enzyme using carboxylic acids from C4 to C18, as well as the effects of alcohol chain, organic solvents, biocatalyst content, reaction temperature and substrate concentration. Caprylic and myristic acids were those with the highest reaction rates and yields, using ethanol as substrate. The shorter the alcohol chain, the higher the enzyme activity. Regarding the secondary alcohols, while MCI-Lecitase had no activity versus isopropanol, using 2-pentanol the activity was similar to that with 1-pentanol. Comparing the agitation systems, MCI-Lecitase presented an initial reaction rate more than 2-times higher in the ultrasound-assisted reaction than under traditional mechanical stirring. Moreover, under ultrasonic energy the maximum rate was achieved using 0.5 M of substrates, while under mechanical stirring the maximum enzyme activity was reached at 0.3 M of substrates. Concerning the operational stability, MCI-Lecitase was quite unstable, losing its activity after 6 reaction cycles. By adding molecular sieves in the reaction medium, MCI-Lecitase retained 30% of its initial activity after 6 cycles.
Enzyme-support strategies are increasingly replacing conventional chemical methods in both laboratories and industries with attributes including efficiency, higher performance and multifarious use, where silica surfaces show potential due to the chemical bonds based on the presence of hydroxyl groups which can be modified with different additives. Surface-modified silica is a novel class of materials capable of improving enzyme stability and reusability that can be applied to support several immobilization techniques. This review describes the use of innovative modified supports to improve the state of enzyme immobilization and provide the industrial sector with new perspectives.
A series of novel isomannide-based fatty acid monoesters 3(a-f) were synthesized by employing a highly active, water resistant and easily recoverable carbon-based solid acid catalyst derived from glycerol. The mannitol was reacted with decanoic, lauric, myristic, palmitic, stearic and oleic acids in presence of carbon acid catalyst under solvent free conditions to obtain corresponding isomannide fatty acid monoesters involving in situ dehydration of mannitol to isomannide followed by acylation. The optimized reaction conditions for obtaining isomannide monoesters are: fatty acid to mannitol mole ratio (1:1.5), catalyst 20 wt% of mannitol, temperature 180 °C and reaction time of 12 h. The carbon acid catalyst was recovered by filtration and reused for five cycles without losing its catalytic activity. The use of a recyclable solid acid catalyst makes this method more convenient, simple, and cost effective in addition to high selectivity with good yields. All the synthesized compounds were further evaluated for their surface active properties such as, critical micelle concentration (CMC), surface tension at the CMC (γCMC), surfactant concentration required to reduce the surface tension of the solvent by 20 mN/m (pC20), maximum surface excess (τmax), and the interfacial area occupied by the surfactant molecules (Amin) using surface tension measurements. The micellization (∆G°mic) and adsorption free energies (∆G°ads) were calculated. Isomannide monomyristate (3c) and isomannide monolaurate (3b) exhibited superior surface active properties followed by isomannide monopalmitate (3d) compared to other isomannide monoesters.
Sugar esters are considered as surfactants due to its amphiphilic balance that can lower the surface tension in oil/water mixtures. Enzymatic syntheses of these compounds are interesting both from economic and environmental considerations. A study was carried out to evaluate the effect of four solvents, temperature, substrate molar ratio, biocatalyst source and immobilization methodology on the yield and specific productivity of lactulose palmitate monoester synthesis. Lipases from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL), immobilized in porous silica functionalized with octyl groups (adsorption immobilization, OS) and with glyoxyl-octyl groups (both adsorption and covalent immobilization, OGS) were used. The highest lactulose palmitate yields were obtained at 47ºC in acetone, for all biocatalysts, while the best lactulose: palmitic acid molar ratio differed according to the immobilization methodology, being 1:1 for AsL-OGS biocatalyst (20.7 ± 3%) and 1:3 for the others (30-50%).
A block copolymer of 2-dimethylaminoethyl methacrylate (DMAEMA) and glycidyl methacrylate (GMA) was grafted onto the surface of magnetic nanoparticles (Fe3O4) via atom transfer radical polymerization. The resultant PGMA-b-PDMAEMA-grafted-Fe3O4 magnetic nanoparticles with amino and epoxy groups were employed to immobilize the lipase from Burkholderia cepacia successfully by physical adsorption and covalent bonding. Biochemical characterization shows that the immobilized lipase exhibits improved thermal stability, good tolerance to organic solvents with high log P, and higher pH stability than the free lipase at pH 9.0. After six consecutive cycles, the residual activity of the immobilized lipase is still over 55% of its initial activity.
Oils, fats, carbohydrates, lignin, and amino acids are all important raw materials for the production of biorenewables. These compounds already play an important role in everyday life in the form of wood, fabrics, starch, paper and rubber. Enzymatic reactions do, in principle, allow the transformation of these raw materials into biorenewables under mild and sustainable conditions. There are a few examples of processes using immobilised enzymes that are already applied on an industrial scale, such as the production of High-Fructose Corn Syrup, but these are still rather rare. Fortunately, there is a rapid expansion in the research efforts that try to improve this, driven by a combination of economic and ecological reasons. This review focusses on those efforts, by looking at attempts to use fatty acids, carbohydrates, proteins and lignin (and their building blocks), as substrates in the synthesis of biorenewables using immobilised enzymes. Therefore, many examples (390 references) from the recent literature are discussed, in which we look both at the specific reactions as well as to the methods of immobilisation of the enzymes, as the latter are shown to be a crucial factor with respect to stability and reuse. The applications of the renewables produced in this way range from building blocks for the pharmaceutical and polymer industry, transport fuels, to additives for the food industry. A critical evaluation of the relevant factors that need to be improved for large-scale use of these examples is presented in the outlook of this review.
Lipase from Candida rugosa was immobilized onto natural kaolin by physical adsorption method. About 77% of protein content was immobilized onto the support. The activities of the immobilized lipase were determined by the esterification activities using oleic acid and 1-butanol as substrates and hexane as reaction medium. The effects of reaction temperature, thermostability, stability in organic solvent, leaching and storage studies of immobilized lipase were investigated. Kaolin-immobilized lipase exhibited activities higher by fourfolds than the native lipase after thermal stability test at 70 °C. Immobilized lipase was found to be stable in hexane at room temperature up to 12 days and also showed higher stability than native lipase in the storage study. Leaching studies showed that the immobilized lipase remained full activity even after being washed by 20 ml of solvent. The experimental results showed that physical adsorption is suitable for the attachment of enzyme onto kaolin.
Aspergillus terreus lipase has exhibited novel capability of catalyzing esterification reaction between fatty acids (C 4 -C 18) and primary, secondary and tertiary monohydric alcohols. Although, the lipase efficiently catalyzed the esterification of saturated stearic acid (C 18:0), it failed to accept the monounsaturated oleic acid (C 18:1) as the substrate which is also a C 18 acid, but has a double bond. Thus, this enzyme has a great potential to be used as a selective catalyst for the separation of almost identical saturated and unsaturated fatty acids in their mixtures. Extending this work, highly efficient and regioselective conversion of sorbitol to its 1(6) -monostearate has been achieved by Aspergillus terreus lipase -mediated esterification in n-hexane. Ninety four per cent conversion to the ester was achieved in 12 h on optimizing four physico-chemical factors, i.e. molar ratios of substrates, temperature, solvent and water activity. Aspergillus terreus lipase immobilized on Accurel was efficient in the synthesis of sorbitol stearate and was reused three times without any significant decline in its activity, yields were further enhanced to 96 % on using the immobilized enzyme.
With current developments in enzyme-catalyzed reactions and techniques available for rational redesign of natural biocatalysts,
the enzymatic biosynthesis can become one of the most valuable synthetic methods. Enzymatic regioselective catalysis in organic
media has played a key role in pursuing asymmetric synthesis for active chiral compounds. Here, we shortly describe some historical
issues of the rapidly growing area, enzymatic catalysis in synthetic organic chemistry and then review researches that have
been carried out in the regioselective enzymatic catalysis for the past two decades. An application of this technology to
the modification of some potential target drug compound will be also presented.
The immobilization of Penicillin acylase (PGA) enzyme (which belongs to the Ntn hydrolase super family) into the amino-functionalized SBA-15 mesoporous molecular sieve is carried out to see the effect of silica as a host matrix on the enzyme kinetics. Physicochemical characterization by nitrogen adsorption, powder XRD and TEM methods indicate that the characteristic hexagonal features and the original pore structure of the parent SBA-15 is retained even after the incorporation of PGA. The adsorption of PGA on SBA-15 shows a dependence on the pore volume and the composition of the adsorbent. The maximum loading of the enzyme was observed at pH 7.8, slightly below the isoelectric point of the enzyme. The loading capacity of immobilized PGA is 34 mg protein per 0.5 g of SBA-15. The trapped enzyme is more stable than the soluble form to temperature and pH environments and retained 73% of its activity after immobilization. This enhanced stability is attributed to the protective nature of the cage itself and to the rigidity of the SiO2 matrix, which reduces the freedom of peptide-chain refolding of molecular motions that occur in denaturation processes. The strength of binding is very strong; however, the activity of the immobilized enzyme is then simply restored with very little leakage of enzyme from the support. An important feature of the immobilized PGA enzyme is the excellent reusability without significant loss in activity, which indicates potentially exciting industrial/biomedical application of this support.
The synthesis of wax ester using refined, bleached and deodorized (RBD) palm oil and oleyl alcohol catalyzed by lipozyme IM was carried out. Response surface methodology (RSM) based on a five-level, four-variable central composite rotatable design (CCRD) was used to evaluate the interactive effects of synthesis, of reaction time (2.5–10 h), temperature (30–70 °C), amount of enzyme (0.1–0.2 g) and substrate molar ratio (palm oil to oleyl alcohol, 1:1–1:5) on the percentage yield of wax esters. The optimum conditions derived via RSM were: reaction time 7.38 h, temperature 53.9 °C, amount of enzyme 0.149 g, and substrate molar ratio 1:3.41. The actual experimental yield was 84.6% under optimum condition, which compared well to the maximum predicted value of 85.4%.
Bentonite is an inexpensive matrix for enzyme immobilization and has been frequently utilized for this purpose. In addition to its low cost, bentonite has several advantages for use as a support, including its lack of toxicity and chemical reactivity, allowing easy fixation of enzymes. In this article, Candida rugosa lipase was non-covalently immobilized on bentonite. The properties of immobilized enzyme were defined. Optimum pH and water content were determined as 7.5 and 100 μL, respectively. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. Half-lives of the immobilized enzyme at 50, 60, and 70 °C were 45, 15, and 4 min, respectively, whereas for the soluble free lipase it was 17 min at 50 °C. Kinetics were tested at 37 °C following the hydrolysis of olive oil and obeys the Lineweaver–Burk type of rate equation. The Km was 4 × 10−2 mM and the Vmax was 15 mmol h−1 mg−1.
An enzymatic synthesis was developed on a new set of D-glucuronic acid esters and particularly the tetradecyl-D-glucopyranosiduronate also named tetradecyl D-glucuronate. Chromatographic analyses revealed the presence of the ester as a mixture of anomeric forms for carbon chain lengths superior to 12. TOF/MS and MS/MS studies confirmed the synthesis of glucuronic acid ester. The NMR study also confirmed the structure of glucuronic acid esters and clearly revealed an anomeric (α/β) ratio equivalent to 3/2
Immobilization of biologically active proteins on nanosized surfaces is a key process in bionanofabrication. Carbon nanotubes with their high surface areas, as well as useful electronic, thermal and mechanical properties, constitute important building blocks in the fabrication of novel functional materials.
Lipases from Candida rugosa (CRL) were found to be adsorbed on the multiwalled carbon nanotubes with very high retention of their biological activity (97%). The immobilized biocatalyst showed 2.2- and 14-fold increases in the initial rates of transesterification activity in nearly anhydrous hexane and water immiscible ionic liquid [Bmim] [PF6] respectively, as compared to the lyophilized powdered enzyme. It is presumed that the interaction with the hydrophobic surface of the nanotubes resulted in conformational changes leading to the 'open lid' structure of CRL. The immobilized enzyme was found to give 64% conversion over 24 h (as opposed to 14% with free enzyme) in the formation of butylbutyrate in nearly anhydrous hexane. Similarly, with ionic liquid [Bmim] [PF6], the immobilized enzyme allowed 71% conversion as compared to 16% with the free enzyme. The immobilized lipase also showed high enantioselectivity as determined by kinetic resolution of (+/-) 1-phenylethanol in [Bmim] [PF6]. While free CRL gave only 5% conversion after 36 h, the immobilized enzyme resulted in 37% conversion with > 99% enantiomeric excess. TEM studies on the immobilized biocatalyst showed that the enzyme is attached to the multiwalled nanotubes.
Successful immobilization of enzymes on nanosized carriers could pave the way for reduced reactor volumes required for biotransformations, as well as having a use in the construction of miniaturized biosensensor devices.
Unusually versatile substrate specificity is shown by lipases. Not only do they hydrolyze triacylglycerols—for example, in the stomach and intestine during digestion of dietary fat—and various synthetic esters and amides, but their high stability in organic solvents permits their use in transesterification reactions and ester synthesis as well. Reactions based on lipase catalysis usually proceed with high regio- and enantioselectivity. Thus, the Ca²⁺ antagonist diltiazem (1) was obtained with lipase from Serratia marcescens. Over 30 lipases have been cloned in the last few years. Since the tertiary structure of 12 lipases is known, there are presently significant efforts to improve this class of enzymes by protein engineering techniques, in view of their use in detergents and other fields of industrial application.
Three enzymes were immobilized onto polyionic hydrogel, ChitoXan, obtained by complexation between chitosan and xanthan. The biocatalysts used were two proteases (protease type XIX from Fungal d’Aspergillus sojae and the trypsin type II.S from Porcine Pancreas) and a lipase (lipase Type VII from Candida rugosa). The immobilization efficiencies and the relative activities were investigated for these enzymes. The immobilization efficiencies changed with each enzyme and varied between 53 and 80%. Good relative activities were found for the lipase Type VII from Candida rugosa and the protease type XIX from Fungal d’Aspergillus sojae. For the latter, the influence of several factors were studied: molarity of the storage buffer, storage temperature and time of hydrogel, and the enzyme concentration. For the immobilized lipase, hydrolysis of olive oil in aqueous and organic media has been compared. This study confirmed that the lipase modified the external and internal structure of the hydrogel from fibrillar to the formation of globular structures in the presence of lipases.
The lipase-catalyzed esterification of partially acetylated sucrose has been studied. It was shown that the chemical acetylation increased the reaction rate of the subsequent enzymatic acylation. Thus it was possible to perform the enzymatic acylation in the absence of solvents while underivatized sucrose did not react. From HMBC NMR spectroscopy, it was concluded that the main site of acylation was the 6-position of the fructose moiety.
We have investigated the use of the glutaraldehyde chemistry to stabilize glutaryl acylase (GAC). GAC has been immobilized on different aminated supports (ethylendiamine (EA) or polyethylenimine (PEI) coated supports) and the effect of the treatment with glutaraldehyde on both stability and activity has been analyzed. It was determined that immobilization on aminated supports increased the enzyme stability, and that this stabilization increased with the size of the polyethylenimine. The treatment with glutaraldehyde presented a low impact on the enzyme activity (activity recoveries were over 80%) and greatly improved the enzyme stability. A similar treatment using the enzyme immobilized on supports that cannot react with glutaraldehyde did not give rise to stabilization, suggesting that this stabilization was due to a reaction between the enzyme and the support through the glutaraldehyde chemistry. Curiously, the larger the PEI, the lower the stabilization observed. Therefore, final differences between different immobilized GAC preparations treated with glutaraldehyde were not very significant. Other variables, such as glutaraldehyde concentration, were found to have a great impact on the final results (optimal concentration was in the range 0.5–0.65%). After optimization, the stability of the best immobilized GAC was over 250-fold higher than that of the soluble enzyme, retaining 90% of the immobilized activity and much more stable than commercially available GAC preparations or the enzyme immobilized on pre-activated supports. The simple preparation of this immobilized GAC, its good activity and stability, make this strategy very suitable for its industrial implementation.
In this review, a comprehensive and illustrative survey is made of the regioselective synthesis of esters of sugars and related compounds using lipases. The main emphasis has been given to the screening and use of commercially available lipases for the enzymatic esterification of neutral monosaccharides, disaccharides, sugar alcohols and their selected ether and ester derivatives. The effect of solvents and solubilizing agents in improving the yields of the resultant sugar fatty acid esters has been incorporated. Further, solvent‐free esterification with molten fatty acids, use of ionic liquids and microwave radiations for improvement in the methodology have also been discussed. Copyright © 2006 Society of Chemical Industry
Formation of oligosaccharides, especially disaccharides, from heating lactose in solid state and in molten state was examined. In heated lactose in solid state, lactulose, 3-0-β-D-galactopyranosyl-D-glucose, and 6-0-β-D-galactopyranosyl-D-glucose were detected. In the molten state, 6-0-β-D-galactopyranosyl-D-galactose was detected in addition to the former three disaccharides. The identity of these compounds produced from lactose was established by methylation analysis and hydrolysis.
The aim of this work was to determine the enzymatic activities resulting from the adsorption of Rhizomucor miehei lipase (RML) and Candida cylindracea lipase (CCL) onto three different phyllosilicates (sepiolite, palygorskite and montmorillonite), comparing the resultant activities with those obtained following similar immobilisation technique on a widely used resin (Duolite A-568). Due to the different adsorption mechanisms produced, different derivatives with higher hydrolytic activities can be obtained. Comparing the clays tested, the results showed that, in comparison with the laminar silicate (montmorillonite sample) and Duolite A-568 (spherical particles), fibrous materials (palygorskite and sepiolite) resulted in derivatives with higher hydrolytic activities in the hydrolysis of different ethyl esters. Moreover, according to the data obtained with the electrophoresis, the selectivity of immobilisation for RML in the case of fibrous silicates was optimal. As a conclusion, and according to the activities and selectivities measured, at least two out of the four studied materials (sepiolite and palygorskite) would be useful as supports for immobilisation for proteins of relatively low molecular weight (such as RML) for further use in biotransformations, while for C. cylindracea the immobilisation onto duolite rendered a derivative specially active in the hydrolysis of ethyl formiate (esterasic activity). (C) 2001 Elsevier Science B.V. All rights reserved.
Regioselective esterification and acetalation reactions of lactose have produced partly protected derivatives which have significant value in the further modification of lactose and as precursors for the synthesis of higher oligosaccharides.
Unusually versatile substrate specificity is shown by lipases. Not only do they hydrolyze triacylglycerols—for example, in the stomach and intestine during digestion of dietary fat—and various synthetic esters and amides, but their high stability in organic solvents permits their use in transesterification reactions and ester synthesis as well. Reactions based on lipase catalysis usually proceed with high regio- and enantioselectivity. Thus, the Ca2+ antagonist diltiazem (1) was obtained with lipase from Serratia marcescens. Over 30 lipases have been cloned in the last few years. Since the tertiary structure of 12 lipases is known, there are presently significant efforts to improve this class of enzymes by protein engineering techniques, in view of their use in detergents and other fields of industrial application.
The enantioselective hydrolysis of (3RS,4RS)-trans-4-(4′-fluorophenyl)-6-oxo-piperidin-3-ethyl carboxylate (±)-2 was effected using a commercial preparation of lipase from C. antarctica A (CAL-A). We found that the hydrolytic activity of the lipase (immobilized on a number of very different supports) with this substrate was negligible. However, a contaminant esterase with Mw of 52 KDa from this commercial preparation exhibited much higher activity with (±)-2. This enzyme was purified and immobilized on PEI-coated support and the resulting enzyme preparation was highly enantioselective in the hydrolysis of (±)-2 (E >100), hydrolyzing only the (3S,4R)-(−)-3, which is a useful intermediate for the synthesis of pharmaceutically important (−)-paroxetine. Optimization of the reaction system was performed using a racemic mixture with a substrate concentration of 50 mM. This enzyme preparation was used in three reaction cycles and maintained its catalytic properties.
Synthesis of layered double hydroxides (LDHs) of Mg/Al-NO3− and Mg/Al-sodium dodecyl sulphate (SDS) of molar ratio of Mg/Al (4:1) were done by co-precipitation through continuous agitation. Their structures were determined using X-ray diffractometer (XRD). The spectra showed that basal spacing for synthesized Mg/Al-NO3− and Mg/Al-SDS were around 7.8 and 34.3 Å, respectively. The expansion of layered structure was observed to accommodate the surfactant anion between the interlayer for the Mg/Al-SDS. Lipase from Candida rugosa was immobilised onto these materials by physical adsorption method. The percentage of protein loaded into Mg/Al-SDS (70.8%) is higher than Mg/Al-NO3− (36.9%). The synthesis of butyl oleate using immobilised lipase as biocatalyst was investigated through esterification of oleic acid and 1-butanol as substrate and hexane as reaction medium. The effects of surface area, reaction temperature, thermal stability, leaching study, stability in organic solvent and storage were investigated. The stability was found to be the highest in the Mg/Al-SDS.
The acylation of several pyranoses and fructose with fatty acids by lipases in a solid-phase system was investigated. Highest selectivity was found using lipase from Candida antarctica B immobilized on polypropylene EP 100. For β-D(+)-glucose 98% mono- and 2% diacylation using palmitic acid in t-butanol were obsd. Much lower selectivities were achieved with D-(+)-mannose (75% monoacylation) and D(+)-galactose (50% monoacylation), which was explained by the different orientation of the C2-hydroxyl groups. The selectivity in the acylation of fructose was significantly influenced by the fatty acid chain length and the org. solvent. Monoacylation was favored in less hydrophobic solvents (e.g. t-butanol) using long chain fatty acids. [on SciFinder(R)]
Selective lipase-catalyzed synthesis of glucose fatty acid esters in two-phase systems consisting of an ionic liq. (1-butyl-3-Me imidazolium tetrafluoroborate [BMIM][BF4] or 1-butyl-3-Me imidazolium hexafluorophosphate [BMIM][PF6]) and tert-butanol as org. solvent was investigated. The best enzyme was com. available lipase B from Candida antarctica (CAL-B), but also lipase from Thermomyces lanuginosa (TLL) gave good conversion. After thorough optimization of several reaction conditions (chain-length and type of acyl donor, temp., reaction time, percentage of co-solvent) conversions up to 60% could be achieved using fatty acid vinyl ester as acyl donors in [BMIM][PF6] in the presence of 40% t-BuOH with CAL-B at 60°C. [on SciFinder(R)]
This review describes a recently developed method for enzymatic synthesis with mainly undissolved substrates at very high concentrations. At the end of the reaction, up to 80% (w/w) reaction mixture is product, a fact which is promising in terms of industrial applications and large-scale systems. In comparison with other approaches for enzymatic peptide synthesis, this method gives a very high mass of product combined with good reaction yields and rates. We will analyze the historical development from two different directions which have been reported; these are described with terms like “solid-to-solid conversion” and “heterogenous eutectic mixtures”. The reaction requires and takes place in a liquid phase which, however, may be of very small volume. This review analyzes in detail the function of additional water or organic solvents (around 10% w/w). The liquid phase formed is usually dependent on these third components; however, it can also result from a eutectic two-substrate mixture. We summarize what is known so far about the thermodynamics and kinetics. Finally, the possibility for and main problems of scale up are discussed.
The enzymatic synthesis of methyl adipate via green esterification of adipic acid and methanol in hexane has been studied. Lipase from Candida rugosa immobilised onto various layered double hydroxides (LDHs) by a reproducible and simple method of physical adsorption was used as biocatalyst with promising result. Mg/Al–NO3−, Zn/Al–NO3− and Ni/Al–NO3− of LDHs with molar ratio of M2+/M3+ = 4:1 were synthesised by co-precipitation method with continuous agitation. The percentages of protein loading on Mg/Al–NO3−, Zn/Al–NO3− and Ni/Al–NO3− were 71%, 67% and 58%, respectively, due to the larger surface area, porosity and basal spacing of the supports. Parameter studies of reaction time, reaction temperature, water activity, thermostability, storage, leaching and reusability were investigated and optimised. Optimum conditions to produce adipate ester upto 80 % were reaction time; 2.5 h, temperature; 50 °C, and water activity; 0.53, respectively. Increased in optimisation conditions and enhanced stability properties were found after immobilisation compared to the native lipase.
Candida rugosa lipase was immobilized in the polymer of polyvinyl alcohol (PVA), alginate and boric acid. Calcium alginate improved the surface properties whereas PVA contributed strength. A percentage ratio of 12.5:0.05 PVA:sodium alginate not only prevented agglomeration, but produced beads of high gel strength. The performance of the immobilized biocatalyst was evaluated for the synthesis of ethyl hexanoate in isooctane. The thermal stability of the enzyme increased ten times upon immobilization. The beads showed nearly complete retention of activity in reuse upto 10 cycles and possessed shelf life of 10 months. The immobilized lipase had higher esterification ability as compared to its free counterpart.
Mica was modified either by acid treatment, grafting with aminopropyl-, octyl-, vinyl-, mercapto- and glycidoxy-triethoxysilanes, and activation of pre-treated support with glutaraldehyde (Glu). The derivatives were characterized by X-ray diffraction (XRD), infra-red spectroscopy (FTIR), surface area and porosity analysis, scanning electron microscopy coupled with energy dispersive X-ray (SEM-EDX) and transmission electron microscopy (TEM) techniques. The modified micas were used for immobilization of lipase from Candida rugosa (CRL). Activity of the lipase was determined by esterification and exhibited the improved activity than the free enzyme following the order; Amino-CRL > Glu-Amino-CRL > Octyl-CRL > Vinyl-CRL > Glycidoxy-CRL > Mercapto-CRL > Mica-CRL. Lipase immobilized mica showed enhanced protein loading (up to 8.22 mg protein/g support) and immobilization (up to 78%) compared to the free lipase and unmodified mica.
Candida rugosa lipase was entrapped in hybrid organic–inorganic sol-gel powder prepared by acid-catalyzed polymerization of tetramethoxysilane (TMOS) and alkyltrimethoxysilanes, and used in catalyzing esterification reactions between ethanol and butyric acid in hexane. Optimum preparation conditions were studied, which are gels made from propyltrimethoxysilane (PTMS)/TMOS molar ratio=4:1, hydrolysis time of silane precursor=30 min, water/silane molar ratio=24, enzyme loading=6.25% (w/w) of gel, and 1 mg PVA/mg lipase. The percentage of protein immobilization was 95% and the resulting lipase specific activity was 59 times higher than that of a non-immobilized lyophilized lipase. To prepare magnetic lipase-immobilized sol-gel powder (MLSP) for easier recovery of the biocatalyst, Fe3O4 nanoparticles were prepared and co-entrapped with lipase during gel formation. This procedure induced surface morphological change of the sol-gel powder and showed adverse effect on enzyme activity. Hence, although only 9% decrease in protein immobilization efficiency was observed, the corresponding reduction in enzyme activity could be up to 45% when sol-gel powder was doped with 25% (v/v) Fe3O4 magnetic nanoparticles solution. Lipase-immobilized sol-gel polymer was also formed within the pores of different porous supports to improve its mechanical stability. Non-woven fabric, with a medium pore size of all the supports tested, was found to be the best support for this purpose. The thermal stability of lipase increased 55-fold upon entrapment in sol-gel materials. The half-lives of all forms of sol-gel-immobilized lipase were 4 months at 40 °C in hexane.
Lipase from Candida rugosa adsorbed on polypropylene powder (CR/PP) was subjected to activation pre-treatments in order to enhance its activity in solvent-free ethyl oleate synthesis. The lipase activation achieved upon adsorption onto a hydrophobic support like PP was further enhanced through oil–water interfacial activation and bioimprinting of the immobilized catalyst. Several aliphatic hydrocarbons/buffer pH 7 mixtures were used in the pre-activation of CR/PP with specific activity increments of up to 29%. Molecular bioimprinting was also performed, with specific activity enhancement of near 70% with respect to non-treated CR/PP. The effect of several fatty acids used as templates and the water present in the reaction medium was studied. The oil–water activation and bioimprinting treatments that led to the best activities were assayed at the immobilization step. Instead of pre-treating CR/PP adsorbed in buffer medium, interfacial activation with octane/buffer and bioimprinting with a mix of fatty acids were carried out in the immobilization vial. The best results were found for CR/PP immobilized in 5/95 octane/buffer (v/v, %) medium. In that way, a biocatalyst with enhanced specific activity is obtained right from the immobilization vial with no need of further activation steps prior to reaction.
Palm esters were synthesized through enzymatic transesterification of various palm oil fractions with oleyl alcohol using Lipozyme RM IM as the catalyst. At the optimized alcoholysis reaction condition, after 5 h reaction time all palm oil fractions exhibited a high percentage yields of esters (>80%). Simultaneous differential scanning calorimeter-thermal gravity analysis showed a high thermal stability profile of palm esters. Other physicochemical properties of palm esters such as refractive index, density, surface tension, slip melting point, saponification value, iodine value and acid value were analyzed following standard test methods modified from the American Oil Chemists’ Society standards. The dermal irritation assay of palm oil esters shows the non-irritancy of the esters with a Human Irritancy Equivalent (HIE) score below 0.9. Furthermore, an increase in skin hydration of 40.7% after 90 min after application in an acute moisturizing test, has proven the suitably of palm oil esters to be used in the cosmetics formulation.
Lipase-catalyzed synthesis of different sugar fatty acid esters was performed in high yields in 2-methyl-2-butanol at atmospheric pressure and in supercritical carbon dioxide (SC CO2) at 10 MPa. Influence of molecular sieves concentration on conversion in SC CO2 was studied. Growth inhibitory effect of commercial sucrose fatty acid esters and enzymatically synthesized sucrose and fructose fatty acid esters on Gram-positive and Gram-negative micro-organisms, as well as on yeast was tested. Sucrose laurate inhibited the growth of Bacillus cereus food poisoning bacteria at a concentration of 9.375 mg/ml.
A support for enzyme immobilization was prepared by functionalization of mesoporous silica with octyltriethoxysilane. The features of the surface enables the adsorption of lipase from Candida antarctica B via strong hydrophobic interactions, enhancing the stability of the adsorbed enzyme molecules. Derivatives with a high enzyme loading (200 mg protein/g of silica) can be obtained due to the high porosity and surface properties of the support while the immobilization occurs in a monolayer fashion. The lack of inactive enzyme aggregates, together with the high enzyme loading, are responsible for the high catalytic activity achieved by these species. Derivatives were prepared with different lipase loading, and the activities were tested and compared to the commercial derivative Novozym 435. The stability of the catalyst and hence its industrial applicability were tested by performing subsequent reaction cycles of acylation of ethanolamine with lauric acid in acetonitrile. Conversion was quantitative even after 15 reaction cycles.
The lipase-catalyzed synthesis of ascorbyloleate was studied using different preparations of lipases from Candida antarctica. Conversion to ascorbyloleate was influenced by the type of lipase, the carrier used for immobilization, water activity and organic solvent. The highest conversion (63%) was found using Chirazyme L-2 immobilized on carrier C2, in acetone, using methyloleate as acyl donor (4-fold excess), at water activity 0.11 and in the presence of molecular sieves. (c) 2005 Elsevier Ltd. All rights reserved.
The lipases from Thermomyces lanuginosus (immobilized by granulation with silica) and Candida antarctica B (adsorbed on Lewatit, “Novozym 435”) were comparatively assayed for the synthesis of sugar esters by transesterification of sugars with fatty acid vinyl esters in 2-methyl-2-butanol:dimethylsulfoxide mixtures. We found that lipase from C. antarctica B is particularly useful for the preparation of 6,6′-di-acylsucrose, whereas T. lanuginosus lipase catalyzes selectively the synthesis of 6-O-acylsucrose. The granulated T. lanuginosus lipase retained more than 80% of its initial activity after 20 cycles of 6 h. Both lipases were similarly effective for the regioselective synthesis of 6′-O-palmitoylmaltose and 6-O-lauroylglucose. The effect of the synthesized sugar esters on the growth in liquid medium of various microorganisms (Gram-positive, Gram-negative and yeasts) was evaluated. 6-O-lauroylsucrose and 6′-O-lauroylmaltose inhibited the growth of Bacillus sp. at a concentration of 0.8 mg/ml, and of Lactobacillus plantarum at 4 mg/ml. Sucrose dilaurates and 6-O-lauroylglucose did not show antimicrobial activity, probably due to their low aqueous solubility. As regards the inhibition of yeasts, none of the tested carbohydrate esters inhibited significantly the growth of Zygosaccharomyces rouxii and Pichia jadinii.