Biophysical characterization and structural determination of the potent cytotoxic Psathyrella asperospora lectin

A lectin with strong cytotoxic effect on human colon cancer HT29 and monkey kidney VERO cells was recently identified from the Australian indigenous mushroom Psathyrella asperospora and named PAL. We herein present its biochemical and structural analysis using a multidisciplinary approach. Glycan arrays revealed binding preference towards N‐acetylglucosamine (GlcNAc) and, to a lesser extent, towards sialic acid (Neu5Ac). Submicromolar and millimolar affinity was measured by surface plasmon resonance for GlcNAc and NeuAc, respectively. The structure of PAL was resolved by X‐ray crystallography, elucidating both the protein's amino acid sequence as well as the molecular basis rationalizing its binding specificity. Proteins 2017; 85:969–975. © 2016 Wiley Periodicals, Inc.     

A Calcium Ion-Dependent Dimeric Bean Lectin with Antiproliferative Activity Toward Human Breast Cancer MCF-7 Cells

In this study, a 60.8-kDa dimeric lectin was isolated from the Phaseolus vulgaris cv. jade bean and characterized. The lectin was bound on Blue Sepharose 6 and Q Sepharose and was finally purified by size exclusion chromatography on Superdex 200. Its hemagglutinating activity toward rabbit erythrocytes was dependent on divalent cations, especially calcium ions. Various carbohydrates tested were devoid of any effect on the hemagglutinating activity. The lectin was stable at pH between 4.5 and 9.4 and temperatures between 30 and 70 °C. It did not exert antifungal activity toward Valsa mali, Setosphaeria turcica, Mycosphaerella arachidicola, Fusarium oxysporum and Bipolaris maydis. The IC₅₀ of the antiproliferative activity of the lectin toward MCF-7 human breast cancer cells was 174 μM. It did not inhibit proliferation of WRL-68 human normal embryonic hepatocytes. The lectin was dependent on calcium ions for hemagglutinating activity and possessed a blocked N-terminus. These two characteristics make the lectin unique among Phaseolus lectins.     

Multi-specificity of a Psathyrella velutina mushroom lectin: heparin/pectin binding occurs at a site different from the N-acetylglucosamine/N-acetylneuraminic acid-specific site.

An N-acetylglucosamine (GlcNAc)/N-acetylneuraminic acid-specific lectin from the fruiting body of Psathyrella velutina (PVL) is a useful probe for the detection and fractionation of specific carbohydrates. In this study, PVL was found to exhibit multispecificity to acidic polysaccharides and sulfatides. Purified PVL and a counterpart lectin to PVL in the mycelium interact with heparin neoproteoglycans, as detected by both membrane analysis and solid phase assay. The pH-dependencies of the binding to heparin and GlcNAc5-6 differ. The heparin binding of PVL is inhibited best by pectin, polygalacturonic acid, and highly sulfated polysaccharides, but not by GlcNAc, colominic acid, or other glycosaminoglycans. Sandwich affinity chromatography indicated that PVL can simultaneously interact with heparin- and GlcNAc-containing macromolecules. Extensive biotinylation was found to suppress the binding activity to heparin while the GlcNAc binding activity is retained. On the other hand, biotinyl PVL binds to sulfatide and the binding is not inhibited by GlcNAc, N-acetylneuraminic acid, or heparin. These results indicate that PVL is a multi-ligand adhesive lectin that can interact with various glycoconjugates. This multispecificity needs to be recognized when using PVL as a sugar-specific probe to avoid misleading information about the nature of glycoforms.     

Terminal N-Acetylgalactosamine-Specific Leguminous Lectin from Wisteria japonica as a Probe for Human Lung Squamous Cell Carcinoma

Millettia japonica was recently reclassified into the genus Wisteria japonica based on chloroplast and nuclear DNA sequences. Because the seed of Wisteria floribunda expresses leguminous lectins with unique N-acetylgalactosamine-binding specificity, we purified lectin from Wisteria japonica seeds using ion exchange and gel filtration chromatography. Glycan microarray analysis demonstrated that unlike Wisteria floribunda and Wisteria brachybotrys lectins, which bind to both terminal N-acetylgalactosamine and galactose residues, Wisteria japonica lectin (WJA) specifically bound to both α- and β-linked terminal N-acetylgalactosamine, but not galactose residues on oligosaccharides and glycoproteins. Further, frontal affinity chromatography using more than 100 2-aminopyridine-labeled and p-nitrophenyl-derivatized oligosaccharides demonstrated that the ligands with the highest affinity for Wisteria japonica lectin were GalNAcβ1-3GlcNAc and GalNAcβ1-4GlcNAc, with K _a values of 9.5 × 10⁴ and 1.4 × 10⁵ M^-1, respectively. In addition, when binding was assessed in a variety of cell lines, Wisteria japonica lectin bound specifically to EBC-1 and HEK293 cells while other Wisteria lectins bound equally to all of the cell lines tested. Wisteria japonica lectin binding to EBC-1 and HEK293 cells was dramatically decreased in the presence of N-acetylgalactosamine, but not galactose, mannose, or N-acetylglucosamine, and was completely abrogated by β-hexosaminidase-digestion of these cells. These results clearly demonstrate that Wisteria japonica lectin binds to terminal N-acetylgalactosamine but not galactose. In addition, histochemical analysis of human squamous cell carcinoma tissue sections demonstrated that Wisteria japonica lectin specifically bound to differentiated cancer tissues but not normal tissue. This novel binding characteristic of Wisteria japonica lectin has the potential to become a powerful tool for clinical applications.     

Cytotoxicity and Glycan-Binding Profile of a <Emphasis Type="SmallCaps">d</Emphasis>-Galactose-Binding Lectin from the Eggs of a Japanese Sea Hare (<Emphasis Type="Italic">Aplysia kurodai</Emphasis>)

A divalent cation-independent 16 kDa d-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized d-galactose and d-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl d-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galβ1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k _ass and k _diss values are 2.4 × 10³ M^?1 s^?1 and 3.8 × 10^?3 s^?1, respectively. AKL-2 appeared cytotoxicity against both Burkitt’s lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.     

Expression, Purification, and Refolding of Active Recombinant Human E-selectin Lectin and EGF Domains in Escherichia coli

Attempts to obtain active E-selectin from Escherichia coli (E. coli) have not yet been successful. In this study, we succeeded in expressing the recombinant lectin and epidermal growth factor domain fragments of human E-selectin (rh-ESLE) in E. coli on a large-scale. The rh-ESLE protein was expressed as an inactive form in the inclusion bodies. The inactive form of rh-ESLE was denatured and solubilized by 6 M guanidine hydrochloride and then purified by Ni²⁺ affinity chromatography under denaturing conditions. Denatured rh-ESLE was then refolded by a rapid-dilution method using a large amount of refolding buffer, which contained arginine and cysteine/cystine. The refolded rh-ESLE showed binding affinity for sLe^X (K _d = 321 nM, B_max = 1.9 pmol/μg protein). This result suggests that the refolded rh-ESLE recovered its native and functional structure.     

Binding specificity of R-10G and TRA-1-60/81, and substrate specificity of keratanase II studied with chemically synthesized oligosaccharides

Recently, we established a mouse monoclonal antibody specific to hiPS/ hES cells, R-10G, which recognizes a type of keratan sulfate. Keratan sulfates (KS) comprise a family of glycosaminoglycans consisting of the repeating unit of [Gal-GlcNAc(6S)]. However, there is a diversity in the degree of sulfation at Gal and GlcNAc residues, and also in the mode of linkage, Galβ1 ? 3GlcNAc (type 1) or Galβ1 ? 4GlcNAc (type 2). To gain more insight into the binding specificity of R-10G, we carried out an ELISA test on avidin-coated plates using polyethylene glycol (PEG)₃-biotinylated derivatives of a series of N-acetyllactosamine tetrasaccharides (keratan sulfates (KSs)). The results suggested that the minimum epitope structure is Galβ1 ? 4GlcNAc(6S)β1 ? 3Galβ1 ? 4GlcNAc(6S)β1 (type 2- type 2 keratan sulfate). Removal of sulfate from GlcNAc(6S) or addition of sulfate to Gal abolished the binding activity almost completely. We also examined the binding specificity of TRA-1-60/81 in the same assay system. The minimum epitope structure was shown to be Galβ1 ? 3GlcNAcβ1 ? 3Galβ1 ? 4GlcNAcβ1 in agreement with the previous study involving glycan arrays (Natunen et al., Glycobiology, 21, 1125–1130 (2011)). Interestingly, however, TRA-1-60/81 was shown to bind to Galβ1 ? 3GlcNAc(6S)β1 ? 3Galβ1 ? 4GlcNAc(6S)β1 (type 1- type 2 keratan sulfate) dose-dependently, being more than one-third the binding activity toward Galβ1 ? 3GlcNAcβ1 ? 3Galβ1 ? 4GlcNAcβ1 than in the case of TRA-1-60. In addition, a substrate specificity study on keratanase II revealed that keratanase II degraded not only “type 2-type 2 keratan sulfate” but also “type 1-type 2 keratan sulfate”, significantly.     

Periplasmic expression,purification, and characterization of an anti-epidermal growth factor receptor antibody fragment in <Emphasis Type="Italic">Escherichia coli</Emphasis>

New structural designs of antibody fragments have considerable biotechnological and therapeutic potential. In this study, we describe the construction and functional expression of a cetuximab-based antibody fragment (scFv-C_H3, minibody) that exhibits activity against human colon cancer. Heterologous expression in Escherichia coli (E. coli) was improved by optimizing the host cells, signal peptides, induction conditions, and culture media. The recombinant minibody was expressed successfully in the periplasm of E. coli BL21(DE3) and purified by immobilized metal affinity chromatography using a Ni²⁺-NTA resin. The purified minibody showed high binding affinity to cell-surface epidermal growth factor receptor (EGFR) and exhibited inhibition of EGFR-mediated signal transduction in the human colon cancer cell line HT29 in a similar way by the cetuximab. The minibody also showed significant level of anti-cancer ability in the HT29 colorectal cancer xenograft model, which was lower than that by the cetuximab.     

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.     

Characterization of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) lectin for biological activity

Lectins are proteins that are subject of intense investigations. Information on lectin from chickpea (Cicer arietinum L.) with respect to its biological activities are very limited. In this study, we purified lectin from the seeds of chickpea employing DEAE-cellulose and SP-Sephadex ion exchange chromatography and identified its molecular subunit mass as 35 kDa. The free radical scavenging activity of lectin measured by the DPPH assay has IC₅₀ of 0.88 µg/mL. Lectin exerted antifungal activity against Candida krusei, Fusarium oxysporium oxysporium, Saccharomyces cerevisiae and Candida albicans, while antibacterial activity against E. coli, B. subtilis, S. marcescens and P. aeruginosa. The minimum inhibitory concentrations were 200, 240, 160 and 140 µg for C. krusei, F. oxysporium, S. cerevisiae and C. albicans respectively. Lectin was further examined for its antiproliferative potential against cancerous cell line. The cell viability assay indicated a high inhibition activity on Ishikawa, HepG2, MCF-7 and MDA-MB-231 with IC₅₀ value of 46.67, 44.20, 53.58 and 37.46?µg/mL respectively. These results can provide a background for future research into the benefits of chickpea lectin to pharmacological perspective.     

Analysis of agalacto-IgG in rheumatoid arthritis using surface plasmon resonance

It is well established that IgG from rheumatoid arthritis (RA) patients are less galactosylated than IgG from normal individuals. Determination of agalacto-IgG may therefore aid in diagnosis and treatment of RA. The decrease in galactosylation of IgG leads to an increase in terminal N-acetylglucosamine residues, which can be detected using a specific lectin from Psathyrella velutina. In the present study IgG from RA and control serum was purified using affinity chromatography. The samples were then, after reduction, analyzed on a BIOCORE® 2000 system with immobilized Psathyrella velutina lectin. Using this technique it was possible to discriminate between IgG from RA patients and IgG from control individuals with respect to its content of IgG with terminal N-acetylglucosamine. The affinity biosensor technique makes it possible to detect binding without labeling or using secondary antibodies.     

Purification and partial characterization of a chymotrypsin-like serine fibrinolytic enzyme from Bacillus amyloliquefaciens FCF-11 using corn husk as a novel substrate

A non-toxic, direct-acting fibrinolytic enzyme, FCF-11, from a newly isolated Bacillus amyloliquefaciens FCF-11 was purified, characterized and assayed both in vitro and in vivo for its thrombolytic potential. Corn husk was used as for the first time as the sole carbon/nitrogen source for enzyme production. The molecular weight of the purified enzyme was 18.2 kDa and purification increased its specific activity 443.5-fold with a recovery of 17 %. Maximal activity was attained at a temperature of 40 °C and pH of 8.0. Additionally the isoelectric point of this protein was 10 ± 0.2. Tosyl lysine chloromethyl ketone, phenylmethylsulphonyl fluoride, soybean trypsin inhibitor, and aprotinin highly repressed this activity. The presence of ethylenediaminetetraacetic acid, and two metalloprotease inhibitors, 2,2′-bipyridine and o-phenanthroline, didn’t affect the enzymatic activity. Furthermore, it was found to exhibit a higher specificity for the chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like serine protease. Its apparent K _m and V _max for the synthetic substrate N-Suc-Phe-pNA were 0.45 mM and 8.26 μmoles/mg/min, respectively. FCF-11 showed direct action upon blood clots in vitro and prolonged the blood clotting time to 4.1-fold, suggesting this enzyme be a beneficial thrombolytic agent especially, with regard with low molecular weight and non specificity to other plasma proteins. FCF-11 could not degrade collagen and was non-cytotoxic to HT29 cells or mammalian erythrocytes. Further, enzyme at a dose of 2 mg/kg was devoid of toxicity as well as hemorrhagic activity on BALB/c mouse model, supporting its suitability for the development of a better and safer thrombolytic drug.     

Purification and properties of an N-acetylglucosamine-specific lectin from Psathyrella velutina mushroom

A lectin in the fruiting bodies of Psathyrella velutina was purified by affinity chromatography on a chitin column and subsequent ion-exchange chromatography. P. velutina lectin (PVL) tends to aggregate irreversibly in buffered saline, but the addition of glycerol (10%, v/v) to lectin solutions was found to prevent aggregate formation. PVL is assumed to occur as a monomer of a polypeptide of Mr = 40,000 as determined by gel filtration and by gel electrophoresis in the presence of sodium dodecyl sulfate. PVL is specific for N-acetylglucosamine (GlcNAc). It was determined by equilibrium dialysis to have four binding sites/polypeptide molecule showing an average intrinsic association constant of K0 = 6.4 x 10(3) M-1 toward this sugar. The binding specificity of the lectin was studied by hemagglutination inhibition assays and by avidin-biotin-mediated enzyme immunoassays using various GlcNAc-containing saccharides. The results indicate that methyl N-acetyl beta-glucosaminide was a slightly better inhibitor than the corresponding alpha-anomer. PVL binds well to oligosaccharides bearing nonreducing terminal beta-GlcNAc linked 1----6 or 1----3 but poorly to those having a 1----4 linkage, such as N-acetylated chito-oligosaccharides. It also binds to the subterminal GlcNAc moiety when it is substituted at the C-6 position but does not interact with the moiety when substituted either at C-3 or C-4. Thus, these results show that PVL is quite different in its binding specificity from other GlcNAc-binding lectins of higher plants since they bind preferentially to beta-GlcNAc in 1----4 linkage and they have a high affinity for chitin oligosaccharides.     

A novel carbohydrate binding activity of annexin V toward a bisecting N-acetylglucosamine

A bisecting GlcNAc-binding protein was purified from a Triton X-100 extract of a porcine spleen microsomal fraction using affinity chromatography, in conjunction with an agalacto bisected biantennary sugar chain-immobilized Sepharose. Since the erythroagglutinating phytohemagglutinin (E-PHA) lectin preferentially binds to sugar chains which contain the bisecting GlcNAc, during purification the binding activity of the protein was evaluated by monitoring the inhibition of lectin binding to the N-acetylglucosaminyltransferase III (GnT-III)-transfected K562 cells which express high levels of the bisecting GlcNAc. The molecular mass of the purified protein was found to be 33 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By sequencing analysis, the isolated protein was identified as annexin V. Flow cytometric analysis showed that fluorescein-labeled annexin V binds to the GnT-III-transfected cells but not to mock cells, and that the binding was not affected by the addition of phospholipids. Furthermore, surface plasmon resonance measurements indicated that annexin V binds to the agalacto bisected biantennary sugar chain with a K(d) of 200 microM while essentially no binding was observed in the case of the corresponding non-bisected sample. These results suggest that annexin V has a novel carbohydrate binding activity and may serve as an endogenous lectin for mediating possible signals of bisecting GlcNAc, which have been implicated in a variety of biological functions.     

Cloning, expression and characterization of phenylalanine ammonia-lyase from Rhodotorula glutinis

The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg^?1 and the k _cat/K _m was 1.9 × 10⁴ mM^?1 s^?1, exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry.     

A glucose/mannose binding lectin from litchi (Litchi chinensis) seeds: Biochemical and biophysical characterizations

BackgroundLectins are highly important biomolecules to study several biological processes. A novel α-D-glucose/mannose specific lectin was isolated from the seeds of litchi fruits (Litchi chinensis) and its various biophysical and biochemical properties were studied.MethodsPurification was done by successive Sephadex G 100 and Con A-Sepharose 4B affinity chromatography. SDS-PAGE, Surface Plasmon Resonance (SPR), steady state absorbance, fluorescence, time-correlated single-photon counting, circular dichroism and antibiofilm activity by measuring total protein estimation and azocasein degradation assay have been performed.ResultsThe purified lectin is a homodimer of molecular mass ~ 54 kDa. The amount of lectin required for hemagglutination of normal human O erythrocytes was 6.72 µg/ml. Among the saccharides tested, Man-α-(1,6)-Man was found to be the most potent inhibitor (0.01 mM) determined by hemagglutination inhibition assay. Steady state and time resolved fluorescence measurements revealed that litchi lectin formed ground state complex with maltose (K_a=4.9 (±0.2)×10⁴ M^?1), which indicated static quenching (Stern-Volmer (SV) constant K_sv=4.6 (±0.2)×10⁴ M^?1). CD measurements demonstrated that litchi lectin showed no overall conformational change during the binding process with maltose. The lectin showed antibiofilm activity against Pseudomonus aeruginosa.ConclusionsA novel homodimeric lectin has been purified from the seeds of litchi fruits (Litchi chinensis) having specificity for α-d-glucose/mannose. The thermodynamics and conformational aspects of its interaction with maltose have been studied in detail. The antibiofilm activity of this lectin towards Pseudomonus aeruginosa has been explored.General significanceThe newly identified litchi lectin is highly specific for α-d-glucose/mannose with an important antibiofilm activity towards Pseudomonus aeruginosa.     

Purification,characterization and fine sugar specificity of a N-Acetylgalactosamine specific lectin from <Emphasis Type="Italic">Adenia hondala</Emphasis>

Plant lectins are gaining interest because of their interesting biological properties. Several Adenia species, that are being used in traditional medicine to treat many health ailments have shown presence of lectins or carbohydrate binding proteins. Here, we report the purification, characterization and biological significance of N-Acetyl galactosamine specific lectin from Adenia hondala (AHL) from Passifloraceae family. AHL was purified in a single step by affinity chromatography on asialofetuin Sepharose 4B column, characterized and its fine sugar specificity determined by glycan array analysis. AHL is human blood group non specific and also agglutinates rabbit erythrocytes. AHL is a glycoprotein with 12.5% of the carbohydrate, SDS-PAGE, MALDI-TOF-MS and ESI-MS analysis showed that AHL is a monomer of 31.6 kDa. AHL is devoid of DNase activity unlike other Ribosome inactivating proteins (RIPs). Glycan array analysis of AHL revealed its highest affinity for terminal lactosamine or polylactosamine of N- glycans, known to be over expressed in hepatocellular carcinoma and colon cancer. AHL showed strong binding to human hepatocellular carcinoma HepG2 cells with MFI of 59.1 expressing these glycans which was effectively blocked by 93.1% by asialofetuin. AHL showed dose and time dependent growth inhibitory effects on HepG2 cells with IC₅₀ of 4.8 μg/ml. AHL can be explored for its clinical potential.     

Functional expression and characterization of a chitinase from the marine archaeon Halobacterium salinarum CECT 395 in Escherichia coli

The HschiA1 gene of the archaeon Halobacterium salinarum CECT 395 was cloned and overexpressed as an active protein of 66.5 kDa in Escherichia coli. The protein called HsChiA1p has a modular structure consisting of a glycosyl hydrolase family 18 catalytic region, as well as a N-terminal family 5 carbohydrate-binding module and a polycystic kidney domain. The purified recombinant chitinase displayed optimum catalytic activity at pH 7.3 and 40 °C and showed high stability over broad pH (6–8.5) and temperature (25–45 °C) ranges. Protein activity was stimulated by the metal ions Mg⁺², K⁺, and Ca⁺² and strongly inhibited by Mn⁺². HsChiA1p is salt-dependent with its highest activity in the presence of 1.5 M of NaCl, but retains 20 % of its activity in the absence of salt. The recombinant enzyme hydrolysed p-NP-(GlcNAc)₃, p-NP-(GlcNAc), crystalline chitin, and colloidal chitin. From its sequence features and biochemical properties, it can be identified as an exo-acting enzyme with potential interest regarding the biodegradation of chitin waste or its bioconversion into biologically active products.     

UFEIII, a fibrinolytic protease from the marine invertebrate, Urechis unicinctus

A new serine protease with fibrinolytic activity from a marine invertebrate, Urechis unicinctus, was purified to electrophoretic homogeneity using column chromatography. SDS-PAGE of the purified enzyme showed a single polypeptide chain with MW ~20.8 kDa. Its N-terminal sequence was IIGGSQAAITSY. The purified enzyme, UFEIII, was stable at pH 6–10 below 60 °C with an optimum pH of 8.5 at approx. 55 °C. The enzyme activity was significantly inhibited by PMSF and SBTI suggesting that it was a serine protease. In fibrin plate assays, UFEIII was contained 1.46 × 10³ U (urokinase units) mg^?1 total fibrinolytic activity, which consisted of 692 U mg^?1 direct fibrinolytic activity and 769 U mg^?1 plasminogen-activator activity. K_m and V_max values for azocasein were 1 mg ml^?1 and 43 μg min^?1 ml^?1, respectively.     

Characterization of ergothionase from Burkholderia sp. HME13 and its application to enzymatic quantification of ergothioneine

We identified ergothionase, which catalyzes conversion of ergothioneine to thiolurocanic acid and trimethylamine, in a newly isolated ergothioneine-utilizing strain, Burkholderia sp. HME13. The enzyme was purified and its N-terminal amino acid sequence was determined. Based on the amino acid sequence, the gene encoding the enzyme was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity and characterized. The enzyme consisted of four identical 55-kDa subunits. The enzyme showed maximum activity at pH 8.0 and 65 °C and was stable between pH 7.0 and pH 10.0 and up to 60 °C. The enzyme acted on ergothioneine (K _m: 19 μM, V _max: 270 μmol/min/mg), but not d-histidine, l-histidine, d-tyrosine, l-tyrosine, d-phenylalanine, or l-phenylalanine. The enzyme was activated by BaCl₂ and strongly inhibited by CuSO₄, ZnSO₄, and HgCl₂. The amino acid sequence of ergothionase showed 23 % similarity to histidine ammonia-lyase (HAL) from Pseudomonas putida and 17 % similarity to phenylalanine ammonia-lyase (PAL) from parsley. However, the tripeptide sequence, Ala-Ser-Gly, which is important for catalysis in both HAL and PAL, was not conserved in ergothionase. The application of ergothionase for the quantification of ergothioneine contained in practical food and blood samples was investigated by performing a recovery test. Satisfactory recovery data (98.7–104 %) were obtained when ergothioneine was added to extract of tamogitake and hemolysis blood.