Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei

The effects of important reaction parameters for enhancing isoamyl acetate formation through lipase-catalyzed esterification of isoamyl alcohol were investigated in this study. Increase in substrate (acid) concentration led to decrease in conversions. A critical enzyme concentration of 3 g l(-1) was detected for a substrate concentration of 0.06 M (each of alcohol and acid). Solvents with partition coefficient higher than 1000 (log P>3.0) supported enzyme activity to give high conversions. Acetic acid at higher concentrations could not be esterified easily probably owing to its role in lowering the microaqueous pH of the enzyme. Extraneous water/buffer addition decreased the isoamyl acetate yields slightly ( approximately 10%) at 0.005-0.01% v/v of the reaction mixture and drastically (>40%) at above 0.01% v/v. Buffer saturation of the organic solvent employed improved esterification (upto two-fold), particularly at moderately higher substrate concentrations (>0.18 M). Employing acetic anhydride instead of acetic acid resulted in a two-fold increase in the yields (at 0.25 M substrate). Use of excess nucleophile (alcohol) concentration by increasing the alcohol/acid molar ratio resulted in higher conversions in shorter duration (upto eight-fold even at 1.5 M acetic acid). Yields above 80% were achieved with substrate concentrations as high as 1.5 M and more than 150 g l(-1) isoamyl acetate concentrations were obtained employing a relatively low enzyme concentration of 10 g l(-1). The operational stability of lipase was also observed to be reasonably high enabling ten reuses of the biocatalyst.     

Enzymatic synthesis of isoamyl butyrate using immobilized Rhizomucor miehei lipase in non-aqueous media

Isoamyl butyrate, an important fruity flavor ester, was synthesized using Rhizomucor miehei lipase immobilized on a weak anion exchange resin (Lipozyme IM-20) by the esterification of isoamyl alcohol and butyric acid. The effects of various reaction parameters such as substrate and enzyme concentrations, substrate molar ratio, temperature and incubation time have been investigated. Yields above 90% were obtained with substrate concentrations as high as 2.0 M. No evidence of enzyme inhibition by butyric acid was present up to 1.0 M concentration. Acid inhibition and, to a small extent, alcohol inhibition were evident above 1.0 M substrate concentration. Conversions reached a saturation value by the end of 24–48 h of incubation due to the accumulation of the water of reaction. The equilibrium was successfully pushed forward towards esterification by removing the accumulated water using a molecular sieve.Journal of Industrial Microbiology & Biotechnology (2000) 25, 147–154. Received 09 February 2000/ Accepted in revised form 24 June 2000     

Highly enantioselective esterification of racemic ibuprofen in a packed bed reactor using immobilised Rhizomucor miehei lipase

A systematic study of the enantioselective resolution of ibuprofen by commercial Rhizomucor miehei lipase (Lipozyme(R) IM20) has been carried out using isooctane as solvent and butanol as esterificating agent. The main variables controlling the process (temperature, ibuprofen concentration, ratio butanol:ibuprofen) have been studied using an orthogonal full factorial experimental design, in which the selected objective function was enantioselectivity. This strategy has resulted in a polynomial function that describes the process. By optimizing this function, optimal conditions for carrying out the esterification of racemic ibuprofen have been determined. Under these conditions, enantiomeric excess and total conversion values were 93.8% and 49.9%, respectively, and the enantioselectivity was 113 after 112 h of reaction. These conditions have been considered in the design of a continuous reactor to scale up the process. The esterification of ibuprofen was properly described by pseudo first-order kinetics. Thus, a packed bed reactor operating as a plug-flow reactor (PFR) is the most appropriate in terms of minimizing the residence time compared with a continuous stirred tank reactor (CSTR) to achieve the same final conversion. This reactor shows a similar behavior in terms of enantioselectivity, enantiomeric excess, and conversion when compared with batch reactors. A residence-time distribution (RTD) shows that the flow model is essentially a plug flow with a slight nonsymmetrical axial dispersion (Peclet number = 43), which was also corroborated by the model of CSTR in series. The stability of the system (up to 100 h) and the possibility of reutilization of the enzyme (up to four times) lead to consider this reactor as a suitable configuration for scale up of the process.     

Improvement of the stability and selectivity of Rhizomucor miehei lipase immobilized on silica nanoparticles: Selective hydrolysis of fish oil using immobilized preparations

We report the effect of random and oriented immobilization of Rhizomucor miehei lipase (RML) on its functional properties. For this purpose, silica nanoparticles (MCM-41 and SBA-15) were prepared, characterized and functionalized by glycidyloxypropyl trimethoxysilane. Direct immobilization of RML on these supports was performed via the variety of amino acid residues on the surface of RML which promotes random immobilization. To perform oriented immobilization, partial modification of epoxy functionalized supports was carried out by introducing iminodiacetic acid groups followed by addition of Cu²⁺. In this way, immobilization is mainly directed via the most accessible histidine group, followed by intramolecular reaction of the other nucleophilic residues of the enzyme and the remaining epoxy groups on the support. The results showed higher thermal stability for immobilized derivatives compared to the soluble enzyme. Co-solvent stability of the derivatives was also studied in presence of six polar organic solvents (DMSO, THF, acetonitrile, 1-propanol, 2-propanol and dioxane). Influence of the immobilization procedure on activity and selectivity of the immobilized preparations was studied in selective hydrolysis of fish oil. All the derivatives discriminate between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA. Remarkable improvement in selectivity was obtained using oriented immobilization of RML.     

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.     

Enantioselective enzymatic hydrolysis of racemic glycidyl esters by using immobilized porcine pancreas lipase with improved catalytic properties

The crude porcine pancreas lipase (PPL) extract is a mixture of several proteins (mainly lipases and esterases). In order to develop enzymatic catalysts with good catalytic properties for hydrolytic enantioselective reactions in aqueous homogeneous medium, we studied the immobilization of the different enzymes contained in the crude PPL extracts by selective sequential adsorption on hydrophobic supports bearing octyl, octadecyl and phenyl groups. Some minor proteins were selectively adsorbed on octyl and octadecyl supports while the most abundant lipase was adsorbed on the support bearing phenyl groups. The enantioselectivity of the different lipase derivatives were tested considering the hydrolysis of esters of 1,2-epoxi-1-propanol (glycidol). The most abundant lipase contained in the commercial crude PPL extract resulted almost inactive while some lipases contained in low concentrations displayed high activities and enantioselectivities. The most interesting results were obtained with a 28-kDa protein selectively adsorbed on octyl-agarose. With this enzyme derivative, the residual butyric ester of glycidol was recovered with 96% enantiomeric excess at 55% conversion.     

Synthesis of ethyl isovalerate using Rhizomucor miehei lipase: optimization

Immobilized lipase from Rhizomucor miehei (Lipozyme IM-20) was used to catalyze the esterification reaction between isovaleric acid and ethanol to synthesize ethyl isovalerate in n-hexane. Response surface methodology based on five-level four-variable central composite rotatable design was employed to optimize four important reaction variables such as enzyme/substrate E/S ratio, substrate concentration, incubation time, and temperature affecting the synthesis of ethyl isovalerate. The optimum conditions predicted for achieving maximum ester yield (500 mM) are as follows: E/S ratio, 48.41 g/mol; substrate concentration, 1 M; reaction time, 60 h; temperature, 60 degrees C. The predicted value matched well with experimentally obtained value of 487 mM.     

Hyperactivation of Rhizomucor miehei lipase by hydrophobic xerogels

Although a variety of approaches exist for the immobilization of enzymes, the "science" of enzyme immobilization is still in its infancy. In recent years, considerable interest has developed regarding the use of xerogels for enzyme immobilization. There are several advantages to xerogels for enzyme immobilization, including the opportunity to produce them in defined shapes or thin films and the ability to manipulate their physical characteristics (e.g., porosity, hydrophobicity, and optical properties). In this study we examined the effect of xerogel hydrophobicity on the activity of lipase (EC 3.2.2.3) from Rhizomucor miehei. The hydrophobicity of the xerogels was manipulated by generating xerogels with various molar ratios of propyltrimethoxysilane (PTMS) to tetramethoxysilane (TMOS), from 1:1 to 10:1. The belief was that, by increasing the proportion of propyl groups, the hydrophobicity of the resulting xerogel would be increased. Differences in the hydrophobicity of the resulting xerogels were confirmed using water-affinity studies. Two approaches were taken for water-affinity determinations by examining the ability of the xerogels to remove water from air (controlled humidity) and from water-saturated isopropyl ether. Xerogels with higher propyl content showed a reduced affinity for water. A crude lipase preparation from Rhizomucor miehei was then contacted with sized xerogel particulates and the effect of the xerogel on lipase activity was determined. The presence of the xerogel resulted in hyperactivation of the lipase. Analysis of the protein adsorption revealed changes in the profile of proteins adsorbed to the xerogel based on the hydrophobicity of the xerogel. Based on estimations of the specific activity of the hyperactivated lipase, a minimum hyperactivation of 207% was observed. Part of the hyperactivation may be attributable to xerogel-lipase interactions, but also to the adsorption of a component from the crude lipase preparation that may complex with the lipase and the xerogel producing a stabilizing effect. Further improvements in hyperactivation and selectivity of the xerogels is likely possible by working at lower PTMS:TMOS ratios than those investigated in this study.     

Synthesis of acetone glycerol acyl esters by immobilized lipase ofMucor miehei

Summary The applications of immobilized lipase ofMucor miehei for the synthesis of acetone glycerol acyl ester from acetone glycerol and fatty acid, which is the first step for monoglyceride production was investigated. With a high oleic acid to acetone glycerol ratio (O/A, mol/mol), a high catalytic activity was observed under low water content in the reaction mixture. By the combination of high O/A ratio (>3) and removal of water which was produced during the reaction, the conversion degree was increased to almost 100%. With the O/A ratio of 3, the approximate half-life of the immobilized lipase and productivity of ester was estimated to be 20 days and 869 g product/g immobilized enzyme per 2 half-lives, respectively.     

Enzymatic esterification of ethanol by an immobilised Rhizomucor miehei lipase in a perforated rotating disc bioreactor

A perforated rotating disc bioreactor was developed to perform the esterification of ethanol with oleic acid, catalyzed by a lipase from Rhizomucor miehei immobilized by adsorption on to a hydrophobic support-Accurel EP700. The bioreactor with total recirculation operated at an optimum agitation rate of 400 rev./min. The experimental results, in this condition, were predict by a kinetic model using the constants obtained in the batch (Erlenmeyer flasks) assays: a catalytic constant, k(cat) = 5.78 mmol/h. mg protein; a Michaelis constant for ethanol, K(m(Et)) = 1.20 M; a Michaelis constant for oleic acid, K(m(Ol)) = 1.16 x 10(-8) M, and a dissociation constant of the ethanol-lipase complex, K((Et)) = 9.46 x 10(7) M. The efficiency of conversion gradually decreased during continuous operation of the reactor. The enzymatic activity decayed according to a first order deactivation model and the integrated equations of a continuous stirred tank reactor (CSTR) and a plug flow reactor (PFR). A half-life time of the lipase of about 10 days and a deactivation constant of 0.003 h(-1) were obtained in the present system.     

Optimisation of n-octyl oleate enzymatic synthesis over Rhizomucor miehei lipase

Octyl oleate is a useful organic compound with several applications in cosmetic, lubricant and pharmaceutical industry. At first, the enzymatic synthesis of n-octyl oleate by direct lipase-catalysed esterification of oleic acid and 1-octanol was investigated in a stirred batch reactor in solvent-free system. A systematic screening and optimisation of the reaction parameters were performed to gain insight into the kinetics mechanism. Particularly, enzyme concentration, reaction temperature, stirrer speed, water content, substrates concentration and molar ratio were optimised with respect to the final product concentration and reaction rate. The kinetics mechanism of the reaction was investigated. Finally, a comparison of the experimental results obtained in a solvent free-system with those using two different solvents, supercritical carbon dioxide (SC-CO₂) and n-hexane, was proposed. It resulted that in SC-CO₂ higher concentration of the desired product was attained, requiring lower enzyme concentrations to achieve comparable conversion of free fatty acid into fatty acid ester.     

Computational analysis of chain flexibility and fluctuations in Rhizomucor miehei lipase.

We have performed molecular dynamics simulation of Rhizomucor miehei lipase (Rml) with explicit water molecules present. The simulation was carried out in periodic boundary conditions and conducted for 1. 2 ns in order to determine the concerted protein dynamics and to examine how well the essential motions are preserved along the trajectory. Protein motions are extracted by means of the essential dynamics analysis method for different lengths of the trajectory. Motions described by eigenvector 1 converge after approximately 200 ps and only small changes are observed with increasing simulation time. Protein dynamics along eigenvectors with larger indices, however, change with simulation time and generally, with increasing eigenvector index, longer simulation times are required for observing similar protein motions (along a particular eigenvector). Several regions in the protein show relatively large fluctuations and in particular motions in the active site lid and the segments Thr57-Asn63 and the active site hinge region Pro101-Gly104 are seen along several eigenvectors. These motions are generally associated with glycine residues, while no direct correlations are observed between these fluctuations and the positioning of prolines in the protein structure. The partial opening/closing of the lid is an example of induced fit mechanisms seen in other enzymes and could be a general mechanism for the activation of Rml.     

Immobilization of Rhizomucor miehei lipase on a polymeric film for synthesis of important fatty acid esters: kinetics and application studies

Bioprocess and Biosystems Engineering - The present work deals with the designing of biocompatible hybrid blend of cellulosic copolymers made of hydroxypropyl methylcellulose (HMC) and chitosan...     

Immobilization of Rhizomucor miehei lipase onto montmorillonite K-10 and polyvinyl alcohol gel

Abstract

Immobilization of enzymes from different sources on various supports in designed systems increases enzymes’ stability by protecting the active site of it from undesired effect of reaction environment. Also, immobilization decreases the cost of separation and facilities the reuse of the enzymes. Therefore, the design of new immobilization enzyme preparations has been an inevitable area of modern biotechnology. Herein, Rhizomucor miehei lipase (RML) was immobilized on montmorillonite K-10 (MMT-RML) by adsorption and in polyvinyl alcohol (PVA-RML) by entrapment to obtain a more stable and active lipase preparation. The free and immobilized lipase preparations were characterized for p-nitrophenyl palmitate hydrolysis. The apparent Michaelis–Menten (K_mapp) constant was almost the same for the free RML and PVA-RML, whereas the corresponding value was 17.7-fold lower for MMT-RML. PVA-RML and MMT-RML have shown a 1.1 and 23.8 folds higher catalytic efficiency, respectively, than that of the free RML. The half-lives of PVA-RML and MMT-RML were found to be 7.4 and 3.4 times longer than the free RML at 35?°C, respectively. PVA-RML and MMT-RML maintained 65% and 87% of their initial activities after four reuses. These results showed that the catalytic performance of RML has improved significantly by immobilization.     

High-throughput screening of B factor saturation mutated Rhizomucor miehei lipase thermostability based on synthetic reaction

Conventional lipase screening methods are mostly based on hydrolytic activity, which may not always be the best method to assess the enzyme activity, especially for evaluating synthetic activity. Here we developed a high throughput and visual method to screen clones with high synthetic activity and used it to assess lipases thermostability. All mutants' lipase synthetic activity were identified through esterification of caprylic acid and ethanol with methyl red as the pH indicator adding in the substrates on according to the color change halo around the colony on culture plates since synthetic reaction was often accompanied with a rise in pH. After two rounds operation with the pH indicator screening method, we obtained a double mutant Asn120Lys/Lys131Phe from the Rhizomucor miehei lipase saturation mutated library based on amino acid residue B factors. The mutant's initial synthetic activity was a little higher than wild type and its thermostability in synthetic reaction was enhanced, which remained 63.1% residual activity after being heated at 70°C for 5h comparing to 51.0% of wild type. The double mutant with the two residue replacements balanced well between stability and activity. Yeast surface display technology and the pH indicator method, combined with colony screening were shown to facilitate high-throughput screening for lipase synthetic activity.     

Physical crosslinking of lipase from Rhizomucor miehei immobilized on octyl agarose via coating with ionic polymers: Avoiding enzyme release from the support

Lipase from Rhizomucor miehei (RML) was immobilized on octyl-agarose (OC) at different loadings. Using low enzyme loadings (1/7 of the maximum loading), the incubation of the enzyme with polyethylenimine (PEI) increased the resistance to enzyme desorption in the presence of Triton X-100. However, more than 10% of the enzyme activity could be released from the OC-RML-PEI. The same treatment using fully loaded biocatalyst reduced the enzyme desorption to less than 5%. Further treatment with dextran sulfate (DS) of the PEI treaded immobilized enzyme fully avoids the enzyme desorption even in presence of a Triton X-100 concentration higher than that required for the complete enzyme release from OC-RML. This treatment produced a high stabilization of OC-RML in thermal or organic solvent inactivations, reducing the enzyme release under these drastic conditions. Nevertheless, the support could be recovered by incubation under adequate conditions, and reused in several adsorption/desorption cycles. Thus, the strategy permitted to avoid enzyme desorption, very likely by physical intermolecular crosslinking improving enzyme stability, while still maintaining the reversibility of the immobilization.     

Polyhydric alcohol protective effect on Rhizomucor miehei lipase deactivation enhanced by pressure and temperature treatment

The influence of polyhydric alcohols (sorbitol, xylitol, erythritol, glycerol) on the thermal stability of Rhizomucor miehei lipase has been studied at high hydrostatic pressure (up to 500 MPa). In the absence of additives, a protective effect (PE) (the ratio between the residual activities determined at 480 MPa for the enzyme in the presence or absence of polyhydric alcohols) of low-applied pressures (from 50 MPa to 350 MPa) against thermal deactivations (at 50°C and 55°C) has been noticed. In the presence of additives, a strong correlation between PE and the total hydroxyl group concentration has been obtained, for the first time, under treatments of combining denaturing temperatures and high hydrostatic pressures. This relationship does not seem to be dependent on the nature polyhydric alcohols as the same effect could be observed with 1 M sorbitol and 2 M glycerol. This PE, against thermal and high pressure combined lipase deactivation, increases with polyhydric alcohol concentrations, and when temperature increases from 25°C to 55°C.     

Enhanced activity of Rhizomucor miehei lipase by directed evolution with simultaneous evolution of the propeptide

Propeptides are short sequences that facilitate the folding of their associated proteins. The present study found that the propeptide of Rhizomucor miehei lipase (RML) was not proteolytically removed in Escherichia coli. Moreover, RML was not expressed if the propeptide was removed artificially during the cloning process in E. coli. This behavior in E. coli permitted the application of directed evolution to full-length RML, which included both propeptide and catalytic domain, to explore the role played by the propeptide in governing enzyme activity. The catalytic rate constant, k (cat), of the most active mutant RML protein (Q5) was increased from 10.63?±?0.80 to 71.44?±?3.20?min(-1) after four rounds of screening. Sequence analysis of the mutant displayed three mutations in the propeptide (L57V, S65A, and V67A) and two mutations in the functional region (I111T and S168P). This result showed that improved activity was obtained with essential involvement by mutations in the propeptide, meaning that the majority of mutants with enhanced activity had simultaneous mutations in propeptide and catalytic domains. This observation leads to the hypothesis that directed evolution has simultaneous and synergistic effects on both functional and propeptide domains that arise from the role played by the propeptide in the folding and maturation of the enzyme. We suggest that directed evolution of full-length proteins including their propeptides is a strategy with general validity for extending the range of conformations available to proteins, leading to the enhancement of the catalytic rates of the enzymes.     

Casein hydrolysis in stirred tank reactors using chymotrypsin immobilized on magnetic supports

Nickel-NiO-BSA-chymotrypsin has been used to hydrolyze casein in both batch and continuous stirred tank reactors. The kinetics of hydrolysis in both types of reactor were considered. Some operational problems encountered using fine powder catalysts in batch stirred tank reactors are discussed. High gradient magnetic separation was found to be a powerful catalysts retention method for magnetic support particles, particularly when using a ferromagnetic collection matrix. Nickel particles of diameter 3–7 μm were easily separated from water at a processing velocity of 39 mm/sec. Nickel powder and precipitated Fe₃O₄ were also separated satisfactorily using a rotating drum magnetic separator.