Enzymatic Biodiesel Production from Sunflower Oil by Candida antarctica Lipase in a Solvent-free System

Methanolysis (transesterification with methanol) of sunflower oil by lipase from Candida antarctica (Novozym 435) in a solvent-free system has been studied. Stepwise as well as continuous methanol feeding was applied to avoid strong substrate inhibition. Glycerol was found to cause strong product inhibition on the enzymatic reaction, therefore glycerol removal by dialysis was investigated using a flat sheet membrane module.     

Enzymatic Biodiesel Production from Sunflower Oil by Candida antarctica Lipase in a Solvent-free System

Methanolysis (transesterification with methanol) of sunflower oil by lipase from Candida antarctica (Novozym 435) in a solvent-free system has been studied. Stepwise as well as continuous methanol feeding was applied to avoid strong substrate inhibition. Glycerol was found to cause strong product inhibition on the enzymatic reaction, therefore glycerol removal by dialysis was investigated using a flat sheet membrane module.     

Mechanistic modeling of biodiesel production using a liquid lipase formulation

In this article, a kinetic model for the enzymatic transesterification of rapeseed oil with methanol using Callera? Trans L (a liquid formulation of a modified Thermomyces lanuginosus lipase) was developed from first principles. We base the model formulation on a Ping‐Pong Bi‐Bi mechanism. Methanol inhibition, along with the interfacial and bulk concentrations of the enzyme was also modeled. The model was developed to describe the effect of different oil compositions, as well as different water, enzyme, and methanol concentrations, which are relevant conditions needed for process evaluation, with respect to the industrial production of biodiesel. The developed kinetic model, coupled with a mass balance of the system, was fitted to and validated on experimental results for the fed‐batch transesterification of rapeseed oil. The confidence intervals of the parameter estimates, along with the identifiability of the model parameters were presented. The predictive capability of the model was tested for a case using 0.5% (wt. Enzyme/wt. Oil), 0.5% (wt. Water /wt. Oil) and feeding 1.5 times the stoichiometric amount of methanol in total over 24 h. For this case, an optimized methanol feeding profile that constrains the amount of methanol in the reactor was computed and the predictions experimentally validated. Monte‐Carlo simulations were then used to characterize the effect of the parameter uncertainty on the model outputs, giving a biodiesel yield, based on the mass of oil, of 90.8 ± 0.55 mass %. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1277–1290, 2014     

Putting thought to paper: a microARCS protease screen

In micro-arrayed compound screening (microARCS), an agarose gel is used as a reaction vessel that maintains humidity and compound location as well as being a handling system for reagent addition. Two or more agarose gels may be used to bring test compounds, targets, and reagents together, relying on the pore size of the gel matrix to regulate diffusion of reactants. It is in the microenvironment of the agarose matrix that all the components of an enzymatic reaction interact and result in inhibitable catalytic activity. In an effort to increase the throughput of microARCS-based screens, reduce the effort involved in manipulating agarose gels, and reduce costs, blotter paper was used rather than a second agarose gel to introduce a substrate to a gel containing a target enzyme. In this assay, the matrix of the blotter paper did not prevent the substrate from diffusing into the enzyme gel. The compound density of the microARCS format, the ease of manipulating sheets of paper for reagent addition, and a scheduled protocol for running multiple gels allowed for a throughput capacity of more than 200,000 tests per hour. A protease assay was developed and run in the microARCS format at a rate of 200,000 tests per hour using blotter paper to introduce the substrate. Picks in the primary screen were retested in the microARCS format at a density of 384 compounds per sheet. IC(50) values were confirmed in a 96-well plate format. The screen identified several small molecule inhibitors of the enzyme. The details of the screening format and the analysis of the hits from the screen are presented.     

Immobilization and biocatalysis of chlorophyllase in selected organic solvent systems

Chlorophyllase extract from Phaeodactylum tricornutum was immobilized by physical adsorption on DEAE-cellulose and silica gel as well as by covalent binding on Eupergit C, Eupergit C250L, Eupergit C/ethylenediamine (EDA) and Eupergit C250L/EDA. Although the highest immobilization yield (83-93%) and efficiency (51-53%) were obtained when chlorophyllase extract was immobilized on DEAE-cellulose and silica gel, there was no improvement in the thermal stability of chlorophyllase as compared to that of the free one. The immobilization of chlorophyllase extract on Eupergit C250L/EDA resulted by a high recovery of enzymatic activity, with an immobilization efficiency of 44%, and promoted a higher stabilization of chlorophyllase (four times) in the aqueous/miscible organic solvent medium. On the other hand, the inhibitory effect of refined bleached deodorized (RBD) canola oil was reduced by immobilization of chlorophyllase extract onto silica gel as compared to those obtained with other enzyme preparations. However, the re-cycled chlorophyllase extract immobilized on Eupergit C250L/EDA retained more than 75% of its initial enzyme activity after 6 cycles, whereas that immobilized on silica gel was completely inactivated. The highest catalytic efficiency, for both free and immobilized chlorophyllase on Eupergit C250L/EDA, was obtained in the ternary micellar system as compared to the aqueous/miscible organic solvent and biphasic media.     

Production of biodiesel by lipase-catalyzed transesterification of vegetable oils: a kinetics study

Kinetics of production of biodiesel by enzymatic methanolysis of vegetable oils using lipase has been investigated. A mathematical model taking into account the mechanism of the methanolysis reaction starting from the vegetable oil as substrate, rather than the free fatty acids, has been developed. The kinetic parameters were estimated by fitting the experimental data of the enzymatic reaction of sunflower oil by two types of lipases, namely, Rhizomucor miehei lipase (RM) immobilized on ion-exchange resins and Thermomyces lanuginosa lipase (TL) immobilized on silica gel. There was a good agreement between the experimental results of the initial rate of reaction and those predicted by the proposed model equations, for both enzymes. From the proposed model equations, the regions where the effect of alcohol inhibition fades, at different substrate concentrations, were identified. The proposed model equation can be used to predict the rate of methanolysis of vegetable oils in a batch or a continuous reactor and to determine the optimal conditions for biodiesel production.     

Effects of environmental conditions and methanol feeding strategy on lipase-mediated biodiesel production using soybean oil

Methanol is a commonly used acyl acceptor for lipase-driven biodiesel production, but a high concentration of methanol is detrimental for lipase activity. To overcome this drawback, a simple fed-batch process was developed by optimization of the methanol feeding strategy and reaction conditions. For the feeding strategy, an equal volume of pure methanol was fed twice with specified time intervals into a reactor initially containing a 1:1 molar ratio of soybean oil to methanol in order to adjust the net molar ratio of the oil to methanol to 1:3. In contrast with the batch reaction, a higher agitation speed in the fed-batch process elevated the conversion yield of soybean oil to biodiesel. An agitation speed of 600 rpm and a reaction temperature of 70°C were chosen as the optimal environmental conditions. Residual lipase activities for the fed-batch operation at 40 ∼ 70°C and 600 rpm were 7.1 ± 1.4 times higher than that of the batch method at 40°C with the same agitation speed, indicating that methanol feeding can prevent significant deactivation of lipase. Finally, two times feeding methanol at 2 and 6 hr resulted in a biodiesel productivity of 10.7%/h and 94.9% final conversion yield under the optimal conditions.     

Hydrolysis of crambe oil by enzymatic catalysis: An evaluation of the operational conditions

Abstract

In this work, the enzymatic hydrolysis of the crambe oil by using a commercial immobilized lipase Lipozyme RM IM was evaluated. The effect of the operational conditions, such as temperature, water/oil molar ratio, enzyme/substrate mass ratio and stirring speed were assessed based on the experimental designs. The experiments were performed in a closed and batch system with controlled temperature and stirring speed. In addition, the kinetics of the process was studied in the best operational conditions, wherein the experimental data were obtained and described by a mathematical model. The influence of the operational conditions was assessed based on the measured values of the free fatty acids (FFA) produced by the enzymatic hydrolysis. In 4?h of reaction, a yield of 42.6% was observed and the most significant operational conditions were the enzyme/substrate mass ratio and stirring speed. By the kinetic investigation, an initial reaction rate of 3.5?×?10⁴?mol?mL^?1?h^?1 and a maximum yield of 74% were observed after 40?h of reaction (in the equilibrium condition). The mathematical model was not only able to adequately describe the experimental data of FFA concentrations profiles but also showed predictive capacity to independents assays in different operational conditions. Therefore, based on the simulation analysis of the enzymatic hydrolysis of the crambe oil, the model can be useful for process optimization and phenomenological studies.     

On-line monitoring of methanol in n-hexane by an organic-phase alcohol biosensor

An organic-phase alcohol biosensor has been developed by co-entrapping alcohol oxidase and horseradish peroxidase within an ionotropy polymer hydrogel matrix fabricated from silica gel particles, hydroxyethyl carboxymethylcellulose, an adduct of 3-methoxy-4-ethoxybenzaldehyde and 4-tert-butylpyridinium acetohydrazone, and octadecylsilica particles. The viability of the immobilised enzymes for the biocatalytic reaction of methanol in n-hexane was comparatively studied by using a bulk cell or a volume-changeable flow-through cell coupled with an oxygen optical transducer. It was found that the microenvironment around the enzyme, the deterioration property of the enzyme, the substrate throughput and the mass transfer process of the reactant in the bioreactor were the crucial parameters affecting the performance of the alcohol organic-phase biosensor. Our optimal biosensor was constructed from a flow-through cell packed with small particles of immobilised enzymes and it could maintain the biocatalytic reaction at high and stable rate for on-line detection of methanol in n-hexane under flow operation mode. The biosensor had an analytical working range of 2.3-90 mM methanol in n-hexane. The response times (t95) were 4.5 and 7.5 min for 60 and 10 mM methanol, respectively. The operational lifetime of the biosensor was more than 45 assays and the shelf lifetime was longer than 2 weeks. The biosensor has been successfully applied to determine the methanol content in a commercial gasoline-methanol blend sample with good recovery.     

Enzymatic production of biodiesel from cotton seed oil using t-butanol as a solvent

The enzymatic production of biodiesel by methanolysis of cottonseed oil was studied using immobilized Candida antarctica lipase as catalyst in t-butanol solvent. Methyl ester production and triacylglycerol disappearance were followed by HPLC chromatography. It was found, using a batch system, that enzyme inhibition caused by undissolved methanol was eliminated by adding t-butanol to the reaction medium, which also gave a noticeable increase of reaction rate and ester yield. The effect of t-butanol, methanol concentration and temperature on this system was determined. A methanolysis yield of 97% was observed after 24h at 50 degrees C with a reaction mixture containing 32.5% t-butanol, 13.5% methanol, 54% oil and 0.017 g enzyme (g oil)(-1). With the same mixture, a 95% ester yield was obtained using a one step fixed bed continuous reactor with a flow rate of 9.6 mlh(-1) (g enzyme)(-1). Experiments with the continuous reactor over 500 h did not show any appreciable decrease in ester yields.     

Purification and kinetic properties of sialidase from Clostridium perfringens

Clostridium perfringens sialidase was isolated from a culture medium of bacterial cells by ammonium sulfate precipitation (42-85%), followed by purification through Sephadex G-75 gel chromatography, DEAE A-50 anion exchange chromatography, FPLC medium pressure anion exchange chromatography and finally FPLC medium pressure isochromatofocussing. From 9 l culture medium 1.17 mg sialidase was isolated with a specific activity of 295 U/mg. The enzyme appeared to be homogeneous by analytical polyacrylamide gel electrophoresis. The molecular mass was measured to be 66 kDa. Km values ranging from 0.6 to 1.6mM were determined for several oligosaccharides as substrates. The enzyme catalyzed transglycosylation reactions with methanol as a nucleophilic reagent competitive with water. In the enzymatic hydrolysis of the (3'-methoxyphenyl)glycoside of alpha-N-acetylneuraminic acid, increase of methanol concentration had no effect on the release of 3-methoxyphenol. This finding suggests that the formation of the enzyme-glycon intermediate is the rate-determining step for this substrate.     

Analysis of conversion and operation strategies for enzymatic hydrolysis of lignocellulosic biomass in a series of CSTRs with distributed feeding

Design considerations for enzymatic hydrolysis of lignocellulosic biomass in two and three continuous stirred tank reactors (CSTRs) in series with distributed feeding of substrate and enzyme, followed by a series of CSTRs, are discussed. A previously developed, fitted, and validated kinetic model is extended to accommodate distributed feeding and used along with the micromixing limiting situations of macrofluid and microfluid to describe the reaction system. The capabilities of the reaction system proposed are explored for a range of cumulative substrate concentration from 5 to 20% w/w (dry basis). Continuous distributed feeding does not show advantages in terms of cellulose conversion when compared with the operation where an equivalent mass of substrate is added at the first reactor of the series, but the potential to increase substrate concentration beyond the concentrations that can be handled in conventional CSTRs, and therefore, the volumetric productivity of reactors, is evident.     

Efficient immobilization of epoxide hydrolase onto silica gel and use in the enantioselective hydrolysis of racemic para-nitrostyrene oxide

Epoxide hydrolase from Aspergillus niger (E.C. 3.3.2.3) was immobilized by covalent linking to epoxide-activated silica gel under mild conditions. A very easy procedure allowed to prepare an immobilized biocatalyst with more than 90% retention of the initial enzymatic activity. Immobilized and free enzyme showed very similar behaviour with respect to the effect of pH on activity and stability. One benefit of immobilizing epoxide hydrolase from A. niger on silica gel was the enhanced enzyme stability in the presence of 20% DMSO. The kinetic resolution of racemic para-nitrostyrene oxide was investigated by using this new immobilized biocatalyst. The enantioselectivity of the enzyme was not altered by the immobilization reaction: both unreacted epoxide and formed diol were obtained with very high ee (99 and 92%, respectively). In addition, the biocatalyst could be easily separated from the reaction mixture and re-used for over nine cycles without any noticeable loss of enzymatic activity or change in the enantioselectivity extent. The activity of immobilized AnEH was retained for several months.     

Highly efficient transformation of waste oil to biodiesel by immobilized lipase from Penicillium expansum

An inexpensive self-made immobilized lipase from Penicillium expansum was shown to be an efficient biocatalyst for biodiesel production from waste oil with high acid value in organic solvent. It was revealed that water from the esterification of free fatty acids and methanol prohibited a high methyl ester yield. Adsorbents could effectively control the concentration of water in the reaction system, resulting in an improved methyl ester yield. Silica gel was proved to be the optimal adsorbent, affording a ME yield of 92.8% after 7 h. Moreover, the enzyme preparation displayed a higher stability in waste oil than in corn oil, with 68.4% of the original enzymatic activity retained after being reused for 10 batches.     

Comparative study of controlled pore glass, silica gel and poraver for the immobilization of urease to determine urea in a flow injection conductimetric biosensor system

This study compared the responses of three enzyme reactors containing urease immobilized on three types of solid support, controlled pore glass (CPG), silica gel and Poraver. The evaluation of each enzyme reactor column was done in a flow injection conductimetric system. When urea in the sample solution passed though the enzyme reactor, urease catalysed the hydrolysis of urea into charged products. A lab-built conductivity meter was used to measure the increase in conductivity of the solution. The responses of the enzyme reactor column with urease immobilized on CPG and silica gel were similar and were much higher than that of Poraver. Both CPG and silica gel reactor columns gave the same limit of detection, 0.5 mM, and the response was still linear up to 150mM. The analysis time was 4-5 min per sample. The enzyme reactor column with urease immobilized on CPG gave a slightly better sensitivity, 4% higher than the reactor with silica gel. The life time of the immobilized urease on CPG and silica gel were more than 310h operation time (used intermittently over 7 months). Good agreement was obtained when urea concentrations of human serum samples determined by the flow injection conductimetric biosensor system was compared to the conventional methods (Fearon and Berthelot reactions). These were statistically shown using the regression line and Wilcoxon signed rank tests. The results showed that the reactor with urease immobilized on silica gel had the same efficiency as the reactor with urease immobilized on CPG.     

Dynamic modeling of an enzymatic membrane reactor for the treatment of xenobiotic compounds

A membrane enzymatic reactor, consisting of a stirred tank coupled to an ultrafiltration membrane was set up for the enzymatic oxidation of xenobiotic compounds. The azo dye Orange II was selected for the model compound and manganese peroxidase for the oxidative enzyme. The ligninolytic cycle was initiated and maintained by the controlled addition of all factors (reactants, mediators, and stabilizers) at suitable rates. Considering the distinctiveness of this process, in which the substrate to be oxidized is not the primary substrate for the enzyme, a kinetic model was developed. The azo dye concentration and hydrogen peroxide addition rate were found to be the main factors affecting the process. The reaction kinetics was defined using a Michaelis-Menten model with respect to the Orange II concentration and a first-order linear dependence relative to the H(2)O(2) addition rate. The dynamic model, which takes into account both the kinetics and the hydraulics of the system, was validated by comparing the experimental results in continuous operation under steady and non-steady state to model predictions. In particular, the model predicted the behavior of the system when unexpected alterations in steady-state operation occurred. Furthermore, the model allowed us to obtain the most appropriate H(2)O(2)/Orange II ratio in the feed to maximize the process efficiency.     

Understanding the influence of temperature change and cosolvent addition on conversion rate of enzymatic suspension reactions based on regime analysis

It is a commonly held belief that enzymatic conversions of substrate in aqueous suspensions can be speeded up by raising the temperature or adding organic solvents to promote dissolution of the substrate. To quantify the impact of such changes, we studied the alpha-chymotrypsin-catalyzed hydrolysis of dimethyl benzylmethylmalonate as a model system. It was found that, upon addition of organic cosolvents, longer process times were actually required, even though the substrate solubility increased severalfold as expected. Upon raising the temperature from 25 degrees C to 37 degrees C, on the other hand, both the substrate solubility, the substrate dissolution rate, and the enzymatic reaction rate increased, leading to shorter process times. A dissolution-reaction model incorporating the kinetics of enzyme deactivation could be developed. A simple relation for the prediction of the overall process time was established by evaluating the time constants for the subprocesses: substrate dissolution; enzymatic conversion; and enzyme deactivation. Using regime analysis, rules of thumb for the optimization of an enzymatic suspension reaction were derived.     

Enzyme Kinetics for Complex System Enables Accurate Determination of Specificity Constants of Numerous Substrates in a Mixture by Proteomics Platform

Many important experiments in proteomics including protein digestion, enzyme substrate screening, enzymatic labeling, etc., involve the enzymatic reactions in a complex system where numerous substrates coexists with an enzyme. However, the enzyme kinetics in such a system remains unexplored and poorly understood. Herein, we derived and validated the kinetics equations for the enzymatic reactions in complex system. We developed an iteration approach to depict the enzymatic reactions in complex system. It was validated by 630 time-course points from 24 enzymatic reaction experiments and was demonstrated to be a powerful tool to simulate the reactions in the complex system. By applying this approach, we found that the ratio of substrate depletion is independent of other coexisted substrates under specific condition. This observation was then validated by experiments. Based on this striking observation, a simplified model was developed to determine the catalytic efficiencies of numerous competing substrates presented in the complex enzyme reaction system. When coupled with high-throughput quantitative proteomics technique, this simplified model enabled the accurate determination of catalytic efficiencies for 2369 peptide substrates of a protease by using only one enzymatic reaction experiment. Thus, this study provided, in the first time, a validated model for the large scale determination of specificity constants which could enable the enzyme substrate screening approach turned from a qualitative method of identifying substrates to a quantitative method of identifying and prioritizing substrates. Data are available via ProteomeXchange with identifier PXD004665.     

Anaerobic ammonium oxidation (anammox) irreversibly inhibited by methanol

Methanol inhibition of anaerobic ammonium oxidation (anammox) activity was characterized. An enrichment culture entrapped in a polyethylene glycol gel carrier was designed for practical uses of wastewater treatment. Batch experiments demonstrated that anammox activity decreased with increases in methanol concentration, and relative activity reached to 29% of the maximum when 5 mM methanol was added. Also, batch experiments were conducted using anammox sludge without immobilization. Anammox activity was evaluated by quantifying ¹⁴N¹⁵N (²⁹N) emission by combined gas chromatography-quadrupole mass spectrometry, and the anammox activity was found to be almost as sensitive to methanol as in the earlier trials in which gel carriers were used. These results indicated that methanol inhibition was less severe than previous studies. When methanol was added in the influent of continuous feeding system, relative activity was decreased to 46% after 80 h. Although the addition was halted, afterwards the anammox activity was not resumed in another 19 days of cultivation, suggesting that methanol inhibition to anammox activity was irreversible. It is notable that methanol inhibition was not observed if anammox activity was quiescent when substrate for anammox was not supplied. These results suggest that methanol itself is not inhibitory and may not directly inhibit the anammox activity.     

二十二碳六烯酸甲酯标准样品的研制

本制备采用以深海鱼油为基础原料,在碱性条件下皂化水解为游离脂肪酸,经尿素－甲醇包合分离后,以硫酸－甲醇甲酯化,再经硝酸银－硅胶柱层析分离,从而得到高纯度的二十二碳六烯酸甲酯标准物质,并经定值测试,确认该标准物质的含量≥98％以上。