Stability of subtilisin and lysozyme under high hydrostatic pressure

The stabilities of subtilisin and lysozyme under hydrostatic pressures up to 200 MPa were investigated for up to 7 days at 25 degrees C. Methods were chosen to assess changes in tertiary and secondary protein structure as well as aggregation state. Tertiary structure was monitored in situ with second derivative UV spectroscopy and after pressure treatment by dynamic light scattering and second derivative UV spectroscopy. Secondary structure and potential secondary structural changes were characterized by second derivative FTIR spectroscopy. Changes in aggregation state were assessed using dynamic light scattering. Additionally, protein concentration balances were carried out to detect any loss of protein as a function of pressure. For the conditions tested, neither protein shows measurable changes in tertiary or secondary structure or signs of aggregation. Lysozyme concentration balances show no dependence on pressure. Subtilisin concentration balances at high protein concentration (4 mg/mL and higher) do not show pressure dependence. However, the concentration balances carried out at 0.4 mg/mL show a clear sign of pressure dependence. These results may be explained by protein interaction with the vial surface and appear to be rate limited by the equilibrium between active and inactive protein on the surface. Pressure increases protein loss, and the estimated partial molar volume change between the two states is estimated to be -20 +/- 10 mL/mol.     

超高压对植物乳杆菌能量代谢影响的研究

以植物乳杆菌ATCC8014为试材,研究超高压对其能量代谢的影响。建立了用氯化碘硝基四唑紫测定ATCC8014的INT代谢还原活性的比色法。用比色法测定了超高压对ATCC8014的INT代谢还原活性与葡萄糖利用的影响。试验结果表明,150~250MPa作用15min在MRS琼脂培养基上随着压力的增大菌落数显著降低,INT代谢还原活性降低显著,葡萄糖的利用变化不明显;超过300MPa后,葡萄糖的利用才显著降低;400MPa处理15min,尽管在MRS琼脂培养基上菌落数低于检测限,INT代谢还原活性为0%,而葡萄糖的利用能力仍为对照组的56.1%,超高压作用下ATCC8014的灭活与INT代谢还原活性的降低的相关性较好。说明ATCC8014的细胞膜上参与葡萄糖的吸收和运输的酶、糖酵解的酶与调节系统比三羧酸循环的酶与调节系统较耐压。三羧酸循环比糖酵解对超高压敏感,三羧酸循环的抑制是超高压灭活其的重要原因,这为了探讨超高压杀灭植物乳杆菌的机制提供了一定的理论依据。     

Changes in rabbit skeletal myosin and its subfragments under high hydrostatic pressure

The pressure-induced denaturation of rabbit skeletal myosin and its subfragments under hydrostatic pressure were investigated. Four nanometer of red shift of the intrinsic fluorescence spectrum was observed in myosin under a pressure of 400 MPa. The ANS fluorescence of myosin increased with elevating pressure. Changes in the intrinsic fluorescence spectra of myosin and its subfragments were quantified and expressed as the center of spectral mass. The center of spectral mass of myosin and its subfragments linearly decreased with elevating pressure, and increased with lowering pressure. The fluorescence intensity of the ANS-labeled rod did not change during pressure treatment. The present results indicate that the most pressure-sensitive portion of myosin molecule is the head. Hysteresis of the center of spectral mass of S1 appeared under pressures above 300 MPa. Changes in the center of spectral mass of S1 above 350 MPa showed stronger hysteresis. The center of spectral mass did not decrease above 350 MPa during the compression process, indicating that S1 was stable in a partially denatured state at 350 MPa under pressure. The changes in the relative intensities of ANS fluorescence of S1 were measured under pressures up to 400 MPa, and the ANS fluorescence intensity increased with elevating pressure but it did not change after pressure release. The ANS fluorescence intensity increased under constant pressure suggesting that the pressure-induced denaturation of myosin was accelerated during pressurization.     

Sub-zero temperature inactivation of carboxypeptidase Y under high hydrostatic pressure.

High hydrostatic pressure induced cold inactivation of carboxypeptidase Y. Carboxypeptidase Y was fully active when exposed to subzero temperature at 0.1 MPa; however, the enzyme became inactive when high hydrostatic pressure and subzero temperature were both applied. When the enzyme was treated at pressures higher than 300 MPa and temperatures lower than -5 degrees C, it underwent an irreversible inactivation in which nearly 50% of the alpha-helical structure was lost as judged by circular dichroism spectral analysis. When the applied pressure was limited to below 200 MPa, the cold inactivation process appeared to be reversible. In the presence of reducing agent, this reversible phenomenon, observed at below 200 MPa, diminished to give an inactive enzyme; the agent reduces some of disulfide bridge(s) in an area of the structure that is newly exposed area because of the cold inactivation. Such an area is unavailable if carboxypeptidase Y is in its native conformation. Because all the disulfide bridges in carboxypeptidase Y locate near the active site cleft, it is suggested that the structural destruction, if any, occurs preferentially in this disulfide rich area. A possible mechanism of pressure-dependent cold inactivation of CPY is to destroy the alpha-helix rich region, which creates an hydrophobic environment. This destruction is probably a result of the reallocation of water molecules. Experiments carried out in the presence of denaturing agents (SDS, urea, GdnHCl), salts, glycerol, and sucrose led to a conclusion consistent with the idea of water reallocation.     

An improved sampling technique for bacterial cultures under high hydrostatic pressure

A sampling technique for bacterial cultures subjected to high hydrostatic pressure is described. A sample-receiving vessel with a motor driven interface-piston is employed. By precisely matching the pressures in the bulk culture and the sample-receiving vessel, none of the sample is subjected to the high shear forces common to other desings of high pressure sampler. The use of the technique was illustrated by the growth of an anaerobic culture at 300 bar and 75°C.     

High pressure microscopy--a powerful tool for monitoring cells and macromolecules under high hydrostatic pressure.

A high pressure chamber, which withstands a pressure up to 300 MPa has been developed. The so-called HPDS (Hartmann, Pfeifer, Dornheim, Sommer) High Pressure Cell in combination with an inverted microscope and an analysis system allows brilliant microscopic colour pictures with an optical resolution better than 0.56 microm. The pressure chamber allows the in situ observation of dynamic changes of microscopic structures in bright field, phase contrast and fluorescence microscopy. This publication should demonstrate the capabilities of the system using results of experiments with two types of Spirogyra algae. The pictures have shown significant variations of the chloroplasma and the cell wall membrane at pressures of up to 120 MPa. The new system provides a simple way to perform microscopic analyses at pressures of up to 300 MPa.     

Flying-patch patch-clamp study of G22E-MscL mutant under high hydrostatic pressure

High hydrostatic pressure (HHP) present in natural environments impacts on cell membrane biophysical properties and protein quaternary structure. We have investigated the effect of high hydrostatic pressure on G22E-MscL, a spontaneously opening mutant of Escherichia coli MscL, the bacterial mechanosensitive channel of large conductance. Patch-clamp technique combined with a flying-patch device and hydraulic setup allowed the study of the effects of HHP up to 90 MPa (as near the bottom of the Marianas Trench) on the MscL mutant channel reconstituted into liposome membranes, in addition to recording in situ from the mutant channels expressed in E. coli giant spheroplasts. In general, against thermodynamic predictions, hydrostatic pressure in the range of 0.1–90 MPa increased channel open probability by favoring the open state of the channel. Furthermore, hydrostatic pressure affected the channel kinetics, as manifested by the propensity of the channel to gate at subconducting levels with an increase in pressure. We propose that the presence of water molecules around the hydrophobic gate of the G22E MscL channel induce hydration of the hydrophobic lock under HHP causing frequent channel openings and preventing the channel closure in the absence of membrane tension. Furthermore, our study indicates that HHP can be used as a valuable experimental approach toward better understanding of the gating mechanism in complex channels such as MscL.     

Differential polarized phase fluorometric studies of phospholipid bilayers under high hydrostatic pressure

Differential polarized phase fluorometry was used to quantify the rotational rate ($\text{R}$) and limiting anisotropy ($\text{r}{}_{\mathrm{\infty }}$) of the membrane probe diphenylhexatriene (DPH) in solvents and lipid vesicles exposed to hydrostatic pressures ranging from 1 bar to 2 kbar. These measurements reveal the effect of pressure on the phase-transition temperatures of the phosphatidylcholine vesicles, and the effects of pressure on order parameter of the acyl side-chain region of the membranes, the latter as indicated by $\text{r}{}_{\mathrm{\infty }}$. In addition to the well-known elevation of the transition temperature ($\text{T}{}_{\mathrm{<mtext>c</mtext>}}$) with pressure, our results demonstrate that increased pressure restores the order of the bilayers to that representative of temperatures below the transition temperature. We also found that solvents which allowed free isotropic rotation of DPH at 1 bar no longer allowed free rotation when sufficiently compressed; moreover, the apparent DPH rotational rate increased with $\text{r}{}_{\mathrm{\infty }}$. Pressure studies using both DPH and the charged DPH analogue, trimethylammonium DPH (TMA-DPH) indicated that the $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ of dipalmitoylphosphatidylcholine vesicles increased 23 K/kbar and an apparent volume change of 0.036 ml/mol lipid at the phase transition. Assuming, as has been proposed, that TMA-DPH is localized near the glycerol backbone region of the bilayers, these results indicate a similar temperature- and pressure-dependent phase transition in this region and the acyl side-chain region of the membrane.     

Partial alignment and measurement of residual dipolar couplings of proteins under high hydrostatic pressure

High-pressure NMR spectroscopy has emerged as a complementary approach for investigating various structural and thermodynamic properties of macromolecules. Noticeably absent from the array of experimental restraints that have been employed to characterize protein structures at high hydrostatic pressure is the residual dipolar coupling, which requires the partial alignment of the macromolecule of interest. Here we examine five alignment media that are commonly used at ambient pressure for this purpose. We find that the spontaneous alignment of Pf1 phage, d(GpG) and a C12E5/n-hexnanol mixture in a magnetic field is preserved under high hydrostatic pressure. However, DMPC/DHPC bicelles and collagen gel are found to be unsuitable. Evidence is presented to demonstrate that pressure-induced structural changes can be identified using the residual dipolar coupling.     

Subsampling technique for measuring growth of bacterial cultures under high hydrostatic pressure.

A method is presented for measuring growth of bacteria under high hydrostatic pressure in subsamples taken without pressure change in the incubation vessel. Subsamples may be withdrawn rapidly (5 s) and are not subjected to shear forces. Vice versa, nutrient media, labeled substrates, etc., may be introduced into the culture while under pressure. Chemical fixation of subsamples for electron microscopy or adenosine 5'-triphosphate determinations under pressure is also possible without affecting the growing culture. Data are given of growth experiments demonstrating the feasibility of the method. Problems of oxygen depletion are discussed.     

Numerical simulation of the mechanics of a yeast cell under high hydrostatic pressure

The mechanical effects of the compression of a yeast cell (Saccharomyces cerevisiae) under high hydrostatic pressure used for the processing of food and food ingredients are modelled and simulated with the finite-element method. The cell model consists of a cell wall, cytoplasm a lipid filled vacuole and the nucleus. Material parameters have been taken from literature or have been derived from thermodynamic relationships of water and lipids under high hydrostatic pressure. The model has been validated for a pressure load up to 250 MPa. Comparison of the volume reduction to in situ experimental observations reveals very good agreement. Dimensional analysis of the governing equations shows that transient pressure application in a high-pressure food process does not enhance structural inactivation (mechanical damage), unless pressure oscillation frequencies of 700 MHz are applied. The deformation of the cell under pressure deviates strongly from isotropic volume reduction. Especially, organelle membranes exhibit large effective strain values. Hydrostatic stress conditions are preserved in the interior part of the cell. A pressure load of 400 MPa, which is critical upon disruption of cell organelle membranes, generates an effective strain up to 80%. In the cell wall, the stress state is heterogeneous. Von-Mises stress reaches the critical value upon failure of the cell wall of 70+/-4 MPa at a pressure load between 415 and 460 MPa.     

Inactivation of Campylobacter jejuni by high hydrostatic pressure

AIMS: To investigate the response of Campylobacter jejuni ATCC 35919 and 35921 to high pressure processing (HPP) while suspended in microbiological media and various food systems. METHODS AND RESULTS: Campylobacter jejuni 35919 and 35921 were subjected to 10-min pressure treatments between 100 and 400 MPa at 25 degrees C suspended in Bolton broth, phosphate buffer (0.2 m, pH 7.3), ultra-high temperature (UHT) whole milk, UHT skim milk, soya milk and chicken pureé. The survivability of C. jejuni was further investigated by inoculated pack studies. HPP at 300-325 MPa for 10 min at 25 degrees C was sufficient to reduce viable numbers of both strains to below detectable levels when cells were pressurized in Bolton broth or phosphate buffer. All food products examined offered a protective effect in that an additional 50-75 MPa was required to achieve similar levels of inactivation when compared with broth and buffer. Inoculated pack studies showed that the survivability of C. jejuni following pressurization improved with decreasing post-treatment storage temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: These data demonstrated that HPP at levels of 相似文献   

Hydrolysis of β-lactoglobulin by thermolysin and pepsin under high hydrostatic pressure

Hydrolysis of β-lactoglobulin with thermolysin and pepsin at pressures ranging between 0.1 and 350 MPa showed a significant increase of cleavage rates. Pressure-induced changes of susceptibility to hydrolysis of β-lactoglobulin proteolytic sites were also observed. The pressure, raised to 200 MPa, accelerates the hydrolysis of β-lactoglobulin by thermolysin and changes obtained peptide profiles. Initially, higher pressure makes the N-terminal, and to a smaller extent, C-terminal peptide fragments of β-lactoglobulin molecule, more susceptible to removal by thermolysin. This indicates combined influence of pressure-induced thermolysin activation and partial unfolding of β-lactoglobulin by compression at neutral pHs. The rates of hydrolysis of β-lactoglobulin by pepsin (negligible at 0.1 MPa) are increased considerably with pressure up to 300 MPa. The Susceptibility of β-lactoglobulin proteolytic sites to peptic cleavage remains constant over all the studied pressure range. The lack of significant qualitative changes in the peptic peptide profiles produced at different pressures and at clearly pressure-dependent rates points to negative reaction volume changes as the major factor in peptic hydrolysis of β-lactoglobulin under high pressure. Thus the β-lactoglobulin molecule resists pressure-induced unfolding in acid pHs and yields to it in neutral pHs. © 1995 John Wiley & Sons, Inc.     

Dissociation and aggregation of lactic dehydrogenase by high hydrostatic pressure

As shown by earlier experiments high hydrostatic pressure affects the catalytic function of lactic dehydrogenase from rabbit muscle. In the presence of substrates denaturation occurs, whereas in the absence of substrates and --SH-protecting reagents oxidation of sulfhydryl groups takes place [Schmid, G., Lüdemann, H.-D. & Jaenicke, R. (1975) Biophys. Chem. 3, 90--98; (1978) Eur. J. Biochem. 86, 219--224]. Avoiding oxidation effects by reducing conditions in the solvent medium and by chelation of heavy metal ions, the remaining high-pressure effects consist of dissociation of the native quaternary structure into subunits followed by aggregation. Both reactions are influenced by temperature and enzyme concentration. Short incubation (less than or equal to 10 min) at pH 6.0--8.5 and pressures of 0.3--1.0 kbar causes dissociation which is reversed at normal pressure. At 5 degrees C the activation volume is found to be delta V not equal to = -62 +/- 3cm3 . mol-1. Above 1.2 kbar irreversible aggregation takes place; the reaction is favoured by low temperature and decreased pH. The activation volume for the aggregation process at 5 degress C is delta V not equal to = -97 +/- 3cm3 . mol-1. The results may be described by a reaction sequence comprisign pressure-induced dissociation of the native enzyme into its subunits followed by subunit aggregation to form inactive high-molecular-weight particles.