Kinetics of enzymatic synthesis of L-ascorbyl acetate by Lipozyme TLIM and Novozym 435

L-ascorbyl acetate was synthesized through lipase-catalyzed esterification using Lipozyme TLIM and Novozym 435. Four solvents, including methanol, ethanol, acetonitrile, and acetone were investigated for the reaction, and acetone and acetonitrile were found to be suitable reaction media. The influences of several parameters such as water activity (a _w), substrate molar ratio, enzyme loading, and reaction temperature on esterification of L-ascorbic acid were systematically and quantitatively analyzed. Through optimizing the reaction, lipase-catalyzed esterification of L-ascorbic acid gave a maximum conversion of 99%. The results from using Lipozyme TLIM and Novozym 435 as biocatalysts both showed that a _w was an important factor for the conversion of L-ascorbic acid. The effect of pH value on lipase-catalyzed L-ascorbic acid esterification in acetone was also investigated. Furthermore, results from a kinetic characterization of Lipozyme TLIM were compared with those for Novozym 435, and suggested that the maximum reaction rate for Lipozyme TLIM was greater than that for Novozym 435, while the enzyme affinity for substrate was greater for Novozym 436.     

Optimization and kinetic study on the synthesis of palm oil ester using Lipozyme TL IM

Enzymatic synthesis of palm oil esters (POE) was carried out via alcoholysis of palm oil (PO) and oleyl alcohol (OA) catalyzed by Lipozyme TL IM. The optimum reaction conditions were: temperature: 60 °C; enzyme load: 24.7 wt%; substrate ratio: 1:3 (PO/OA), impeller speed: 275 rpm and reaction time: 3 h. At the optimum condition, the conversion of POE was 79.54%. Reusability study showed that Lipozyme TL IM could be used for 5 cycles with conversion above 50%. The alcoholysis reaction kinetic follows the Ping-Pong Bi-Bi mechanism characterized by the V_max, K_m(PO), and K_m(OA) values of 32.7 mmol/min, 0.3147 mmol/ml and 0.9483 mmol/ml, respectively. The relationship between initial reaction rate and temperature was also established based on the Arrhenius law.     

Novozym 435 for production of biodiesel from unrefined palm oil: Comparison of methanolysis methods

Biodiesel (BD) is commonly produced from refined vegetable oils by alkali-catalyzed methanolysis. Unrefined vegetable oils are economically attractive but not suitable for alkali catalysis because of their high content of free fatty acids (FFAs). Novozym 435 (immobilized Candida antarctica lipase B), which accepts both FFA and oil as substrates, was, therefore, employed to convert unrefined palm oil to BD. Three different methanolysis methods, namely, t-butanol mediated system (method-1), LiCl solution based controlled release system for methanol (method-2) and solvent-free system with three successive additions of methanol (method-3), were compared. The optimal methanol to oil molar ratios in the method-1, -2 and -3 are 6:1, 3:1 and 3:1, respectively. BD yield at an optimal methanol concentration reaches 91–92% after 10, 20 and 24 h in the method-1, -2 and -3, respectively. BD yield remains the same over five repeated cycles in the method-1, while it drops to 68 and 71% by the fifth cycle in the method-2 and -3, respectively. The results show that the method-1 is the most effective for production of BD from a low cost feedstock like unrefined palm oil.     

Ultrasonic irradiation with vibration for biodiesel production from soybean oil by Novozym 435

The production of biodiesel with soybean oil and methanol through transesterification by Novozym 435 (Candida antarctica lipase B immobilized on polyacrylic resin) were conducted under two different conditions—ultrasonic irradiation and vibration to compare their overall effects. Compared with vibration, ultrasonic irradiation significantly enhanced the activity of Novozym 435. The reaction rate was further increased under the condition of ultrasonic irradiation with vibration (UIV). Effects of reaction conditions, such as ultrasonic power, water content, organic solvents, ratio of solvent/oil, ratio of methanol/oil, enzyme dosage and temperature on the activity of Novozym 435 were investigated under UIV. Under the optimum conditions (50% of ultrasonic power, 50 rpm vibration, water content of 0.5%, tert-amyl alcohol/oil volume ratio of 1:1, methanol/oil molar ratio of 6:1, 6% Novozym 435 and 40 °C), 96% yield of fatty acid methyl ester (FAME) could be achieved in 4 h. Furthermore, repeated use of Novozym 435 after five cycles showed no obvious loss in enzyme activity, which suggested this enzyme was stable under the UIV condition. These results indicated that UIV was a fast and efficient method for biodiesel production.     

Enzymatic synthesis of perlauric acid using Novozym 435

For the biocatalytic synthesis of perlauric acid, Novozym 435 (Candida antarctica lipase immobilized on polyacrylic resin) was found to be the most active catalyst and toluene was the most suitable solvent. The effects of various parameters on conversion and rates of reaction were studied in the absence of mass transfer limitations. Reusability studies indicated that there was enzyme deactivation and from a preliminary study of the deactivation, it was observed that the deactivation obeys a pseudo-first-order model. Lineweaver–Burk plots indicated the formation of a ternary complex. A model based on ordered bi–bi mechanism was found to fit the initial rate data very well and using this model, some kinetic parameters were calculated by non-linear regression analysis.     

Conversion of Soybean Oil to Biodiesel Fuel Using Lipozyme TL IM in a Solvent-free Medium

The conversion of soybean oil to biodiesel fuel was investigated in the presence of a lipase from Thermomyces lanuginosus (commercially called Lipozyme TL IM) in a solvent-free medium. The lipase was inactivated when more than 1.5 molar equivalent of methanol was added to the oil mixture. To fully convert the oil to its corresponding methyl esters, the reaction was performed successfully by a three-step addition of 1 molar equivalent of methanol and under the optimized conditions (40°C, 150 rpm, 10% enzyme quantity based on oil weight), the maximum methyl ester (ME) yield was 98% after 12 h reaction. By-product glycerol had a negative effect on enzymatic activity and iso-propanol was found to be effective for glycerol removal, in the presence of which lipase expressed relatively high activity and more than 94% of the ME yield was maintained after being used repeatedly for 15 batches.     

Conversion of Soybean Oil to Biodiesel Fuel Using Lipozyme TL IM in a Solvent-free Medium

The conversion of soybean oil to biodiesel fuel was investigated in the presence of a lipase from Thermomyces lanuginosus (commercially called Lipozyme TL IM) in a solvent-free medium. The lipase was inactivated when more than 1.5 molar equivalent of methanol was added to the oil mixture. To fully convert the oil to its corresponding methyl esters, the reaction was performed successfully by a three-step addition of 1 molar equivalent of methanol and under the optimized conditions (40°C, 150 rpm, 10% enzyme quantity based on oil weight), the maximum methyl ester (ME) yield was 98% after 12 h reaction. By-product glycerol had a negative effect on enzymatic activity and iso-propanol was found to be effective for glycerol removal, in the presence of which lipase expressed relatively high activity and more than 94% of the ME yield was maintained after being used repeatedly for 15 batches.     

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.     

A practical approach to obtain high yield lipase-mediated synthesis of octyl caprylate with Novozym 435

Abstract

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

Improved synthesis of 1,3-diolein by Novozym 435-mediated esterification of monoolein with oleic acid

We successfully developed an efficient and promising bioprocess for 1,3-diolein synthesis by performing Novozym 435-mediated esterification of oleic acid with monoolein in this work. Under the optimized conditions (60 °C, molar ratio of oleic acid to monoolein 1.2:1), a 1,3-diolein yield of 93.7% could be achieved, and the yield of 1,2-diolien was low (2.6%). The high yield of 1,3-diolein and the optimum reaction time were improved remarkably compared with the results of our previous study, which involved the enzymatic esterification of oleic acid with glycerol. An additional advantage of the new process is the fact that 90% original activity of the enzyme was maintained after being used for 100 reactions. The present work could be seen as a useful enzyme-catalyzed process for the industrial production of 1,3-diacylglycerol.     

Novel and highly regioselective route for synthesis of 5-fluorouridine lipophilic ester derivatives by lipozyme TL IM

For the first time, lipozyme TL IM, an inexpensive lipase from Thermomyces lanuginosa, was successfully applied to the regioselective synthesis of lipophilic 5-fluorouridine ester derivatives. The ESI-MS and (13)C NMR analysis confirmed that the end products of the acylation were 5'-O-acyl 5-fluorouridines, more powerful anti-tumor drugs than 5-fluorouridine itself. Notably, the chain length of acyl donors had an obvious effect on the initial rate and the maximum substrate conversion of the regioselective acylation. The acylation of 5-fluorouridine with vinyl laurate was used as a model to explore the influence of various factors on the reaction with respect to the initial rate, the maximum substrate conversion and the regioselectivity. The optimum water activity, the molar ratio of vinyl laurate to 5-fluorouridine, reaction temperature and shaking rate were 0.07, 15/1, 45 degrees C and 200rpm, respectively, under which the maximum substrate conversion and the regioselectivity were as high as 98.4 and >99%, respectively, after a reaction time of around 6h.     

Reactor Design for the Novozym 435 ® -catalysed Enantioselective Esterification of 4-methyloctanoic Acid

The bench scale Novozym 435 ® catalysed esterification of 4-methyloctanoic acid with ethanol was studied at 35°C. Esterification in a batch reactor (molar ratio of 1:8 (acid:EtOH)) resulted in the isolation of the enantiomerically enriched product (ee p =81%) and substrate (ee s =93%). In order to integrate reaction and separation, liquid-vapour equilibria calculations were performed showing that an excess of ethanol results in a very low ester fraction in the vapour phase. Since this is undesirable for an integrated process of reaction and product removal, a repeated batch reaction was performed using a molar ratio of 10:1 (acid:EtOH). After six cycles (45% conversion) the ee of 4-methyloctanoic acid ethyl ester turned out to be 80%. For different E values the ee p was calculated for batch and repeated batch reactions. It was shown that in all cases the ee p was higher for the repeated batch reaction. However, the product is not enantiopure since the E value of the reaction is rather low at the low ethanol concentration used. An alternative approach would be the continuous separation of the product during the reaction. A mathematical model was developed to describe esterification in a packed bed reactor integrated with product separation. This model shows that integration of reaction and product removal in advance is not suitable either to obtain an enantiomerically pure product. Since the optimal reaction conditions (high ethanol concentration) and the optimal separation system (low ethanol concentration) do not match in this reaction, the preference is given to the batch reaction at high ethanol concentrations because in that case the highest enantioselectivity of the enzyme is obtained.     

Ring-opening bulk polymerization of epsilon-caprolactone and trimethylene carbonate catalyzed by lipase Novozym 435.

The affects of lipase concentration on ring-opening bulk polymerizations of epsilon-caprolactone and trimethylene carbonate were studied by using Novozym 435 (immobilized form of lipase B from Candida antarctica) as biocatalyst. The polymerization of epsilon-caprolactone was carried out in bulk at 70 degrees C. Three lipase concentrations of 9.77, 1.80 and 0.50 mg/mmol epsilon-CL were used in the experiment. The results showed that increasing the lipase concentration used in the polymerization system resulted in an increased rate of monomer consumption. For an enzyme concentration of 9.8 mg lipase per mmol monomer, an 80% monomer conversion was achieved in a 4-h time period, while for the lower enzyme concentration of 1.8 mg lipase per mmol monomer, 48 h were needed to reach monomer conversion. Linear relationships between Mn and monomer conversions were observed in all three enzyme concentrations, suggesting that the product molecular weight may be controlled by the stoichiometry of the reactants for these systems. At the same monomer conversion level, however, Mn decreased with increasing enzyme concentration. After correcting for the amount of monomer consumed in initiation, the plot of ln[([M]o - [M]i)/([Mt] - [M]i)] versus reaction time was found to be linear, suggesting that the monomer consumption followed a first-order rate law and no chain termination occurred. For the TMC systems, the polymerization was carried out in bulk at 55 degrees C. Similar to the epsilon-CL systems, increasing the Novozym 435 concentration from 8.3 to 23.6 mg/mmol TMC increased the rate of monomer conversion. Unlike the epsilon-CL systems, however, nonlinear relationships were obtained between Mn and monomer conversion, indicating that possible chain transfer and/or slow initiation had taken place in these systems. Consistent with the above result, nonlinear behavior was observed for the plot of ln[[M]o/[M]t] versus reaction time.     

FTIR-ATR characterization of free Rhizomucor meihei lipase (RML), Lipozyme RM IM and chitosan-immobilized RML

The synthesis and characterization of biocatalysts based on lipase from Rhizomucor miehei (RML) immobilized on chitosan-based supports were investigated. The enzyme was immobilized on chitosan following two strategies: (i) physical adsorption; and (ii) covalent bonding using glutaraldehyde. The content of enzyme bound in the supports, as precipitable protein, was analyzed using UV/visible methods. FTIR-ATR spectroscopy was employed to characterize the prepared biocatalysts, as well the native enzyme and a commercial biocatalyst Lipozyme RM IM, used as reference materials. Analysis of the amide I′ signal allowed to follow the changes in the secondary structure of the enzyme after binding to the support and its thermal stability. The hydrolysis of ethyl stearate monitored in situ by FTIR-ATR was used as a test reaction. Results showed that RML was bound to Chit and Glut–Chit with minor changes in its secondary structure, thermal stability and enzymatic activity in a selected reaction test.     

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.     

Comparison of the requirements for ribonucleic acid synthesis with the requirements for deoxyribonucleic acid synthesis in animal tissues

Ribonucleic acid polymerase and deoxyribonucleic acid polymerase have been partially purified from bovine lymphosarcoma, lymph node, and thymus. An examination of the deoxyribonucleic acid requirements of the two enzymes indicates that “native” deoxyribonucleic acid is the preferred template for ribonucleic acid synthesis; heat-denatured deoxyribonucleic acid is considerably less active. The primer requirements for deoxyribonucleic acid synthesis differ: “native” deoxyribonucleic acid is usually inactive, while denatured deoxyribonucleic acid is active. The two enzymes also differ in pH optima and in their requirements for metal cofactors.     

Yeast PAPS reductase: properties and requirements of the purified enzyme

The enzymatic mechanism of sulphite formation in Saccharomyces cerevisiae was investigated using a purified 3-phosphoadenylsulphate (PAPS) reductase and thioredoxin. The functionally active protein (M_R 80–85 k) is represented by a dimer which reduces 3-phosphoadenylyl sulphate to adenosine-3,5-bisphosphate and free sulphite at a stoichiometry of 1:1. Reduced thioredoxin is required as cosubstrate. Examination of the reaction products showed that free anionic sulphite is formed with no evidence for bound-sulphite(s) as intermediate. V _max of the enriched enzyme was 4–7 nmol sulphite · min^-1 · mg^-1 using the homologous thioredoxin from yeast. The velocity of reaction decreased to 0.4 nmol sulphite · min^-1 · mg^-1 when heterologous thioredoxin (from Escherichia coli) was used instead. The K _m of homologous thioredoxin was 0.6 · 10^-6 M, for the heterologous cosubstrate it increased to 1.4 · 10^-6 M. The affinity for PAPS remained practically unaffected (K _{m PAPS}: 19 · 10^-6 M in the homologous, and 21 · 10^-6 M in the heterologous system). From the kinetic data it is concluded that the enzyme followed an ordered mechanism with thioredoxin as first substrate followed by PAPS as the second. Parallel lines in the reciprocal and a common intersect in the Hanes-plots for thioredoxin were seen as indication of a ping-pong (with respect to thioredoxin) uni-bi (with respect to PAPS) mechanism.Abbreviations APS adenylyl sulphate - DTE dithioerythritol - DTT dithiothreitol - HPLC high performance liquid chromatography - IEF isoelectric focusing - LSC liquid scintillation counting - 3,5-PAP adenosine-3,5-bisphosphate - PAPS 3-phosphoadenylyl sulphate - PEP phospho-(enol)pyruvate - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - Tris 2-amino-2-hydroxymethyl-1,3-propanediol     

Pancreatic enzyme requirements for the dissociation of rat hearts for culture

Summary Nine crude commercially available samples of pancreatic enzyme preparations were examined in an effort to establish enzyme requirements for dissociation of rat heart to single cells for culture. Disc gel electrophoresis, using purified enzymes as references, indicated the presence of at least five enzymes. The levels of these enzymes, trypsin, chymotrypsin, elastase, lipase, and amylase were quantified in the commercial samples by established enzyme assays. The crude enzyme preparations were compared for their abilities to provide good yields of viable cells from the hearts of neonatal white rats. The abilities of the cells to thrive in culture and to beat rhythmically were also used in the comparison. Commercial purified samples of the five enzymes were used singly and in various combinations in preparing cultures to establish minimal pancreatic enzyme requirements for dissociation of rat hearts. It was concluded that at least trypsin, chymotrypsin, and elastase were required to obtain viable rat heart cells in high yield. Amylase and lipase activities were shown not to be necessary for dissociation. Most commercial samples of trypsin, commonly used to dissociate heart tissue, contain trypsin, chymotrypsin, and elastase in concentrations sufficient to release heart cells in good yield with minimal damage. The destruction of cells observed with some of the commercial samples examined was not due to improper levels of trypsin, chymotrypsin, or elastase. Work is in progress to identify the destructive agent(s). This work was supported in part by United States Public Health Service Grant HL10018 and The Pennsylvania State University Agricultural Experiment Station Grant 1858. Authorized for publication on December 28, 1973 as paper 4602 in the journal series of the Pennsylvania Agricultural Experiment Station.