Enzyme-catalysed production of n-butyl palmitate using ultrasound-assisted esterification of palmitic acid in a solvent-free system

Bioprocess and Biosystems Engineering - This work exhibits the implementation of ultrasound technology in solvent-free synthesis study of n-butyl palmitate using Fermase CALB™10000....     

Optimized enzymatic synthesis of ascorbyl esters from lard using Novozym 435 in co-solvent mixtures

A mild and efficient method for the conversion of fatty acid methyl esters from lard into ascorbyl esters via lipase-catalyzed transesterification in co-solvent mixture is described. A solvent engineering strategy was firstly applied to improve fatty acid ascorbyl esters production. The co-solvent mixture of 30% t-pentanol:70% isooctane (v/v) was optimal. Response surface methodology (RSM) and central composite design (CCD) were employed to estimate the effects of reaction parameters, such as reaction time (12–36 h), temperature (45–65 °C), enzyme amount (10–20%, w/w, of fat acid methyl esters), and substrate molar ratio of fatty acid methyl esters to ascorbic acid (8:1–12:1) for the synthesis of fatty acid ascorbyl esters in co-solvent mixture. Based on the RSM analysis, the optimal reaction conditions were determined as follows: reaction time 34.32 h, temperature 54.6 °C, enzyme amount 12.5%, substrate molar ratio 10.22:1 and the maximum conversion of fatty acid ascorbyl esters was 69.18%. The method proved to be applicable for the synthesis of ascorbyl esters using Novozym 435 in solvent.     

Markedly improving Novozym 435-mediated regioselective acylation of 1-beta-D-arabinofuranosylcytosine by using co-solvent mixtures as the reaction media

A comparative study was made of Novozym 435-catalyzed regioselective acylation of 1-beta-D-arabinofuranosylcytosine with vinyl propionate for the preparation of the 5'-O-monoester in eleven co-solvent mixtures and three pure polar solvents. Novozym 435 displayed low or no acylation activity toward 1-beta-D-arabinofuranosylcytosine in pure polar solvents, although those solvents can dissolve the nucleosides well. When a hexane-pyridine co-solvent system was adopted, both the initial rate and the substrate conversion were enhanced markedly. The polarity of co-solvent mixtures had significant effect on the reaction. Among the solvent mixtures investigated, the higher the polarity of the solvent mixture, the lower the initial reaction rate and the substrate conversion. It was also found that the acylation was dependent on the hydrophobic solvent content, the water activity and the reaction temperature. The most suitable co-solvent, initial water activity, and reaction temperature were hexane-pyridine (28:72, v/v), 0.07, and 50 degrees C, respectively. Under these conditions, the initial rate, the substrate conversion and the regioselectivity were as high as 91.1 mM h(-1), >97% and >98%, respectively, after a reaction time of 6 h. Among the reaction mediums examined, the lowest apparent activation energy was achieved with hexane-pyridine (28:72, v/v), in which Novozym 435 also exhibited good thermal stability.     

Efficient lipase catalysed production of a lubricant and surfactant formulation using a continuous solvent-free process

The transesterification of sunflower oil with a high oleic acid residue content (typically 83.5%) with butanol-1 by immobilised Lipozyme was carried out in a solvent free system and in a continuous way. During the first 6 h of reactor operation, a transition phase was observed, in which the main products were butyl ester and glycerol. This latter being insoluble in the reaction mixture, it is adsorbed onto the enzyme support thus leading to a decrease in enzyme performance. Step by step, less and less glycerol is produced and finally when glycerol is no longer produced a steady state is attained. The product composition is a mixture of butyl ester (65 molar%), monoglyceride (26 molar%), diglyceride (6 molar%) and residual triglyceride (3 molar%). This mixture has interesting lubricant and surfactant properties. The reactor was maintained without any loss in activity for a period of 3 months. This result is very different to that obtained using an organic solvent (n-hexane) which leads to a total loss of enzyme activity within a few hours.     

Enzymatic synthesis of n-butyl palmitate in a solvent-free system: RSM optimization and kinetic studies

The present paper demonstrates application of biocatalysis to the synthesis of n-butyl palmitate, a cosmetic emollient ester in a solvent-free system (SFS). Fermase CALB?10000, a commercial Candida antarctica lipase B was used to accomplish the synthesis. In order to evaluate the effect of various process parameters on the synthesis, one factor at a time methodology (OFAT) and response surface methodology (RSM) complimented with central composite design (CCD) were employed. On the basis of the results obtained in one factor at one time studies, temperature, enzyme dose, and molar ratio were chosen as significant parameters and their range was selected for RSM study. The optimized factors suggested by RSM model were, temperature –60.12?°C, enzyme dose –5% w/w, and alcohol: acid ratio –2.25:1. Under these optimized factors, the experimental conversion observed was 91.25% which was in close agreement to the model predicted conversion of 92% and the enzymes were reusable up to four cycles. A separate study was carried out in order to study the effect of palmitic acid on n-butyl palmitate synthesis and to understand the kinetic profile of n-butyl palmitate synthesis reaction. Ordered bi-bi model showed a good experimental fit to the kinetic data.     

Enzymatic production of enantiopure ketoprofen in a solvent-free two-phase system

Enzymatic hydrolysis conducted in a medium composed of solely substrate is considered to resolve racemic ketoprofen esters. In a system composed of two components, the pure liquid substrate (organic phase) and water (aqueous phase), hydrolysis products can be efficiently removed from the reaction mixtures. Accordingly, in this study we designed a solvent-free two-phase system for the enantioselective enzymatic hydrolysis of ketoprofen esters. In order to further optimize this system, the influences of various factors, such as the pH of the aqueous phase, temperature, enzyme content, and the alcohol chain length of esters, were examined on conversion and enantiomeric excess. 1N NaHCO₃ was identified as the most efficient aqueous phase for the extraction of ketoprofen. Changes in the amount of enzyme did not significantly affect the maximum conversion or the enantiomeric excess. On the other hand, ketoprofen esters with shorter alcohol chains displayed higher initial reaction rates and conversions in solventless media. In the case of ketoprofen propyl ester, for example, the productivity of the solvent-free two-phase system was about 10–100 times higher than that obtained to date for ketoprofen esterification with alcohols in organic solvents. The enantioselectivities obtained in solvent-free media were similar to those obtained for the enantioselective esterification of ketoprofen in organic solvents.     

Solvent-free methanolysis of canola oil in a packed-bed reactor with use of Novozym 435 plus loofa

Enzymatic methanolysis of canola oil in the solvent-free system was studied in a packed-bed reactor (PBR) using small pieces of loofa plus Novozym 435. Response surface methodology (RSM) was applied to determine the effect of the transesterification conditions, namely flow rate of substrate (x₁), temperature (x₂) and methanol to canola oil molar ratio (x₃) as the regressors, on the methyl ester production. A central composite design (CCD) was employed to optimize the reaction. A second-order polynomial multiple regression model was chosen and analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.996, thus adjustment of the model with experimental data was ensured. The methyl ester yield increased as the flow rate of the reaction mixture in the PBR increased from its low to the middle level thereafter, increasing the flow rate corresponded to decreasing the yield. The same trends of changes were observed for the other two factors. The optimum process conditions for biodiesel production in the PBR were found to be: x₁ = 6.3 mL/min, x₂ = 38 °C and x₃ = 4.3. The same batch was successfully used repeatedly in the PBR for six enzymatic cycles (432 h), where the methyl ester yield was maintained above 97%.     

Effect of process variables on photosynthetic algal hydrogen production

Chlamydomonas reinhardtii is a green alga that can use the sun's energy to split water into O(2) and H(2). This is accomplished by means of a two-phase cycle, an aerobic growth phase followed by an anaerobic hydrogen production phase. The effects of process variables on hydrogen production are examined here. These variables include cell concentration, light intensity, and reactor design parameters that affect light transport and mixing. An optimum cell concentration and light intensity are identified, and two reactor designs are compared. The maximum hydrogen production observed in this study was 0.29 mL of hydrogen per milliliter of suspension. This was measured at atmospheric pressure during a 96 h production cycle. This corresponds to an average hydrogen production rate of 0.12 mmol/mL.h.     

Enzymatic production of linalool esters in organic and solvent-free system

This work investigated the influence of temperature, enzyme concentration, substrates molar ratio, in the absence and presence of organic solvent, at two molar ratios of the substrates on the enzymatic production of linalil esters using the immobilized lipase Novozym 435 as catalyst, different acids and linalool and Ho-Sho essential oil as substrates. The best reaction conversion was obtained at the highest temperature (70 °C), for both solvent free (3.81%) and with solvent addition (2.25%), for a solvent to substrates molar ratio of 2:1, enzyme concentration of 5 wt% and acid to alcohol molar ratio of 1:1. The reaction kinetics revealed that Ho-Sho essential oil afforded the greatest conversions when compared with pure linalool. Higher linalil esters production were achieved after 10 h reaction (5.58%) in 2:1 solvent to substrates molar ratio, with enzyme concentration of 5 wt%, at 70 °C and anhydride to alcohol molar ratio of 1:1 using Ho-Sho essential oil as substrate.     

A continuous process for biodiesel production in a fixed bed reactor packed with cation-exchange resin as heterogeneous catalyst

Continuous esterification of free fatty acids (FFA) from acidified oil with methanol was carried out with NKC-9 cation-exchange resin in a fixed bed reactor with an internal diameter of 25 mm and a height of 450 mm to produce biodiesel. The results showed that the FFA conversion increased with increases in methanol/oil mass ratio, reaction temperature and catalyst bed height, whereas decreased with increases in initial water content in feedstock and feed flow rate. The FFA conversion kept over 98.0% during 500 h of continuous esterification processes under 2.8:1 methanol to oleic acid mass ratio, 44.0 cm catalyst bed height, 0.62 ml/min feed flow rate and 65°C reaction temperature, showing a much high conversion and operational stability. Furthermore, the loss of sulfonic acid groups from NKC-9 resin into the production was not found during continuous esterification. In sum, NKC-9 resin shows the potential commercial applications to esterification of FFA.     

Rapid enzymatic production of acylglycerols from conjugated linoleic acid and glycerol in a solvent-free system

Rates and product distributions have been determined for the consecutive esterification reactions of conjugated linoleic acid (CLA) with glycerol in the presence of an immobilized Candida antarctica lipase from Novo Nordisk (Lipozyme IM). In a solvent-free environment, both rates and product distributions are affected by the ratio of reactants, temperature, and hydration level. For mole ratios of CLA to glycerol ranging from 1/1 to 3/1, incorporation of at least 95% of the original CLA into the product acylglycerols was achieved in less than 7 h of reaction at 50°C. © Rapid Science Ltd. 1998     

Optimization of reaction variables for sucrose monoester production using lipase in a solvent free system by Taguchi's method

Taguchi's method was attempted successfully to optimize the reaction variables and their ranges for sucrose monoester production. Optimal conditions determined by Taguchi's method were 35°C, 200rpm, capric acid (40mmol) and sucrose (3mmol), 24h, 0.5g lipase, 0.72g Ba(OH)2 ·8H2O, 0.72g Ba(OH)2·1H2O. A product yield of 21% was obtained. © Rapid Science Ltd. 1998     

Soy protein recovery in a solvent-free process using continuous liquid-solid circulating fluidized bed ion exchanger

Soy protein concentrates and soy protein isolates act as ingredients in bakery, meat and dairy products, baby formulas, starting materials for spun textured vegetable products, and other nutritional supplements. In this study, the effectiveness of a liquid-solid circulating fluidized bed (LSCFB) ion exchanger is demonstrated for the recovery of soluble soy proteins from full fat and defatted soy flour. Under steady-state operating conditions, about 50% of the proteins could be recovered from the feed streams entering the ion exchanger. The LSCFB was shown to be a promising system for the recovery of soy protein from both defatted and full fat soy flour solutions. As the ion exchange process captures dissolved proteins, the system may offer a less damaging form of processing compared with the acid precipitation process where soy protein aggregates form and functionality is affected. In addition, the LSCFB allows simultaneous adsorption and desorption of the proteins allowing for a continuous operation. No prefiltration of feed containing suspended particles is required as well, because fluidization is used in place of packed bed technology to improve on current ion exchange processes.     

Optimization of key process variables for enhanced hydrogen production by Enterobacter aerogenes using statistical methods

The individual and mutual effects of glucose concentration, temperature and pH on the hydrogen production by Enterobacter aerogenes were investigated in a batch system. A Box-Behnken design and response surface methodology (RSM) were employed to determine the optimum condition for enhanced hydrogen production. The hydrogen production rate was investigated by simultaneously changing the three independent variables, which all had significant influences on the hydrogen production rate. The maximum hydrogen production rate of 425.8 ml H(2)(g dry cell h)(-1) was obtained under the optimum condition of glucose concentration 118.06 mM, temperature 38 degrees C and pH 6.13. The experimental results showed that the RSM with the Box-Behnken design was a useful tool for achieving high rate of hydrogen production by E. aerogenes.     

Enzymatic transesterification in a solvent-free system: synthesis of sn-2 docosahexaenoyl monoacylglycerol

Abstract

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

Production of 10(S)-hydroxy-8(E)-octadecenoic and 7,10(S,S)-hydroxy-8(E)-octadecenoic ethyl esters by Novozym 435 in solvent-free media

Novozym 435, lipase B from Candida antarctica, was used in this study for the production of ethyl esters. For the first time, trans-hydroxy-fatty acid ethyl esters were synthesized in vitro in solvent-free media. We studied the effects of the substrate–ethanol molar ratio and enzyme synthetic stability of the biocatalyst. To determine the structure of the formed compounds, Fourier transformed infrared spectroscopy, nuclear magnetic resonance, and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry were used, three less time-consuming structural techniques. trans-Hydroxy-fatty acid ethyl esters were synthesized with a reaction yield of 90 % or higher with optimal reaction conditions.     

Performance of a continuous foam separation column as a function of process variables

Enrichment and recovery of bovine serum albumin has been examined in a continuous foam separation column. The effects of the operating factors, superficial air velocity, feed flow rate, feed concentration and pH on the above characteristics was investigated. The protein enrichment decreased with the increase in the value of each of these parameters. Protein recovery increased with increasing air velocity, decreased with increasing feed flow rate and did not change very much with increasing feed concentration. Maximum protein recovery was obtained at the isoelectric point (pH 4.8) of the protein. Maximum protein recovery was found to be a strong function of the air velocity in the range 0.05-0.15 cm/s. Further increase in air velocity did not have much effect on recovery because of very large bubbles formed as a result of coalescence. Bubble size was determined as a function of the above factors in the liquid and foam sections of the column. It was found to be dependent on protein concentration, feed flow rate and solution pH. The effect was more significant in the foam section of the column. The bubbles in the foam section were significantly larger (about 3-10 times) than those in the liquid, with a sharp change at the foam-liquid interface. The bubble size measurements were used to calculate the interfacial area and it was shown that the rate of protein removal increases with increasing interfacial area.     

Lipase-catalyzed synthesis of structured phenolic lipids in solvent-free system using flaxseed oil and selected phenolic acids as substrates

Structured phenolic lipids (PLs) were obtained by lipase-catalyzed transesterification of flaxseed oil, in a solvent-free system (SFS), with selected phenolic acids, including hydroxylated and/or methoxylated derivatives of cinnamic, phenyl acetic and benzoic acids. A bioconversion yield of 65% was obtained for the transesterification of flaxseed oil with 3,4-dihydroxyphenyl acetic acid (DHPA). However, the effect of the chemical structure of phenolic acids on the transesterification of flaxseed oil in SFS was of less magnitude as compared to that in organic solvent system (OSS). Using DHPA, the APCI-MS analysis confirmed the synthesis of monolinolenyl, dilinolenyl, linoleyl linolenyl and oleyl linolenyl dihydroxyphenyl acetates as phenolic lipids. A significant increase in the enzymatic activity from 200 to 270 nmol of PLs/g solid enzyme/min was obtained upon the addition of the non-ionic surfactant Span 65. However, upon the addition of the anionic surfactant, sodium bis-2-ethylhexyl sulfosuccinate (AOT), and the cationic one, hexadecyltrimethylammonium bromide (CTAB), the enzymatic activity was decreased slightly from 200 to 192 and 190 nmol of PLs/g solid enzyme/min, respectively. The results also showed that the increase in DHPA concentration from 20 to 60 mM resulted in a significant increase in the volumetric productivity (P(V)) from 1.61 to 4.74 mg PLs per mL reaction mixture per day.     

Pseudomonas aeruginosa LBI production as an integrated process using the wastes from sunflower-oil refining as a substrate

Pseudomonas aeruginosa LBI produced surface active rhamnolipids when cultivated on waste from the sunflower-oil process under different conditions. These biosurfactants, which reduce the superficial and interfacial tensions between fluids, offer advantages over their chemical counterparts, especially because of their ecological acceptability. These molecules can be used in fields as diverse as chemical, pharmaceutical and petrochemical industries. In this work, we present the effect of C/N ratio on growth and production yield. The best production yields (Y P/S) were achieved for C/N ratios (in g/g) of 8/1 (0.22) and 6.4/1 (0.23). The product concentration was very satisfactory (7.3g/L) at C/N ratio of 8/1, especially when considering that the substrate was basically composed of wastes that would otherwise constitute an environmental disposal problem.