Evaluation of diatomaceous earth as a support for sol–gel immobilized lipase for transesterification

Enzymatic production of biodiesel by triglyceride transesterification is a promising alternative to chemically catalyzed biodiesel production despite the challenges involved with using enzymes. Celite^® supported lipase sol–gels were investigated as an option for solving some of the challenges associated with the use of enzymes for biodiesel production addressing such problems as activity, stability and reusability of the enzyme. Three types of Celite^® were considered (R633, R632, and R647) and compared to unsupported lipase sol–gels. Various factors were considered with regard to comparing the support materials. They included surface morphology characterized using surface area analysis and scanning electron microscopy, physical properties including adhesion of the sol–gel to the Celite^® and the protein loading on the Celite^®, and finally enzymatic properties based on the conversion of methanol to methyl oleate and the enzymatic activity of lipase. All the sol–gels showed good conversion and initial lipase activity, and all the Celite^® supports had similar sol–gel adhesion and protein loading. Sol–gel immobilized lipase supported on Celite^® R632 had an average 6-h percent conversion of approximately 60%, and an average initial lipase activity comparable to that of the unsupported sol–gel formulation.     

Kinetic modelling of the production of methyl oleate by Celite® supported lipase sol–gels

This study illustrates the benefits of Celite^® supported lipase sol–gels for the transesterification of triolein to produce methyl oleate. A ping–pong bi–bi kinetic model was developed and validated taking into account the inhibition effects of methanol and glycerol as well as the effect of temperature. Although initial reaction rate models are useful for predicting the kinetics in the absence of products, a kinetic model beyond the initial conditions that considers glycerol inhibition is important. The model developed was consistent with the experimental data (R² = 0.95) predicting an increase in methyl oleate production with increasing methanol concentration up to an optimal range of 1.3 M to 2.0 M depending on the temperature. In general, increasing the temperature increased the initial reaction rate for the immobilized lipase over the temperature range of 40–60 °C. Based on the kinetic constants, the maximum velocity of the reverse reaction is about 25% slower than that of the forward reaction and glycerol inhibition has a more significant effect on the reaction kinetics than methanol inhibition. The model developed would be useful for understanding the effects of methanol and glycerol inhibition as well as temperature on the production of methyl oleate using lipase-mediated enzymatic transesterification.     

An organic solvent-tolerant alkaline lipase from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CS268 and its application in biodiesel production

In an effort to identify a microbial lipase that can catalyze transesterification reactions used in biodiesel production, an organic solvent-tolerant lipase was purified from Streptomyces sp. CS268. The molecular weight of the purified lipase was estimated to be 37.5 kDa by SDS-PAGE. The lipase showed highest activity at a temperature of 30°C and pH 8.0 while it was stable in the pH range 4.0 ∼ 9.0 and at temperatures ≤ 50°C. It showed the highest hydrolytic activity towards medium-length acyl chain p-nitrophenyl decanoate with K _m and V _max values of 0.59 mM and 319.5 mmol/mg/min, respectively. Also, the lipase showed non-position specificity for triolein hydrolysis. The purified lipase catalyzed transesterification reaction of soybean oil with methanol, suggesting that it can be a potential enzymatic catalyst for biodiesel production.     

Combination of two lipases more efficiently catalyzes methanolysis of soybean oil for biodiesel production in aqueous medium

A combination of two lipases was employed to catalyze methanolysis of soybean oil in aqueous medium for biodiesel production. The two lipase genes were cloned from fungal strains Rhizomucor miehei and Penicillium cyclopium, and each expressed successfully in Pichia pastoris. Activities of the 1,3-specific lipase from R. miehei (termed RML) and the non-specific mono- and diacylglycerol lipase from P. cyclopium (termed MDL) were 550 U and 1545 U per ml respectively, and enzymatic properties of these supernatant of fermentation broth (liquid lipase) were stable at 4 °C for >3 months. Under optimized conditions, the ratio of biodiesel conversion after 12 h at 30 °C, using RML alone, was 68.5%. When RML was assisted by addition of MDL, biodiesel conversion ratio was increased to >95% under the same reaction conditions. The results suggested that combination of lipases with different specificity, for enzymatic conversion of more complex lipid substrates, is a potentially useful strategy for biodiesel production.     

Immobilization of lipase Eversa Transform 2.0 on poly(urea–urethane) nanoparticles obtained using a biopolyol from enzymatic glycerolysis

In this work, the free lipase Eversa^® Transform 2.0 was used as a catalyst for enzymatic glycerolysis reaction in a solvent-free system. The product was evaluated by nuclear magnetic resonance (¹H NMR) and showed high conversion related to hydroxyl groups. In sequence, the product of the glycerolysis was used as stabilizer and biopolyol for the synthesis of poly(urea–urethane) nanoparticles (PUU NPs) aqueous dispersion by the miniemulsion polymerization technique, without the use of a further surfactant in the system. Reactions resulted in stable dispersions of PUU NPs with an average diameter of 190 nm. After, the formation of the PUU NPs in the presence of concentrated lipase Eversa^® Transform 2.0 was studied, aiming the lipase immobilization on the NP surface, and a stable enzymatic derivative with diameters around 231 nm was obtained. The hydrolytic enzymatic activity was determined using ρ-nitrophenyl palmitate (ρ-NPP) and the immobilization was confirmed by morphological analysis using transmission electron microscopy and fluorescence microscopy.

     

An organic solvent–tolerant lipase from Streptomyces sp. CS133 for enzymatic transesterification of vegetable oils in organic media

The enzymatic route for biodiesel production has been noted to be cost ineffective due to the high cost of biocatalysts. Reusing the biocatalyst for successive transesterification cycles is a potential solution to address such cost inefficiency. However, when organic solvent like methanol is used as acyl-acceptor in the reaction, the biocatalyst (lipase) gets severely inactivated due to the inhibitory effect of undissolved methanol in the reaction medium. Thus, organic solvent–tolerant lipase is highly desirable for enzymatic transesterification. In response to such desirability, a lipase (LS133) possessing aforesaid characteristic was extracted from Streptomyces sp. CS133. Relative molecular mass of the purified LS133 was estimated to be 39.8 kDa by SDS-PAGE. Lipase LS133 was stable in pH range 5.0–9.0 and at temperature lower than 50 °C while its optimum lipolytic activity was achieved at pH 7.5 and 40 °C. It showed the highest hydrolytic activity towards long chain p-nitrophenyl palmitate with K_m and V_max values of 0.152 mM and 270.2 mmol min^?1 mg^?1, respectively. It showed non-position specificity for triolein hydrolysis. The first 15 amino acid residues of its N-terminal sequence, AIPLRQTLNFQAXYQ, were noted to have partial similarity with some of the previously reported microbial lipases. Its catalytic involvement in biodiesel production process was confirmed by performing enzymatic transesterification of vegetable oils with methanol.     

Kinetics of lipase recovery from the aqueous phase of biodiesel production by macroporous resin adsorption and reuse of the adsorbed lipase for biodiesel preparation

A commercial macroporous resin (D3520) was screened for lipase recovery by adsorption from the aqueous phase of biodiesel production. The influences of several factors on the adsorption kinetics were investigated. It was found that the kinetic behavior of lipase adsorption by macroporous resin could be well described by pseudo-first-order model. Temperature had no significant effects on lipase adsorption, while resin-to-protein ratio (R) significantly affected both rate constant (k₁) and equilibrium adsorption capacity (Q_e). No lipase was adsorbed when mixing (shaking) was not performed; however, protein recovery reached 98% after the adsorption was conducted at 200 rpm for 5 h in a shaker. The presence of methanol and glycerol showed significant negative influence on lipase adsorption kinetics. Particularly, increasing glycerol concentration could dramatically decrease k₁ but not impact Q_e. Biodiesel was found to dramatically decrease Q_e even present at a concentration as low as 0.02%, while k₁ was found to increase with biodiesel concentration. The adsorbed lipase showed a relatively stable catalytic activity in tert-butanol system, but poor stability in solvent-free system when used for biodiesel preparation. Oil and biodiesel were also found to adsorb onto resin during transesterification in solvent-free system. Therefore, the resin had to be washed by anhydrous methanol before re-used for lipase recovery.     

Immobilization of <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B onto Purolite<Superscript>®</Superscript> MN102 and its application in solvent-free and organic media esterification

The aim of this study was to develop simple and efficient method for immobilization of Candida antarctica lipase B onto hydrophobic anion exchange resin Purolite^® MN102 and to apply immobilized catalyst for the enzymatic synthesis of two valuable esters—isoamyl acetate and l-ascorbyl oleate. At optimized conditions (1 M phosphate buffer pH = 7, 7 h at 25 °C, and 18.75 mg of offered proteins g^?1 of support), immobilized lipase with hydrolytic activity of 888.4 p-nitrophenyl butyrate units g^?1 was obtained. Afterwards, preparation was applied for the solvent-free synthesis of isoamyl acetate from triacetin and isoamyl alcohol. At 75 °C, 1 M of isoamyl alcohol, and 6 mg ml^?1 of enzyme 100 % yield was achieved in 6 h, while at prolonged reaction times, complete conversion was enabled even at lower temperatures, lower lipase loadings, and higher substrate concentrations. After 15 consecutive reuses (60 h), activity of catalyst dropped to 50 % of its initial value and total amount of 1.31 mol (170.55 g) of ester with 1 g of biocatalyst was produced. Even higher operational stability of lipase (25 % loss of activity in 200 h) was observed in the synthesis of l-ascorbyl oleate performed in organic solvent (t-butanol). Multiple use of one batch of immobilized biocatalyst in both cases led to a significant process cost reduction and substantial increment of corresponding productivities.     

An anion-exchange radioassay for glucose 6-phosphate phosphatase: use in topological studies with endoplasmic reticulum vesicles.

A simple procedure is presented for the enzymatic preparation of [2-3H]mannose 6-phosphate (Man 6-P) with purified yeast hexokinase and unlabeled ATP. The enzymatically synthesized [2-3H]Man 6-P is utilized as the radiolabeled substrate in a new rapid assay for glucose 6-phosphate (Glc 6-P) phosphatase. The principle of the assay procedure is that the unreacted substrate, [2-3H]Man 6-P, is retained by the anion-exchange resin, AG 1-X8 (acetate), while the enzymatic product, [2-3H]-mannose, is eluted directly into a scintillation counting vial. When Glc 6-P phosphatase activity associated with mouse liver endoplasmic reticulum (ER) vesicles is assayed by the new chromatographic assay, the same characteristic latency and properties are observed, as determined by the commonly used colorimetric assay of inorganic phosphate produced. The anion-exchange radioassay described should be useful for a variety of topological studies on enzymes associated with membrane vesicles derived from liver and kidney ER.     

Ectoine improves yield of biodiesel catalyzed by immobilized lipase

To improve the production of biodiesel by enzymatic conversion of triglycerides in cottonseed oil, compatible solutes were added to the solvent-free methanolysis system to prevent competitive methanol inhibition on the immobilized lipase (Novozym^® 435). The results indicated that the addition of ectoine increased biodiesel synthesis using a three-step methanol addition process. The concentration of methyl ester (ME) reached a maximum of 95.0% in the presence of 1.1 mmol/l ectoine, an increase of 20.9% compared to that in the absence of ectoine. On the other hand, excess ectoine decreased the ME concentration. Ectoine was also shown to enhance reuse of the immobilized lipase, significantly improving ME concentrations in each recycling test. Total concentrations of ME with added ectoine were about 1.5 times that without ectoine during five recycling tests (molar ratio of cottonseed oil to methanol, 1:4). Enzymatic reaction kinetics showed, in the concentration ranges of 0.8–1.14 mol/l and 0.03–8 mol/l for triglyceride and methanol, respectively, that ectoine had no effect on the initial reaction rates when methanol concentrations were below 0.5 mol/l. When methanol concentration exceeded 0.5 mol/l, the addition of 0.8 mmol/l ectoine increased the initial reaction rates, and the lipase exhibited a lower affinity for methanol and higher affinity for triglyceride (kinetic parameters of K_mA increase, K_mTG decrease). However, the initial reaction rates decreased significantly when 8 mmol/l ectoine was added, with the lipase having higher affinity for methanol and lower affinity for triglyceride (K_mA decrease, K_mTG increase). The supplementation of ectoine provided a new method for the purpose of improving yield of biodiesel catalyzed by enzyme.     

Immobilization of lipase on epoxy-activated Purolite® A109 and its post-immobilization stabilization

In this study, Purolite^® A109, polystyrenic macroporous resin, was used as immobilization support due to its good mechanical properties and high particle diameter (400 μm), which enables efficient application in enzyme reactors due to lower pressure drops. The surface of support had been modified with epichlorhydrine and was tested in lipase immobilization. Optimized procedure for support modification proved to be more efficient than conventional procedure for hydroxy groups (at 22 °C for 18 h), since duration of procedure was shortened to 40 min by performing modification at 52 °C resulting with almost doubled concentration of epoxy groups (563 μmol g⁻¹). Lipase immobilized on epoxy-modified support showed significantly improved thermal stability comparing to both, free form and commercial immobilized preparation (Novozym^® 435). The highest activity (47.5 IU g⁻¹) and thermal stability (2.5 times higher half-life than at low ionic strength) were obtained with lipase immobilized in high ionic strength. Thermal stability of immobilized lipase was further improved by blocking unreacted epoxy groups on supports surface with amino acids. The most efficient was treatment with phenylalanine, since in such a way blocked immobilized enzyme retained 65% of initial activity after 8 h incubation at 65 °C, while non-blocked derivative retained 12%.     

Cloning,expression and characterization of a new lipase from <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Bioinformatic analysis of the Yarrowia lipolytica CLIB122 genome has revealed 18 putative lipase genes all of which were expressed in Escherichia coli and screened for hydrolyzing activities against p-nitrophenyl-palmitate. One positive transformant containing an ORF of 1,098 bp encoding a protein of 365 amino acids was obtained. To characterize its enzymatic properties, the lipase gene was functionally expressed in Pichia pastoris. The resulting lipase exhibited the highest activity towards p-NP-decanoate at pH 7 and 35°C. In addition, the new lipase had a lower optimal temperature and pH compared to other Y. lipolytica lipases. It was noticeably enhanced by Ca²⁺, but was inhibited by PMSF, Hg²⁺ and Ni²⁺. The new lipase displayed the 1,3-specificity for triolein.     

In vivo biostability of four types of arterial grafts with impervious walls; their haemodynamic and pathological characteristics

We describe the haemodynamic and pathological characteristics of four types of impervious arterial prostheses, two alloplastic (Milrathane^® and Gore-Tex^®), and two chemically processed bovine heterografts (Solcograft^® and Solco P^®). They were implanted in the thoracic aortae of dogs for durations of 24 hours, 48 hours, one weeks, two weeks, one month, three months and six months. Haemodynamic analyses showed no relation between the shear rate index, I·_Y, and compliance, C_D. The observed shear rates are 6.5 times lower than those likely to damage the endothelial cell layer. Macroscopic and microscopic observations of explanted grafts showed the presence of obstructive thrombi at the anastomoses of Mitrathane^® grafts as early as one week. Gore-Tex^® grafts develop in the area of anastomoses parietal-thrombi which reorganize and become covered with pseudo-endothelial cells. The bovine heterografts show a similar behaviour. However, whereas Solcograft^® has an irregular thin wall, Solco P^® had improved characteristics except in the graft implanted for three months which demonstrated some manufacturing weaknesses. Both types showed the development of anastomotic pannus covered with endothelial-like cells. All grafts, whether alloplastic or chemically processed, suffered from an absence of healing of the middle part of the prosthesis. The cause of this problem will be found in the analysis of the biochemical and enzymatic reactations between the material used and its physiological surrounding.     

A novel control of enzymatic enantioselectivity through the racemic temperature influenced by reaction media

The influence of reaction media on the racemic temperature (T_r) in the lipase-catalyzed resolution of ketoprofen vinyl ester was investigated. An effective approach to the control of the enzymatic enantioselectivity and the prediction of the increasing tendency was developed based on the T_r influenced by reaction media. The T_r for the resolution catalyzed by Candida rugosa lipase (CRL) was found at 29 °C in aqueous and S-ketoprofen was obtained predominantly at 40 °C. However, CRL showed R-selectivity at 40 °C in diisopropyl ether because the T_r was changed to 56 °C. CRL, lipase from AYS Amano^® and Mucor javanicus lipase were further applied for the investigation of the enzymatic enantioselectivity in dioxane, DIPE, isooctane and their mixed media with water. The effects of the reaction medium on T_r could be related to the solvent hydrophobicity, the lipase conformational flexibility and the interaction between the enantiomers and the lipase.     

Incorporation of cholesterol into serum high and low density lipoproteins

Excess cholesterol (CHL) was incorporated into serum lipoproteins by incubation with Celite^®-dispersed CHL or CHL crystals. The initial rates of uptake were 15 and 7 mol CHL/mol lipoprotein × h^? for low (LDL) and high (HDL) density lipoprotein, respectively. Saturation values were obtained after 48 h and were 90 and 65 mol CHL/mol LDL, and 42 and 32 mol/mol HDL using CHL on Celite and CHL cyrstals, respectively. Characterization of the lipo-proteins showed a small change in electrophoretic mobility and an increase in molecular masses, especially after incubation with CHL on Celite. Spectroscopic methods showed only minor effects on the protein moieties.     

Production,purification and characterization of an extracellular lipase from Aureobasidium pullulans HN2.3 with potential application for the hydrolysis of edible oils

Lipase production (8.02 ± 0.24 U/ml) by the yeast Aureobasidium pullulans HN2.3 isolated from sea saltern was carried by using time-dependent induction strategy. The lipase in the supernatant of the yeast cell culture was purified to homogeneity with a 3.4-fold increase in specific lipase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography and anion-exchange chromatography. According to the data on SDS polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 63.5 kDa. The optimal pH and temperature of the purified enzyme were 8.5 and 35 °C, respectively. The enzyme was greatly inhibited by Hg²⁺, Fe²⁺ and Zn²⁺. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride, not inhibited by ethylene diamine tetraacetic acid (EDTA), but weakly inhibited by iodoacetic acid. It was found that the purified lipase had the highest hydrolytic activity towards peanut oil.     

Genetic organization of the putative salbostatin biosynthetic gene cluster including the 2-<Emphasis Type="Italic">epi</Emphasis>-5-<Emphasis Type="Italic">epi</Emphasis>-valiolone synthase gene in <Emphasis Type="Italic">Streptomyces albus</Emphasis> ATCC 21838

In this paper, we provide the first report of utilizing recombinant fungal whole cells in enzymatic biodiesel production. Aspergillus oryzae, transformed with a heterologous lipase-encoding gene from Fusarium heterosporum, produced fully processed and active forms of recombinant F. heterosporum lipase (FHL). Cell immobilization within porous biomass support particles enabled the convenient usage of FHL-producing A. oryzae as a whole-cell biocatalyst for lipase-catalyzed methanolysis. The addition of 5% water to the reaction mixture was effective in both preventing the lipase inactivation by methanol and facilitating the acyl migration in partial glycerides, resulting in the final methyl ester content of 94% even in the tenth batch cycle. A comparative study showed that FHL-producing A. oryzae attained a higher final methyl ester content and higher lipase stability than Rhizopus oryzae, the previously developed whole-cell biocatalyst. Although both FHL and R. oryzae lipase exhibit 1,3-regiospecificity towards triglyceride, R. oryzae accumulated a much higher amount of sn−2 isomers of partial glycerides, whereas FHL-producing A. oryzae maintained a low level of the sn−2 isomers. This is probably because FHL efficiently facilitates the acyl migration from the sn−2 to the sn−1(3) position in partial glycerides. These findings indicate that the newly developed FHL-producing A. oryzae is an effective whole-cell biocatalyst for enzymatic biodiesel production.     

Enhanced Performance of Rhizopus oryzae Lipase Immobilized on Hydrophobic Carriers and Its Application in Biorefinery of Rapeseed Oil Deodorizer Distillate

In this study, hydrophobic macroporous resin NKA was employed as matrix for immobilization of free Rhizopus oryzae lipase (ROL). The performance of the immobilized ROL was significantly enhanced. The recovery activity was up to 1,293.78 % and the specific activity increased to 152,914 U/g-protein, which was 46-fold higher than that of the free lipase. Moreover, the immobilized lipase showed higher thermostability and better pH-resistance than its free counterpart. Additionally, three different nonaqueous modification strategies (including bioimprinting, lecithin coating, and lyophilization protection) were further utilized to improve the performance of the immobilized lipase. The corresponding enhancements were 33.68 %, 31.98 %, and 99.86 %. When these modifications were combined together, the activity improved 209.51 %. In order to confirm its practical application, the modified ROL was used to biorefine rapeseed oil deodorizer distillate (RODD) for biodiesel production. The highest conversion yield reached 98.23 %, much close to that (97.46 %) of Novozym 435. The results suggest that the prepared lipase in this study is a promising biocatalyst with high stability, efficiency and operational reusability.     

Kinetics of enzymatic transesterification and thermal deactivation using immobilized Burkholderia lipase as catalyst

The most effective way of enzymatic synthesis of biodiesel is through lipase-catalyzed transesterification, while its performance and economic feasibility should still be improved. In this study, lipase produced by an isolated Burkholderia sp. was immobilized on microsize Celite materials functionally modified with long alkyl groups. The specific activity of the immobilized lipase was 1,154 U/g. The methanolysis of olive oil catalyzed by the immobilized lipase obeyed Ping Pong Bi Bi model with an estimated V _max, K _m,TG, K _m,M and K _i,M value of 0.61 mol/(L min), 7.93 mol/L, 1.01 mol/L, and 0.24 mol/L, respectively. The activation energy of the enzymatic reaction is estimated as 15.51 kJ/mol. The immobilized lipase exhibits high thermal stability with thermal deactivation energy of 83 kJ/mol and a long half-life. The enthalpy, Gibb’s free energy, and entropy of the immobilized lipase were in the range of 80.02–80.35 kJ/mol, 88.35–90.13 kJ/mol, and ?28.22 to ?25.11 J/(mol K), respectively.     

Biodiesel production with immobilized lipase: A review

Fatty acid alkyl esters, also called biodiesel, are environmentally friendly and show great potential as an alternative liquid fuel. Biodiesel is produced by transesterification of oils or fats with chemical catalysts or lipase. Immobilized lipase as the biocatalyst draws high attention because that process is “greener”. This article reviews the current status of biodiesel production with immobilized lipase, including various lipases, immobilization methods, various feedstocks, lipase inactivation caused by short chain alcohols and large scale industrialization. Adsorption is still the most widely employed method for lipase immobilization. There are two kinds of lipase used most frequently especially for large scale industrialization. One is Candida antartica lipase immobilized on acrylic resin, and the other is Candida sp. 99–125 lipase immobilized on inexpensive textile membranes. However, to further reduce the cost of biodiesel production, new immobilization techniques with higher activity and stability still need to be explored.