Miniemulsion as efficient system for enzymatic synthesis of acid alkyl esters

The aim of this work is to devise an efficient enzymatic process for the production of linear alkyl esters in aqueous miniemulsion systems. The esterification reactions of linear alcohols and carboxylic acids were performed with three different enzymes, commercial Amano lipase PS from Pseudomonas cepacia, Lipase type VII from Candida rugosa, and lyophilized Fusarium solani pisi cutinase expressed in Saccharomyces cerevisiae SU50. The miniemulsion system shows a high potential for the synthesis of linear alkyl esters, for example, hexyl octanoate, which could be synthesized with an ester yield of 94% using Amano lipase PS. Even with hydrophilic alcohols as ethanol, ethyl decanoate could be obtained with a concentration of 0.45 M and a yield of 62% using F. s. pisi cutinase as catalyst. High esterification rates for ethyl‐ and hexyloleate in miniemulsion showed a significant shift in cutinase selectivity towards longer chain length carboxylic acids. The stepwise addition of the alcohol led to an increase of the esterification yield. Moreover, increasing the amount of dispersed organic phase, mainly consisting of the substrates, led to a significant increase of the final ester concentration (e.g., concentration of 1.4 M for ethyl decanoate for the esterification with Amano Lipase PS). Biotechnol. Bioeng. 2010;106: 507–515. © 2010 Wiley Periodicals, Inc.     

Enzymatic synthesis of ethyl esters from waste oil using mixtures of lipases in a plug‐flow packed‐bed continuous reactor

This work describes the continuous synthesis of ethyl esters via enzymatic catalysis on a packed‐bed continuous reactor, using mixtures of immobilized lipases (combi‐lipases) of Candida antarctica (CALB), Thermomyces lanuginosus (TLL), and Rhizomucor miehei (RML). The influence of the addition of glass beads to the reactor bed, evaluation of the use of different solvents, and flow rate on reaction conditions was studied. All experiments were conducted using the best combination of lipases according to the fatty acid composition of the waste oil (combi‐lipase composition: 40% of TLL, 35% of CALB, and 25% of RML) and soybean oil (combi‐lipase composition: 22.5% of TLL, 50% of CALB, and 27.5% of RML). The best general reaction conditions were found to be using tert‐butanol as solvent, and the flow rate of 0.08 mL min^?1. The combi‐lipase reactors operating at steady state for over 30 days (720 h), kept conversion yields of ～50%, with average productivity of 1.94 g_{ethyl esters} h^?1, regardless of the type of oil in use. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:952–959, 2018     

Enzymatic synthesis of the acrylic esters: a comparative study

Various acrylic esters were synthesized by Candida cylindracea lipase (CCL) catalysed transesterification with different alcohols. A comparative study was carried out using 2,3-butanedione mono-oxime acrylate and vinyl acrylate as acylating agents. The rate of conversion was faster when oxime acrylate was used as acylating agent as compared to vinyl acrylate. Effect of solvents on the rate of conversion was studied and diisopropyl ether (DIPE) was proved to be a better solvent as compared to CHCl₃ and THF. The effect of various structural aspects of the substrates on the rate of conversion was studied. Among the linear alcohols studied, ease of conversion was found to be in the order of n-octanol>n-hexanol>n-butanol. Up to 80% conversion was achieved in the case of cyclohexyl methanol.     

Enzymatic synthesis of trehalose esters having lipophilicity

To improve trehalose lipophilicity, trehalose was regioselectively esterified with vinyl fatty acid esters in dimethyl formamide by protease from Bacillus subtilis to give 6-O-lauroyltrehalose, 6-O-myristoyltrehalose, 6-O-palmitoyltrehalose, 6-O-stearoyltrehalose, 6-O-oleoyltrehalose and 6-O-linoleoyltrehalose. The influence of structural variation by changing fatty acid substitute was examined by measurement of the surface tension and biodegradability.     

Enzymatic preparation of fatty acid esters of sugar alcohols by condensation in acetone using a packed-bed reactor with immobilized Candida antarctica lipase

Mono- and dilauroyl arabitols, ribitols, xylitols and sorbitols were synthesized batchwise or continuously at 50°C or 60°C by condensation catalyzed by an immobilized Candida antarctica lipase in acetone. Continuous production was realized using a system where a column packed with sugar alcohol and a packed-bed reactor with the immobilized lipase were connected in series. The concentrations of the mono- and dilauroyl esters of each sugar alcohol became almost constant at mean residence times of 15 min or longer in the packed-bed reactor. The monolauroyl, monomyristoyl and monopalmytoyl arabitols, ribitols, xylitols and sorbitols were continuously produced using the reactor system at 60°C, and the productivity was in the range of 1.3–2.0 kg L^?1-reactor·day except for the fatty acid esters of sorbitol, the productivity of which was 0.6–0.8 kg L^?1-reactor·day.     

Enzymatic preparation of fatty acid esters of sugar alcohols by condensation in acetone using a packed-bed reactor with immobilized Candida antarctica lipase

Mono- and dilauroyl arabitols, ribitols, xylitols and sorbitols were synthesized batchwise or continuously at 50°C or 60°C by condensation catalyzed by an immobilized Candida antarctica lipase in acetone. Continuous production was realized using a system where a column packed with sugar alcohol and a packed-bed reactor with the immobilized lipase were connected in series. The concentrations of the mono- and dilauroyl esters of each sugar alcohol became almost constant at mean residence times of 15 min or longer in the packed-bed reactor. The monolauroyl, monomyristoyl and monopalmytoyl arabitols, ribitols, xylitols and sorbitols were continuously produced using the reactor system at 60°C, and the productivity was in the range of 1.3-2.0 kg L^-1-reactor·day except for the fatty acid esters of sorbitol, the productivity of which was 0.6-0.8 kg L^-1-reactor·day.     

Enzymatic synthesis of a 6-sialyl lactose analogue using a pH-responsive water-soluble polymer support

The Letter describes a strategy for the enzymatic synthesis of glycans based on a pH-responsive water-soluble polymer. In neutral condition, the polymer is water-soluble and convenient for in-solution enzymatic synthesis, whereas in acidic condition (pH lower than 4.0), the polymer disconnects with the product and becomes insoluble, which can be easily removed. A 6-Sialyl lactose analogue was synthesized as a model reaction using this approach.     

Column flow reactor using acetohydroxyacid synthase I from Escherichia coli as catalyst in continuous synthesis of R-phenylacetyl carbinol

We tested the possibility of utilizing acetohydroxyacid synthase I (AHAS I) from Escherichia coli in a continuous flow reactor for production of R-phenylacetyl carbinol (R-PAC). We constructed a fusion of the large, catalytic subunit of AHAS I with a cellulose binding domain (CBD). This allowed purification of the enzyme and its immobilization on cellulose in a single step. After immobilization, AHAS I is fully active and can be used as a catalyst in an R-PAC production unit, operating either in batch or continuous mode. We propose a simplified mechanistic model that can predict the product output of the AHAS I-catalyzed reaction. This model should be useful for optimization and scaling up of a R-PAC production unit, as demonstrated by a column flow reactor.     

Long-Term continuous culture of hepatocytes in a packed-bed reactor utilizing porous resin

As part of our attempt to develop a hybrid artificial liver support system using cultured hepatocytes, we investigated the long-term metabolic function of hepatocytes incubated in a packed-bed type reactor using reticulated polyvinyl formal (PVF) resin as a supporting material. Long-term (up to 1 week) perfusion culture experiments using the packed-bed reactor (20 mm i.d.) loaded with 500 PVF resin cubes (mean pore size 250 mum, 2 x 2 x 2 mm), together with conventional monolayer culture experiments as controls, were performed in serum-free or serum-containing medium. Ammonium metabolism and urea synthesis activities were evaluated quantitatively based on reaction kinetic analyses. Initial rates of ammonium metabolism and urea-N synthesis, as well as GPT enzyme activities, were adopted as indexes of the metabolic performance of the reactor and activities of the cultured hepatocytes.When serum-free medium was used in the perfusion cultures, ammonium metabolic and urea-N synthetic rates showed significant decay with elapse of the culture period, being less than 10% of those measured on day 1. This loss of activity was more prominent in the perfusion culture than in the monolayer cultures using this medium. In contrast, when serum-containing medium was used, approximately 50% of these activities obtained on day 1 were maintained even at the end of the cultures both in the perfusion and monolayer culture experiments.We concluded that the packed-bed reactor using PVF resin enabled high-density culture of hepatocytes, and showed a satisfactory ability to maintain the metabolic function of immobilized hepatocytes for relatively long periods of up to 1 week. This type of reactor is thus considered to represent a breakthrough in overcoming the difficulties involved in the development of a hybridtype artificial liver support system. (c) 1994 John Wiley & Sons, Inc.     

Optimization of alkyl ester production from grease using a phyllosilicate sol-gel immobilized lipase**,***

Simple alkyl ester derivatives of restaurant grease were prepared using a lipase from Pseudomonoas cepacia immobilized within a phyllosilicate sol-gel matrix as biocatalyst. Alcoholysis reactions of grease were carried out in solvent-free media using a one-step addition of alcohol to the reaction mixture. The immobilized lipase was active from 40 to 70 °C. Ester yields (60–97%) were highest when using a ratio of reactants of 2 mmol grease to 8 mmol alcohol and the biocatalyst was 10% (w/w) of grease in the presence of molecular sieves.     

Enzymatic synthesis of esters using an immobilized lipase

Various esters were synthesized in nearly anhydrous hexane from alcohols and carboxylic acids using a lipase from Candida cylindracea. The enzyme was immobilized on a nylon support and protein loadings as high as 10 mg/g were obtained. The activity of the immobilized enzyme was maximum in a range of temperatures from 25 to 37 degrees C. Ethylpropionate was formed from ethanol and propionic acid at a rate of 0.017 mol/h g immobilized protein. Different esters were formed at comparable rates and equilibrium conversions could generally be approached in less than 10 h in a batch reaction system. The immobilized lipase catalyst was quite stable and retained about one third of the initial activity after repeated experiments during the course of 72 days. A stirred tank continuous flow reactor was used successfully for the continuous production of esters.     

Enzymatic synthesis of UDP-galactose on a gram scale

Uridine 5′-diphospho-- -galactose (UDP-Gal) was synthesized on a gram scale from uridine 5′-diphospho-- -glucose and - -galactose 1-phosphate using the enzymes galactose-1-phosphate uridyltransferase (EC 2.7.7.12), phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.27). The synthesis was performed in a repetitive batch mode in which the enzymes, some of which are expensive, were used in 16 subsequent batches without any loss of enzyme activity. The space time yield of the synthesis was 7.1 g/l d. The overall yield of the synthesis amounted to 40% and 1.1 gram of pure UDP-Gal was obtained.     

Preparation of astaxanthin by lipase-catalyzed hydrolysis from its esters in a slug-flow microchannel reactor

Natural astaxanthin is widely used as a food and cosmetics additive because of its multiple biological activities. However, astaxanthin produced by Haematococcus pluvialis is generally esterified, and its activity is far less than that of free astaxanthin. Hydrolysis of astaxanthin esters to free astaxanthin by enzymes can overcome the drawbacks of chemical saponification methods. In this paper, a slug-flow microchannel reactor was constructed and tested in enzymatic hydrolysis of astaxanthin esters. The reactor consists of a “T” slug-flow generator, a stainless-steel microchannel, two constant-flow pumps, and a temperature controller. The reactor has the advantages of simple configuration and easy scale-up, and is suitable for two-phase biochemical reactions. Using the microchannel reactor, astaxanthin esters in H. pluvialis oil were efficiently hydrolyzed to free astaxanthin by lipase from Aspergillus niger. After hydrolysis, the content of free astaxanthin in H. pluvialis oil was 18.8 mg/L, 7.83-times higher than that before hydrolysis (2.13 mg/L). The hydrolysis rate reached 75.4 %. These results indicate that the microchannel reactor can be useful for the production of free astaxanthin from its esters.     

Lipase-catalyzed selective synthesis of monolauroyl maltose using continuous stirred tank reactor

Monolauroyl maltose was synthesized by an immobilized lipase that catalyzed condensation of maltose and lauric acid in acetone using a batch reactor or a continuous stirred tank reactor. Mono- and di-lauroyl maltoses were identified by FT-IR, ¹H NMR, ¹³C NMR and MS. Monolauroyl maltose was selectively synthesized in a continuous stirred tank reactor and no diester was detected. The highest concentration of monolauroyl maltose at 28 mmol/l was obtained in 250 ml acetone when maltose was added at 4 g/d and the molar ratio of lauric acid to maltose was fixed at 4:1 at a flow rate of 0.15 ml/min for both influx and effluent without supplement of fresh molecular sieve.     

Photoelectrochemical treatment of landfill leachate in a continuous flow reactor

Leachate from a municipal landfill site, which has been treated by biological process, was treated by photoelectrochemical oxidation in a pilot scale flow reactor, using DSA anode and UV light irradiation. At a current density of 67.1 mA/cm² and 2.5 h reaction time, the removal rates achieved were for 74.1% COD, 41.6% for TOC, and 94.5% for ammonium in the electrolysis process with UV light irradiation. In comparison, the removal rates of COD, TOC, and ammonium were decreased in the individual electrolysis process, respectively. The increase induced by the UV light irradiation was analyzed. The removal rates increased with the increase of current densities in the photoelectrochemical process. Combined with UV–vis spectra and gas chromatography–mass spectroscopy analysis, it is believed that the organic contaminants were efficiently mineralized into small molecular acids. At the meantime, the concentrations of metal ions in the landfill leachate were largely reduced.     

Kinetics of BTEX degradation in a packed-bed anaerobic reactor

The ever-increasing diversity of industrial activity is responsible for the discharge of compounds that are toxic or difficult to degrade into the environment. Some of the compounds found in surface and ground waters, usually deriving from the contamination of oil-based products, are benzene, toluene, ethylbenzene and xylenes (BTEX). To remove these compounds from contaminated water, a bench-scale horizontal-flow anaerobic immobilized biomass reactor, containing anaerobic biomass from various sources immobilized in polyurethane foam matrices, was employed to treat a synthetic substrate composed of protein, carbohydrates and BTEX solution in ethanol, as well as a BTEX solution in ethanol as the sole carbon source. The reactor removed up to 15.0 mg/l of each BTEX compound over a hydraulic detention time of 11.4 h. A first-order kinetic model fitted the experimental data well, showing correlation coefficients higher than 0.994. The apparent first-order coefficient values, , ranged from 8.4±1.5 day⁻¹ for benzene to 10.7±1.4 day⁻¹ for o-xylene in the presence of ethanol, protein and carbohydrates, and from 10.0±2.0 day⁻¹ for benzene to 13.0±1.7 day⁻¹ for o-xylene in the presence of ethanol. The BTEX degradation rates estimated here were 10- to 94-fold higher than those found in reports on microcosm studies.     

Ultrasound-accelerated enzymatic synthesis of sugar esters in nonaqueous solvents

Comparative studies of enzymatic synthesis of glucose esters under ultrasound and shaking were carried out in nonaqueous media. The influence of solvents, enzymes, chain length of the acyl donors, the power of the ultrasound bath, and intermittent ultrasound on the enzymatic synthesis was investigated. Among the eight solvents selected, pyridine was the most appropriate with alkaline protease from Bacillus subtilis whether under ultrasound or shaking. The acceleration effect of ultrasound with Novozym 435 and the alkaline protease from B. subtilis-catalyzed transesterification increased with the chain length of acyl donors, decreasing from C(10) to C(4). We also investigated the influence of the power (50, 100, and 120 W) of the ultrasound irradiation and the manner of operation (continuous ultrasound, 10 min ultrasound/20 min shaking without ultrasound) on the transesterification. The results showed that higher power and continual operational gave the better acceleration. Ultrasound did not change the character and selectivity of the enzyme in the transesterification.     

Enzymatic synthesis of a new inhibitor of alpha-amylases: acarviosinyl-isomaltosyl-spiro-thiohydantoin

Synthesis of acarviosinyl-isomaltosyl-spiro-thiohydantoin in yields up to 20%, has been achieved by Bacillus stearothermophilus maltogenic amylase (BSMA). BSMA is capable of transferring the acarviosine-glucose residue from an acarbose donor onto glucopyranosylidene-spiro-thiohydantoin. Reactions were followed using HPLC and MALDI-TOF MS. 1H and 13C NMR studies revealed that the enzyme reserved its stereoselectivity. Glycosylation took place mainly at C-6 resulting in alpha-acarviosinyl-(1-->4)-alpha-D-glucopyranosyl-(1-->6)-D-glucopyranosylidene-spiro-thiohydantoin. This compound was found to be a much more efficient salivary amylase inhibitor than glucopyranosylidene-spiro-thiohydantoin with kinetic constants of K(EI)=0.19 microM and K(ESI)=0.24 microM.     

Enzymatic synthesis of a novel glycolipid biosurfactant, mannosylerythritol lipid-D and its aqueous phase behavior

Mannosylerythritol lipids (MELs) produced by yeasts are one of the most promising glycolipid biosurfactants. In this study, we succeeded in the preparation of a novel MEL homolog having no acetyl groups, namely MEL-D. MEL-D was synthesized by lipase-catalyzed hydrolysis of acetyl groups from a known MEL, and identified as 4-O-[2′,3′-di-O-alka(e)noyl-β-d-mannopyranosyl]-(2R,3S)-erythritol. The obtained MEL-D showed a higher critical aggregation concentration (CAC = 1.2 × 10⁻⁵ M) and hydrophilicity compared to known MELs, retaining an excellent surface tension lowering activity (the surface tension at the CAC was 24.5 mN/m). In addition, we estimated the binary phase diagram of the MEL-D–water system based on a combination of visual inspection, polarized optical microscopy, and SAXS measurement. From these results, MEL-D was found to self-assemble into a lamellar (L_α) structure over all ranges of concentration. Meanwhile, the one-phase L_α region of MEL-D was extended wider than those of known MELs. MEL-D might keep more water between the polar layers in accordance with the extension of the interlayer spacing (d). These results suggest that the newly obtained MEL-D would facilitate the application of MELs in various fields as a lamellar-forming glycolipid with higher hydrate ability.