High hydrostatic pressure increased stability and activity of immobilized lipase in hexane

Lipases are important to high value product synthesis, modification, and enhancement. However, they are often unstable above 40 °C. While most current applications of high hydrostatic pressure (HHP) are for inactivating deleterious enzymes, there is evidence that HHP can stabilize and increase activity of some enzymes. This study examines the apparent kinetics of immobilized lipase-catalyzed synthesis of isoamyl acetate at HHP in hexane. HHP reduced thermal inactivation of lipase by up to 152% after 4 h at 80 °C and 400 MPa when compared to incubations at low pressure. No significant differences were found in activation energy (E_a) at different pressures, irrespectively of the pressurization and heating sequence, and were between 35.7 ± 3.5 and 47.8 ± 8.2 kJ mol^?1, depending on the method. In all methods utilized, activity at 63.5 and 80 °C at 400 MPa was greater (from about 20 to 96% increase) than at low pressure. Activity increased by 110% at low pressure versus a 239% increase at 350 MPa when the temperature was increased from 40 to 80 °C. Increasing pressure up to 350 MPa increased lipase activity while pressures greater than 350 MPa maintained or decreased lipase activity. Activation volume (ΔV^≠) appeared negative between ambient pressure and 200 MPa in contrast to a positive ΔV^≠ between 300 and 600 MPa. Apparent ΔV^≠ was 14.3 ± 1.7 or 15.2 ± 2.2 cm³ mol^?1 at 40 or 80 °C, respectively, between 300 and 500 MPa.     

Gene cloning and molecular characterization of the Talaromyces thermophilus lipase catalyzed efficient hydrolysis and synthesis of esters

A genomic bank from Talaromyces thermophilus fungus was constructed and screened using a previously isolated fragment lipase gene as probe. From several clones isolated, the nucleotide sequence of the lipase gene (TTL gene) was completed and sequenced. The TTL coding gene consists of an open reading frame (ORF) of 1083 bp encoding a protein of 269 Aa with an estimated molecular mass of 30 kDa. The TTL belongs to the same gene family as Thermomyces lanuginosus lipase (TLL, Lipolase®), a well known lipase with multiple applications. The promoter sequence of the TTL gene showed the conservation of known consensus sequences PacC, CreA, Hap2-3-4 and the existence of a particular sequence like the binding sites of Oleate Response Element (ORE) and Fatty acids Responsis Element (FARE) which are similar to that already found to be specific of lipolytic genes in Candida and Fusarium, respectively. Northern blot analysis showed that the TTL expression was much higher on wheat bran than on olive oil as sole carbon source. Compared to the Lipolase®, this enzyme was found to be more efficient for the hydrolysis and the synthesis of esters; and its synthetic efficiency even reached 91.6% from Waste Cooking Oil triglycerides.     

Immobilized Talaromyces thermophilus lipase as an efficient catalyst for the production of LML-type structured lipids

Bioprocess and Biosystems Engineering - LML-type structured lipids are one type of medium- and long-chain triacylglycerols. LML was synthesized using immobilized Talaromyces thermophilus lipase...     

Esterification by immobilized lipase in solvent-free media: kinetic and thermodynamic arguments

The aim of this study is to characterize, in solvent-free systems (SFS), the kinetic and thermodynamic performance of batch lipase-catalyzed esterification. SFS are compared to a conventional organic solvent, n-hexane. The esterification of oleic acid with ethanol was chosen as a model reaction. The TABEK (thermodynamic activity-based enzyme kinetics) approach was used to rationally analyze kinetics. Influence of the reaction medium on final conversions was also studied. Several factors, such as initial molar ratio of substrates, reactant availability, initial water content, and quantity of immobilized enzyme, were examined. Special attention was also turned to enzyme stability and reuse after reaction, this last item being a prerequisite in the development of industrial processes. SFS proved to be almost as efficient as n-hexane from a kinetic and thermodynamic point of view and offered a better volumetric production.     

Catalytic properties of lipases immobilized onto ultrasound-treated chitosan supports

Ultrasound sonication has been utilized to produce fragmentation of chitosan polymer and hence increase the chitosan surface area, making it more accessible to interactions with proteins. In this context, we have investigated the catalytic properties of lipases from different sources immobilized onto ultrasound-treated chitosan (ChiS) pre-activated with glutaraldehyde (ChiS-G). Atomic force microscopy indicated that ChiS-G displays a more cohesive frame without the presence of sheared/fragmented structures when compared with ChiS, which might be attributed to the cross-linking of the polysaccharide chains. The immobilization efficiency onto ChiS-G and ChiS were remarkably higher than using conventional beads. In comparison with the free enzymes, lipases immobilized onto ChiS show a slight increase of apparent K_m and decrease of apparent V_max. On the other hand, immobilization onto ChiS-G resulted in an increase of V_max, even though a slight increase of K_m was also observed. These data suggest that the activation of chitosan with glutaraldehyde has beneficial effects on the activity of the immobilized lipases. In addition, the immobilization of the lipases onto ChiS-G displayed the best reusability results: enzymes retained more than 50% of its initial activity after four reuses, which might be attributed to the covalent attachment of enzyme to activated chitosan. Overall, our findings demonstrate that the immobilization of lipases onto ultrasound-treated chitosan supports is an effective and low-cost procedure for the generation of active immobilized lipase systems, being an interesting alternative to conventional chitosan beads.     

Enzymatic activity and stability of D-fructose dehydrogenase and sarcosine dehydrogenase immobilized onto giant vesicles

Stable vesicles with diameters between about 1 and 10 mum were prepared by a particular emulsification technology that involved the use of the surfactants Span 80 and Tween 80 and the phospholipid lecithin (phosphatidylcholine from soybeans). Two membrane enzymes, d-fructose dehydrogenase from Gluconobacter sp. (FDH) and sarcosine dehydrogenase from Pseudomonas putida (SDH), were for the first time immobilized onto the bilayer membranes of these type of vesicles; and the catalytic activity and enzymatic stability were measured and compared with the enzymes in a vesicle-free solution. The enzyme activity as well as stability considerably increased upon immobilization. In particular, immobilized FDH at 25 degrees C was stable for at least 20 days, while the activity of the free enzyme dropped to about 20% of its initial value during the same period of time.In contrast to FDH and SDH, immobilization of sorbitol dehydrogenase from Gluconobacter suboxydans (SODH) was not successful, as no improved activity or stability could be obtained.     

固定化脂肪酶性质及其应用研究

利用以四甲氧基硅烷(TMOS)和甲基三甲氧基硅烷(MTMS)为前驱体的溶胶-凝胶法(sol-gel)固定洋葱假单胞菌属脂肪酶,考查了固定化酶和游离酶的酶学性质及催化不同油脂酯交换合成生物柴油的情况。结果表明,80℃以下固定化酶能保持80%以上的酶活,而游离酶在50℃以后活力急剧下降,到80℃残余酶活约为10%;固定化酶在体积分数50%的甲醇中处理48 h能保持85%的酶活,在体积分数90%的乙醇中处理48h能保持31%的酶活,而游离酶残余酶活只有69%和0;在酯交换反应中固定化酶的催化效率比游离酶高10%~20%,且固定化酶重复使用11次后仍能保持60%的酶活。结果显示,酶经过固定化后稳定性和催化活性显著提高。     

Two additives to improve stability of immobilized lipase

Abstract

The presence of two different additives during non-covalent immobilization of lipase was studied. Lipase was immobilized via hydrophobic interactions on an amorphous silica with large pore size bearing octyl groups on the surface. Polyethyleneglycol (PEG) with different molecular weights (MW: 1500, 3000 and 10,000) were added to the suspension during enzyme immobilization, in an enzyme to PEG molar ratio of 1:10, and also 1:20 in the case of PEG1500. The activity after 15 d increased from 10% (absence of PEG) to values close to 40% in samples with PEG except the catalyst immobilized in the presence of 1:10 PEG1500, which kept fully active after 15 d incubation in toluene at 70?°C. The presence of water during storage of immobilized enzymes leads to significant activity loss. Saturated solutions of salts controlling the water activity of the systems were used to reduce in a controlled fashion the moisture of the systems: CaCl₂ (a_w=?0.037), MgCl₂ (a_w=?0.328), Mg(NO₃)₂ (a_w=?0.529), Na₂PO₄.12H₂O (a_w=?0.74) and KCl (a_w=?0.84). The immobilized lipase was suspended in saturated solutions of these salts, and then filtered and incubated in desiccators in the presence of the corresponding saturated salt solutions. Catalysts suspended and incubated in KCl or only suspended in phosphate kept some 20% activity after 33 d incubation whereas the maximal stability was achieved when the catalyst was suspended in phosphate and kept in a desiccator without salt solution. This catalyst kept around 50% activity after 33 d incubation. An inversely proportional relationship can be established between the stability achieved by the enzyme and the water content of the system.     

Expression of Talaromyces thermophilus lipase gene in Trichoderma reesei by homologous recombination at the cbh1 locus

Journal of Industrial Microbiology & Biotechnology - CBH1 (cellobiohydrolase) comprises the majority of secreted proteins by Trichoderma reesei. For expression of Talaromyces thermophilus...     

Inhibition of lipase activity by low-molecular-weight chitosan

Inhibition of enzymatic activity of lipase (EC 3.1.1.3) from the fungus Candida rugosa and wheat (Triticum aestivum L.) germ by low-molecular-weight chitosan with an average molecular weight of 5.7 kDa in reactions of p-nitrophenyl palmitate cleavage was studied. Preincubation of lipases with chitosan, prior to addition of the substrate to solution, showed that equilibrium during the lipase-inhibitor complex formation was reached within 30 min. The inhibition constants for C. rugosa lipase and wheat germ lipase were 1.4 and 0.9 mM, respectively. The contribution of electrostatic interactions to the complex formation between chitosan and lipases is insignificant.     

Inhibition of lipase activity by low-molecular-weight chitosan

Inhibition of enzymatic activity of lipase (EC 3.1.1.3) from the fungus Candida rugosa and wheat (Triticum aestivum L.) germ by low-molecular-weight chitosan with an average molecular weight of 5.7 kDa in reactions of p-nitrophenyl palmitate cleavage was studied. Preincubation of lipases with chitosan, prior to addition of the substrate to solution, showed that equilibrium during the lipase-inhibitor complex formation was reached within 30 min. The inhibition constants for C. rugosa lipase and wheat germ lipase were 1.4 and 0.9 mM, respectively. The contribution of electrostatic interactions to the complex formation between chitosan and lipases is insignificant.     

Inhibition of lipase activity by low-molecular-weight chitosan

Inhibition of enzymatic activity of lipase (EC 3.1.1.3) from the fungus Candida rugosa and wheat (Triticum aestivum L.) germ by low-molecular-weight chitosan with an average molecular weight of 5.7 kDa in reactions of p-nitrophenyl palmitate cleavage was studied. Preincubation of lipases with chitosan, prior to addition of the substrate to solution, showed that equilibrium during the lipase-inhibitor complex formation was reached within 30 min. The inhibition constants for C. rugosa lipase and wheat germ lipase were 1.4 and 0.9 mM, respectively. The contribution of electrostatic interactions to the complex formation between chitosan and lipases is insignificant.     

Preparation and characterization of urease immobilized onto porous chitosan beads for urea hydrolysis

Urease was covalently immobilized onto porous chitosan beads via primary amine groups connected to the backbone via a six-carbon linear alkyl spacer. The optimum conditions for enzyme immobilization are activating the beads with 1%(w/w) glutaraldehyde, reacting the activated beads in pH 7 buffer with the enzyme, using an enzyme to bead weight ratio of 25, and without lyophilization. Chitosan-bound urease was found to fully retain its specific activity. Properties of the immobilized urease were characterized under batch and flow conditions. Increased optimum reaction temperature, enhanced thermal stability and storage stability, and excellent reusability were found after enzyme immobilization. Continuous hydrolysis of urea solution was studied in a column packed with the enzyme-containing beads for its possible application in regenerating dialysate solution during hemodialysis.     

Activity and storage stability of immobilized glucose oxidase onto magnesium silicate

Glucose oxidase (GOD) was covalently immobilized onto florisil (magnesium silicate) carrier via glutaraldehyde. Immobilization conditions were optimized: the amount of initial GOD per grams of carrier as 5 mg, pH as 5.5, immobilization time as 120 min and temperature as 10 °C. Under the optimized reaction conditions activities of free and immobilized GOD were measured. Free and immobilized GOD samples were characterized with their kinetic parameters, and thermal and storage stabilities. K_M and V_max values were 68.2 mM and 435 U mg GOD⁻¹ for free and 259 mM and 217 U mg GOD⁻¹ for immobilized enzymes, respectively. Operational stability of the immobilized enzyme was also determined by using a stirred batch type column reactor. Immobilized GOD was retained 40% of its initial activity after 50 reuses. Storage stabilities of the immobilized GOD samples stored in the mediums with different relative humidity in the range of 0–100% were investigated during 2 months. The highest storage stability was determined for the samples stored in the medium of 60% relative humidity. Increased relative humidity from 0% to 60% caused increased storage stability of immobilized GODs, however, further increase in relative humidity from 80% to 100% caused a significant decrease in storage stability of samples.     

Biodiesel production from pomace oil by using lipase immobilized onto olive pomace

In the present work, microbial lipase from Thermomyces lanuginosus was immobilized by covalent binding onto olive pomace. Immobilized support material used to produce biodiesel with pomace oil and methanol. The properties of the support and immobilized derivative were evaluated by scanning electron microscopy (SEM). The maximum immobilization of T. lanuginosus was obtained as 18.67 mg/g support and the highest specific activity was 10.31 U/mg protein. The properties of immobilized lipase were studied. The effects of protein concentration, pH and buffer concentration on the immobilization and lipase activity were investigated. Biodiesel production using the immobilized lipase was realized by a three-step addition of methanol to avoid strong substrate inhibition. Under the optimized conditions, the maximum biodiesel yield was 93% at 25 °C in 24 h reaction. The immobilized enzyme retained its activity during the 10 repeated batch reactions.     

Enhanced activity and stability of ionic liquid-pretreated lipase

The activity and stability of Mucor javanicus lipase pretreated with various ionic liquids (ILs) were investigated. The results show that the activity and stability of lipase pretreated with ILs were higher than those of untreated lipase for the hydrolysis reaction in an aqueous medium. The activities of lipase pretreated with ILs such as [Bmim][PF₆], [Emim][Tf₂N], [Bmim][BF₄] and [Emim][BF₄] were 1.81, 1.66, 1.56 and 1.60 times higher than that of untreated lipase, respectively. Furthermore, activities of lipase in ILs were well maintained even after 7 days of incubation in ILs at 60 °C, while untreated lipase in phosphate buffer was fully inactivated only after 12 h of incubation at the same temperature. These results suggest that pretreatment of lipase with ILs might form IL-coated lipase which causes the structural change of lipase, and thus, enhances the activity and stability of lipase in aqueous solution.     

Sequential enzymatic reactions and stability of biomolecules immobilized onto phospholipid polymer nanoparticles

Polymer nanoparticles for sequential enzymatic reactions were prepared by combining a phospholipid polymer shell with a polystyrene core. The active ester groups for the bioconjugation and phospholipid polar groups were incorporated into the phospholipid polymer backbone using a novel active ester monomer and 2-methacryloyloxyethyl phosphorylcholine. For the sequential enzymatic reactions, acetylcholinesterase, choline oxidase, and horseradish peroxidase-labeled IgG were immobilized onto the nanoparticles. As substrates, acetylcholine chloride, choline chloride, and tetramethylbenzidine were added to the nanoparticle suspension, the acetylcholine chloride was converted to choline chloride, the choline chloride was oxidized by choline oxidase, and hydrogen peroxide was then formed as an enzymatic degradation product. The hydrogen peroxide was used for the next enzymatic reaction (oxidized by peroxidase) with tetramethylbenzidine. The sequential enzymatic reactions on the nanoparticles via degradation products (hydrogen peroxide) were significantly higher than that of the enzyme mixture. This result indicated that the diffusion pathway of the enzymatic products and the localization of the immobilized enzyme were important for these reactions. These nanoparticles were capable of facilitating sequential enzymatic reactions.     

Cysteine enhances activity and stability of immobilized papain

Immobilization of papain on Sepharose 6B in the presence of different concentrations of cysteine affected the enzyme activity depending on cysteine concentration. The maximum specific activity was observed when papain was immobilized with 200 mM cysteine. The immobilization process brought significant enhancement of stability to temperature and extreme pH values with respect to free papain. After immobilization, the optimum temperature of papain activity increased by 20°C (from 60 to 80°C) and its optimum pH activity shifted from 6.5 to 8.0. Catalytic efficiency (k_cat/K_m) and specific activity of the immobilized enzyme do not significantly change after immobilization. The temperature profile of this form of immobilized papain showed a broad range of activity compared with both free and immobilized form of papain in the absence of cysteine. This significant behavior in terms of activation energy is also discussed.     

Use of stable emulsion to improve stability, activity, and enantioselectivity of lipase immobilized in a membrane reactor

The enantiocatalytic performance of immobilized lipase in an emulsion membrane reactor using stable emulsion prepared by membrane emulsification technology was studied. The production of optical pure (S)-naproxen from racemic naproxen methyl ester was used as a model reaction system. The O/W emulsion, containing the substrate in the organic phase, was fed to the enzyme membrane reactor from shell-to-lumen. The enzyme was immobilized in the sponge layer (shell side) of capillary polyamide membrane with 50 kDa cut-off. The aqueous phase was able to permeate through the membrane while the microemulsion was retained by the thin selective layer. Therefore, the substrate was kept in the enzyme-loaded membrane while the water-soluble product was continuously removed from the reaction site. The results show that lipase maintained stable activity during the entire operation time (more than 250 h), showing an enantiomeric excess (96 +/- 2%) comparable to the free enzyme (98 +/- 1%) and much higher compared to similar lipase-loaded membrane reactors used in two-separate phase systems (90%). The results demonstrate that immobilized enzymes can achieve high stability as well as high catalytic activity and enantioselectivity.     

Esterification of phenolic acids from green coffee with an immobilized lipase from Candida antarctica in solvent-free medium

Phenolic acid esters were synthesized by direct esterification catalysed by an immobilized lipase obtained from Candida antarctica. Esterification yields ranged from 3 to 98%; yields seem to be linked to the electronic distribution of phenolic acids which affects the reactivity of the carboxylic function. The carbon chain length of the alcohols also plays an important role. It was observed that even for high yields, reaction times were two days or more.