Isolation and Characterization of Isolectins from Talisia esculenta Seeds

Four isolectins (TEL-I, TEL-II, TEL-III and TEL-IV) were isolated from seeds of Talisia esculenta by reverse-phase high-performance liquid chromatography. RP-HPLC was performed on a u-Bondapack C18 column (0.78 cm × 30 cm) (Waters 991-PDA system) at room temperature. Rechromatography of the four fractions on a C18 column under the same conditions yielded lectins with two dissimilar subunits (M_r 20 kDa and 40 kDa) bound noncovalently. The isolectins showed very similar characteristics, such as molecular masses, N-terminal sequences, and hemagglutinating activity, but differed in their isoelectric points and in inhibition by carbohydrates.     

Purification and primary structure of a mannose/glucose‐binding lectin from Parkia biglobosa Jacq. seeds with antinociceptive and anti‐inflammatory properties

Parkia biglobosa (subfamily Mimosoideae), a typical tree from African savannas, possess a seed lectin that was purified by combination of ammonium sulfate precipitation and affinity chromatography on a Sephadex G‐100 column. The P. biglobosa lectin (PBL) strongly agglutinated rabbit erythrocytes, an effect that was inhibited by d ‐mannose and d ‐glucose‐derived sugars, especially α‐methyl‐d ‐mannopyranoside and N‐acetyl‐d ‐glucosamine. The hemagglutinating activity of PBL was maintained after incubation at a wide range of temperature and pH and also was independent of divalent cations. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis, PBL exhibited an electrophoretic profile consisting of a single band with apparent molecular mass of 45 kDa. An analysis using electrospray ionization–mass spectrometry indicated that purified lectin possesses a molecular average mass of 47 562 ± 4 Da, and the analysis by gel filtration showed that PBL is a dimer in solution. The complete amino acid sequence of PBL, as determined using tandem mass spectrometry, consists of 443 amino acid residues. PBL is composed of a single non‐glycosylated polypeptide chain of three tandemly arranged jacalin‐related domains. Sequence heterogeneity was found in six positions, indicating that the PBL preparations contain highly homologous isolectins. PBL showed important antinociceptive activity associated to the inhibition of inflammatory process. Copyright © 2013 John Wiley & Sons, Ltd.     

Enzymatic synthesis of β‐glucosylglycerol using a continuous‐flow microreactor containing thermostable β‐glycoside hydrolase CelB immobilized on coated microchannel walls

β‐Glucosylglycerol (βGG) has potential applications as a moisturizing agent in cosmetic products. A stereochemically selective method of its synthesis is kinetically controlled enzymatic transglucosylation from a suitable donor substrate to glycerol as acceptor. Here, the thermostable β‐glycosidase CelB from Pyrococcus furiosus was used to develop a microstructured immobilized enzyme reactor for production of βGG under conditions of continuous flow at 70°C. Using CelB covalently attached onto coated microchannel walls to give an effective enzyme activity of 30 U per total reactor working volume of 25 µL, substrate conversion and formation of transglucosylation product was monitored in dependence of glucosyl donor (2‐nitrophenyl‐β‐D ‐glucoside (oNPGlc), 3.0 or 15 mM; cellobiose, 250 mM), the concentration of glycerol (0.25–1.0 M), and the average residence time (0.2–90 s). Glycerol caused a concentration‐dependent decrease in the conversion of the glucosyl donor via hydrolysis and strongly suppressed participation of the substrate in the reaction as glucosyl acceptor. The yields of βGG were ≥80% and ≈60% based on oNPGlc and cellobiose converted, respectively, and maintained up to near exhaustion of substrate (≥80%), giving about 120 mM (30 g/L) of βGG from the reaction of cellobiose and 1 M glycerol. The structure of the transglucosylation products, 1‐O‐β‐D ‐glucopyranosyl‐rac‐glycerol (79%) and 2‐O‐β‐D ‐glucopyranosyl‐sn‐glycerol (21%), was derived from NMR analysis of the product mixture of cellobiose conversion. The microstructured reactor showed conversion characteristics similar to those for a batchwise operated stirred reactor employing soluble CelB. The advantage of miniaturization to the microfluidic format lies in the fast characterization of full reaction time courses for a range of process conditions using only a minimum amount of enzyme. Biotechnol. Bioeng. 2009;103: 865–872. © 2009 Wiley Periodicals, Inc.     

Occurrence of natural lectin with bacterial agglutination property in the serum of lepidopteran pest,Parasa lepida

Insects depend on lectins for non‐self recognition and clearance of invading pathogens. Naturally occurring lectin showing specificity for galactose was purified from the serum of lepidopteran pest Parasa lepida by affinity chromatography using Sepharose 6B coupled with galactose as a gel matrix. Preliminary studies on crude serum agglutinin revealed that the agglutinin molecule showed varying degrees of specificity to avian and mammalian red blood cells tested. Among them, the highest titer of 128 was recorded against rabbit red blood cell type. The agglutinin molecule in the crude serum was stable up to 60°C and at pH between 6 and 9. Also, the hemagglutinating activity was neither dependent on divalent cations nor sensitive to ethylenediaminetetraacetic acid treatment. Galactose inhibited the hemagglutinating activity at minimum inhibitory concentration of 12.5 mM and hence it was used as a ligand for affinity chromatography. Native polyacrylamide gel electrophoresis analysis revealed a single band and the molecular weight of the lectin was found to be approximately 90 kDa. Bacterial agglutination activity of the purified lectin with two significant toxin bacteria, namely Salmonella typhi and Bacillus thuringiensis, was observed.     

Mannose-specific isolectins with different hemagglutinating potencies isolated from Chinese daffodil (Narcissus tazetta var. chinensis) leaves

Three mannose-specific lectins exhibiting considerable similarities in NH₂-terminal amino acid sequence were isolated from leaves of the Chinese daffodil Narcissus tazetta (Family Amaryllidaceae). The purification protocol involved extraction with an aqueous buffer, anion exchange chromatography on DEAE-cellulose using stepwise elution with increasing salt concentrations, affinity chromatography on mannose-agarose, and FPLC-gel filtration on Superose 12. From the peak unadsorbed on DEAE-cellulose, and two peaks adsorbed on the ion exchanger and eluted respectively with 0.2 M Tris-HCl buffer and 0.5 M NaCl, were prepared fractions which yielded isolectins 1, 2, and 3 after adsorption on mannose-agarose and FPLC-gel filtraton. All three isolectins were homodimers with a molecular weight of 26 kDa. The lectin unadsorbed on DEAE-cellulose had the lowest, while the most strongly adsorbed lectin had the highest hemagglutinating activity.     

Chromatographic profiles of tryptophan and kynurenine enantiomers derivatized with (S)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole using LC–MS/MS on a triazole‐bonded column

d ‐ and l ‐Tryptophan (Trp) and d ‐ and l ‐kynurenine (KYN) were derivatized with a chiral reagent, (S )‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS), and were separated enantiomerically by high‐performance liquid chromatography (HPLC) equipped with a triazole‐bonded column (Cosmosil HILIC) using tandem mass spectrometric (MS/MS) detection. Effects of column temperature, salt (HCO₂NH₄) concentration, and pH of the mobile phase in the enantiomeric separation, followed by MS detection of (S )‐DBD‐PyNCS‐d ,l ‐Trp and ‐d ,l ‐KYN, were investigated. The mobile phase consisting of CH₃CN/10 mM ammonium formate in H₂O (pH 5.0) (90/10) with a column temperature of 50–60 °C gave satisfactory resolution (R s) and mass‐spectrometric detection. The enantiomeric separation of d ,l ‐Trp and d ,l ‐KYN produced R s values of 2.22 and 2.13, and separation factors (α) of 1.08 and 1.08, for the Trp and KYN enantiomers, respectively. The proposed LC–MS/MS method provided excellent detection sensitivity of both enantiomers of Trp and KYN (5.1–19 nM).     

Purification and molecular characterization of a novel mannose‐specific lectin from Dioclea reflexa hook seeds with inflammatory activity

A novel lectin present in Dioclea reflexa seeds (DrfL) was discovered and described in this study. DrfL was purified in a single step by affinity chromatography in a Sephadex G‐50 column. The lectin strongly agglutinated rabbit erythrocytes and was inhibited by α‐methyl‐d ‐mannoside, d ‐mannose, and d ‐glucose. The hemagglutinating activity of DrfL is optimum at pH 5.0–7.0, stable up to 50 °C, and dependent on divalent cations. Similar to other lectins of the subtribe Diocleinae, the analysis by mass spectrometry indicated that DrfL has three chains (α, β, and γ) with masses of 25 562, 12 874, and 12 706 Da, respectively, with no disulfide bonds or glycosylation. DrfL showed inflammatory activity in the paw edema model and exhibited low cytotoxicity against Artemia sp. Copyright © 2015 John Wiley & Sons, Ltd.     

Biological Activities of Triterpenoids and Phenolic Compounds from Myrica cerifera Bark

Seven triterpenoids, 1  –  7 , two diarylheptanoids, 8 and 9 , four phenolic compounds, 10  –  13 , and three other compounds, 14  –  16 , were isolated from the hexane and MeOH extracts of the bark of Myrica cerifera L. (Myricaceae). Among these compounds, betulin ( 1 ), ursolic acid ( 3 ), and myricanol ( 8 ) exhibited cytotoxic activities against HL60 (leukemia), A549 (lung), and SK‐BR‐3 (breast) human cancer cell lines (IC₅₀ 3.1 – 24.2 μm ). Compound 8 induced apoptotic cell death in HL60 cells (IC₅₀ 5.3 μm ) upon evaluation of the apoptosis‐inducing activity by flow cytometric analysis and by Hoechst 33342 staining method. Western blot analysis on HL60 cells revealed that 8 activated caspases‐3, ‐8, and ‐9 suggesting that 8 induced apoptosis via both mitochondrial and death receptor pathways in HL60. Upon evaluation of the melanogenesis‐inhibitory activity in B16 melanoma cells induced with α‐melanocyte‐stimulating hormone (α‐MSH), erythrodiol ( 7 ), 4‐hydroxy‐2‐methoxyphenyl β‐d ‐glucopyranoside ( 13 ), and butyl quinate ( 15 ) exhibited inhibitory effects (65.4 – 86.0% melanin content) with no, or almost no, toxicity to the cells (85.9 – 107.4% cell viability) at 100 μm concentration. In addition, 8 , myricanone ( 9 ), myricitrin ( 10 ), protocatechuic acid ( 11 ), and gallic acid ( 12 ) revealed potent DPPH radical‐scavenging activities (IC₅₀ 6.9 – 20.5 μm ).     

Purification and partial characterization of a new mannose/glucose‐specific lectin from Dialium guineense Willd seeds that exhibits toxic effect

A new mannose/glucose‐specific lectin, named DigL, was purified from seeds of Dialium guineense by a single step using a Sepharose 4b‐Mannose affinity chromatography column. DigL strongly agglutinated rabbit erythrocytes and was inhibited by d ‐mannose, d ‐glucose, and derived sugars, especially α‐methyl‐d ‐mannopyranoside and N‐acetyl‐d ‐glucosamine. DigL has been shown to be a stable protein, maintaining its hemagglutinating activity after incubation at a wide range of temperature and pH values and after incubation with EDTA. DigL is a glycoprotein composite by approximately 2.9% of carbohydrates by weight. By sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, the purified DigL exhibited an electrophoretic profile consisting of a broad band of 28–30 kDa. Analysis using electrospray ionization mass spectrometry indicated that purified DigL possesses a molecular average mass of 28 452 ± 2 Da and shows the presence of possible glycoforms. In addition, DigL exhibited an intermediary toxic effect on Artemia sp. nauplii, and this effect was both dependent on native structure and mediated by a carbohydrate‐binding site. Copyright © 2013 John Wiley & Sons, Ltd.     

Engineered disulfide bonds improve thermostability and activity of L‐isoleucine hydroxylase for efficient 4‐HIL production in Bacillus subtilis 168

4‐Hydroxyisoleucine, a promising drug, has mainly been applied in the clinical treatment of type 2 diabetes in the pharmaceutical industry. l ‐Isoleucine hydroxylase specifically converts l‐ Ile to 4‐hydroxyisoleucine. However, due to its poor thermostability, the industrial production of 4‐hydroxyisoleucine has been largely restricted. In the present study, the disulfide bond in l ‐isoleucine hydroxylase protein was rationally designed to improve its thermostability to facilitate industrial application. The half‐life of variant T181C was 4.03 h at 50°C, 10.27‐fold the half‐life of wild type (0.39 h). The specific enzyme activity of mutant T181C was 2.42 ± 0.08 U/mg, which was 3.56‐fold the specific enzyme activity of wild type 0.68 ± 0.06 U/mg. In addition, molecular dynamics simulation was performed to determine the reason for the improvement of thermostability. Based on five repeated batches of whole‐cell biotransformation, Bacillus subtilis 168/pMA5‐ido^T181C recombinant strain produced a cumulative yield of 856.91 mM (126.11 g/L) 4‐hydroxyisoleucine, which is the highest level of productivity reported based on a microbial process. The results could facilitate industrial scale production of 4‐hydroxyisoleucine. Rational design of disulfide bond improved l ‐isoleucine hydroxylase thermostability and may be suitable for protein engineering of other hydroxylases.     

Proteolytic Activity of Lectins from the Nitrogen-Fixing Bacterium Bacillus polymyxa

Two lectins (LI and LII) stripped from the surface of Bacillus polymyxa1460 cells were found to possess proteolytic activity, which was associated with their hemagglutinating activity. The inhibition of the hemagglutinating activity of lectins by specific carbohydrate haptens changed their enzyme activities. The inhibition of hemagglutinating activity by glucuronic acid or fructose 1,6-diphosphate decreased the proteolytic activities of both lectins, whereas the blocking of this activity with D-glucosamine or D-galactosamine increased the proteolytic activity of LII. The molecules of the B. polymyxalectins are suggested to contain two active centers responsible for hemagglutinating and proteolytic activities.     

Cytotoxic and Apoptosis‐inducing Activities of Taraxastane‐type Triterpenoid Derivatives in Human Cancer Cell Lines

Twenty‐eight taraxastane‐type triterpenoid derivatives 4  –  31 were prepared from the naturally occurring triterpenoids faradiol ( 1 ) and heliantriol C ( 3 ). The cytotoxic activities of these compounds and arnidiol ( 2 ) were evaluated in leukemia (HL60), lung (A549), duodenal (AZ521), and breast (SK‐BR‐3) cancer cell lines. 21‐Oxoarnidiol ( 18 ) and faradiol 3,16‐di‐O‐l ‐alaninate ( 31 ) exhibited potent cytotoxicity, with 50% inhibitory concentrations of 0.5 – 2.7 μm . In particular, flow cytometric analysis indicated that compound 31 induced typical apoptotic cell death in HL60 cells. These results suggested that taraxastane‐type triterpenoid derivatives might provide useful antitumor agents with apoptosis‐inducing activity.     

Purification,characterization and partial sequence of a pro‐inflammatory lectin from seeds of Canavalia oxyphylla Standl. & L. O. Williams

Recent studies have shown that lectins are promising tools for use in various biotechnological processes, as well as studies of various pathological mechanisms, isolation, and characterization of glycoconjugates and understanding the mechanisms underlying pathological mechanisms conditions, including the inflammatory response. This study aimed to purify, characterize physicochemically, and predict the biological activity of Canavalia oxyphylla lectin (CoxyL) in vitro and in vivo. CoxyL was purified by a single‐step affinity chromatography in Sephadex® G‐50 column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the pure lectin consists of a major band of 30 kDa (α‐chain) and two minor components (β‐chain and γ‐chain) of 16 and 13 kDa, respectively. These data were further confirmed by electrospray ionization mass spectrometry, suggesting that CoxyL is a typical ConA‐like lectin. In comparison with the average molecular mass of α‐chain, the partial amino acid sequence obtained corresponds to approximately 45% of the total CoxyL sequence. CoxyL presented hemagglutinating activity that was specifically inhibited by monosaccharides (D‐glucose, D‐mannose, and α‐methyl‐D‐mannoside) and glycoproteins (ovalbumin and fetuin). Moreover, CoxyL was shown to be thermostable, exhibiting full hemagglutinating activity up to 60°C, and it was pH‐sensitive for 1 h, exhibiting maximal activity at pH 7.0. CoxyL caused toxicity to Artemia nauplii and induced paw edema in rats. This biological activity highlights the importance of lectins as important tools to better understand the mechanisms underlying inflammatory responses. Copyright © 2014 John Wiley & Sons, Ltd.     

Oleanane‐type Saponins from Glochidion hirsutum and Their Cytotoxic Activities

Five new oleanane‐type saponins, hirsutosides A – E, were isolated from the leaves of Glochidion hirsutum (Roxb .) Voigt . Their structures were elucidated as 21β‐benzoyloxy‐3β,16β,23,28‐tetrahydroxyolean‐12‐ene 3‐O‐β‐d ‐glucopyranoside ( 1 ), 21β‐benzoyloxy‐3β,16β,23,28‐tetrahydroxyolean‐12‐ene 3‐O‐β‐d ‐glucopyranosyl‐(1 → 3)‐β‐d ‐glucopyranoside ( 2 ), 21β‐benzoyloxy‐3β,16β,23,28‐tetrahydroxyolean‐12‐ene 3‐O‐6‐acetyl‐[β‐d ‐glucopyranosyl‐(1 → 3)]‐β‐d ‐glucopyranoside ( 3 ), 21β‐benzoyloxy‐3β,16β,23,28‐tetrahydroxyolean‐12‐ene 3‐O‐β‐d ‐glucopyranosyl‐(1 → 3)‐〈‐l ‐arabinopyranoside ( 4 ), and 21β‐benzoyloxy‐3β,16β,23‐trihydroxyolean‐12‐ene‐28‐al 3‐O‐β‐d ‐glucopyranosyl‐(1 → 3)‐α‐l ‐arabinopyranoside ( 5 ). All isolated compounds were evaluated for cytotoxic activities on four human cancer cell lines, HepG‐2, A‐549, MCF‐7, and SW‐626 using the SRB assay. Compounds 1 , 2 , 4 , and 5 showed significant cytotoxic activities against all human cancer cell lines with IC₅₀ values ranging from 3.4 to 10.2 μm . Compound 3 containing acetyl group at glc C(6″) exhibited weak cytotoxic activity with IC₅₀ values ranging from 47.0 to 54.4 μm .     

Hydrolytic properties of a thermostable α‐l‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus

Aims: To characterize of a thermostable recombinant α‐l ‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino‐oligosaccharides to l ‐arabinose. Methods and Results: A recombinant α‐l ‐arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi‐Trap anion exchange chromatography with a specific activity of 28·2 U mg^?1. The native enzyme was a 58‐kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of α‐l ‐arabinofuranosidases were completely conserved in α‐l ‐arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5·5 and 80°C with a half‐life of 49 h at 75°C. Among aryl‐glycoside substrates, the enzyme displayed activity only for p‐nitrophenyl‐α‐l ‐arabinofuranoside [maximum k_cat/K_m of 220 m(mol l^?1)^?1 s^?1] and p‐nitrophenyl‐α‐l ‐arabinopyranoside. This substrate specificity differs from those of other α‐l ‐arabinofuranosidases. In a 1 mmol l^?1 solution of each sugar, arabino‐oligosaccharides with 2–5 monomer units were completely hydrolysed to l ‐arabinose within 13 h in the presence of 30 U ml^?1 of enzyme at 75°C. Conclusions: The novel substrate specificity and hydrolytic properties for arabino‐oligosaccharides of α‐l ‐arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of l ‐arabinose in concert with endoarabinanase and/or xylanase. Significance and Impact of the Study: The findings of this work contribute to the knowledge of hydrolytic properties for arabino‐oligosaccharides performed by thermostable α‐l ‐arabinofuranosidase.     

Occurrence of highly yielded lectins homologous within the genus Eucheuma

We previously reported that the red alga Eucheuma serra contains large amounts of mitogenic isolectins (ESA-1 and ESA-2), the hemagglutinating activities of which were strongly inhibited by glycoproteins bearing high mannose-type N-glycans. We therefore further examined two other species, E. amakusaensis and E. cottonii. Several lectins were isolated easily by a combination of extraction with aqueous ethanol, precipitation with cold ethanol, gel filtration, and ion exchange chromatography from both species, respectively. The purified lectins were designated as EAA-1, EAA-2, EAA-3, ECA-1 and ECA-2 after the specific names of both algae. The yields of EAAs and ECAs were as high as 2.8 and 2.7 mg g⁻¹ of dry tissue, respectively, indicating that both species would also be good sources for high lectin yields. The five purified lectins shared the same properties in hemagglutinating activity, mitogenic activity, and hemagglutination-inhibition test in which glycoproteins bearing high mannose-type N-glycans were the most inhibitory. They also had almost identical molecular weight and 20 N-terminal amino acid sequence to each other and to those of ESAs, and only differed in the isoelectric point, indicating that they are isolectins to each other. The study thus demonstrated that several species of Eucheuma contain high yields of lectins homologous between species, suggesting that the genus as a whole may be considered as a valuable source of lectin proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina

Plant seeds, a rich source of proteins, are considered important for their application as functional ingredients in a food system. A novel ribosome-inactivating protein (RIP), balsamin was purified from the seeds of Balsam apple, Momordica balsamina. Balsamin was purified by ion exchange chromatography on CM Sepharose and gel filtration on superdex-75. It has a molecular weight of 28 kDa as shown by SDS-PAGE analysis. Balsamin inhibits protein synthesis in a rabbit reticulocyte lysate-based cell free translation assay with an IC(50) of 90.6 ng ml(-1). It has RNA N-glycosidase activity and releases a 400-base long fragment termed the Endo fragment from 28S rRNA in the same manner as does saporin-6 from Saponaria officinalis. The N-terminal sequence analysis of the first 12 amino acids of balsamin revealed that it shares 83% similarity with type I RIP α-MMC from Momordica charantia and 50% similarity with β-MMC (from Momordica charantia), bryodin I (from Bryonia dioica) and luffin a (from Luffa cylindrica). Balsamin was further characterized by mass spectrometry. CD spectroscopic studies indicate that secondary structure of balsamin contains helix (23.5%), β-strand (24.6%), turn (20%) and random coil (31.9%). Thus RIPs activity expressed in vegetables like Momordica sp. advocates its usage in diet.     

High‐level conversion of L‐lysine into 5‐aminovalerate that can be used for nylon 6,5 synthesis

L ‐Lysine is a potential feedstock for the production of bio‐based precursors for engineering plastics. In this study, we developed a microbial process for high‐level conversion of L ‐lysine into 5‐aminovalerate (5AVA) that can be used as a monomer in nylon 6,5 synthesis. Recombinant Escherichia coli WL3110 strain expressing Pseudomonas putida delta‐aminovaleramidase (DavA) and lysine 2‐monooxygenase (DavB) was grown to high density in fed‐batch culture and used as a whole cell catalyst. High‐density E. coli WL3110 expressing DavAB, grown to an optical density at 600 nm (OD₆₀₀) of 30, yielded 36.51 g/L 5AVA from 60 g/L L ‐lysine in 24 h. Doubling the cell density of E. coli WL3110 improved the conversion yield to 47.96 g/L 5AVA from 60 g/L of L ‐lysine in 24 h. 5AVA production was further improved by doubling the L ‐lysine concentration from 60 to 120 g/L. The highest 5AVA titer (90.59 g/L; molar yield 0.942) was obtained from 120 g/L L ‐lysine by E. coli WL3110 cells grown to OD₆₀₀ of 60. Finally, nylon 6,5 was synthesized by bulk polymerization of ?‐caprolactam and δ‐valerolactam prepared from microbially synthesized 5AVA. The hybrid system demonstrated here has promising possibilities for application in the development of industrial bio‐nylon production processes.     

PURIFICATION AND CHARACTERIZATION OF A LECTIN,BRYOHEALIN, INVOLVED IN THE PROTOPLAST FORMATION OF A MARINE GREEN ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA) 1

When the coenocytic green alga Bryopsis plumosa (Huds.) Ag. was cut open and the cell contents were expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. Aggregation of cell organelles in seawater was mediated by a lectin–carbohydrate complementary system. Two sugars, N‐acetyl‐d ‐glucosamine and N‐acetyl‐d ‐galactosamine inhibited aggregation of cell organelles. The presence of these sugars on the surface of chloroplasts was verified with their complementary fluorescein isothiacyanate‐labeled lectins. An agglutination assay using human erythrocytes showed the presence of lectins specific for N‐acetyl‐d ‐galactosamine and N‐acetyl‐d ‐glucosamine in the crude extract. One‐step column purification using N‐acetyl‐d ‐glucosamine‐agarose affinity chromatography yielded a homogeneous protein. The protein agglutinated the cell organelles of B. plumosa, and its agglutinating activity was inhibited by the above sugars. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed that this protein might be composed of two identical subunits cross‐linked by two disulfide bridges. Enzyme and chemical deglycosylation experiments showed that this protein is deficient in glycosylation. The molecular weight was determined as 53.8 kDa by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The N‐terminal 15 amino acid sequence of the lectin was Ser–Asp–Leu–Pro–Thr–X–Asp–Phe–Phe–His–Ile–Pro–Glu–Arg–Tyr, and showed no sequence homology to those of other reported proteins. These results suggest that this lectin belongs to a new class of lectins. We named this novel lectin from B. plumosa“bryohealin.”