Permeation High-performance Liquid Chromatography Research Articles

Relationship between the cholesterol and triglyceride content of lipoprotein subclasses and carotid intima-media thickness: A cross-sectional population-based study

BackgroundThe association between lipoprotein subclasses and carotid intima-media thickness (cIMT) progression has yet to be fully evaluated. We assessed which lipoprotein subclasses were associated with maximum cIMT levels in the general population. MethodsIn this study, cholesterol and triglyceride content of 20 lipoprotein subclasses were analyzed using gel permeation high-performance liquid chromatography (GP-HPLC) in 864 Japanese women and men (mean age 57 y, free of chronic liver or kidney diseases and off lipid-lowering, hormone replacement, or adrenocorticosteroid medications). Univariate and multivariate regression analyses and univariate and partial correlation analyses were performed to examine the relationships between lipoprotein subclasses and maximum cIMT levels. ResultsAfter adjusting for age, sex, systolic blood pressure, smoking, diabetes, and anti-hypertensive agents, elevated low-density lipoprotein (LDL)-2 and −3 cholesterol (particle diameter 25.5 nm and 23.0 nm, respectively; medium and small LDL) were associated with higher maximum cIMT levels in both women and men (all p for trend < 0.05). These associations were significant even after participants taking anti-diabetic or anti-hypertensive agents were excluded. No significant associations were found between any triglyceride subclasses and maximum cIMT levels. ConclusionsSmaller LDL particle cholesterol values are the most atherogenic lipoprotein parameter.

In vitro angiotensin-converting enzyme and dipeptidyl peptidase-IV inhibitory, and antioxidant activity of blue mussel (Mytilus edulis) byssus collagen hydrolysates

AbstractLarge quantities of mussel byssus are generated annually as a co-product of the mussel-processing industry. This fibrous material is a rich source of collagen, which when extracted has potential uses as an alternative source of collagen for food applications. However, due the complex structure of the material, the extraction of the collagenous components using food-friendly strategies has proved challenging to date. An enzyme-aided method, using a proline endoproteinase, was employed for the extraction of collagen from mussel byssus yielding 138.82 ± 2.25 mg collagen/g dry weight. Hydrolysates of the collagen extract were generated using five food-grade enzyme preparations with Corolase® PP giving the highest extent of hydrolysis. Reversed-phase and gel permeation high-performance liquid chromatography of the extracted collagen and its enzymatic hydrolysates showed significant hydrolysis of collagen. The hydrolysates generated with Corolase® PP showed the highest in vitro bioactivities: angiotensin-converting enzyme (ACE) IC50 = 0.79 ± 0.17 mg/ml, dipeptidyl peptidase-IV (DPP-IV) IC50 = 0.66 ± 0.17 mg/ml and oxygen radical absorbance capacity (ORAC) activity = 311.23 ± 13.41 µmol trolox equivalents (TE)/g. The results presented herein indicate that in addition to acting as an alternative source of collagen for food applications, mussel byssus collagen-derived hydrolysates have potential applications as functional food ingredients for the management of metabolic diseases such as type II diabetes and hypertension.

Abstract 9167: Relationship Between Dietary Vitamin D Intake, Obesity, and Twenty Lipoprotein Subclasses: Results from the Kyushu and Okinawa Population Study

Introduction: The correlation between serum vitamin D deficiency, obesity, and various diseases has been reported, however, there were few investigations on the role of dietary vitamin D intake on obesity and dyslipidemia. Objective: To examine whether dietary vitamin D intake affects obesity and dyslipidemia. Methods: Dietary vitamin D intake amounts calculated using obtained data of food frequency questionnaire and 20 lipoprotein cholesterol and triglycerides subclasses using gel permeation high performance liquid chromatography were analyzed in 945 men and women (mean age 58.3 years, free of thyroid or chronic liver diseases and off osteoporosis, cholesterol lowering, or adrenocorticosteroid medications). Univariate and multivariate regression, and partial correlation analyses were performed to examine the associations between dietary vitamin D intake, obesity, and lipoprotein subclasses. Results: Dietary vitamin D intake had a significant negative association with body mass index in both post- and pre-menopausal women groups ( r = -0.1053 and -0.1761, P = 0.02 and 0.04, respectively). Regarding 20 lipoprotein subclasses, dietary vitamin D intake were negatively associated with very low-density lipoprotein-3 cholesterol in premenopausal women ( r = -0.2099, P =0.02) and positively associated with total high-density lipoprotein (HDL) cholesterol in men ( r = 0.1159, P = 0.04). In addition, dietary vitamin D intake also showed a marginally positive correlation with total HDL cholesterol in premenopausal women ( r = 0.1323, P = 0.13). Those association were also found in the frequency of fish intake, a major source of vitamin D in Japan. Conclusions: Our results indicate that proactive dietary vitamin D intake may help prevent or eliminate obesity and dyslipidemia.

Abstract 9165: Relationship Between the Cholesterol and Triglyceride Content of Lipoprotein Subclasses and Carotid Imtima-Media Thickness: Results from the Kyushu and Okinawa Population Study

Introduction: The association between 20 lipoprotein cholesterol and triglycerides subclasses and carotid intimal medial thickness (cIMT) progression has not been evaluated fully. Objective: Our goal was to assess which lipoprotein subclasses were associated with max cIMT levels. Methods: Levels of cIMT and 20 lipoprotein cholesterol and triglycerides subclasses using gel permeation high-performance liquid chromatography (GP-HPLC) were analyzed in 864 men and women (mean age 57 years, free of chronic liver or kidney diseases and off cholesterol-lowering, hormone replacement, or adrenocorticosteroid medications). Univariate and multivariate regression analyses were performed to examine the relationships between lipoprotein subclasses and max cIMT levels. Results: Max cIMT levels were 0.90 mm in women and 0.97 mm in men. After adjustment for age, gender, systolic blood pressure, smoking habits, diabetes, and hypertension treatment, elevated low-density lipoprotein cholesterol-2 and -3 were associated with higher max cIMT levels in both women and men. Decreased high-density lipoprotein cholesterol-7 were associated with higher max cIMT levels only in women (all P for trend &lt; 0.05). However, no associations were found between any triglyceride subclasses and max cIMT levels. Conclusions: Our data indicate that elevated smaller LDL cholesterol levels separated by GP-HPLC were significantly associated with max cIMT in both men and women. Our results support the concept that small dense LDL cholesterol is the most atherogenic lipoprotein parameter.

In Vitro Digestibility and Antioxidant Activity of Plant Protein Isolate and Milk Protein Concentrate Blends

The replacement of animal with plant proteins in human diets has been increasing in recent years. The impact of blending milk protein concentrate (MPC) with protein isolates from soy (SPI), rice (RPI) and pea (PPI) on the in vitro digestibility and antioxidant activity of the resultant blends was investigated. Different plant protein–MPC blends (i.e., SPI–MPC (25:75), RPI–MPC (50:50) and PPI–MPC (25:75)) were analyzed. The lowest protein digestibility corrected amino acid score (PDCAAS) was associated with RPI (0.70), while the blends had PDCAAS values above 1.00 demonstrating the high digestibility of the proteins in the blends studied. An in vitro simulated gastrointestinal digestion was carried out on the samples. The degree of hydrolysis and gel permeation high performance liquid chromatography profiles showed that the SPI–MPC blend was more extensively digested in the gastric phase compared with the two other blends, while the PPI–MPC and RPI–MPC blends were mainly digested during the intestinal phase. The SPI–MPC digested blend had the highest 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity having a half maximal effective concentration (EC50) of 0.10 ± 0.01 mg/mL. The findings show that blends of plant protein with MPC had higher in vitro digestibility and antioxidant activity compared to the individual plant protein isolates.

Chemical structure of a novel heteroglycan polysaccharide isolated from the biomass of Ophiocordyceps Sobolifera

This study aims at identifying the structure of polysaccharide (PS) extracted from the biomass of Vietnam-grown Ophiocordyceps sobolifera for the first time. The molecular weight of the obtained polysaccharide (denoted as PS-T100) is found to be 2.29 × 105 Da using gel permeation high-performance liquid chromatography. The structural investigations of the sample PS-T100 unveil a novel structure composed of a heteropolysaccharide with the appearance of two main sugars, D-glucose and D-mannose, in the backbone. Indeed, methylation analysis indicates that PS-T100 is mainly composed of (1→3)-linked glucosyl and (1→3)-linked mannosyl units. Moreover, 1D and 2D nuclear magnetic resonance spectroscopy interpretations of the sample PS-T100 prove the repeating unit of the isolated polysaccharide is [→3)-β-D-Glcp-(1→3)-α-D-Manp-(1→3)-β-D-Glcp-(1→3)-α-D-Manp-(1→3)-β-D-Glcp-(1→]n. Such a structure appears to be a novel polysaccharide. The obtained results contribute to enriching the database of Ophiocordyceps sobolifera- extracted PSs and open up a new pathway toward the production of PS-T100 for biomedical applications.

Administration of eicosapentaenoic acid may alter lipoprotein particle heterogeneity in statin-treated patients with stable coronary artery disease: A pilot 6-month randomized study

BackgroundWe hypothesized that the addition of eicosapentaenoic acid (EPA) to ongoing statin therapy could change the particle heterogeneity of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles, even in stable coronary artery disease (CAD) patients. MethodsWe assigned CAD patients already receiving statin therapy to one of two groups: an EPA group (1800 mg/day; n = 30) and a control group (n = 30). A gel permeation high-performance liquid chromatography method was used to measure the particle concentration and number of lipoprotein subclasses. ResultsIn the EPA group, significant decreases of both the concentration and number of medium LDL (p = 0.0002 and 0.0001), small LDL (p = 0.0004 and 0.0005) and very small LDL (p = 0.0005 and 0.002) particles were observed. Conversely, the concentration and number of large HDL particles increased significantly (p = 0.024 and 0.048). The concentration of very large HDL particles also increased significantly (p = 0.028). Furthermore, significant correlations between the variables that showed significant changes in the LDL and HDL particle subclasses, and the EPA/arachidonic acid (AA) ratio were found. No other significant associations of lipoprotein particle heterogeneity with the serum EPA/AA ratio were noted in either the control group or the EPA group. Interestingly, univariate and multivariate regression analyses revealed that increased serum lecithin–cholesterol acyltransferase activity, a key enzyme of HDL cholesterol efflux, was a predictor for increased above-mentioned HDL particles subclasses. ConclusionsAdministration of EPA might alter both LDL and HDL particle heterogeneity, causing decreased concentration and number of smaller LDL particles and increased concentration and number of larger HDL particles. Furthermore, addition of EPA to ongoing statin therapy appears to be capable of increasing the EPA/AA ratio, which might have an anti-atherosclerotic effect on lipoprotein particle heterogeneity, even in stable CAD patients with well-controlled serum lipid levels. Clinical Trial RegistrationUMIN (http://www.umin.ac.jp/) Study ID: UMIN000010452.

Separation and Purification of Lipase Inhibitory Peptide from Fermented Milk by Lactobacillus plantarum Q180

In this study, we separated and purified lipase inhibitory peptide from fermented milk by Lactobacillus plantarum Q180 with the aim of developing a new functional anti-lipase activity yogurt product. L. plantarum 180 was inoculated into 10% reconstituted skimmed milk and incubated at 37°C until the pH of the culture reached pH 4.4. The lipase activity was measured using porcine pancreatic lipase. The lipase inhibitory peptides were gradually isolated by ultrafiltration, reversed phase column chromatography (RPC), reversed phase high-performance liquid chromatography (RP-HPLC), and gel permeation high-performance liquid chromatography (GP-HPLC) from the fermented milk by L. plantarum Q180. An ODS-AQ column was used for the RPC, a Vydac C18 column for the RP-HPLC, and a Superdex Peptide HR column for the GP-HPLC. The peptide was composed of Asp, Thr, Ile, Ser, Ala, and Gln, and the anti-lipase activity (IC50) was 2,817 μg/mL.

Particle number analysis of lipoprotein subclasses by gel permeation HPLC in patients with cholesteryl ester transfer protein deficiency.

ObjectiveWe previously reported that patients with cholesteryl ester transfer protein (CETP) deficiency (CETP-D) have a higher prevalence of atherosclerotic cardiovascular disease, in spite of increased HDL-C levels. However, characterization of HDL in CETP-D has not been well described. Therefore, we examined HDL particle number (PN) rather than HDL-C level.Approach and resultsNine patients with CETP-D and 9 normolipidemic subjects were enrolled. We performed gel permeation high-performance liquid chromatography (GP-HPLC) analysis, determined the cholesterol and triglyceride composition of all lipoprotein subclasses, and calculated the PN of each subclass, which consisted of 3 VLDL (large, medium, and small), 4 LDL (large, medium, small, and very small), and 5 HDL (very large, large, medium, small, and very small) subclasses. The PNs of large and medium LDL were significantly lower in CETP-D than that in healthy subjects (0.66- and 0.63-fold decrease, respectively; p<0.001), whereas the PN of very small LDL, which is known to be atherogenic, was significantly higher (1.36-fold increase, p = 0.016). The PNs of very large and large HDL in CETP-D were markedly higher than that in healthy subjects (19.9- and 4.5-fold increase, respectively; p<0.001), whereas the PNs of small and very small HDL, which have more potent anti-atherogenic functions, were significantly lower (0.76- and 0.61-fold decrease, respectively; p<0.001).ConclusionWe have assessed the PNs of detailed subclasses of patients with CETP-D for the first time. The PN of larger HDL was markedly increased, that of smaller HDL was decreased, and that of very small LDL was increased, suggesting that CETP-D has pro-atherogenic lipoprotein properties.

Recent Advances in Analytical Methods on Lipoprotein Subclasses: Calculation of Particle Numbers from Lipid Levels by Gel Permeation HPLC Using “Spherical Particle Model”

Recently, we developed an analytical method for determining the lipid levels and particle numbers in lipoprotein subclasses covering a wide size range from chylomicrons to small high density lipoproteins, by using gel permeation high-performance liquid chromatography (GP-HPLC). The challenges in analytical methods on lipoprotein subclasses have been addressed from 1980 by Hara and Okazaki using commercial TSK gel permeation columns. Later, the improvements in the hardware, separation and detection of lipoproteins, and the data processing software, using a Gaussian distribution approximation to calculate lipid levels of lipoprotein subclasses, have been extensively utilized in these analytical methods for over thirty years. In this review, we describe on the recent advances in analytical methods on lipoprotein subclasses based on various techniques, and the calculation of particle numbers from lipid levels by GPHPLC using the "spherical particle model". Free/ester ratio of cholesterol in particular lipoprotein subclass was accurately estimated from triglyceride, total cholesterol (free and esterified) and the size of the particle based on this model originally proposed by Shen and Kezdy.

Recent developments in analytical methods for tracing gluten

According to Codex only foods not exceeding a level of 20 mg gluten/kg may bear a gluten-free label. This also sets the standard for analytical methods for gluten detection. In this paper the currently used methods for gluten analysis are reviewed and new developments are discussed. At the moment, the most commonly used methods are ELISA-based, but also PCR-based methods have been successfully employed. Proteomics-based methods such as reversed-phase (RP-) or gel permeation (GP-) high-performance liquid chromatography (HPLC) have been widely used for characterisation of cereal proteins. Methods combining mass spectrometry and liquid chromatography (LC-MS/MS) are the most promising non-immunological approaches for accurate quantitation of gluten traces. However, due to its requirement of expensive equipment and expertise it is not widely used for routine analysis. New developments include immunosensors, aptamers, microarrays, and multianalyte profiling. Despite the merits and challenges of the different methods, the need for an independent reference method and a generally applicable reference material remain.

Separation, purification and primary reverse cholesterol transport study of Cordyceps militaris polysaccharide

The authors designed to separate, purify and determine the monosaccharide composition of the polysaccharide from Cordyceps militaris, and study its effect on reverse cholesterol transport in vivo by isotope tracing assay. Polysaccharides were separate and purify by ion exchange column Q-sepharose Fast Flow and size exclusion column Sephacryl S200HR; the molecular weight and monosaccharide composition of the polysaccharides were determined by high performance gel permeation chromatography and high performance liquid chromatography coming with pre-column derivation, respectively. Finally, three purified polysaccharides CMBW1, CMBW2 and CMYW1 were obtained, their total carbohydrate contents were 87%, 89%, 95%, respectively; their protein contents were 6.5%, 1.3%, 2.8%, respectively; their molecular weights were 772.1, 20.9, 13.2 kDa, respectively; CMBW1 was composed of mannose, glucosamine, rhamnose, glucuronic acid, glucose, galactose and arabinose with a molar ratio of 7.25: 0.17: 1.29: 0.23: 6.30: 11.08: 0.79; CMBW2 was composed of mannose, glucosamine, galactose and arabinose with a molar ratio of 2.40: 0.16: 2.92: 0.24; CMYW1 was composed of mannose, glucosamine, glucuronic acid and glucose with a molar ratio of 0.59: 0.57: 0.45: 25.61. Polysaccharide at 50 mg x kg(-1) could significantly improve the transport of 3H- cholesterol to blood and excretion from feces. All of the three purified polysaccharides CMBW1, CMBW2 and CMYW1 were heteropolysaccharide; and they could improve reverse cholesterol transport in vivo, the underlying mechanisms are being studied.

Effects of the Carboxyl Density on the Properties of Polycarboxylate-Type Superplasticizer

The effect of carboxyl density (the ratio of acrylic acid and polyoxyethylene ether) upon the performance, molecular weight, and monomer conversion rate of polycarboxylate (PCE) were investigated by slump test, Gel Permeation Chromatography (GPC) and High Performance Liquid Chromatography (HPLC), respectively. The results showed that with carboxyl density increased from 2:1 to 7:1, molecular weight of PCE increased from 7968.5 to 13930.1 and the molecular weight distribution coefficient increased from 1.5 to 2. The HPLC date indicated carboxyl density does not affect the rate of monomer conversion. Test of the fluidity of cement pastes displayed that as carboxyl density was 4.0:1, PCE show the optimal dispersion and dispersion-retaining ability.

Ultrafiltration recovery of sericin from the alkaline waste of silk floss processing and controlled enzymatic hydrolysis

Abstract This study was designed to improve the traditional scouring procedure using a solution of sodium silicate, sodium carbonate, H2O2, surfactant etc. as a refining agent for degumming machine-drawn raw silk sheet (RSS) and raw silk from the cocoon shell of the silkworm Bombyx mori. The RSS was scoured firstly in strongly alkaline electrolyzed water (SAEW, pH ≥ 11.5) consisting mainly of hydroxyl ions by boiling for 30 min and then treated by the traditional scouring procedure (without alkaline compounds), resulting in refined silk floss. The recycled SAEW containing sericin was separated by ultrafiltration into a retentate with a range of high molecular mass values accounting for 10% (w/w) of the cocoon shell and the filtrate was separated by nanofiltration into a purified H2O filtrate and a retentate containing oligopeptides and free amino acids, which can be used directly as food additives or in biological growth media. The ultrafiltration retentate was hydrolyzed by papain under three different conditions into three groups of sericin peptides with high, middle or low molecular mass. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel permeation high-performance liquid chromatography (GP-HPLC) showed the molecular mass of these sericin peptides was in the range 0.2–60, 0.2–30 and 0.2–15 kDa. These clean products have a variety of applications, including coating materials for surface modification, cell culture media and food additives. The procedure described here could be applied to the manufacture of silk floss quilts and the process of refining raw silk, which results in three clean products of the silk protein fibroin (i.e. stripped of sericin) and reduces environmental pollution from scouring waste containing sericin.

LC-MS/MS Analysis of Canine Lipoproteins Fractionated Using the Ultracentrifugation-Precipitation Method

ABSTRACTDue to the lack of a gold standard method in canine lipoprotein analysis, it is unclear whether canine high-density lipoprotein (HDL) and low-density lipoprotein (LDL) can be accurately evaluated by the lipoprotein analysis methods used for dogs. This study investigated whether the ultracentrifugation-precipitation (U-P) method was suitable as a gold standard method for analyzing canine lipoprotein. First, the U-P method was compared with a gel permeation high-performance liquid chromatography system (GP-HPLC). The concentrations of canine HDL cholesterol (HDL-C) and LDL cholesterol (LDL-C) determined by the U-P method correlated closely with those determined by GP-HPLC. However, the canine HDL-C concentration determined by the U-P method was lower than that determined by GP-HPLC, and the canine LDL-C concentration determined by the U-P method was higher than that determined by GP-HPLC. This study showed that some canine HDL could be precipitated with heparin manganese chloride solution. Second, the HDL and LDL fractions separated by the U-P method were analyzed by LC-MS/MS. The HDL fraction was found to contain only apolipoprotein A-I, which is an apolipoprotein of HDL, whereas the LDL fraction contained both apolipoprotein A-I and apolipoprotein B-100, which is an apolipoprotein of LDL. This data showed that a certain lipoprotein that includes apolipoprotein A-I might precipitate with canine LDL when using heparin manganese chloride solution. These results indicated that the U-P method is not currently a gold standard method for analyzing canine lipoproteins.

The synthesis and characterization of a side-by-side iron phthalocyanine dimer

The QCA paradigm is one of the approaches to decrease the size scale of computing devices. When molecules are used as QCA cells, they may be able to perform computing at room temperature. This paper describes a novel molecular QCA cell candidate which is a side-by-side iron phthalocyanine dimer, and an investigation of its optical and redox properties. The new dodeca(pentyloxy) substituted side-by-side iron phthalocyanine dimer, along with the octa(pentyloxy) iron phthalocyanine monomer, are soluble in non-polar organic solvents. These compounds were isolated by gel permeation chromatography (GPC) and high-performance liquid chromatography (HPLC) to final purities of 98% and 99%, respectively. The NMR spectra of both compounds in CDCl3 are broad due to aggregation, but become well resolved after the addition of the coordinating solvent pyridine-d5. Addition of pyridine also gives changes in the UV-vis spectra and electrochemical peaks of both monomer and dimer in dichloromethane indicative of axial iron coordination. The electrochemical data indicates the loss of pyridine ligands from the oxidized products of both monomer and dimer. The comproportionation constant of side-by-side phthalocyanine dimer shows that its oxidized and reduced mixed-valence complexes are fairly stable. The dimer is thus a candidate for molecular QCA systems.

Relationship of Cholesterol Levels in LDL Subclasses Measured by Gel Permeation High-Performance Liquid Chromatography and Metabolic Syndrome Risk Items in Healthy Population

Relationship of Cholesterol Levels in LDL Subclasses Measured by Gel Permeation High-Performance Liquid Chromatography and Metabolic Syndrome Risk Items in Healthy Population

Changes of Sunflower Oil Polyenoic Fatty Acids under High Temperatures

Heat induced &lt;I&gt;cis-trans&lt;/I&gt; isomerisation of sunflower oils depending on temperature, reaction time and original content of linoleic acid was investigated. The content of isomeric fatty acids was determined by gas chromatography and the content of polymers by gel permeation high-performance liquid chromatography. The content of &lt;I&gt;trans&lt;/I&gt; fatty acids increased with time and with temperature and a rate of &lt;I&gt;cis-trans&lt;/I&gt; isomerisation and polymerisation depends on the temperature according to Arrhenius equation. The content of polymers was significantly lower in sunflower oil with high content of oleic acid because of the low concentration of linoleic acid in oil. In both oils the content of conjugated linoleic acid initially increased depending on time and temperature, however after certain time the stationary state occurred. Polymerisation of polyenoic fatty acids takes place directly with heat induced &lt;I&gt;cis-trans&lt;/I&gt; isomerisation.

Evaluation of a gel-permeation high-performance liquid chromatography for determining triglyceride levels in serum major lipoproteins, compared with the ultracentrifugation/precipitation method

Objectives To evaluate the gel permeation high-performance liquid chromatography (GP-HPLC) method for determination of triglyceride (TG) levels in low-density lipoprotein (LDL) and high-density lipoprotein (HDL). Design and methods The GP-HPLC and the ultracentrifugation(UC)/precipitation methods were used and compared. Results There was no significant difference in measured levels of LDL-triglyceride between the two methods, but the HDL-triglyceride levels measured by the GP-HPLC technique were significantly higher than the UC/precipitation one (145 ± 47 mg/L and 121 ± 45 mg/L respectively, n = 38, p < 0.0001). Conclusions A GP-HPLC technique provides LDL-TG levels comparable to those obtained by the UC/precipitation method.

Production, partial characterization and mass spectrometric studies of the extracellular laccase activity from Fusarium proliferatum

Benzyl alcohol and starch-free commercial wheat bran were effective inducers of the laccase activity in cultures of Fusarium proliferatum (MUCL 31970). Initial pH value in the cultures was also an overriding factor for increasing its production. By gel permeation high-performance liquid chromatography, the enzyme eluted as an apparently homogeneous peak with a molecular mass of 54 kDa, but by isoelectrofocusing, two proteins with pI values of 5.17 and 5.07 were revealed. Two different phenoloxidase activities were also detected after nondenaturing polyacrylamide gel electrophoresis. By matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), both proteins showed unique fingerprints, so they were classifiable as isozymes, and were named laccase 1 (Lac1, pI 5.17) and laccase 2 (Lac2, pI 5.07). No clear matches were found when compared with other proteins. The tandem mass spectrometry of some peptides from both isozymes reanalyzed by nanoelectron ionization-ion trap-mass spectrometry (nESI-IT-MS) confirmed their unique character. The following interesting properties, particularly its stability at alkaline pH, make this laccase a promising industrial enzyme for biotechnological applications: maximum activity at 60 degrees C, thermal stability for 2 h at 40 degrees C, optimum pH 3.5 (km=62 microM) measured on 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonate), and pH stability 4-8 (75% stability at pH levels 2.2 and 9) for 2 h at 25 degrees C.