Purification,Characterization, Sequencing and Biological Chemistry of Galectin-1 Purified from <Emphasis Type="Italic">Capra hircus</Emphasis> (goat) Heart

A soluble β-galactoside binding 14.5 kDa lectin was purified from the heart of Capra hircus. Its metal independent nature, preferential affinity for β-d-lactose and 90–94% homology with carbohydrate recognition domain of previously reported galectin-1 confirmed its inclusion in galectin-1 subfamily. The secondary structures of the deduced amino acid sequences were generally conserved with previously reported Gal-1. Exposure of the purified protein to varying temperature and pH, oxidant, thiol blocking reagents, denaturants and detergents resulted in significant changes in UV (ultraviolet), fluorescence, CD (circular dichroism) and FTIR (fourier transform infra red) spectra, thus strongly emphasizing the vitality of regular secondary structure of galectins for maintaining their active conformation. Bioinformatics studies corroborated the results obtained in wet lab. Our findings based on physico-chemical properties, oxidative inactivation and structural analysis of the goat heart galectin-1 suggests significant implications in potential biological and clinical applications.     

Physicochemical Properties and Oxidative Inactivation of Soluble Lectin from Water Buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) Brain

Lectins are carbohydrate-binding proteins present in a wide variety of plants and animals, which serve various important physiological functions. A soluble β-galactoside binding lectin has been isolated and purified to homogeneity from buffalo brain using ammonium sulphate precipitation (40–70%) and gel permeation chromatography on Sephadex G_50–80 column. The molecular weight of buffalo brain lectin (BBL) as determined by SDS-PAGE under reducing and non-reducing conditions was 14.2 kDa, however, with gel filtration it was 28.5 kDa, revealing the dimeric form of protein. The neutral sugar content of the soluble lectin was estimated to be 3.3%. The BBL showed highest affinity for lactose and other sugar moieties in glycosidic form, suggesting it to be a β-galactoside binding lectin. The association constant for lactose binding as evidenced by Scatchard analysis was 6.6 × 10³ M⁻¹ showing two carbohydrate binding sites per lectin molecule. A total inhibition of lectin activity was observed by denaturants like guanidine HCl, thiourea and urea at 6 M concentration. The treatment of BBL with oxidizing agent destroyed its agglutination activity, abolished its fluorescence, and shifted its UV absorption maxima from 282 to 250 nm. The effect of H₂O₂ was greatly prevented by lactose indicating that BBL is more stable in the presence of its specific ligand. The purified lectin was investigated for its brain cell aggregation properties by testing its ability to agglutinate cells isolated from buffalo and goat brains. Rate of aggregation of buffalo brain cells by purified protein was more than the goat brain cells. The data from above study suggests that the isolated lectin may belong to the galectin-1 family but is glycosylated unlike those purified till date.     

Surface plasmon resonance imaging for real-time, label-free analysis of protein interactions with carbohydrate microarrays

Plant lectin recognition of glycans was evaluated by SPR imaging using a model array of N-biotinylated aminoethyl glycosides of β-d-glucose (negative control), α-d-mannose (conA-responsive), β-d-galactose (RCA₁₂₀-responsive) and N-acetyl-β-d-glucosamine (WGA-responsive) printed onto neutravidin-coated gold chips. Selective recognition of the cognate ligand was observed when RCA₁₂₀ was passed over the array surface. Limited or no binding was observed for the non-cognate ligands. SPR imaging of an array of 40 sialylated and unsialylated glycans established the binding preference of hSiglec7 for α2-8-linked disialic acid structures over α2-6-sialyl-LacNAcs, which in turn were recognized and bound with greater affinity than α2-3-sialyl-LacNAcs. Affinity binding data could be obtained with as little as 10–20 μg of lectin per experiment. The SPR imaging technique was also able to establish selective binding to the preferred glycan ligand when analyzing crude culture supernatant containing 10–20 μg of recombinant hSiglec7-Fc. Our results show that SPR imaging provides results that are in agreement with those obtained from fluorescence based carbohydrate arrays but with the added advantage of label-free analysis.     

Purification and characterization of a novel β-galactosidase from Bacillus sp MTCC 3088

An extracellular β-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was harvested from the late stationary-phase of Bacillus sp MTCC 3088. The enzyme was purified 36.2-fold by ZnCl₂ precipitation, ion exchange, hydrophobic interaction and gel filtration chromatography with an overall recovery of 12.7%. The molecular mass of the purified enzyme was estimated to be about 484 kDa by gel filtration on a Sephadex G-200 packed column and the molecular masses of the subunits were estimated to be 115, 86.5, 72.5, 45.7 and 41.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the native enzyme, determined by polyacrylamide gel electrofocusing, was 6.2. The optimum pH and temperature were 8 and 60°C, respectively. The Michaelis–Menten constants determined with respect to o-NO₂-phenyl-β-D-galactopyranoside and lactose were 6.34 and 6.18 mM, respectively. The enzyme activity was strongly inhibited (68%) by galactose, the end product of lactose hydrolysis reaction. The β-galactosidase was specific for β-D anomeric linkages. Enzyme activity was significantly inhibited by metal ions (Hg²⁺, Cu²⁺ and Ag⁺) in the 1–2.5 mM range. Mg²⁺ was a good activator. Catalytic activity was not affected by the chelating agent EDTA. Journal of Industrial Microbiology & Biotechnology (2000) 24, 58–63. Received 09 February 1999/ Accepted in revised form 24 September 1999     

A chitotetrose specific lectin fromLuffa acutangula: Physico-chemical properties and the assignment of orientation of sugars in the lectin binding site

A chitooligosaccharide specific lectin (Luffa acutangula agglutinin) has been purified from the exudate of ridge gourd fruits by affinity chromatography on soybean agglutinin-glycopeptides coupled to Sepharose-6B. The affinity purified lectin was found homogeneous by polyacrylamide gel electrophoresis, in sodium dodecyl sulphate-polyacrylamide gels, by gel filtration on Sephadex G-100 and by sedimentation velocity experiments. The relative molecular weight of this lectin is determined to be 48,000 ±1,000 by gel chromatography and sedimentation equilibrium experiments. The sedimentation coefficient (S₂₀, w) was obtained to be 4.06 S. The Stokes’ radius of the protein was found to be 2.9 nm by gel filtration. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis the lectin gave a molecular weight of 24,000 in the presence as well as absence of 2-mercaptoethanol. The subunits in this dimeric lectin are therefore held by non-covalent interactions alone. The lectin is not a glycoprotein and circular dichroism spectral studies indicate that this lectin has 31% α-helix and no β-sheet. The lectin is found to bind specifically to chitooligosaccharides and the affinity of the lectin increases with increasing oligosaccharide chain length as monitored by near ultra-violet-circular dichroism and intrinsic fluorescence titration. The values of ΔG, ΔH and ΔS for the binding process showed a pronounced dependence on the size of the oligosaccharide. The values for both ΔH and ΔS show a significant increase with increase in the oligosaccharide chain length showing that the binding of higher oligomers is progressively more favoured thermodynamically than chitobiose itself. The thermodynamic data is consistent with an extended binding site in the lectin which accommodates a tetrasaccharide. Based on the thermodynamic data, blue shifts and fluorescence enhancement, spatial orientation of chitooligosaccharides in the combining site of the lectin is assigned.     

Purification and characterization of an endoglucanase from <Emphasis Type="Italic">Aspergillus terreus</Emphasis> highly active against barley β-glucan and xyloglucan

An endoglucanase (1, 4-β-d glucan glucanohydrolase, EC 3.2.1.4) which was catalytically more active and exhibited higher affinity towards barley β-glucan, xyloglucan and lichenin as compared to carboxymethylcellulose (CMC) was purified from Aspergillus terreus strain AN₁ following ion-exchange and hydrophobic interaction chromatography and gel filtration. The purified enzyme (40-fold) that apparently lacked a cellulose-binding domain showed a specific activity of 60 μmol mg⁻¹ protein⁻¹ against CMC. The purified enzyme had a molecular weight of 78 and 80 KDa as indicated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and gel filtration, respectively, and a pI of 3.5. The enzyme was optimally active at temperature 60°C and pH 4.0, and was stable over a broad range of pH (3.0–5.0) at 50°C. The endoglucanase activity was positively modulated in the presence of Cu²⁺, Mg²⁺, Ca²⁺, Na⁺, DTT and mercaptoethanol. Endoglucanase exhibited maximal turn over number (K _cat) and catalytic efficiency (K _cat/km) of 19.11 × 10⁵ min⁻¹ and 29.7 × 10⁵ mM⁻¹ min⁻¹ against barley β-glucan as substrate, respectively. Hydrolysis of CMC and barley β-glucan liberated cellobiose, cellotriose, cellotetraose and detectable amount of glucose. The hydrolysis of xyloglucan, however, apparently yielded positional isomers of cellobiose, cellotriose and cellotetraose as well as larger oligosaccharides.     

Purification and characterization of two beta-glucosidases from the thermophilic fungus Thermoascus aurantiacus

β-Glucosidase from the fungusThermoascus aurantiacus grown on semi-solid fermentation medium (using ground corncob as substrate) was partially purified in 5 steps—ultrafiltration, ethanol precipitation, gel filtration and 2 anion exchange chromatography runs, and characterized. After the first anion exchange chromatography, β-glucosidase activity was eluted in 3 peaks (Gl-1, Gl-2, Gl-3). Only the Gl-2 and Gl-3 fractions were adsorbed on the gel matrix. Gl-2 and Gl-3 exhibited optimum pH at 4.5 and 4.0, respectively. The temperature optimum of both glucosidases was at 75–80°C. The pH stability of Gl-2 (4.0–9.0) was higher than Gl-3 (5.5–8.5); both enzyme activities showed similar patterns of thermostability. Under conditions of denaturing gel chromatography the molar mass of Gl-2 and Gl-3 was 175 and 157 kDa, respectively. Using 4-nitrophenyl β-d-glucopyranoside as substrate,K _m values of 1.17±0.35 and 1.38±0.86 mmol/L were determined for Gl-2 and Gl-3, respectively. Both enzymes were inhibited by Ag⁺ and stimulated by Ca²⁺.     

Purification and properties of a thermostable extracellular β-D-xylosidase produced by a thermotolerant Aspergillus phoenicis

A β-D-xylosidase was purified from cultures of a thermotolerant strain of Aspergillus phoenicis grown on xylan at 45°C. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-100. The purified enzyme was a monomer of molecular mass 132 kDa by gel filtration and SDS-PAGE. Treatment with endoglycosidase H resulted in a protein with a molecular mass of 104 kDa. The enzyme was a glycoprotein with 43.5% carbohydrate content and exhibited a pI of 3.7. Optima of temperature and pH were 75°C and 4.0–4.5, respectively. The activity was stable at 60°C and had a K _m of 2.36 mM for p-nitrophenyl-β-D-xylopiranoside. The enzyme did not exhibit xylanase, cellulase, galactosidase or arabinosidase activities. The purified enzyme was active against natural substrates, such as xylobiose and xylotriose. Journal of Industrial Microbiology & Biotechnology (2001) 26, 156–160. Received 23 June 2000/ Accepted in revised form 29 September 2000     

Purification and characterization of a lectin from seeds and cotyledonary callus of Zizyphus mauritiana

A lectin from Zizyphus mauritiana lamk. has been purified from the 25–50% (NH₄)₂SO₄ fraction of crude seed and cotyledonary leaf callus extracts. The lectins purified from the two sources had the same structure and properties. Upon specific adsorption on Sephadex G-100, the lectin (ZML) could be displaced with 0.1 m d-glucose. ZML yielded a single band corresponding to a M_r of 66 kDa both in the absence and presence of β-mercaptoethanol on native- as well as in SDS-PAGE. It is thermostable but pH sensitive and agglutinates human erythrocytes only. Lectin activity could also be detected in the cotyledons, leaf, and stem, and in their cultures, as well as in in vitro regenerants. Cotyledons, cotyledonary leaf and its callus showed much higher lectin activity than seeds. Received: 5 April 1997 / Revision received: 30 June 1997 / Accepted: 31 October 1997     

A mannose-specific tetrameric lectin with mitogenic and antibacterial activities from the ovary of a teleost,the cobia (<Emphasis Type="Italic">Rachycentron canadum</Emphasis>)

A tetrameric lectin, with hemagglutinating activity toward rabbit erythrocytes and with specificity toward d-mannosamine and d(+)-mannose, was isolated from the ovaries of a teleost, the cobia Rachycentron canadum. The isolation protocol comprised ion exchange chromatography on CM-cellulose and Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q, and finally gel filtration by FPLC on Superose 12. The lectin was adsorbed on all ion exchangers used. It exhibited a molecular mass of 180 kDa in gel filtration on Superose 12 and a single 45-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is a tetrameric protein. The hemagglutinating activity of the lectin was stable up to 40°C and between pH 4 and pH 10. All hemagglutinating activity disappeared at 60°C and at pH 1 and pH 13. The hemagglutinating activity was doubled in the presence of 0.1 μM FeCl₃. The lectin exerted antibacterial activity against Escherichia coli with 50% inhibition at 250 μg. There was no antifungal activity toward Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, and Rhizoctonia solani at a dose of 300 μg. The lectin exhibited maximal mitogenic response from mouse splenocytes at a concentration of 14 μM.     

Heterologous expression and characterization of a glucose-stimulated β-glucosidase from the termite <Emphasis Type="Italic">Neotermes koshunensis</Emphasis> in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

Neotermes koshunensis is a lower termite that secretes endogenous β-glucosidase in the salivary glands. This β-glucosidase (G1NkBG) was successfully expressed in Aspergillus oryzae. G1NkBG was purified to homogeneity from the culture supernatant through ammonium sulfate precipitation and anion exchange, hydrophobic, and gel filtration chromatographies with a 48-fold increase in purity. The molecular mass of the native enzyme appeared as a single band at 60 kDa after gel filtration analysis, indicating that G1NkBG is a monomeric protein. Maximum activity was observed at 50 °C with an optimum pH at 5.0. G1NkBG retained 80% of its maximum activity at temperatures up to 45 °C and lost its activity at temperatures above 55 °C. The enzyme was stable from pH 5.0 to 9.0. G1NkBG was most active towards laminaribiose and p-nitrophenyl-β-d-fucopyranoside. Cellobiose, as well as cello-oligosaccharides, was also well hydrolyzed. The enzyme activity was slightly stimulated by Mn²⁺ and glycerol. The K _m and V _max values were 0.77 mM and 16 U/mg, respectively, against p-nitrophenyl-β-d-glucopyranoside. An unusual finding was that G1NkBG was stimulated by 1.3-fold when glucose was present in the reaction mixture at a concentration of 200 mM. These characteristics, particularly the stimulation of enzyme activity by glucose, make G1NkBG of great interest for biotechnological applications, especially for bioethanol production.     

Conjugates of COL-1 monoclonal antibody and β-D-galactosidase can specifically kill tumor cells by generation of 5-fluorouridine from the prodrug β-D-galactosyl-5-fluorouridine

5′-O-β-d-galactosyl-5-fluorouridine is a prodrug that can be converted by the enzyme β-d-galactosidase to the potent antineoplastic drug 5-fluorouridine. The prodrug is more than 100x less toxic than the drug to bone marrow cells in Balb/c mice. The ratio of the IC₅₀ of the prodrug to that of the drug determined on a variety of tumor cell lines in vitro ranged from 500∶1–1000∶1. An antibody-enzyme conjugate (AEC) was synthesized and purified. Maleimide-substituted COL-1 anti-CEA monoclonal antibody was linked to free thiol groups of β-d-galactosidase. The conjugate was purified by size exclusion and ion exchange chromatography. It retained full immunoreactivity and enzyme activity. After binding to antigen-positive tumor cells, the conjugate was able to activate the prodrug and specifically kill the cells. We are continuing to investigate this model for its potential use in antibody-directed enzyme prodrug therapy (ADEPT).     

Purification and Characterization of a Novel β-<Emphasis Type="SmallCaps">d</Emphasis>-Galactosides-Specific Lectin from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A lectin present in seeds of Clitoria ternatea agglutinated trypsin-treated human B erythrocytes. The sugar specificity assay indicated that lectin belongs to Gal/Gal NAc-specific group. Hence the lectin, designated C. ternatea agglutinin (CTA), was purified by the combination of acetic acid precipitation, salt fractionation and affinity chromatography. HPLC gel filtration, SDS-polyacrylamide gel electrophoresis and mass spectrometry indicated that the native lectin is composed of two identical subunits of molecular weight 34.7 kDa associated by non covalent bonds. The N-terminal sequence of CTA shared homology with Glycine max and Pisum sativum. Complete sequence was also found to be homologous to S-64 protein of Glycine max, suggesting that CTA probably exhibits both hemagglutination and probably sugar uptake activity. The carbohydrate binding specificity of the lectin was investigated by quantitative turbidity measurements, and percent inhibition assays. Based on these assays, we conclude that CTA binds β-d-galactosides, and also may has an extended specificity towards non-reducing terminal Neu5Acα2,6Gal.     

Purification and characterization of a highly thermostable α-<Emphasis Type="SmallCaps">l</Emphasis>-Arabinofuranosidase from <Emphasis Type="Italic">Geobacillus caldoxylolyticus</Emphasis> TK4

The gene encoding an α-l-arabinofuranosidase from Geobacillus caldoxylolyticus TK4, AbfATK4, was isolated, cloned, and sequenced. The deduced protein had a molecular mass of about 58 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with α-l-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. A histidine tag was introduced at the N-terminal end of AbfATK4, and the recombinant protein was expressed in Escherichia coli BL21, under control of isopropyl-β-D-thiogalactopyranoside-inducible T7 promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 236 kDa, suggesting a homotetrameric structure. AbfATK4 was active at a broad pH range (pH 5.0–10.0) and at a broad temperature range (40–85°C), and it had an optimum pH of 6.0 and an optimum temperature of 75–80°C. The enzyme was more thermostable than previously described arabinofuranosidases and did not lose any activity after 48 h incubation at 70°C. The protein exhibited a high level of activity with p-nitrophenyl-α-l-arabinofuranoside, with apparent K _m and V _max values of 0.17 mM and 588.2 U/mg, respectively. AbfATK4 also exhibited a low level of activity with p-nitrophenyl-β-d-xylopyranoside, with apparent K _m and V _max values of 1.57 mM and 151.5 U/mg, respectively. AbfATK4 released l-arabinose only from arabinan and arabinooligosaccharides. No endoarabinanase activity was detected. These findings suggest that AbfATK4 is an exo-acting enzyme.     

Characterization of a novel ginsenoside-hydrolyzing α-l-arabinofuranosidase, AbfA, from Rhodanobacter ginsenosidimutans Gsoil 3054T

The gene encoding an α-l-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-[α-l-arabinofuranosyl-(1–6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054^T, and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K _m values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V _max values of 27.1 ± 1.7 and 49.6 ± 4.1 μmol min⁻¹ mg⁻¹ of protein for p-nitrophenyl-α-l-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar groups. These results are the first report of a glycoside hydrolase family 51 α-l-arabinofuranosidase that can transform ginsenoside Rc to Rd.     

Purification and carbohydrate-binding specificity of Agrocybe cylindracea lectin

A lectin was isolated from fruiting bodies of Agrocybe cylindracea by two ion-exchange chromatographies and gel filtration on Toyopearl HW55F. The lectin was homogeneous on polyacrylamide gel electrophoresis and its molecular mass was determined to be 30 000 by gel filtration, and 15 000 by sodium dodecylsulfate polyacrylamide gel electrophoresis, signifying a dimeric protein. Its carbohydrate-binding specificity was investigated both by sugar-hapten inhibition of hemagglutination and by enzyme-linked immunosorbent assay. The inhibition tests showed the affinity of the lectin to be weakly directed toward sialic acid and lactose, and the enhanced affinity toward trisaccharides containing the NeuAcα2,3Galβ-structure. Importantly, the lectin strongly interacted with glycoconjugates containing NeuAcα2,3Galβ1,3GlcNAc-/GalNAc sequences. This revised version was published online in November 2006 with corrections to the Cover Date.     

Expression, Purification and Characterization of a Recombinant β-Glucosidase from Volvariella Volvacea

For the first time, a β-glucosidase gene from the edible straw mushroom, Volvariella volvacea V1-1, has been over-expressed in E. coli. The gene product was purified by chromatography showing a single band on SDS-PAGE. The recombinant enzyme had a molecular mass of 380 kDa with subunits of 97 kDa. The maximum activity was at pH 6.4 and 50 °C over a 5 min assay. The purified enzyme was stable from pH 5.6–8.0, had a half life of 1 h at 45 °C. The β-glucosidase had a K_m of 0.2 mM for p-nitrophenyl-β-D-glucopyranoside.     

Genetic assessment of the importance of galectin-3 in cancer initiation, progression, and dissemination in mice

The galectin family of β-galactoside binding lectins isinvolved in normal and pathological processes. Altered expressionof galectin-3 has been described in many cancers, and studiesof cancer cell lines have implicated this lectin in variousaspects of the tumorigenic cascade. The goal of this reportwas to directly assess the importance of galectin-3 in tumorbiology by introducing the galectin-3 null mutation (galectin-3^–/–)into mouse lines genetically programmed to develop cancers.We used two mouse models of human intestinal cancer, the Apc^Minand Apc^1638N lines, to study tumor initiation and tumor progression.We also crossed the galectin-3^–/– mice with PyMTtransgenic animals, a model in which primary mammary gland tumorsgive rise to lung metastases at high frequency. Unexpectedly,we show that the absence of galectin-3 does not affect the evolutionof the disease in any of these three situations.     

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 45 kDa 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 55.6 μM, with an apparent V_max of 0.083 μmol/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.     

Isolation and characterization of a new phycoerythrin from the cyanobacterium <Emphasis Type="Italic">Synechococcus</Emphasis> sp. ECS-18

A new phycoerythrin, SCH-phycoerythrin, was purified from Synechococcus sp. ECS-18 by DEAE-Sephacel anion exchange chromatography and Sephacryl S-300 gel filtration. The protein pigment had an absorbance maximum at 542 nm and a fluorescence maximum at 565 nm. The native molecular mass was approximately 219 kDa as determined by gel filtration, and sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated the presence of two subunits, with molecular mass of 19 and 17.9 kDa. These observations are consistent with the (αβ)₆ subunit composition that is characteristic of phycoerythrins. The α- and β-subunits showed immunological identity by Ouchterlony double immunodiffusion with an anti-phycoerythrin antiserum. The DNA sequence of the SCH-phycoerythrin gene was determined by PCR amplification using primers based on the conserved N-terminal amino acid sequence of the α- and β-subunits of phycoerythrins.