Post-translationally modified human lens crystallin fragments show aggregation in vitro

BackgroundCrystallin fragments are known to aggregate and cross-link that lead to cataract development. This study has been focused on determination of post-translational modifications (PTMs) of human lens crystallin fragments, and their aggregation properties.MethodsFour crystallin fragments-containing fractions (Fraction I [～3.5 kDa species], Fraction II [～3.5–7 kDa species], Fraction III [～7–10 kDa species] and Fraction IV [>10–18 kDa species]), and water soluble high molecular weight (WS-HMW) protein fraction were isolated from water soluble (WS) protein fraction of human lenses of 50–70 year old-donors. The crystallin fragments of the Fractions I–IV were separated by two-dimensional (2D)-gel electrophoresis followed by analysis of their gel-spots by mass spectrometry. The Fractions I–IV were examined for their molecular mass, particle-diameters, amyloid fibril formation, and for their aggregation by themselves and with WS-HMW proteins.ResultsCrystallin fragments in Fractions I–IV were derived from α-, β- and γ-crystallins, and their 2D-gel separated spots contained multiple crystallins with PTMs such as oxidation, deamidation, methylation and acetylation. Crystallin fragments from all the four fractions exhibited self-aggregated complexes ranging in M_r from 5.5×10⁵ to 1.0×10⁸ Da, with diameters of 10–28 nm, and amyloid fibril-like formation, and aggregation with WS-HMW proteins.ConclusionThe crystallin fragments exhibited several PTMs, and were capable of forming aggregated species by themselves and with WS-HMW proteins, suggesting their potential role in aggregation process during cataract development.General significanceCrystallin fragments play a major role in human cataract development.     

Optimization of Candida sp. 99-125 lipase catalyzed esterification for synthesis of monoglyceride and diglyceride in solvent-free system

Esterification of glycerol and oleic acid catalyzed by lipase Candida sp. 99-125 was carried out to synthesize monoglyceride (MAG) and diglyceride (DAG) in solvent-free system. Beta-cyclodextrin as an assistant was mixed with the lipase powder. Six reaction variables, initial water content (0–14 wt% of the substrate mass), the glycerol/oleic acid molar ratio (1:1–6:1), catalyst load (3–15 wt% of the substrate mass), reaction temperature (30–60 °C), agitator speed (130–250 r/min) and beta-cyclodextrin/lipase mass ratio (0–2) were optimized. The optimal conditions to the synthesis of MAG and DAG were different: the optimal glycerol/oleic acid molar ratio, beta-cyclodextrin/lipase mass ratio, catalyst load and reaction temperature were 6:1, 0, 5%, 50 °C for MAG, and 5:1, 1.5, 10%, 40 °C for DAG, respectively. The optimal water content and agitator speed for both MAG and DAG were 10% and 190 r/min, respectively. Under the optimal conditions, 49.6% MAG and 54.3% DAG were obtained after 8 h and 4 h, respectively, and the maximum of 81.4% MAG plus DAG (28.1% MAG and 53.3% DAG) was obtained after 2 h under the DAG optimal condition. Above 90% purity of MAG and DAG can be obtained by silica column separation.     

A novel angiotensin I-converting enzyme (ACE) inhibitory peptide from a marine Chlorella ellipsoidea and its antihypertensive effect in spontaneously hypertensive rats

Marine Chlorella ellipsoidea protein was hydrolyzed using Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, trypsin, α-chymotrypsin, pepsin and papain. Alcalase-proteolytic hydrolysate exhibited the highest ACE inhibitory activity among them and was fractionated into three ranges of molecular weight (below 5 kDa, 5–10 kDa and above 10 kDa). The below 5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. During consecutive purification, a potent ACE inhibitory peptide from marine C. ellipsoidea, which was composed of 4 amino acids, Val–Glu–Gly–Tyr (MW: 467.2 Da, IC₅₀ value: 128.4 μM), was isolated. Lineweaver–Burk plots suggest that the peptide purified acts as a competitive inhibitor against ACE and stable against gastrointestinal enzymes of pepsin, trypsin and α-chymotrypsin. Furthermore, antihypertensive effect in spontaneously hypertensive rats (SHRs) also revealed that oral administration of purified peptide can decrease systolic blood pressure significantly. The results suggest that marine C. ellipsoidea would be an attractive raw material for the manufacture of antihypertensive nutraceutical ingredients.     

Cuticle expansion during feeding in the tick Amblyomma hebraeum (Acari: Ixodidae): The role of hydrostatic pressure

Female Amblyomma hebraeum ticks (Acari: Ixodidae) increase their weight ∼10-fold during a ‘slow phase of engorgement’ (7–9 days), and a further 10-fold during the ‘rapid phase’ (12–24 h). During the rapid phase, the cuticle thins by half, with a plastic (permanent) deformation of greater than 40% in two orthogonal directions. A stress of 2.5 MPa or higher is required to achieve this degree of deformation (Flynn and Kaufman, 2015). Using a dimensional analysis of the tick body and applying the Laplace equation, we calculated that the tick must achieve high internal hydrostatic pressures in order to engorge fully: greater than 55 kPa at a fed:unfed mass ratio of ∼20:1, when cuticle thinning commences (Flynn and Kaufman, 2011). In this study we used a telemetric pressure transducer system to measure the internal hydrostatic pressure of ticks during feeding. Sustained periods of irregular high frequency (>20 Hz) pulsatile bursts of high pressure (>55 kPa) were observed in two ticks: they had been cannulated just prior to the rapid phase of engorgement, and given access to a host rabbit for completion of the feeding cycle. The pattern of periods of high pressure generation varied over the feeding cycle and between the two specimens. We believe that these pressures exceed those reported so far for any other animal.     

One-step purification of YLLIP2 isoforms from Candida sp. 99–125 by polyethyleneimine modified poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolith

The extracellular lipase Yarrowia lipolytica (YLLIP2) crude extract was efficiently separated and purified from Candida sp. 99–125 by one-step ion-exchange chromatography on polyethyleneimine (PEI) functionalized monolithic columns. The preparative conditions for the functionalization of monoliths were optimized, including PEI molecular mass, PEI concentration, modification time and temperature. The monolithic skeleton was prepared in situ by polymerization of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) with a volume ratio of 8:2. Heptane was used as the porogen. PEI 30 kDa with the concentration of 10% (v/v) was applied for the modification of the monolith at 55 °C for 12 h. Lipase (EC.3.1.1.3) from Candida sp. 99–125 was separated to four isoforms (isoform A, isoform B, isoform C and isoform D). As analyzed on non-denaturing PAGE and MALDI-TOF–MS, the four isoforms are homogenous and have the same molecular mass of approximate 38 kDa. The monoliths can afford direct crude lipase loading without increasing too much back pressure, which explores the great potential of the application of monoliths for one-single step fast separation and purification of complicated proteins.     

Cloning and characterization of a recombinant family 5 endoglucanase from Bacillus licheniformis strain B-41361

The gene encoding a family 5 endoglucanase, cel5A, was cloned from the moderate thermophile Bacillus licheniformis strain B-41361. The primary structure of the translated cel5A gene predicts a 49 amino acid putative secretion signal and a 485 residue endoglucanase consisting of an N-terminal family 5 catalytic domain and C-terminal family 3 cellulose binding domain. The endoglucanase portion of the gene was expressed in Escherichia coli, but soluble activity in cell lysates was due to a truncated enzyme with an apparent mass of 42 kDa, the equivalent of the predicted catalytic domain. Insoluble protein renatured from inclusion bodies was protected against truncation, yielding an active holoenzyme (rCel5A) with apparent mass of 62 kDa. The recombinant rCel5A was optimally active at 65 °C and pH 6.0, but retained only 10% activity after 1 h incubation at this temperature. At 55 °C, rCel5A had a broad pH range for activity and stability, with greater than 75% relative activity from pH 4.5–7.0, and retaining greater than 80% relativity activity across the range pH 4.5–8.0 following 1 h incubation at 55 °C. It readily hydrolyzed pNPC, carboxymethylcellulose, barley β-glucan, and lichenan, but despite binding to cellulose, had only weak activity against avicel. Hydrolysis products from soluble polysaccharides included glucose, cellobiose, cellotriose, and cellotetraose. The catalytic properties, broad pH range and thermostability of the recombinant B. licheniformis endoglucanase may prove suitable for industrial applications.     

Wastewater treatment performance of a vermifilter enhancement by a converter slag–coal cinder filter

The study aimed at investigating rural domestic wastewater treatment performance through vermifilter enhancement by a converter slag–coal cinder filter. The research was carried out by column experiments in a lab scale. Results showed the average removal rate of TCOD, BOD, ammonia nitrogen (NH₄⁺-N) and phosphorus removal by the system were 78.0%, 98.4%, 90.3%, 62.4%, respectively at a hydraulic loading rate of 4 m³ m^?2 day^?1. Vermifiltration was effective for insoluble organic matter and suspended solid removal, and the converter slag–coal cinder filter played an important role in phosphorus removal. The molecular weight of particles in the influent and effluents from every unit of the system were distributed in a wide range between 0.1 kDa and 10,000 kDa with predominance of values between 10 kDa and 300 kDa; the major portion of soluble material are high MW compounds. In addition, the optimal design parameters for vermifiltration and converter slag and coal cinder filters, respectively, were studied in the experiments.     

ACE inhibitory,hypotensive and antioxidant peptide fractions from Mucuna pruriens proteins

Hydrolysates and peptide fractions obtained from Mucuna pruriens protein concentrate were studied for their angiotensin converting enzyme (ACE) inhibitory, hypotensive and antioxidant activities. The hydrolysate obtained by pepsin–pancreatin (HPP) was the most active with an ACE IC₅₀ value of 19.5 μg/mL, a Trolox equivalent antioxidant capacity (TEAC) value of 102.8 mM/mg and a ferric reducing power (FRP) IC₅₀ of 67.2 μg/mL. At a dose of 5 mg/kg HPP decrease systolic (32.2%) and diastolic (37%) blood pressure in rats more pronounced than Captopril. The peptide fraction <1 kDa from HPP was the most active with an ACE inhibitory of 10.2 μg/mL (IC₅₀), a TEAC value of 709.8 mM/mg and a FRP IC₅₀ of 54.9 μg/mL. These results indicate that the hydrolysates and peptide fractions of M. pruriens would be used as nutraceuticals ingredients for preventing and providing therapy against hypertension and diseases related to oxidative damage.     

Enhancement of the enzymatic digestibility of sugarcane bagasse by alkali–peracetic acid pretreatment

The enzymatic digestibility of sugarcane bagasse was greatly increased by alkali (NaOH)–peracetic acid (PAA) pretreatment under mild conditions. The effects of several factors affecting the pretreatment were investigated. It was found that when bagasse was pre-pretreated by 10% (based on initial dry materials) NaOH with 3:1 liquid-to-solid ratio at 90 °C for 1.5 h and further delignified by 10% peracetic acid (based on initial dry materials) at 75 °C for 2.5 h, the yield of reducing sugars reached 92.04% by enzymatic hydrolysis for 120 h with cellulase loading of 15 FPU/g solid. Compared with acid and alkali pretreatment, alkali–PAA pretreatment could be conducted under milder conditions and was more effective for delignification with less carbohydrates being degraded in the pretreatment process. Alkaline stage played an important role for partial delignification, swelling fibers and subsequently reducing PAA loading. No loss of cellulase activity (FPA) was observed in the liquid phase for alkali–PAA pretreated bagasse after enzymatic hydrolysis for 120 h.     

Ecological risk assessment of polycyclic aromatic hydrocarbons (PAHs) in the water from a large Chinese lake based on multiple indicators

In the surface water of Lake Chaohu, China, the concentrations of 16 priority polycyclic aromatic hydrocarbons (PAHs) were measured by gas chromatograph–mass spectrometer (GC–MS). Based on the species sensitivity distribution (SSD) model and the probabilistic risk assessment (PRA) model, the indicators were calculated to assess the potential ecological risk of the individual and of multiple congeners of PAHs and their probabilities. The results revealed that the average residual level of the total PAHs (PAH₁₆) in the water ranged from 95.2 to 370.1 ng/L, with a mean value 181.5 ± 70.8 ng/L. The PAH content in the water was dominated by the low-molecular-weight congeners. The multi-substance potentially affected fractions (msPAFs) of the studied PAHs obtained by the SSD model varied from 0.29% (site B3) to 1.58% (site B6), with an average of 0.51 ± 0.34%. The average of the msPAFs (0.93%) for the inflow rivers was greater than that for the western (0.42%) and eastern (0.34%) parts of the lake. The greatest ecological risk probability calculated by the PRA model was found for Pyr (1.55%), followed by Ant (7.07 × 10⁻²%), Fla (2.21 × 10⁻²%), Phe (9.25 × 10⁻⁶%), Nap (1.01 × 10⁻⁵%), Flo (1.16 × 10⁻¹⁴%) and Ace (2.86 × 10⁻¹⁶%). The same order of ecological risks calculated by the two models was found for the studied PAH compounds. The toxicity data might be the primary source of the ecological risk uncertainties, as indicated by the greater values of coefficients of variation (CV) for the toxicity. This study concluded that the combinations of multiple indicators based on the SSD and PRA models for the ecological risk assessment are necessary to provide more general information on the spatial variations and the probabilities of potential ecological risks of the individual and multiple congeners of PAHs.     

Cadmium distribution and its effects on molybdate-containing hydroxylases in Phragmites australis

The influence of cadmium (Cd) on physiological and biochemical parameters was studied to elucidate the mechanism of Cd resistance in Phragmites australis. Cadmium concentrations in roots, stems and leaves increased with exogenous Cd concentration, but Cd content in roots was much higher than in shoots. X-ray microanalysis was used to reveal compartments in which Cd accumulated in root cortex. Cadmium concentrations followed a gradient with the sequence: intercellular space > cell wall > vacuole > cytoplasm, indicating that most Cd was immobilized in the apoplast or sequestered into the vacuolar lumen. Sequential extraction of various Cd chelates revealed that more than half of extractable Cd was bound to proteins, whereas 26% was bound to organic acids. Cd-binding protein fractions were found in the roots after gel filtration chromatography, among which a polypeptide with an apparent molecular mass of 14 kDa bound Cd most avidly. One newly synthesized polypeptide of low molecular mass (1 kDa) appeared under Cd pollution, whereas a prominent fraction of 72 kDa disappeared. Four aldehyde oxidase (AO) isoenzyme activities increased significantly in roots under Cd pollution. Cd stress also enhanced xanthine dehydrogenase (XDH) activities in roots. Two AO polypeptides of different molecular sizes were detected in the roots by Western blot assay. The abundance of the 160 kDa subunit correlated with Cd stress, but the amount of the 90 kDa polypeptide did not change under Cd treatment. Enhanced abscisic acid (ABA) contents were observed in roots of P. australis exposed to Cd. The involvement of Cd distribution in plant tissues and subcellular compartments and of AO and XDH enzymatic activities in the acclimation mechanism of P. australis to Cd pollution is discussed herein.     

Preparation of a diacylglycerol-enriched soybean oil by phosphalipase A1 catalyzed hydrolysis

Partial hydrolysis catalyzed by phospholipase A1 (Lecitase Ultra) in a solvent free system was firstly used to produce diacylglycerols (DAGs)-enriched soybean oil. In this study, five reaction parameters namely agitation speed (100–500 rpm), reaction time (2–10 h), water content (10–50 wt% of oil mass), enzyme load (5–40 U/g of oil mass), and reaction temperature (30–70 °C) were investigated. The reaction was up-scaled to 1 kg of soybean oil at 40 °C of reaction temperature, with 300 rpm of agitation speed, 40 wt% of water content, 6 h of reaction time and 22 U/g of enzyme load. Purification by molecular distillation yielded 70% DAG-enriched oil with 42.64 wt% of DAG. The composition of acylglycerols of soybean oil and the DAG-enriched soybean oil was analyzed and identified by high performance liquid chromatography (HPLC) and HPLC/electrospray ionization/mass spectrometer. The released fatty acid from the partial hydrolysis of soybean oil catalyzed by phospholipase A1 showed a higher saturated fatty acid content than that of the raw material. Compared to the lipase catalyzed process, this new phospholipase A1 catalyzed one showed the advantages of low amount production of byproduct, namely, monoacylglycerols.     

Simultaneous determination of ABT-888, a poly (ADP-ribose) polymerase inhibitor,and its metabolite in human plasma by liquid chromatography/tandem mass spectrometry

A reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of ABT-888 and its major metabolite (M8) in human plasma. Sample preparation involved a liquid–liquid extraction by the addition of 0.25 ml of plasma with 10 μl of 1 M NaOH and 1.0 ml ethyl acetate containing 50 ng/ml of the internal standard zileuton. The analytes were separated on a Waters XBridge C₁₈ column using a gradient mobile phase consisting of methanol/water containing 0.45% formic acid at the flow rate of 0.2 ml/min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the ABT-888 and M8 concentration ranges of 1–2000 ng/ml in human plasma. The lower limits of quantitation (LLOQ) were 1 ng/ml for both ABT-888 and M8 in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully employed to characterize the plasma concentration–time profile of ABT-888 after its oral administration in cancer patients.     

Partitioning of protease from stomach of albacore tuna (Thunnus alalunga) by aqueous two-phase systems

Partitioning of protease from stomach of albacore tuna using an aqueous two-phase system (ATPS) was investigated. The best ATPS conditions for protease partitioning from stomach extract (SE) and acidified counterpart (ASE) were 25% PEG1000–20% MgSO₄ and 15% PEG2000–15% MgSO₄, which increased the purity by 7.2-fold and 2.4-fold with the recovered activity of 85.7% and 89.1%, respectively. Electrophoretic study revealed that SE had a major protein with a molecular weight (MW) of 40.6 kDa, while protein with MW of 32.7 kDa was predominant in ASE and ATPS fractions. Pepsinogen in SE might be activated to pepsin by acidification and partitioning process. SE was quite stable at 0 and 4 °C up to 14 days. The loss in protease activity in ASE and selected ATPS fractions was more pronounced when storage time and temperature increased. Therefore, ATPS can be effectively used to recover and purify protease from albacore tuna stomach.     

Separation,purification and preliminary characterization of sulfated polysaccharides from Sargassum plagiophyllum and its in vitro anticancer and antioxidant activity

Sulfated polysaccharides (SPs) were identified in different portions of the thallus of Sargassum plagiophyllum C. Agardh, with TBO staining. SPs were extracted using a blade and purified by Q sepharose fast flow anion-exchange chromatography, resulting in SP fractions F1, F2 and F3, with molecular weights of 30, 35 and 20 kDa, respectively. An SP yield of 43.1% was obtained in F3, while F2 yielded a sulfate content of 21.9%. Furthermore, the in vitro anticancer and antioxidant activities of the polysaccharide fractions were evaluated. The F2 fraction showed higher anticancer activity against HepG2 and A549 cells than the other two fractions, with IC₅₀ values of 600 μg/mL and 700 μg/mL, respectively. The normal breast epithelial cell line (HBL-100) exhibited IC₅₀ concentrations of 1200 and 1400 μg/mL for crude sulfated polysaccharides (CSPs) and all SP fractions (F1–F3). These results indicated that the anticancer activity of F2 could be related to its sulfate content. However, the antioxidant activities of F1–F3 were low at their tested concentrations.     

Trace quantification of 1-octacosanol and 1-triacontanol and their main metabolites in plasma by liquid–liquid extraction coupled with gas chromatography–mass spectrometry

A method for the simultaneous determination of 1-octacosanol and 1-triacontanol and their main metabolites in rat plasma was developed. The procedure involved ethanolic NaOH saponification of the sample, acidification, liquid–liquid extraction, and derivatization of the analytes to its trimethylsilylether/ester, followed analysis by gas chromatography–mass spectrometry (GC–MS) in selected ion monitoring (SIM) mode. Quantification was performed by the internal standard method using betulin. The method had a good linearity over the range 8.4–540 ng/ml (r ≥ 0.998) and showed an excellent intra-day (R.S.D. = 0.59–3.06%) and inter-day (R.S.D. = 2.99–5.22%) precision according to the acceptance criteria. The detection limits ranged between 1.32 and 3.47 ng/ml. The method was applied successfully to study the total plasmatic concentration of 1-octacosanol, octacosanoic acid, 1-triacontanol, and triacontanoic acid, after an oral dose of policosanols mixture, using plasma samples of 100 μl.     

Three-phase partitioning of trypsin inhibitor from legume seeds

Three-phase partitioning (TPP) was used to isolate trypsin inhibitors from navy bean (NB), red kidney bean (RK) and adzuki bean (AZ) from the Royal Project Foundation in Thailand. The method was to mix the crude extract with solid ammonium sulfate (30% saturation, w/v) and tert-butanol (t-butanol) in order to obtain the three phases. The trypsin inhibitor was purified to 5-, 14- and 7-fold with 315%, 441% and 228% recovery for NB, RK and AZ, respectively. The SDS-PAGE showed the major inhibitor band with the molecular weights (MWs) of 132, 118 and 13 kDa for NB, RK and AZ, respectively. The fractions from NB and AZ showed higher pH stability compared to that of the RK, and they had the optimum pH ranges of 7–9. The highest relative inhibitory activity of the fractions of NB and RK were found at 50 °C, and all fractions were quite stable at 90 °C for 60 min of incubation. Increasing the concentration of salt (up to 3%, w/v) did not significantly decrease the inhibitory activity of all fractions (p > 0.05). The trypsin inhibitors from the three legumes were unable to inhibit the autolysis of Pacific whiting and arrowtooth flounder muscles.     

Electromyographic,but not mechanomyographic amplitude–force relationships,distinguished differences in voluntary activation capabilities between individuals

The purpose of the present study was to examine the influence of activation capabilities on the electromyography (EMG_RMS) and mechanomyography amplitude (MMG_RMS)–force relationships of the vastus lateralis (VL) and rectus femoris (RF). Thirteen men (mean ± SD; age = 22 ± 3 year) performed nine submaximal contractions (10–90% maximal voluntary contraction [MVC]) with the interpolated twitch technique performed during a separate contraction at 90% MVC to calculate percent voluntary activation (%VA). Nine participants with >90% VA were categorized into the high-activated group with the remaining categorized into the moderate-activated group. Slopes (b terms) were calculated from the log-transformed EMG_RMS and MMG_RMS–force relationships. The b terms (collapsed across the VL and RF) for the EMG_RMS–force relationships were greater for the high- (1.29 ± 0.31) than the moderate-activated (1.10 ± 0.20) group. In contrast, there were no differences in the b terms for the MMG_RMS–force relationships between the high- and moderate-activated groups. For the EMG_RMS and MMG_RMS–force relationships, the b terms were greater for the RF (1.38 ± 0.30, 0.81 ± 0.20) than the VL (1.08 ± 0.19, 0.60 ± 0.13) collapsed across groups. The b terms from the EMG_RMS–force relationships, but not the MMG_RMS–force relationships, reflected differences in %VA.     

A flexible and high throughput liquid chromatography–tandem mass spectrometric assay for the quantitation of telcagepant in human plasma

Telcagepant (MK-0974) is a novel oral calcitonin gene-related peptide (CGRP) receptor antagonist and is currently under clinical development. Results from phases II and III clinical trials have suggested that telcagepant is effective for migraine treatment. A reliable and high throughput protein precipitation (PPT) method for determination of telcagepant in human plasma using liquid chromatography coupled with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry has been developed. Clinical samples, internal standard (IS) and acetonitrile are transferred into 96-well plates using a robotic liquid handling system. An aliquot of 10 μL supernatant is directly injected into the LC–MS/MS system where separation is performed on a FluoPhase RP (150 × 2.1 mm, 5 μm) column with an isocratic mobile phase (60% acetonitrile with 0.1% formic acid and 40% water with 0.1% formic acid) at 0.2 mL/min. The interfering 3S-diastereomer of telcagepant, which is observed in clinical samples, is chromatographically resolved from telcagepant. The PPT procedure significantly reduces the time required for sample processing and the assay is sufficiently sensitive for detection using both API 4000 and API 3000 mass spectrometers. The linear calibration range is 5–5000 nM using 200 μL of plasma. Assay intraday validation was conducted using six calibration curves derived from six lots of human control plasma. Calibration standard accuracy did not deviate by more than 3% and 6% of nominal values, and precision did not exceed 4% coefficient of variation (CV) and 10% CV, respectively on the API 4000 and API 3000. Several clinical phases IIb and III studies have been successfully supported with this assay.     

Biochemical characterization of a lichenase from Penicillium occitanis Pol6 and its potential application in the brewing industry

The purification and characterization of an extracellular lichenase from the fungus Penicillium occitanis Pol6 were studied. The strain produced the maximum level of extracellular lichenase (45 ± 5 U ml⁻¹) when grown in a medium containing oat flour (2%, w/v) at 30 °C for 7 days. The purified enzyme EG_L showed as a single protein band on SDS–PAGE with a molecular mass of 20 kDa. Its N-terminal sequence of 10 amino acid residues was determined as LDNGAPLLNV. The purified enzyme showed an optimum activity at pH 3.0 and 50–60 °C. The half-lives of EG_L at 60 °C and 70 °C were 80 min and 21 min, respectively. Substrate specificity studies revealed that the enzyme is a true β-1,3-1,4-d-glucanase. The enzyme hydrolyzed lichenan to yield trisaccharide, and tetrasaccharide as the main products. Under simulated mashing conditions, addition of EG_L (20 U/ml) or a commercial β-glucanase (20 U/ml) reduced the filtration time (25% and 21.3%, respectively) and viscosity (10% and 8.18%, respectively). These characteristics indicate that EG_L is a good candidate in the malting and brewing industry.