Isolation and characterization of a galactose-specific lectin (EantH) with antimicrobial activity from Euphorbia antiquorum L. latex.

A homodimeric 75 kDa lectin with hemagglutination activity (HA) was purified from the crude latex of Euphorbia antiquorum L. by two types of chromatography, on cation exchange (HiTrap SP FF) and hydrophobic HiTrap Phenyl FF (high sub) columns. The purified protein was designated EantH, and is classified as a galactose-specific thermostable lectin. The HA of EantH was stable at pH values of 5-9 and temperature 5-65 °C. The lectin had bacteriostatic action on the Gram-positive bacteria Staphylococcus aureus and S. epidermidis, with a minimum inhibitory concentration (MIC) of 2000 µg/ml and on a Gram-negative bacterium Samonella typhimurium, with a MIC of 1000 µg/ml. EantH inhibited the growth of Propionibacterium acnes and Streptococcus agalactiae with MIC of 125 µg/ml and 250 µg/ml, respectively. EantH killed P. acnes and S. agalactiae with a minimum microbicidal concentration (MMC) of 1000 µg/ml and 2000 µg/ml, respectively. Scanning electron microscopy indicated that binding of EantH to the carbohydrates in the cell walls of P. acnes and S. typhimurium drastically altered the bacterial cells, and led to inhibition of growth and/or cell death. The antimicrobial activity of EantH could be neutralized by d­galactose, indicating that its bactericidal action involves binding to galactose in the cell wall.

Purification and characterization of a chloride ion-dependent α-glucosidase from the midgut gland of Japanese scallop (Patinopecten yessoensis).

Marine glycoside hydrolases hold enormous potential due to their habitat-related characteristics such as salt tolerance, barophilicity, and cold tolerance. We purified an α-glucosidase (PYG) from the midgut gland of the Japanese scallop (Patinopecten yessoensis) and found that this enzyme has unique characteristics. The use of acarbose affinity chromatography during the purification was particularly effective, increasing the specific activity 570-fold. PYG is an interesting chloride ion-dependent enzyme. Chloride ion causes distinctive changes in its enzymatic properties, increasing its hydrolysis rate, changing the pH profile of its enzyme activity, shifting the range of its pH stability to the alkaline region, and raising its optimal temperature from 37 to 55 °C. Furthermore, chloride ion altered PYG's substrate specificity. PYG exhibited the highest Vmax/Km value toward maltooctaose in the absence of chloride ion and toward maltotriose in the presence of chloride ion.

Production of 1,5-anhydro-d-fructose by an α-glucosidase belonging to glycoside hydrolase family 31.

α-1,4-Glucan lyases [glycoside hydrolase family (GH) 31] catalyze an elimination reaction to form 1,5-anhydro-d-fructose (AF), while GH31 α-glucosidases normally catalyze a hydrolytic reaction. We determined that a small amount of AF was produced by GH31 Aspergillus niger α-glucosidase from maltooligosaccharides by elimination reaction, likely via an oxocarbenium ion intermediate.

Different molecular complexity of linear-isomaltomegalosaccharides and ß-cyclodextrin on enhancing solubility of azo dye ethyl red: towards dye biodegradation.

Intermolecular interaction of linear-type α-(1 → 6)-glucosyl megalosaccharide rich (L-IMS) and water-insoluble anionic ethyl red was firstly characterized in a comparison with inclusion complexation by cyclodextrins (CDs) to overcome the problem of poor solubility and bioavailability. Phase solubility studies indicated an enhancement of 3- and 9-fold over the solubility in water upon the presence of L-IMS and ß-CD, respectively. (1)H NMR and circular dichrosim spectra revealed the dye forms consisted of 1:1 stoichiometric inclusion complex within the ß-CD cavity, whereas they exhibited non-specific hydrophobic interaction, identified by solvent polarity changes, with L-IMS. The inclusion complex delivered by ß-CD showed an uncompetitive inhibitory-type effect to azoreductase, particularly with high water content that did not promote dye liberation. Addition of the solid dye dispersed into coupled-enzyme reaction system supplied by L-IMS as the dye solubilizer provided usual degradation rate. The dye intermission in series exhibited successful removal with at least 5 cycles was economically feasible.

A novel metabolic pathway for glucose production mediated by α-glucosidase-catalyzed conversion of 1,5-anhydrofructose.

α-Glucosidase is in the glycoside hydrolase family 13 (13AG) and 31 (31AG). Only 31AGs can hydrate the D-glucal double bond to form α-2-deoxyglucose. Because 1,5-anhydrofructose (AF), having a 2-OH group, mimics the oxocarbenium ion transition state, AF may be a substrate for α-glucosidases. α-Glucosidase-catalyzed hydration produced α-glucose from AF, which plateaued with time. Combined reaction with α-1,4-glucan lyase and 13AG eliminated the plateau. Aspergillus niger α-glucosidase (31AG), which is stable in organic solvent, produced ethyl α-glucoside from AF in 80% ethanol. The findings indicate that α-glucosidases catalyze trans-addition. This is the first report of α-glucosidase-associated glucose formation from AF, possibly contributing to the salvage pathway of unutilized AF.

Exchanging a single amino acid residue generates or weakens a +2 cellooligosaccharide binding subsite in rice ß-glucosidases.

Os3BGlu6, Os3BGlu7, and Os4BGlu12 are rice glycoside hydrolase family 1 ß-glucosidases, the structures of which have been solved by X-ray crystallography. In complex structures, Os3BGlu7 residue Asn245 hydrogen bonds to the second sugar in the +1 subsite for laminaribiose and the third sugar in the +2 subsite for cellotetraose and cellopentaose. The corresponding Os3BGlu6 residue, Met251, appears to block the binding of cellooligosaccharides at the +2 subsite, whereas His252 in this position in Os4BGlu12 could hydrogen bond to oligosaccharides. Mutation of Os3BGlu6 Met251 to Asn resulted in a 15-fold increased k(cat)/K(m) value for hydrolysis of laminaribiose compared to wild type Os3BGlu6 and 9 to 24-fold increases for cellooligosaccharides with degrees of polymerization (DP) of 2-5. On the other hand, mutation of Os3BGlu7 Asn245 to Met decreased the k(cat)/K(m) of hydrolysis by 6.5-fold for laminaribiose and 17 to 30-fold for cellooligosaccharides with DP >2, while mutation of Os4BGlu12 His252 to Met decreased the corresponding k(cat)/K(m) values 2 to 6-fold.

Genomic and expression analysis of glycosyl hydrolase family 35 genes from rice (Oryza sativa L.).

BACKGROUND: Many plant beta-galactosidases (Bgals) have been well characterized and their deduced biological functions mainly involve degradation of structural pectins, xyloglucans or arabinogalactoproteins in plant cell walls. However, gene multiplicity in glycosyl hydrolase family 35 (GH35), to which these proteins belong, implies diverse functions. In this study, the gene multiplicity, apparent evolutionary relationships and transcript expression of rice Bgal genes were examined, in order to predict their biological functions. RESULTS: Fifteen rice Bgal genes were identified in the plant genome, one of which encodes a protein similar to animal Bgals (OsBgal9), and the remaining 14 fall in a nearly plant-specific subfamily of Bgals. The presence of both classes of Bgals in bryophytes, as well as vascular plants, suggests both gene lineages were present early in plant evolution. All 15 proteins were predicted to contain secretory signal sequences, suggesting they have secretory pathway or external roles. RT-PCR and database analysis found two distinct lineages to be expressed nearly exclusively in reproductive tissues and to be closely related to Arabidopsis Bgals expressed most highly in flower and pollen. On the other hand, OsBgal6 is expressed primarily in young vegetative tissues, and alternative splicing in panicle prevents its production of full-length protein in this reproductive tissue. OsBgal11 also showed alternative splicing to produce different length proteins. OsBgal13 produced by recombinant expression in Escherichia coli hydrolyzed alpha-L-arabinoside in addition to beta-D-galactoside and beta-(1-->3)-, beta-(1-->4)- and beta-(1-->6)- linked galacto-oligosaccharides. CONCLUSION: Rice GH35 contains fifteen genes with a diversity of protein sequences, predicted locations and expression and splicing patterns that suggest that OsBgals enzymes may play a variety of roles in metabolism of cell wall polysaccharides, glycoproteins and glycolipids.