Kinetics of lipase catalyzed isoamyl acetate synthesis at high hydrostatic pressure in hexane

Lipase-catalyzed synthesis of isoamyl acetate in hexane at 10–250 MPa at 80°C and 1–100 MPa at 40°C resulted in activation volumes of −12.9 ± 1.7 and −21.6 ± 2.9 cm³ mol⁻¹, respectively. Increasing pressure from 10 to 200 MPa resulted in approximately 10-fold increase in V _max at both 40 and 80°C. Pressure increased the K _m from 2.4 ± 0.004 to 38 ± 0.78 mM at 40°C. In contrast, at 80°C the pressure did not affect the K _m.     

Esterification activity and stability of Talaromyces thermophilus lipase immobilized onto chitosan

The Talaromyces thermophilus lipase (TTL) was immobilized by different methods namely adsorption, ionic binding and covalent coupling, using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the most suitable support material preserving the catalytic activity almost intact and offering maximum immobilization capacity (76% and 91%, respectively). The chitosan-immobilized lipase could be reputably used for ten cycles with more than 80% of its initial hydrolytic activity. Shift in the optimal temperature from 50 to 60 °C and in the pH from 9.5 to 10, were observed for the immobilized lipase when compared to the free enzyme.The catalytic esterification of oleic acid with 1-butanol has been carried out using hexane as organic solvent. A high performance synthesis of 1-butyl oleate was obtained (95% of conversion yield) at 60 °C with a molar ratio of 1:1 oleic acid to butanol and using 100 U (0.2 g) of immobilized lipase. The esterification product is analysed by GC/MS to confirm the conversion percentage calculated by titration.     

Application of high hydrostatic pressure for increasing activity and stability of enzymes

Elevated hydrostatic pressure has been used to increase catalytic activity and thermal stability of alpha-chymotrypsin (CT). For an anilide substrate, characterized by a negative value of the reaction activation volume (DeltaV( not equal)), an increase in pressure at 20 degrees C results in an exponential acceleration of the hydrolysis rate catalyzed by CT reaching a 6.5-fold increase in activity at 4700 atm (4.7 kbar). Due to a strong temperature dependence of DeltaV( not equal), the acceleration effect of high pressure becomes more pronounced at high temperatures. For example, at 50 degrees C, under a pressure of 3.6 kbar, CT shows activity which is more than 30 times higher than the activity at normal conditions (20 degrees C, 1 atm). At pressures of higher than 3.6 kbar, the enzymatic activity is decreased due to a pressure-induced denaturation.Elevated hydrostatic pressure is also efficient for increasing stability of CT against thermal denaturation. For example, at 55 degrees C, CT is almost instantaneously inactivated at atmospheric pressure, whereas under a pressure of 1.8 kbar CT retains its anilide-hydrolyzing activity during several dozen minutes. Additional stabilization can be achieved in the presence of glycerol, which is most effective for protection of CT at an intermediate concentration of 40% (v/v). There has been observed an additivity in stabilization effects of high pressure and glycerol: thermal inactivation of pressure-stabilized CT can be decelerated in a supplementary manner by addition of 40% (v/v) glycerol. The protection effect of glycerol on the catalytic activity and stability of CT becomes especially pronounced when both extreme factors of temperature and pressure reach critical values. For example, at approximately 55 degrees C and 4.7 kbar, enzymatic activity of CT in the presence of 40% (v/v) glycerol is severalfold higher than in aqueous buffer.The results of this study are discussed in terms of the hypotheses which explain the action of external and medium effects on protein structure, such as preferential hydration and osmotic pressure. (c) 1996 John Wiley & Sons, Inc.     

Immobilization of lipase by salts and the transesterification activity in hexane

Lipases from six different sources were immobilized on Celite and five types of salt. The transesterification activities in hexane for lipases immobilized on EDTA-Na₂ increased by 463% for the lipase from Candida rugosa (CRL), 2700% for the lipase from Candida sp. (CSL) and 1215% for the lipase from Pseudomonas sp. (PSL), compared to the salt-free enzyme. With 0.5% sucrose for CRL or 1% sorbitol for PSL as the lyoprotectant during lyophilization process, transesterification activity increased by 100% and 13%, respectively, compared to the immobilized enzyme on EDTA-Na₂ without lyoprotectant.     

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。     

High hydrostatic pressure approach proves RNA catalytic activity without magnesium

High hydrostatic pressure (HHP) technique was used to evaluate a mechanism of RNA hydrolysis with RNA. We showed that hammerhead ribozyme specifically cleaves RNA substrate at HHP in the absence of Mg(2+). A deoxyribozyme "10-23" was active in the same conditions. These results pointed out that the hydrolytic activity of nucleic acid depends on proper tertiary structure of a complex with a substrate. They prove that magnesium ion is not directly involved in catalysis process. On that basis we show the mechanism of RNA hydrolysis catalyzed with nucleic acids at HHP.     

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.     

High hydrostatic pressure in cancer immunotherapy and biomedicine

High hydrostatic pressure (HHP) has been known to affect biological systems for >100?years. In this review, we describe the technology of HHP and its effect macromolecules and physiology of eukaryotic cells. We discuss the use of HHP in cancer immunotherapy to kill tumor cells for generation of whole cell and dendritic cell-based vaccines. We further summarize the current use and perspectives of HHP application in biomedicine, specifically in orthopedic surgery and for the viral, microbial and protozoan inactivation to develop vaccines against infectious diseases.     

Algal photosynthesis at increased hydrostatic pressure and constant pO2

Summary Rates of oxygen evolution, ¹⁴CO₂ uptake, and growth were measured in several species of algae and diatoms exposed to hydrostatic pressures up to 600 atm. All of the parameters measured decreased approximately linearly with increased pressure over the range tested. Oxygen evolution appears to be less sensitive to increased pressure than CO₂ fixation or growth.This paper was supported by Office of Naval Research Contract No. 00014-67-A-0387-0008 and intramural research grants from the University of Hawaii.This work is taken in part from a thesis to be submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Microbiology, University of Hawaii.     

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.     

Residual stability of lipase from Candida rugosain hexane, supercritical CO , and supercritical SF

Lipase from Candida rugosa (EC 3.1.1.3) lost only 15% of its activity when held in supercritical CO and about 10% activity in both supercritical SF and hexane even after two days of incubation at up to 60°C and 82 atm.A pressure of 680 atm resulted in up to 15% loss of enzyme activity in supercritical CO and only about 5% loss of activity in supercritical SF 6 even at 410 atm. There was about 60% decrease in enzyme activity even at 1% water content in supercritical CO . Supercritical SF is a better solvent than supercritical CO and hexane.     

High activity and selectivity immobilized lipase on Fe3O4 nanoparticles for banana flavour synthesis

Lipase (E.C.3.1.1.3) from Thermomyces lanuginosus (TL) was directly bonded, through multiple physical interactions, on citric acid functionalized monodispersed Fe₃O₄ nanoparticles (NPs) in presence of a small amount of hydrophobic functionalities. A very promising scalable synthetic approach ensuring high control and reproducibility of the results, and an easy and green immobilization procedure was chosen for NPs synthesis and lipase anchoring. The size and structure of magnetic nanoparticles were characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD). The samples at different degree of functionalization were analysed through thermogravimetric measurements. Lipase immobilization was further confirmed by enzymatic assay and Fourier transform infrared (FT-IR) spectra. Immobilized lipase showed a very high activity recovery up to 144% at pH = 7 and 323% at pH = 7.5 (activity of the immobilized enzyme compared to that of its free form). The enzyme, anchored to the Fe₃O₄ nanoparticles, to be easy recovered and reused, resulted more stable than the native counterpart and useful to produce banana flavour. The immobilized lipase results less sensitive to the temperature and pH, with the optimum temperature higher of 5 °C and optimum pH up shifted to 7.5 (free lipase optimum pH = 7.0). After 120 days, free and immobilized lipases retained 64% and 51% of their initial activity, respectively. Ester yield at 40 °C for immobilized lipase reached 88% and 100% selectivity.     

Structural stability of human butyrylcholinesterase under high hydrostatic pressure

Human butyrylcholinesterase is a nonspecific enzyme of clinical, pharmacological and toxicological significance. Although the enzyme is relatively stable, its activity is affected by numerous factors, including pressure. In this work, hydrostatic pressure dependence of the intrinsic tryptophan fluorescence in native and salted human butyrylcholinesterase was studied up to the maximum pressure at ambient temperature of about 1200 MPa. A correlated large shift toward long wavelengths and broadening observed at pressures between 200 and 700 MPa was interpreted as due to high pressure-induced denaturation of the protein, leading to an enhanced exposure of tryptophan residues into polar solvent environment. This transient process in native butyrylcholinesterase presumably involves conformational changes of the enzyme at both tertiary and secondary structure levels. Pressure-induced mixing of emitting local indole electronic transitions with quenching charge transfer states likely describes the accompanying fluorescence quenching that reveals different course from spectral changes. All the pressure-induced changes turned irreversible after passing a mid-point pressure of about 400 ± 50 MPa. Addition of either 0.1 M ammonium sulphate (a kosmotropic salt) or 0.1 M lithium thiocyanate (a chaotropic salt) to native enzyme similarly destabilized its structure.     

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.     

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.     

High hydrostatic pressure induces slow contraction in mouse cardiomyocytes

Cardiomyocytes are contractile cells that regulate heart contraction. Ca²⁺ flux via Ca²⁺ channels activates actomyosin interactions, leading to cardiomyocyte contraction, which is modulated by physical factors (e.g., stretch, shear stress, and hydrostatic pressure). We evaluated the mechanism triggering slow contractions using a high-pressure microscope to characterize changes in cell morphology and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in mouse cardiomyocytes exposed to high hydrostatic pressures. We found that cardiomyocytes contracted slowly without an acute transient increase in [Ca²⁺]_i, while a myosin ATPase inhibitor interrupted pressure-induced slow contractions. Furthermore, transmission electron microscopy showed that, although the sarcomere length was shortened upon the application of 20 MPa, this pressure did not collapse cellular structures such as the sarcolemma and sarcomeres. Our results suggest that pressure-induced slow contractions in cardiomyocytes are driven by the activation of actomyosin interactions without an acute transient increase in [Ca²⁺]_i.     

High hydrostatic pressure and biology: a brief history

Pressure as a thermodynamical parameter was successively introduced in physics, hydrometallurgy, geochemistry, and biology. In all cases, the main objective was to recreate a natural phenomenon (gas or liquid compressibility, synthesis or crystal growth of minerals, survival of deep sea microorganisms…). The introduction of high hydrostatic pressure (HHP) in Biology was an important scientific feature over the last hundred years. This paper describes the different steps that have led to the spreading of pressure in biology and the opening of new frontiers either in basic and applied researches due to the specific characteristics of the pressure parameter. Because of the low energy conveyed by this parameter, leading to the preservation of most organoleptic properties of foods, and its ability to inactivate many pathogens, the use of HHP began to spread at the end of the twentieth century into the food industry, in particular for the development of pathogen inactivation processes. Today, even if this field is still the first application domain for HHP, more and more research works have shown that this parameter could be of great interest in health and medicine sciences.