Sephacryl S-200 Gel Filtration Chromatography Research Articles

In silico and biochemical analysis on a newly isolated Trichoderma asperellum l-asparaginase

Microbial l-asparaginase enzyme has been considered as a good source of potential anticancer drugs. The enzyme purified from different microbes has been shown different biochemical and kinetics properties. Thus, our study aims to investigate l-asparaginase from Trichoderma asperellum (MN960163), including in silico characterization of the l-asparaginase encoding genes as well as the biochemical characters of the purified enzyme. In silico, analysis of the T. asp genome database revealed the presence of six protein encoding genes with N-terminal asparaginase domain that has asparaginase production potential as indicated from their physico-chemical properties, verification of three of them was confirmed. T. asperellum l- asparaginase with a specific activity of 200 U/mg and 61.7 fold purification was achieved by ammonium sulphate precipitation (60%) followed by DEAE-sepharose ion exchange and Sephacryl S-200 gel filtration chromatography. The purified enzyme has a molecular mass of 65 kDa and exhibited its maximum activity at pH 8 and 50 °C. The purified enzyme showed high thermal stability up to 50 °C and a high affinity to l- asparagine with a Km of 0.35 mM. The activity was significantly enhanced by Cu, K and Ca ions while Fe and EDTA was completely inhibited l-asparginase activity. In conclusion, the current data highlight new possible genes that could be employed as a promising source in asparaginase production for medical purposes. Additionally, the feature of the purified asparaginase enzyme could help to elucidate the unique properties of T. asp asparaginase.

Characterization of the C-type lectin from the marine sponge (Stylissa flexibilis)

A lectin from the marine sponge Stylissa flexibilis, designated as SFL, was purified by cold ethanol precipitation followed by ion exchange chromatography on DEAE Sepharose column and Sephacryl S-200 gel filtration. SFL is a dimeric glycoprotein of 32 kDa subunits linked by a disulfide bridge with a molecular mass of 64 kDa by SDS-PAGE and 65 kDa by Sephacryl S-200 gel filtration chromatography. The lectin preferentially agglutinated enzyme treated human A erythrocytes, whereas it did not agglutinate any type of rabbit, human B and O erythrocytes, irrespective of treatment with enzymes. The hemagglutination activity of lectin was strongly inhibited by monosaccharide, D-galactose and glycoproteins, asialo-porcine stomach mucin and asialo-fetuin, indicating that lectin is specific for O-glycans. Activity of SFL was stable over a range of pH from 5 to 8, up to 60 °C for 30 min and its activity was Ca2+ dependent, indicating that SFL was belonged to the C-type lectin family and requires metal for biological activity. SFL caused agglutination of Vibrio alginolyticus and V. parahaemolyticus in a dose dependent manner and inhibited the growth rate of these bacterial strains, suggesting that the lectin caused the agglutination through binding to the target receptor(s) on the surface of Vibrios. Thus, SFL can be considered as a good source of lectin(s) being useful as carbohydrate probe and antibacterial reagent.

Purification and Biochemical Characterization of a Lipase Produced by Aspergillus Niger: Interfacial Binding, Activity of Lipase at the Lipid-Water Interface Using Monomolecular Film Techniques

Aspergillus Niger lipase (ANL) was purified from a culture medium. Pure ANL was obtained after ammonium sulfate fractionation, Sephacryl S-200 gel filtration and cation exchange chromatography (Mono-Q). The pure lipase, which is not a glycoprotein, was presented as a monomer having a molecular mass of about 99 kDa. The lipase activity was maximal at pH 8 and at 39°C. ANL hydrolyses the long chains of triacylglycerols more efficiently than the short ones. A specific activity of 3779 U/mg was measured on olive oil as substrate at 39°C and at pH 8 in the presence of 4 mM NaTDC. ANL was inactivated when the enzyme was incubated at 75°C or at pH less than 4. Natural detergent (NaTDC), pH dependence of the catalytic activity of ANL was at pH 8, which confirm that ANL present one pH profile.

Isolation of Nile crocodile (Crocodylus niloticus) serum immunoglobulin M and Y (IgM and IgY)

Crocodile immunity has not been fully characterised with more studies on crocodile innate immunity than cell-mediated or humoral immunity. Crocodile immunoglobulin genes have been described but immunoglobulin proteins have not been isolated or studied biochemically. Two large proteins proposed to be crocodile IgM and IgY were isolated and purified from Crocodylus niloticus sera using two different protocols. A 50% (w/v) ammonium sulfate and a 15% (w/v) polyethylene glycol precipitation step was followed by Cibacron blue F3GA affinity- and Sephacryl-S300 gel filtration chromatography. An alternate purification protocol, with only two steps, involved thiophilic affinity- and Sephacryl-S300 gel filtration chromatography. The purified crocodile IgM resolved on reducing SDS-PAGE with an apparent mass of 180 kDa. Purified crocodile IgY resolved at 180 kDa alongside chicken IgY on a non-reducing SDS-PAGE gel, and is deduced to consist of two 66 kDa heavy and two 23 kDa light chains under reducing conditions. The thiophilic/gel filtration two-step protocol gave three-fold higher yields of isolated protein than the four-step precipitation/chromatography protocol. Antibodies against the isolated crocodile IgM and IgY were raised in chickens and affinity purified. The chicken antibodies differentiated between crocodile IgM and IgY and have the potential for use in the diagnosis of crocodile infections. The purified crocodile antibodies can be biochemically characterised and compared to mammalian and avian antibodies to give a better understanding of crocodile humoral immunity.

Purification and characterization of a novel high molecular weight alkaline protease produced by an endophytic Bacillus halotolerans strain CT2

A protease-producing strain CT2 isolated from Tunisian potatoes, exhibiting a potent protease activity (prot CT2), was identified as Bacillus halotolerans according to 16S ribosomal DNA sequence analysis. Maximum prot CT2 production was obtained in medium supplemented with bean seed proteins. Proteolytic activity was purified by ammonium sulphate precipitation, Sephacryl S-200 gel filtration and SP-sepharose cation-exchange chromatography. Optimal enzyme activity was reached at pH 9 and temperature of 50 °C. Proteolytic activity was enhanced by Ca2+ and Mn2+ ions, completely inhibited by PMSF suggesting a serine protease nature and exhibited high stability in the presence of commercial detergents. Prot CT2 showed broad substrate specificity towards both synthetic and natural substrates, with a high capacity to hydrolyze legume seed proteins. Using electrophoretic analysis, its molecular weight was around 250 kDa with two major subunit showing important homologies with serine proteases belonging to the subtilisin-like serine proteases. Based on the Lineweaver–Burk plots Km and Vmax values were 10 mg/ml and 50,000 U/mg respectively. This newly described prot CT2 displays relevant properties which highlight its potential use in various industrial and biotechnological applications.

Purification and Characterization of Active Aggregates of an Organic Solvent Tolerant Lipase from Marinobacter sp. EMB5

Background: Marinobacter is a halophilic bacterial genus and a bio-geochemically important group in sea water, is generally associated with the bioremediation of hydrocarbon-rich effluents released into the salt-rich environment by various anthropogenic activities. It is a well-known lipase producer-the NCBI Protein database shows nearly 520 Marinobacter lipases. In this context, its lipase and esterase are considered important. However, their enzymatic properties in purified form have been scantily studied. Since hydrocarbons are acted upon on solvent water interface, the stability and catalytic behavior of Marinobacter lipase in non-aqueous media needs to be more specifically investigated. Aim: Marinobacter sp. EMB5 was isolated from Kozhikode, Kerala, India. It was found to be potent lipase producer. The aim of the present work was to obtain a purified preparation and undertake its detailed characterization with respect to above aspects. Work: The crude Marinobacter sp. lipase was purified by Sephacryl S200 gel filtration chromatography. The lipase eluted as a single activity peak in void volume and had a very high molecular mass. Aggregation has not been reported in case of Marinobacter lipase, so far, although it is known in other lipases. The aggregated lipase was mixed with 2-propanol (70%, v/v) and disaggregated lipase was obtained as a single activity peak in Sephacryl S200 gel filtration chromatography corresponding to 82 kDa molecular mass. This monomeric species was less active than aggregated purified preparation. Hence, further characterization and studies were carried out on purified active aggregate lipase. It was stable in a wide range of organic solvents at high concentration up to 24 h. The organic solvent stability was further investigated by Far UV CD spectroscopy and fluorescence spectroscopy. Conclusion: Due to the fact that Marinobacter sp. EMB5 lipase remained stable in organic solvents for prolonged incubation periods, it has potential to be used in industrial organic synthesis.

Interaction of tubulin and protein kinase CK2 in Trypanosoma equiperdum.

A polypeptide band with an apparent molecular weight of 55,000 was phosphorylated in vitro in whole-cell lysates of Trypanosoma equiperdum. This band corresponds to tubulin as demonstrated by immunoprecipitation of the phosphorylated polypeptide from T. equiperdum extracts when anti-α and anti-β tubulin monoclonal antibodies were employed. A parasite protein kinase CK2 was in charge of modifying tubulin given that common mammalian CK2 inhibitors such as emodin and GTP, hindered the phosphorylation of tubulin and exogenously added casein. Interestingly, a divalent cation-dependent translocation of the T. equiperdum tubulin and the CK2 responsible for its phosphorylation was noticed, suggesting a direct interaction between these two proteins. Additionally, this fraction of tubulin and its kinase coeluted using separations based on parameters as different as charge (DEAE-Sepharose anion-exchange chromatography) and size (Sephacryl S-300 gel filtration chromatography). Analyses by non-denaturing polyacrylamide gel electrophoresis and immunoblot of the purified and radioactively labeled fraction containing both tubulin and the CK2 enzyme, established the phosphorylation of a single band that was recognized by anti-CK2 α-subunit and anti-tubulin antibodies. All these findings revealed a physical association between a pool of tubulin and a CK2 in T. equiperdum.

Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity

Crude enzyme from Geotrichum candidum S12 exhibited high activity towards hexanol at pH 4.0, distinguishing it from currently known enzymes. To identify the dominant enzyme contributing to this activity, the crude enzyme extract was separated into different fractions by ammonium sulfate precipitation, MonoQ anion-exchange chromatography, and Sephacryl S-200 gel filtration chromatography. Afraction with high activity towards hexanol at pH 4.0 was obtained, exhibiting 38-fold improved purity and a specific activity of 3802.7 U/mg. After electrophoretic analysis, the fraction showed a molecular weight of 223 kDa by Native-PAGE and 51.4 kDa by SDS-PAGE. The purified fraction was identified as a glutamate dehydrogenase (GDH) by peptide mass fingerprinting data. This fraction showed high activity towards glutamate, α-ketoglutarate, hexanol, and isoamyl alcohol with a Km value of 41.74, 4.01, 20.37, and 19.37 mM, respectively, but with no activity towards methanol, ethanol, 1-propanol, and isobutanol. As a comparison, the GDH from yeast had no activity towards hexanol and other alcohols. Kinetic analysis revealed that glutamate and hexanol served as competitive inhibitors to each other for the purified GDH. The GDH showed the highest activity towards hexanol at pH 4.0 and 30 °C, and was the most stable at pH 2.2–7.0 and ≤40 °C. The presence of ADP, Fe2+, K+, and Zn2+ increased the enzymatic activity towards hexanol and EDTA, Pb2+, Mn2+, ATP, and DTT decreased the activity. These novel characteristics expand the reported properties of GDH and suggest the newly characterized GDH has unique potential for practical application.

Immobilization of Purified Phytase Enzyme from Tirmit (Lactarius volemus) on Coated Chitosan with Iron Nanoparticles and Investigation of Its Usability in Cereal Industry

In this study, phytase enzyme was purified and characterized from Tirmit (Lactarius volemus) mushroom and purified phytase enzyme was immobilized on the surface of modified chitosan with nano-Fe3O4 nanoparticles. Then, free and immobilized enzyme applications had been carried out in cereal crops. Phytase enzyme was purified 120.2-fold and characterized with 11.6 % yield in range of 40–80 % from Tirmit (L. volemus) by using, ammonium sulfate precipitation, DEAE-Sephadex ion exchange chromatography and Sephacryl S200 gel filtration chromatography techniques. The purification fold was calculated by determining the activity and amount of protein for each step. Then, purified phytase enzyme was immobilized on modified chitosan support material with Fe3O4 nanoparticles. For the immobilized and free enzyme, optimum pH was 5.5 and optimum temperature was 50 °C. First, the effects of some metal ions, such as CuCl2, HgCl2, FeCl2, MgCl2, ZnCl2 and CaCl2, were investigated on the bound and free purified phytase enzyme. Second, molecular weight of the enzyme was determined by SDS-PAGE electrophoresis method. Third, substrate specificity was determined for the purified phytase enzyme using some natural polymers, such as locust bean gum, gelatin, xylene, cellulose, phytate and starch. Finally, investigated whether the immobilized and free enzyme could be hydrolyzed the phytic acid in green lentils, red lentils, peas, pinto beans, beans, brass, corn, dried corn, oat, rye, wheat, broad bean, chickpea and peanuts or not.

A thermostable-endo-β-(1,4)-mannanase from Pediococcus acidilactici (M17): purification, characterization and its application in fruit juice clarification

A Turkish fermented sausage was bought from a local market in Erzurum and was used for isolation and characterization of lactic acid bacteria. As the result of 16S rRNA analysis, this bacterium was determined as Pediococcus acidilactici (Genbank number KF895384) at a rate of 99 %. The mannanase from P. acidilactici (M17) was purified using the (NH4)2SO4 precipitation, DEAE-cellulose ion exchange and Sephacryl S200 gel filtration chromatography techniques. The yield and purification fold of mannanase were 10.6 % and 106.1, respectively. The molecular mass of the enzyme (38 kDa) was determined by SDS-PAGE and gel filtration chromatography. In order to identify, the resistance of the purified mannanase to Mn2+, Co2+, Cu2+, Zn2+ and Ca2+ was examined, and all the metal ions activated the enzyme at a significant level. The V max and K m values for locust bean gum were calculated as 78.74 mmol/min mg and 0.592 mM, by Lineweaver–Burk. Purified mannanase was also used to clarify some fruit juices. The studied variables were, the incubation time (from 0 to 60 min), the enzyme concentration (17 EU/L), and the temperature (60 °C). It was observed that the purified mannanase had maximum yield on orange juice with 161.1 %. Also maximal clarification was obtained as 84.65 %, and the clarification for grape juice and sugar reduction was obtained as 19.26 mg/mL for apple juice. Consequently, it was concluded that the P. acidilactici (M17) mannanase could be successfully used in the fruit juice industry.

A Sialic Acid Assay in Isolation and Purification of Bovine .κ-Casein Glycomacropeptide: A Review

Sialic acid is a carbohydrate moiety of k-casein glycomacropeptide (GMP), which is a 64 amino acid residue C-terminal sialylated phosphorylated glycopeptide released from k-casein by the action of chymosin during cheese making. GMP lacks aromatic amino acids including phenylalanine, tyrosine, and tryptophan. Because of its unique amino acid composition and various biological activities, GMP is thought to be a potential ingredient for dietetic foods (e.g., a food for PKU patients) and pharmaceuticals. Thus, increased attention has been given to the development of techniques to purify GMP. In this review, techniques of GMP purification described in patents and scientific research papers were introduced. A sialic acid assay is the important method to track GMP isolation and purification processes, for which the thiobarbituric acid reaction with 1-propanol as a chromophore extracting solvent is an inexpensive, practical and specific technique. Sephacryl S-200 gel filtration chromatography, cellulose acetate electrophoresis, and sodium dodecyl sulfate polyacrylamide gel electrophoresis are the major techniques to identify sialic acid specific to GMP. Sephacryl S-200 chromatography and cellulose acetate electrophoresis are also used to detect GMP sialic acid in whey pearmeate and whey added commercial margarine samples. Future research includes development of an economical industrial scale method to produce high purity GMP.

Purification and characterization of [Formula: see text]-mannanase from Bacillus pumilus (M27) and its applications in some fruit juices.

Thermo alkaline mannanase was purified from the bacteria of Bacillus pumilus (M27) using the techniques of ammonium sulphate precipitation, DEAE-Sephadex ion exchange chromatography and Sephacryl S200 gel filtration chromatography with 111-fold and 36% yield. It was determined that the enzyme had 2 sub-units including 35kDa and 55kDa in gel filtration chromatography and SDS-PAGE electrophoresis systems. The optimum pH and temperature was determined as 8 and 60°C, respectively. It was also noticed that the enzyme did not lose its activity at a wide interval such as pH3-11 and at high temperatures such as 90°C. Additionally, the effects of some metal ions on the mannanase enzyme activity. Moreover, the clarifying efficiency of purified mannanase enzyme with some fruit juices such as orange, apricot, grape and apple was also investigated. Enzymatic treatment was carried out with 1mLL(-1) of purified mannanase for 1h at 60°C. It was determined that the highest pure enzyme was efficient upon clarifying the apple juice at 154% rate.

BACE2의 대량발현 및 리폴딩

BACE2 is a membrane-bound aspartic protease that is highly homologous with BACE1. While BACE1 processes the amyloid precursor protein (APP) at a key step in generating <TEX>${\beta}$</TEX>-amyloid peptide and presumably causes Alzheimer's disease (AD), BACE2 has not been demonstrated to be involved in APP processing directly, and its physiological functions are unknown. To determine its function and to develop inhibitors from marine sources, we constructed an overexpression vector for producing BACE2. The gene encoding human BACE2 protease was amplified using the polymerase chain reaction and cloned into the pET11a expression vector, resulting in pET11a/BACE2. Recombinant BACE2 protease was overexpressed successfully in E. coli as inclusion bodies, refolded using the rapid-dilution method, and purified via two-step fast protein liquid chromatography using Sephacryl S-300 gel filtration and Resource-Q column chromatography. The BACE2 protease produced was an active form. This study provides an efficient method not only for studying the basic properties of BACE2, but also for developing inhibitors from natural marine sources.

Characterization of an organic solvent-tolerant lipase from Idiomarina sp. W33 and its application for biodiesel production using Jatropha oil

A halophilic strain W33 showing lipolytic activity was isolated from the saline soil of Yuncheng Salt Lake, China. Biochemical and physiological characterization along with 16S rRNA gene sequence analysis placed the isolate in the genus Idiomarina. The extracellular lipase was purified to homogeneity by 75% ammonium sulphate precipitation, DEAE-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography. The molecular mass of the purified lipase was estimated to be 67 kDa by SDS-PAGE. Substrate specificity test indicated that it preferred long-chain p-nitrophenyl esters. Optimal lipase activity was found to be at 60 °C, pH 7.0-9.0 and 10% NaCl, and it was highly active and stable over broad temperature (30-90 °C), pH (7.0-11.0) and NaCl concentration (0-25%) ranges, showing excellent thermostable, alkali-stable and halotolerant properties. Significant inhibition by diethyl pyrocarbonate and phenylarsine oxide was observed, implying histidine and cysteine residues were essential for enzyme catalysis. In addition, the lipase displayed high stability and activity in the presence of hydrophobic organic solvents with log P(ow) ≥ 2.13. The free and immobilized lipases produced by Idiomarina sp. W33 were applied for biodiesel production using Jatropha oil, and about 84 and 91% of yields were achieved, respectively. This study formed the basic trials conducted to test the feasibility of using lipases from halophile for biodiesel production.

A New Raw-Starch-Digesting α-Amylase: Production Under Solid-State Fermentation on Crude Millet and Biochemical Characterization

A new Bacillus strain degrading starch, named Bacillus sp. UEB-S, was isolated from a southern Tunisian area. Amylase production using solid-state fermentation on millet, an inexpensive and available agro-resource, was investigated. Response surface methodology was applied to establish the relationship between enzyme production and four variables: inoculum size, moisture-to-millet ratio, temperature, and fermentation duration. The maximum enzyme activity recovered was 680 U/g of dry substrate when using 1.38 × 10(9) CFU/g as inoculation level, 5.6:1 (ml/g) as moisture ratio (86%), for 4 days of cultivation at 37 degrees C, which was in perfect agreement with the predicted model value. Amylase was purified by Q-Sepharose anion-exchange and Sephacryl S-200 gel filtration chromatography with a 14-fold increase in specific activity. Its molecular mass was estimated at 130 kDa. The enzyme showed maximal activity at pH 5 and 70 degrees C, and efficiently hydrolyzed starch to yield glucose and maltose as end products. The enzyme proved its efficiency for digesting raw cereal below gelatinization temperature and, hence, its potentiality to be used in industrial processes.

Purification and physicochemical properties of polygalacturonase from Aspergillus niger MTCC 3323

Polygalacturonases are the pectinolytic enzymes that catalyze the hydrolytic cleavage of the polygalacturonic acid chain. In the present study, polygalacturonase from Aspergillus niger (MTCC 3323) was purified. The enzyme precipitated with 60% ethanol resulted in 1.68-fold purification. The enzyme was purified to 6.52-fold by Sephacryl S-200 gel-filtration chromatography. On SDS–PAGE analysis, enzyme was found to be a heterodimer of 34 and 69kDa subunit. Homogeneity of the enzyme was checked by NATIVE-PAGE and its molecular weight was found to be 106kDa. The purified enzyme showed maximum activity in the presence of polygalacturonic acid at temperature of 45°C, pH of 4.8, reaction time of 15min. The enzyme was stable within the pH range of 4.0–5.5 for 1h. At 4°C it retained 50% activity after 108h but at room temperature it lost its 50% activity after 3h. The addition of Mn2+, K+, Zn2+, Ca2+ and Al3+ inhibited the enzyme activity; it increased in the presence of Mg2+ and Cu2+ ions. Enzyme activity was increased on increasing the substrate concentration from 0.1% to 0.5%. The Km and Vmax values of the enzyme were found to be 0.083mg/ml and 18.21μmol/ml/min. The enzyme was used for guava juice extraction and clarification. The recovery of juice of enzymatically treated pulp increased from 6% to 23%. Addition of purified enzyme increased the %T650 from 2.5 to 20.4 and °Brix from 1.9 to 4.8. The pH of the enzyme treated juice decreased from 4.5 to 3.02.

Purification and Characterization of an Endo-d-arabinase Produced by Cellulomonas

An extracellular endo-D: -arabinase enzyme produced by the bacterial strain of Cellulomonas was purified 77.1-fold with 0.20% recovery for protein by DEAE Sepharose anion exchange, Sephacryl S-300 gel filtration and blue Sepharose affinity chromatography, and designated as CEDAase. The apparent molecular mass of CEDAase was 45kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CEDAase is an endoenzyme for arabinogalactan with the main and specific product of hexa-arabinofuranoside. It reacts optimally with its substrate, arabinogalactan, at approximately pH 8.0 and at 40°C. CEDAase shows stability in the pH range of 6.0-9.0 and at the temperature below 50°C. The K(m) measured for the CEDAase was 55.6μM, with an apparent V(max) of 0.083μmol/min. To our knowledge, for the first time, the current work obtains an extracellular Cellulomonas endo-D: -arabinase enzyme that might be potentially served as a tool enzyme for hydrolyzing specific cell wall such as Mycobacterium cell. It is purified as an important potential initial material basis for mass spectrometric sequencing and chemical gene synthesis. It may make it possible to clone and express this valuable endo-D: -arabinase and make it available to the mycobacteria scientific community.

Monoclonal antibodies recognizing mucus immunoglobulin and surface immunoglobulin-positive cells of flounder (Paralichthys olivaceus)

Mucus immunoglobulin (Ig) of flounder (Paralichthys olivaceus) was purified by the combination of salting-out, Sephacryl S-300 gel filtration chromatography and DEAE Sepharose chromatography. According to the SDS-PAGE and native-PAGE, the purified mucus Ig showed apparent molecular weights of 72kDa (heavy chain) and 26kDa (light chain), and a total molecular weight of 798kDa, which indicated mucus IgM was in tetrameric form. Purified mucus Ig was used to immunize the Balb/C mice, nineteen hybridomas secreting monoclonal antibodies (mAbs) against flounder mucus Ig were obtained by indirect enzyme-linked immunosorbent assay, and three of them designated as 1A-M2, 1C-M10 and 3F-M9 were cloned by limiting dilution. In Western blotting, the three mAbs specifically reacted to the heavy (H) chain of mucus Ig, but not reacted with serum Ig of flounder, whereas mAb 2D8 against serum Ig previously produced could react with the H chain of both mucus and serum Ig, indicating the composition of the mucus and serum Ig H chains was different. Meanwhile, surface Ig positive (sIg+) lymphocytes in the peripheral blood, spleen, skin and gills of healthy flounder, were analyzed by flow cytometry using mAb 1A-M2 and mAb 2D8, and the results revealed that both mAbs were reactive with the sIg+ lymphocytes. The positive reactivity rates for mAb 1A-M2 were 38.64% in the peripheral blood, 23.6% in the spleen, 16.56% in the skin and 6.26% in the gills, while the positive reactivity rates for mAb 2D8 were 48.89%, 33.7%, 15% and 6.02%, respectively, suggesting mucus Ig was similar, but not identical, to serum Ig. These results generated important mucosal immunological information and gave a valuable insight into understanding the mucosal immunity in flounder.

Purification and partial characterization of a thermostable alkaline protease from Bacillus licheniformis LHSB-05 isolated from hot spring

An alkaline protease produced from Bacillus licheniformis LHSB-05 isolated from Nigerian hot spring was purified in a 2-step procedure by ammonium sulphate precipitation and Sephacryl S-300 gel filtration chromatography. The enzyme was purified 13.4-fold with a yield of 6.6%. The molecular weight of the protease was 16 kDa on SDS-PAGE. The purified protease had optimum temperature of 50°C. The enzyme retained 90% of its maximum activity at 60°C and had 45% relative activity at 70°C. This enzyme exhibited high thermostability with 100% stability at 50°C and almost 80% stability at 60°C after 60 min of incubation. The protease had optimum pH of 9.0 and showed relative activity between 81 and 71% in the pH range of 10.0 to 12.0. The enzyme was stable over a broad pH range (6.0 to 12.0). Ca2+ and Mg2+ increased protease activity with 27 and 9%, whereas Hg2+ strongly inhibited its activity by 83%. The enzyme was relatively stable in the presence of Ca2+, Mg2+, Al3+, Zn2+ and ethylene diamine tetra acetic acid (EDTA). These characteristics suggest the suitability of protease from B. licheniformis LHSB-05 for use in detergent industries. Key words: Alkaline protease, Bacillus licheniformis LHSB-05, characterization, purification, thermostability.

PURIFICATION, CHARACTERIZATION AND IN VITRO ANTIOXIDANT ACTIVITIES OF POLYSACCHARIDE FRACTIONS ISOLATED FROM THE FRUITS OF PHYSALIS ALKEKENGI L.

The water-soluble crude polysaccharide PAPS, isolated from the fruits of Physalis alkekengi L. by hot water extraction and ethanol precipitation, was fractionated by diethylaminoethyl-cellulose ion exchange chromatography and Sephacryl S-300 gel filtration chromatography on ATKA explore 100 purifier, giving four polysaccharide fractions coded as PAPSA-a, PAPSA-b, PAPSB-c and PAPSB-d. Their chemical and physical characteristics were determined by chemical methods, gas chromatography and high performance size exclusion chromatography. The in vitro antioxidant activities of polysaccharides were studied by various antioxidant assays. Experiment results showed that all fractions exhibited antioxidant activity in a concentration-dependent manner, and PAPSB-c and PAPSB-d had stronger antioxidant ability than PAPSA-a and PAPSA-b in all the tests. The polysaccharide fractions can be developed as new antioxidant agents. PRACTICAL APPLICATIONS More and more researches are focused on natural products including vegetables and wild fruits sources. Physalis alkekengi is a kind of wild fruit in north of China, famous for its good taste and high nutritional quality. But, at present, the fruits of P. alkekengi are not fully utilized. This work reported on isolation and characterization of polysaccharides from P. alkekengi, and the in vitro antioxidant activities were studied by various antioxidant assays. The objective of this research work was to extend the potential use of P. alkekengi fruits in functional food or antioxidant biomedicine.