Fractional Ammonium Sulfate Precipitation Research Articles

The antifungal substance from Bacillus velezensis B-3 improved the malt quality against premature yeast flocculation induced by Fusarium graminearum infection.

Malt produced from barley contaminated with Fusarium graminearum can cause premature yeast flocculation (PYF) in beer fermentation, which ultimately affects the production efficiency and flavor quality of beer. In this study, a strain of Bacillus velezensis B-3 that was isolated from soil displayed strong antifungal activity against F. graminearum. The antifungal substances were extracted and separated by ammonium sulfate fractional precipitation and semipreparative high-performance liquid chromatography to obtain fraction VI with the strongest antifungal activity. Ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry was used to identify that the fraction contained lipopeptides such as iturin A, surfactant B, and surfactant C. The minimum inhibitory concentration of the fraction was 0.2mg/mL, and confocal laser scanning microscopy showed that the morphology of F. graminearum hyphae was obviously abnormal, with most of them were dead. Antifungal fraction VI could inhibit the growth of F. graminearum and avoid the production of PYF factor during malting but did not affect other qualities of malt. Therefore, the development of antimicrobial agents against Fusarium provides an effective potential strategy to control the production of PYF factors for malt-manufacturing enterprises. PRACTICAL APPLICATION: Antifungal fraction VI could inhibit the growth of F. graminearum and the formation of DON during malting but did not affect other qualities of malt. The levels of soluble nitrogen, free amino nitrogen, and color experienced a substantial reduction compared to the malt infected by F. graminearum. The PYF phenomenon was significantly improved, the number of suspended yeast cells and the degree of fermentation were increased, and the level of residual extract in the wort was low, close to that of control malt.

Modified method for obtaining phycocyanine concentrate of Arthrospira platensis biomass

The biomass of Arthrospira platensis cyanobacteria is a source of bioactive compounds such as chlorophylls, carotenoids, and, particularly, phycobiliproteins: C-phycocyanin and allophycocyanin. The wide range of biological activity shown by extracts with a high content of phycocyanins determines the prospects for their use as dietary supplements and ingredients of special foods. For food purposes, the degree of purity of phycocyanin concentrates, determined by the ratio of optical densities of their aqueous solutions at two wavelengths, namely D620/D280, must be greater than 0.7. Most methods for obtaining phycocyanin concentrates include laborious steps of fractional ammonium sulphate precipitation of protein from A. platensis biomass extracts followed by removal of salts solution. The use of membrane technology, specifically microfiltration, makes it possible to significantly intensify and simplify the process of obtaining phycocyanin concentrates. The aim of this research was to modify the method for obtaining a high-purity A. platensis phycocyanin concentrate by replacing the stage of ammonium sulfate precipitation of the protein by ultrafiltration of the extract followed by microfiltration. Material and methods. A sample of dry A. platensis biomass was used as a feedstock. Extraction of A. platensis biomass was carried out at a temperature of +40 °C for 3 h, the resulting suspension was centrifuged, and the supernatant was separated from the sediment. The obtained extract was subjected to ultrafiltration (membrane with a pore diameter of 30 kDa) followed by removal of the permeate containing low molecular weight impurities. The retentate was subjected to microfiltration (membrane with a pore size of 0.2 μm), concentrated by reverse osmosis and freeze-dried. Results. The content of C-phycocyanin and allophycocyanin in the dry concentrate was 42.0±1.3 and 7.0±0.3%, respectively, the degree of purity was 1.98. Conclusion. The scheme for obtaining A. platensis phycocyanin concentrate has been modified. A concentrate was obtained with a high degree of purity, allowing its use in food.

Biosynthesis of lactosucrose by a new source of β-fructofuranosidase from Bacillus methanolicus LB-1

Lactosucrose (LS) is a prebiotic trisaccharide enzymatically synthesized by transglycosylation from lactose and sucrose with beneficial health effect. The β-fructofuranosidase used for synthesis of LS was produced from Bacillus methanolicus LB-1, which was isolated from traditional rice wine. A maximal yield of 8.63 U/mL of the enzyme was obtained by fermentation with B.methanolicus LB-1 under the optimized conditions: 10g/L of glucose, 5g/L of yeast extract, initial medium pH at 7.0, 37°C, 24h. The enzyme was purified and identified by ammonium sulfate fractional precipitation, Sephadex G-75 gel filtration chromatography and LC-MS, and SDS-PAGE of the purified enzyme showed a major protein band at 45kDa. Biosynthesis of LS was performed using the purified β-fructofuranosidase, and production of LS reached 110g/L under the optimized reaction conditions: pH at 7.0, 37°C, 6.0 U/g sucrose of enzyme, 15% of sucrose, 15% of lactose, 28h. HPLC analysis of the reaction products showed a distinct peak for LS at about 30min of elution, confirming that B.methanolicus LB-1 β-fructofuranosidase had effective transfructosylation activity. Therefore, this new microbial source of β-fructofuranosidase may be a candidate with potential application prospect in biosynthesis of prebiotic LS.

A broad-spectrum novel bacteriocin produced by Lactobacillus sakei in Nanjing Steamed Roast Duck: Purification, antimicrobial characteristics, and antibacterial mechanisms

A new bacteriocin (Y19-2) was isolated, purified, and characterized from Nanjing Steamed Roast Duck, a traditional Chinese fermented meat product. Purification was performed via ammonium sulfate fractional precipitation, diethyl aminoethyl (DEAE)-52 cellulose ion-exchange chromatography, Sephadex G-50 chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC). SDS-PAGE analysis showed the molecular weight of Y19-2 is 20 kDa, making it fall into the third type of bacteriocin based on the classifications of bacteriocins according to their molecular weights. In addition, Y19-2 is heat-resistant as suggested by a merely 14% loss of its antibacterial activity when heated at 121 °C for 30 min. In acidic conditions (pH 3–5), Y19-2 has obvious antibacterial activity against both gram-positive and gram-negative bacteria, and such an activity can be partially inactivated by pepsin and trypsin while being completely inhibited by papain. Scanning electron microscopy and differential thermal scanning analysis showed that Y19-2 could inhibit bacteria growth by destroying the bacterial cell wall and affecting the synthesis of bacteria's nucleic acids. Therefore, Y19-2 is potential as a bioprotective agent in foods.

Purification and Characterization of Trochus radiatus Derived Low Molecular Weight Bactericidal Polypeptide Active Against ESKAPE Pathogens

Antimicrobial peptides comprise core components of innate defense and act as first-line defense molecules in most marine mollusks. Marine natural products have served as templates for the design and development of novel therapeutic molecules against infectious pathogens in the past decades. In this study, we report the isolation, purification and characterization of a bactericidal peptide obtained from a marine top-snail Trochus radiatus. The crude extract of T. radiatus was assessed and found to exert potent activity against the tested fourteen gram-positive and gram-negative clinical pathogens. Three extraction buffers were used and their total protein extraction capacity from the tissue was evaluated. We then analyzed and estimated the total protein concentration and antibacterial capacity of obtained extracts. Aiming to isolate the bioactive peptide that confers antimicrobial activity, the crude extract was subjected to purification by fractional ammonium sulphate precipitation followed by chromatographic purification. The RP-HPLC and Tricine SDS-PAGE gel (15%) revealed the homogeneity of the purified antimicrobial peptide with a relative molecular mass of 7 kDa. This purified antibacterial peptide displayed potent antibacterial activity against both Gram-positive and Gram-negative bacteria’s i.e., Enterococcus faecium, Klebsiella pneumoniae and Shigella dysenteriae. The morphological changes occurred upon treatment was confirmed through electron microscopic imaging. The physicochemical characterization studies confirmed the stability of isolated peptide over variations in pH from 3 to 10, temperature from – 20 to 100 °C and in the presence of human serum. Further, the purified bioactive peptide was found to be less toxic and possess free radical scavenging activity. This is the first report of an antibacterial peptide isolated from the mollusk T. radiatus and this peptide could be developed into a peptide antibiotic alternative to the currently available synthetic or semisynthetic antibiotics.

Purification, characterization, immobilization and kinetic studies of catalase from a novel source Sechium edule

AbstractA catalase from a novel source Sechium edule (squash) has been purified to homogeneity using ammonium sulfate fractional precipitation, dialysis, and anion exchange chromatography on Diethylaminoethyl (DEAE) cellulose in sodium acetate buffer pH 6.0. The purity of the enzyme was analyzed by SDS‐PAGE. The molecular weight of the enzyme using the SDS‐PAGE method was found to be 55 kDa with specific activity 6.35 U/mg. The molecular weight of the enzyme was further confirmed by the INTACT mass spectrometric technique and found to be 52.5 kDa. The Reinheitzahl (Rz) value of the enzyme was 1.5. The MALDI‐TOF analysis of the purified enzyme showed that it contains 529 amino acid residues. The Km, Vmax, optimum pH, and optimum temperature of the free enzyme using H2O2 as the substrate were found to be 0.03 mM, 200 μmol/min, 7.6, and 24°C, respectively. The purified enzyme was immobilized on chitosan beads which was prepared by extracting the chitosan from Cornu aspersum (garden snail) having highest degree of deacetylation%. The kinetic characteristics, Km, Vmax, optimum pH, and optimum temperature of the immobilized catalase were found to be 0.065 mM, 250 μmol/min, 7.6, and 41°C, respectively. The immobilized catalase is more thermostable in comparison to free catalase.

Purification, Characterization and Degradation Performance of a Novel Dextranase from Penicillium cyclopium CICC-4022.

A novel dextranase was purified from Penicillium cyclopium CICC-4022 by ammonium sulfate fractional precipitation and gel filtration chromatography. The effects of temperature, pH and some metal ions and chemicals on dextranase activity were investigated. Subsequently, the dextranase was used to produce dextran with specific molecular mass. Weight-average molecular mass (Mw) and the ratio of weight-average molecular mass/number-average molecular mass, or polydispersity index (Mw/Mn), of dextran were measured by multiple-angle laser light scattering (MALS) combined with gel permeation chromatography (GPC). The dextranase was purified to 16.09-fold concentration; the recovery rate was 29.17%; and the specific activity reached 350.29 U/mg. Mw of the dextranase was 66 kDa, which is similar to dextranase obtained from other Penicillium species reported previously. The highest activity was observed at 55 °C and a pH of 5.0. This dextranase was identified as an endodextranase, which specifically degraded the α-1,6 glucosidic bonds of dextran. According to metal ion dependency tests, Li+, Na+ and Fe2+ were observed to effectively improve the enzymatic activity. In particular, Li+ could improve the activity to 116.28%. Furthermore, the dextranase was efficient at degrading dextran and the degradation rate can be well controlled by the dextranase activity, substrate concentration and reaction time. Thus, our results demonstrate the high potential of this dextranase from Penicillium cyclopium CICC-4022 as an efficient enzyme to produce specific clinical dextrans.

Purification and characteristics of an aflatoxin B1 degradation enzyme isolated from Pseudomonas aeruginosa

Aflatoxin is a highly toxic mycotoxin produced by Aspergillus flavus and Aspergillusparasiticus fungus. Since it contaminates food and grain widely, it has seriously endanger the health of human beings and animals. Some microorganisms that exist in nature can degrade aflatoxin. In this paper, the biological AFB1 (aflatoxin B1)-degradation by Pseudomonas aeruginosa M19 was evaluated. We found that the culture supernatant of P. aeruginosa M19 added with proteinase K (Prok), SDS and heating significantly decreased degradation capacity. Pseudomonas AFB1-degrading enzyme (PADE) was purified from P. aeruginosa M19 by a three-step procedure including ammonium sulfate fractional precipitation and ion exchange chromatography and gel filtration chromatography. PADE was purified 175-fold with a recovery of 35%, and an ultimate specific activity of 6.1×104 U/mg was achieved. The molecular weight of PADE estimated by SDS-PAGE is about 48 kDa. PADE displayed the highest degradation activity for AFB1 at 65°C and pH 6.0. Cu2+ and Fe3+ strongly enhanced the activity, whereas Ca2+ and Zn2+ strongly inhibited the activity. These findings indicate that PADE from P. aeruginosa M19 is a promising candidate for AFB1 biodegradation.

Characteristics and kinetic analysis of β-glucosidase (MaBgl) from Mucor ardhlaengiktus RSC1

Cellulolytic enzymes from fungi are complex compound enzyme structures that generally include three kinds of hydrolytic enzymes, which are called endo-β-1,4-glucanases, exo-β-1,4-glucanases, and β-1,4-glucosidases (β-glucosidases). The fungus Mucor ardhlaengiktus isolated from field rice straw produced a β-glucosidase (MaBgl). Maximal MaBgl production occurred when M. ardhlaengiktus was incubated for 6 days in fermentation liquor at 30 °C and an initial pH of 6. Purified MaBgl was obtained from M. ardhlaengiktus fermentation broth by ammonium sulfate fractional precipitation and DEAE-Sepharose FF ion exchange chromatography. The molecular weight of MaBgl as determined by SDS-PAGE electrophoresis was approximately 72 kDa. The kinetic parameters, Michaelis constant (Km) and maximum velocity (Vmax), of MaBgl were 78.2 μmol/L and 28.5 μmol/(L·min), respectively. Assays of MaBgl produced by M. ardhlaengiktus RSC1 under different conditions were investigated by the 3,5-dinitrosalicylic acid (DNS) assay for glucose. The pH and temperature optima for catalytic activity of MaBgl were pH 4.8 and 50 °C, respectively. MaBgl exhibited good thermal stability in the range of 20 to 30 °C, but the thermal stability of MaBgl decreased rapidly over 60 °C. MaBgl had better pH stability between pH 4.6 and 5.0, and the stability of MaBgl decreased when the pH value was lower or higher than this range.

Purification and characterization of thermostable superoxide dismutase from Anoxybacillus gonensis KA 55 MTCC 12684

A thermostable superoxide dismutase from thermophilic bacterium Anoxybacillus gonensis KA 55 MTCC 12684 which was isolated from Manikaran hotspring of Himachal Pradesh was purified to apparent homogeneity by fractional ammonium sulphate precipitation and anion exchange chromatography. A purification factor of 33.1-fold was achieved, with the purified enzyme exhibiting specific activity of 5758.4 U/mg protein. The purified superoxide dismutase was optimally active at pH 9.0 and displayed stability over a broad pH range of 7.0–10.0 and was stable up to 70 °C. SOD was localized in polyacrylamide gel by activity staining, based on the reduction of nitroblue tetrazolium (NBT) by superoxide ion. The molecular weight of superoxide dismutase was calculated as 31 kDa by SDS-PAGE. The Km and Vmax values of purified enzyme were found to be 1.002 mM and 14,285.71 U/mg of protein respectively. Tests of inhibitors indicated that the enzyme activity was inhibited by hydrogen peroxide and potassium cyanide but not by sodium azide showing that purified SOD was Cu/ZnSod.

Role of Metal Ions, Surfactants and Solvents on Enzymatic Activity of Partial Purified Glucoamylase from Aspergillus niger ER05

The glucoamylase requirement of different industries should vary in their physiochemical and functional properties, so the investigation of new sources for the novel enzymes is the only solution. The current study describe the production of glucoamylase from Aspergillus niger ER05 in a submerged fermentation. The enzyme then partially purified and then effect of thirteen different metal ions (Cs+, K+, Na+, Ba2+, Ca2+ Co2+, Cu2+ , Hg2+, Mg2+, Mn2+, Ni2+, Zn2+ , Fe3+), surfactant as well as organic solvent on catalytic performance of glucoamylase was studied. A newly isolated Aspergillus niger ER05 is hyper producing strain of glucoamylase. Specific activity for the crude enzyme was found to be 6.87 KU/mg. The crude enzyme was partially purified via fractional ammonium sulphate precipitation. Ammonium sulphate saturation from 40-80% was found suitable to precipitate the enzyme. After dialyzing precipitates, the specific activities were found to be 66.33KU/mg with percent yield of 26.5. The inhibitory influence of all metal studies was interestingly found on glucoamylase activity. The strong inhibition was demonstrated in presence of Hg2+, Ni2+, Zn2+, Cu2+, Fe3+. Only Na+ ions were able to maintain the 101% relative activity at 1mM concentration. The SDS completely inhibits the enzyme activity and in presence of tween-80 and triton X-100 glucoamylase exhibited less than 45% relative activity. Furthermore, formaldehyde, isopropanol, ethanol, methanol, and DMSO stabilized the enzyme activity while chloroform inhibits enzyme activity by 48%.

A dual-function chymotrypsin-like serine protease with plasminogen activation and fibrinolytic activities from the GRAS fungus, Neurospora sitophila

In this study, we have isolated and characterized a fibrinolytic enzyme from the GRAS (Generally Recognized as Safe) fungus, Neurospora sitophila. The enzyme was purified by fractional ammonium sulfate precipitation, hydrophobic interaction, ion exchange and gel filtration chromatography to 45.2 fold with a specific activity of 415.6U/mg protein. The native molecular mass of the enzyme was 49kDa, while the denatured molecular mass was 30kDa and 17.5kDa, indicating that the enzyme was a hetero-dimer. It was optimally active at 50°C and pH 7.4 and stable at human physiological temperature and pH. It was found to be a chymotrypsin-like serine protease which cleaved the synthetic chromogenic substrate, N-Succinyl-Ala-Ala-Pro-Phe-pNA for which the apparent Km and Vmax values were 0.24mM and 4.17×10−5mM/s, respectively. The enzyme hydrolyzed all the chains of fibrinogen by cleaving α chain first, followed by β chain and then γ chain. Moreover, the enzyme possessed dual function of direct fibrinolysis as well as plasminogen activation. Due to its attractive biochemical and fibrinolytic properties and being from a GRAS fungus, the fibrinolytic enzyme has application as a safe and efficient thrombolytic drug.

Purification and characterization of a novel tannase produced by Kluyveromyces marxianus using olive pomace as solid support, and its promising role in gallic acid production

Tannase is considered one of the most important industrial enzymes that find great applications in various sectors. Production of tannases through solid state fermentation (SSF) using agro-industrial wastes is an eco-friendly and cheap technology. Tannase was produced by the yeast Kluyveromyces marxianus using olive pomace as a solid support under SSF. It was purified using ammonium sulfate fractional precipitation followed by Sephadex G-200 gel filtration resulting in 64.6% enzyme yield with 1026.12U/mg specific activity and 24.21 purification fold. Pure tannase had molecular weight of 65 KDa and 66.62 KDa by SDS-PAGE and gel filtration, respectively. It showed a maximal activity at 35°C having two different pH optima, one of which is acidic (4.5) and the other one is alkaline (8.5). The enzyme was stable in the acidic range of pH (4.0–5.5) for 30min, and thermostable within the temperature range 30–70°C. Using tannic acid, the enzyme had a Km value of 0.77mM and Vmax of 263.20μmolemin−1ml−1. The effect of different metal ions on enzymatic activity was evaluated. HPLC analysis data indicated that the purified enzyme could carry out 24.65% tannic acid conversion with 5.25 folds increase in gallic acid concentration within 30min only.

Valorization of Fish Viscera for Crude Proteases Production and Its Use in Bioactive Protein Hydrolysate Preparation

Increasing environmental pollution and limited biological resources has emphasized the need for better utilization of fish processing waste. In the present study, visceral proteases from little tuna (Euthynnus affinis), catla (Catla catla) and tilapia (Oreochromis mossambicus) belonging to different habitats using acetone, ethanol and ammonium sulfate fractional precipitation were isolated and characterized. Enzymes precipitated from little tuna and tilapia exhibited higher specific activity at 40% saturation for ammonium sulfate fractional precipitation and the specific activities were 18.19 and 13.67 U/mg for little tuna and tilapia enzymes, respectively. Enzymes precipitated from catla reported higher specific activity of 8.32 U/mg at 60% saturation for ammonium sulfate fractional precipitation. Acetone precipitated fraction exhibited higher recovery for all crude enzymes studied. The effect of different combinations of crude tuna visceral waste enzyme and papain enzyme on hydrolysate properties was optimized using response surface methodology. Second order response surface regression model was used to explain the total variability in terms of quadratic and interaction effects of combination enzyme concentration on the properties of croaker protein hydrolysate with a significant R2 value for protein content (0.92), tyrosine content (0.99), degree of hydrolysis (0.99), DPPH free radical scavenging activity (0.97), ferric reducing antioxidant power (0.93), metal chelating activity (0.88), foaming capacity (0.92) and foam stability (0.88). Based on the desirability score, papain at 0.81% concentration and crude tuna visceral protease at 4.36% was found to be optimal for achieving desired properties of croaker protein hydrolysate.

Purification and characterization of a milk-clotting aspartic protease from Withania coagulans fruit

Withania coagulans fruit has traditionally been used as milk coagulant. The present study reports the purification and characterization of an aspartic protease from W. coagulans fruit. The enzyme was purified via fractional ammonium sulfate precipitation and cation exchange chromatography. SDS-PAGE analysis revealed the presence of a monomeric protein with molecular weight of 31kDa. Proteolytic activity (PA) of the protease was evaluated using casein, and the milk-clotting activity (MCA) was analyzed by skim milk. The Km and Vmax values of the enzyme for casein were obtained to be 1.29mg/ml and 0.035μmol Tyr/min, respectively. Optimal temperature and pH were 65°C and 5.5, respectively. After incubation of enzyme at 65°C for 1h, 73% of PA was remained which demonstrated high thermal stability of the enzyme. Mass spectrometry analysis of the purified protease and enzyme assays in the presence of protease inhibitors indicated that aspartic protease was the only responsible enzyme in milk coagulation. Furthermore, by investigating the effect of salts on enzyme activity, it was observed that both NaCl and CaCl2 reduced enzyme activity. These characteristics of the protease suggest that the enzyme may be suitable for producing low salt content cheeses.

Purification and cytotoxicity of glycoprotein isolated from Dendrobium huoshanense

Dendrobium huoshanense is a rare traditional Chinese medicinal herb, and the anti-tumor activity of its polysaccharides is a research hotspot in traditional Chinese medicine resources domain. This study aims to explore the material basis for the anti-tumor activity of polysaccharide. D. huoshanense was used as raw material in the experiment, and the different protein components were obtained through low salt solution extraction and ammonium sulfate fractional precipitation. Then glycoprotein components were determined by SDS-PAGE electrophoresis staining, and were further isolated and purified by DEAE ion column and Sephadex gel column. At the same time, MTT assay was used in detecting the cytotoxicity of different products on HepG2 cells in vitro. As a result, three kinds of glycoprotein components RG1, RG2, RG3 with relative molecular mass of 22.5, 19.8, 15.6 kDa were gained, and the IC₅₀ of three compounds on human liver cancer cell HepG2 was 534.23 mg•L⁻¹, meanwhile IC₅₀ of single glycoprotein component RG1, RG2 was 432.96, 413.91 mg•L⁻¹ respectively, and glycoprotein component RG3 had no cytotoxicity on HepG2 cells. All in all, the experiment results suggested that two kinds of glycoproteins components with relative molecular mass of 22.5, 19.8 kDa may be one of the material basis for anti-tumor activity of D. huoshanense polysaccharide, and they had a synergistic effect.

Purification, biochemical characterization and structural modelling of alkali-stable β-1,4-xylan xylanohydrolase from Aspergillus fumigatus R1 isolated from soil.

BackgroundAspergillus fumigatus R1 produced xylanase under submerged fermentation which degrades the complex hemicelluloses contained in agricultural substrates. Xylanases have gained considerable attention because of their tremendous applications in industries. The purpose of our study was to purify xylanase and study its biochemical properties. We have predicted the secondary structure of purified xylanase and evaluated its active site residues and substrate binding sites based on the global and local structural similarity.ResultsVarious microorganisms were isolated from Puducherry soil and screened by Congo-red test. The best isolate was identified to be Aspergillus fumigatus R1. The production kinetics showed the highest xylanase production (208 IU/ml) by this organism in 96 h using 1 % rice bran as the only carbon source. The purification of extracellular xylanase was carried out by fractional ammonium sulphate precipitation (30–55 %), followed by extensive dialysis and Bio-Gel P-60 Gel-filtration chromatography. The enzyme was purified 58.10 folds with a specific activity of 38196.22 IU/mg. The biochemical characterization of the pure enzyme was carried out for its optimum pH and temperature (5.0 and 500C), pH and temperature stability, molecular mass (Mr) (24.5 kDa) and pI (6.29). The complete sequence of protein was obtained by mass spectrometry analysis. Apparent Km and Vmax values of the xylanase for birchwood xylan were 11.66 mg/ml and 87.6 μmol min−1 mg−1 respectively.ConclusionPurified xylanase was analyzed by mass-spectrometry which revealed 2 unique peptides. Xylanase under current study showed significant production using agricultural residues and a broad range of pH stability in the alkaline region. Xylanase produced by Aspergillus fumigatus R1 could serve as the enzyme of choice in industries.

Expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for insulin analogue modification

The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important features of insulin derivatives. Insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. The aim of the study was to obtain recombinant PAM and use it for insulin analogue amidation. We stably expressed a recombinant PAM in CHO dhfr-cells in culture. Recombinant PAM was partially purified by fractional ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was used to modify glycine-extended A22(G)-B31(K)–B32(R) human insulin analogue (GKR). Alpha-amidated insulin was analyzed by HPLC and mass spectrometry. Hypoglycemic activity of amidated and non-amidated insulin was compared. The pharmacodynamic effect was based on glucose concentration measurement in Wistar rats with hyperglycemia induced by streptozotocin. The overall glycemic profile up to 36 h was evaluated after subcutaneous single dosing at a range of 2.5–7.5 U/kg b.w. The experiment on rats confirmed with a statistical significance (P < 0.05) hypoglycemic activity of GKR-NH2 in comparison to a control group receiving 0.9% NaCl. Characteristics for GKR-NH2 profile was a rather fast beginning of action (0.5–2.0 h) and quite prolonged return to initial values. GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care. In addition, this work also provides a valuable alternative method for preparing any other recombinant bioactive peptides with C-terminal amidation.

Purification of Δ5-3-ketosteroid isomerase from Digitalis lanata

The isomerization of 5-pregnene-3,20-dione into 4-pregnene-3,20-dione was investigated to shed further light on cardenolide biosynthesis and to characterize the enzymes involved in cardenolide formation. It was shown that the Δ5-3-ketosteroid isomerase of Digitalis lanata, which catalyzes this isomerization, is an individual enzyme and not, as previously thought, associated with Δ5-3β-hydroxysteroid dehydrogenase. The enzyme was purified by fractionated ammonium sulfate precipitation, hydrophobic interaction chromatography and gel filtration. The purification protocol resulted in a 68.1-fold enriched specific enzyme activity with a yield of 2.2%. After an additional chromatofocusing step the 3KSI activity appeared as a single protein band at 17kDa in SDS–PAGE. Plant 3KSI displayed similar properties to microbial 3-ketosteroid isomerases.

Purification and biochemical characterization of manganesecontaining superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3

Thermostable SOD is a promising enzyme in biotechnological applications. In the present study, thermophile Geobacillus sp. EPT3 was isolated from a deep-sea hydrothermal field in the East Pacific. A thermostable superoxide dismutase (SOD) from this strain was purified to homogeneity by steps of fractional ammonium sulfate precipitation, DEAE-Sepharose chromatography, and Phenyl-Sepharose chromatography. SOD was purified 13.4 fold to homogeneity with a specific activity of 3 354 U/mg and 11.1% recovery. SOD from Geobacillus sp. EPT3 was of the Mn-SOD type, judged by the insensitivity of the enzyme to both KCN and H2O2. SOD was determined to be a homodimer with monomeric molecular mass of 26.0 kDa. It had high thermostability at 50°C and 60°C. At tested conditions, SOD was relatively stable in the presence of some inhibitors and denaturants, such as β-mercaptoethanol (β-ME), dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), urea, and guanidine hydrochloride. Geobacillus sp. EPT3 SOD showed striking stability across a wide pH range from 5.0 to 11.0. It could withstand denaturants of extremely acidic and alkaline conditions, which makes it useful in the industrial applications.