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PubMed2006

Gene cloning and characterization of a novel alpha-amylase from alkaliphilic Alkalimonas amylolytica.

A gene encoding an extracellular alpha-amylase (AmyA) was cloned from the alkaliphilic bacterium Alkalimonas amylolytica by enzymatic activity screening in Escherichia coli DH5alpha. The gene amyA consists of 1764 base pairs and was predicted to encode a 587-amino acid protein encompassing a 31-amino acid signal peptide. In addition, a 459-amino acid catalytic domain and a 97-amino acid starch-binding domain (SBD) were found. The SBD showed little similarity to other known SBDs; instead, it contains conserved amino acids typically belonging to the carbohydrate-binding module (CBM) family 20. AmyA could act on both granular and gelatinized starch. The catalytic domain of the enzyme showed little similarity to other known alpha-amylases. Rather, AmyA contains four characteristic conserved regions of glycoside hydrolase family 13. The recombinant enzyme was a liquefying enzyme with the highest activity at 50 degrees C and pH 9.5. The enzyme displayed a unique endo-product profile and action pattern on soluble starch to yield a series of malto-oligosaccharides ranging from maltose to maltoheptaose. The activity of the enzyme was enhanced by Co(2+), but not affected by 5 mM EDTA. Taken together, AmyA from A. amylolytica has potential to be used in paper, textile, detergent and other industries where starch needs to be degraded in an alkaline environment.

Amino Acid Sequence↗
PubMed2003

Synthesis of complex oligosaccharides by using a mutated (1,3)-beta-D-glucan endohydrolase from barley.

Complex oligosaccharides with newly formed (1,3)-beta-glycosidic linkages were obtained in good to excellent yields when substituted or unsubstituted alpha-laminaribiosyl fluorides, acting as donors, were condensed onto mono- and disaccharide beta-D-hexopyranoside acceptors by using a (1,3)-beta-D-glycosynthase. These linear and branched (1,3)-beta-linked oligosaccharides could prove to be important in a range of medical, pharmaceutical, and agricultural applications. Furthermore, the observation that the (1,3)-beta-D-glucan glycosynthase accommodates (1,3)-, (1,4),- and (1,6)-beta-oligosaccharides in its acceptor subsites suggests novel, yet unexpected physiological roles for the wild type (1,3)-beta-D-glucan endohydrolase from higher plants.

Amino Acid Substitution↗
PubMed2006

Chemical and chemoenzymatic synthesis of S-linked ganglioside analogues and their protein conjugates for use as immunogens.

Analogues of the tumor-associated gangliosides GM(3) and GM(2) containing terminal S-linked neuraminic acid residues and an amino terminated, truncated ceramide homologue have been synthesized and conjugated to a protein. The synthesis involved coupling of a S-linked sialyl alpha(2-->3) galactose disaccharide with a glucosyl sphingosine analogue, followed by elaboration and deprotection to give amino-terminated glycosyl ceramide 1. Glycosyltransferase-catalyzed extension of the trisaccharide 1 provided access to the modified GM(2) tetrasaccharide 2 or sulphur-containing GD(3) analogue 30. Owing to their potentially enhanced resistance to endogenous exo-glycoside hydrolases and their inherent non-self character, carbohydrate antigens containing non-reducing terminal thioglycosidic linkages may be more immunogenic than O-linked antigens and may stimulate the production of antibodies capable of recognizing naturally occurring oligosaccharides. Our initial results suggest that in fact these antigens are viable immunogens and furthermore, that immune sera cross reacts with O-gangliosides in the context of a heterologous glycoprotein conjugate.

Animals↗
PubMed1994

Glucose, fructose, mannose and/or glucose-1-phosphate-releasing activity stains for glycosidases and glycosyltransferases in gels after isoelectric focusing.

beta-Fructofuranosidase, alpha-glucosidase, beta-glucosidase, alpha-mannosidase, beta-mannosidase, sucrose phosphorylase, glucosyltransferase and fructosyltransferase were separated by isoelectric focusing and sensitively detected to be slightly diffuse and insoluble spots in thin-layer gels, supported by a glass plate, by release of monosugars or a sugar phosphate, followed by conversion to glucose-6-phosphate (G6P) and then by reduction of NADP+ to NADPH, terminated by the formation of reduced Nitroblue Tetrazolium (NBT). Approximately 1-10 mU of enzyme was focused and the gel, after washing with a buffer, was partially dried and directly stained by uniformly spreading on the gel surface a staining medium containing sucrose or nitrophenyl glycosides as substrates, intermediary enzymes such as hexokinase, mutase and/or isomerase, NADP+, ATP, Mg+, phenazine methosulfate (PMS) and NBT. Specific staining procedures for each of these activities, on sucrose or on the glycosides as substrates, and staining procedures for multiple activities are described, with the conditions necessary for optimal development.

Fructose↗
PubMed1976

[Cleavage of alpha-L-arabinofuranoside, beta-D-glucopyranoside and beta-cellobioside of 4-nitrophenol by enzymes of various fungi - a contribution to increase the selectivity of tumor therapy].

To carry out long-term experiments as part of a therapy concept of malignant tumours using inactive transport forms of cancerostatic substances and their specific cleavage in the acidic pH region of the tumours by application of extraneous enzymes, we require enzymes with similar catalytic and pharmacokinetic properties which differ from each other in immunological respect. In the search for such enzymes, the alpha-L-arabinofuranosidases from 12 different fungi, among them 9 basidiomycetes, were studied. The enzymes mentioned were demonstrable in all fungi. Optimum pH values ranged between 2.5 and 5.5. The Km values for the cleavage of alpha-L-arabinofuranoside were, in most cases, 0.5 to 1.8 moles-liter-1-10(-3). With regard to pH dependence, the alpha-L-arabinofuranosidases of most of the fungi investigated proved adequate for the long-term trials envisaged. 4-nitrophenyl-beta-D-glucopyranoside and -beta-cellobioside were also cleaved by enzyme preparations of all the 11 fungi investigated. The beta-D-glucopyranosidases showed a less favourable pH dependence than the alpha-L-arabinofuranosidases. The cleavage of 4-nitrophenyl-beta-cellobioside, on the contrary, showed mostly a comparatively favourable pH dependence. On the basis of the coinciding optimal pH values and the occurrence of 4-nitrophenyl-beta-D-glucopyranoside as an intermediate product in the cleavage of the corresponding cellobioside, we assume that both substrates are cleaved by beta-glucosidase. Because the occurrence of the glucoside during the cleavage of cellobioside is undesirable for the therapeutic trial, a method is proposed for selection of an appropriate cellobioside splitting enzyme basing on the present studies and the relevant literature.

Aspergillus niger↗
PubMed2005

Cell wall proteins in apoplastic fluids of Arabidopsis thaliana rosettes: identification by mass spectrometry and bioinformatics.

Weakly bound cell wall proteins of Arabidopsis thaliana were identified using a proteomic and bioinformatic approach. An efficient protocol of extraction based on vacuum-infiltration of the tissues was developed. Several salts and a chelating agent were compared for their ability to extract cell wall proteins without releasing cytoplasmic contaminants. Of the 93 proteins that were identified, a large proportion (60%) was released by calcium chloride. From bioinformatics analysis, it may be predicted that most of them (87 out of 93) had a signal peptide, whereas only six originated from the cytoplasm. Among the putative apoplastic proteins, a high proportion (67 out of 87) had a basic pI. Numerous glycoside hydrolases and proteins with interacting domains were identified, in agreement with the expected role of the extracellular matrix in polysaccharide metabolism and recognition phenomena. Ten proteinases were also found as well as six proteins with unknown functions. Comparison of the cell wall proteome of rosettes with the previously published cell wall proteome of cell suspension cultures showed a high level of cell specificity, especially for the different members of several large multigenic families.

Arabidopsis↗
PubMed2005

Structural basis of the substrate subsite and the highly thermal stability of xylanase 10B from Thermotoga maritima MSB8.

The crystal structure of xylanase 10B from Thermotoga maritima MSB8 (TmxB), a hyperthermostable xylanase, has been solved in its native form and in complex with xylobiose or xylotriose at 1.8 A resolution. In order to gain insight into the substrate subsite and the molecular features for thermal stability, we compared TmxB with family 10 xylanase structures from nine microorganisms. As expected, TmxB folds into a (beta/alpha)8-barrel structure, which is common among the glycoside hydrolase family 10. The enzyme active site and the environment surrounding the xylooligosaccharide of TmxB are highly similar to those of family 10 xylanases. However, only two xylose moieties were found in its binding pocket from the TmxB-xylotriose complex structure. This finding suggests that TmxB could be a potential biocatalyst for the large-scale production of xylobiose. The result of structural analyses also indicated that TmxB possesses some additional features that account for its thermostability. In particular, clusters of aromatic residues together with a lack of exposed hydrophobic residues are characteristic of the TmxB structure. TmxB has also a significant number of ion pairs on the protein surface that are not found in other thermophilic family 10 xylanases.

Bacterial Proteins↗
PubMed2007

Towards the molecular understanding of glycogen elongation by amylosucrase.

Amylosucrase from Neisseria polysaccharea (AS) is a transglucosidase from the glycoside-hydrolase family 13 that catalyzes the synthesis of an amylose-like polymer from sucrose, without any primer. Its affinity towards glycogen is particularly noteworthy since glycogen is the best D-glucosyl unit acceptor and the most efficient activator (98-fold k(cat) increase) known for this enzyme. Glycogen-enzyme interactions were modeled starting from the crystallographic AS: maltoheptaose complex, where two key oligosaccharide binding sites, OB1 and OB2, were identified. Two maltoheptaose molecules were connected by an alpha-1,6 branch by molecular modeling to mimic a glycogen branching. Among the various docking positions obtained, four models were chosen based on geometry and energy criteria. Robotics calculations enabled us to describe a back and forth motion of a hairpin loop of the AS specific B'-domain, a movement that assists the elongation of glycogen branches. Modeling data combined with site-directed mutagenesis experiments revealed that the OB2 surface site provides an anchoring platform at the enzyme surface to capture the polymer and direct the branches towards the OB1 acceptor site for elongation. On the basis of the data obtained, a semiprocessive glycogen elongation mechanism can be proposed.

Arginine↗
PubMed2007

Ancestral sequence evolutionary trace and crystal structure analyses of alkaline alpha-amylase from Bacillus sp. KSM-1378 to clarify the alkaline adaptation process of proteins.

The crystal structure of alkaline liquefying alpha-amylase (AmyK) from the alkaliphilic Bacillus sp. KSM-1378 was determined at 2.1 A resolution. The AmyK structure belongs to the GH13 glycoside hydrolase family, which consists of three domains, and bound three calcium and one sodium ions. The alkaline adaptation mechanism of AmyK was investigated by the ancestral sequence evolutionary trace method and by extensive comparisons between alkaline and nonalkaline enzyme structures, including three other protein families: protease, cellulase, and phosphoserine aminotransferase. The consensus change for the alkaline adaptation process was a decrease in the Lys content. The loss of a Lys residue is associated with ion pair remodeling, which mainly consists of the loss of Lys-Asp/Glu ion pairs and the acquisition of Arg ion pairs, preferably Arg-Glu. The predicted replacements of the positively charged amino acids were often, although not always, used for ion pair remodeling.

Amino Acids↗
PubMed2026

Characterization and genomic analysis of Bacillus halotolerans G3-2: a potential biocontrol agent against apple Alternaria leaf blotch disease.

BACKGROUND: Apple Alternaria leaf blotch (ALB) is a devastating disease threatening the apple industry worldwide. Biocontrol offers an effective and environmentally friendly alternative for disease management. RESULTS: Bacillus strain G3-2 exhibits strong antagonistic activity against Alternaria alternata (a major causal pathogen of ALB). In dual-culture assays, G3-2 inhibited A. alternata by 88.39%; in detached-leaf inoculation assays, it reduced the lesion area by >88%. 16S rRNA sequencing and phylogenetic analysis identified this strain as Bacillus halotolerans. Oxford Nanopore Technology (ONT) sequencing generated a 4.18-Mb complete genome (43.8% G + C) containing 4149 protein-coding genes, 30 rRNAs and 86 tRNAs. CAZy annotation identified 182 genes encoding carbohydrate-active enzymes (CAZymes), including glycoside hydrolases, glycosyltransferase, and carbohydrate esterases, suggesting potential for glycosylated secondary metabolite production. AntiSMASH analysis detected nine biosynthetic gene clusters, including those for surfactin, fengycin, bacillaene and laterocidine. Plate assays confirmed that G3-2 has the ability to produce protease, cellulase and siderophore. Moreover, it exhibits ~70% inhibition against several other phytopathogenic fungi. CONCLUSIONS: These findings demonstrate that G3-2 suppresses A. alternata through antibiosis (lipopeptides and polyketides), nutrient competition (siderophores) and cell-wall degradation (proteases and cellulases). Moreover, our study revealed that it has great potential to be used as a broad-spectrum, environmentally friendly biocontrol agent. © 2026 Society of Chemical Industry.

Alternaria↗
PubMed2007

Mass spectrometric identification of covalently bound cell wall proteins from the fission yeast Schizosaccharomyces pombe.

The cell wall of Schizosaccharomyces pombe is bilayered, consisting of an inner layer of mainly polysaccharides and an outer layer of galactomannoproteins. We present a detailed analysis of the cell wall proteome. Six covalently-bound cell wall proteins (CWPs) were identified using tandem mass spectrometry, including four predicted GPI-dependent CWPs (Gas1p, Gas5p, Ecm33p and Pwp1p) and two alkali-sensitive CWPs (Psu1p and Asl1p). Gas1p and Gas5p belong to glycoside hydrolase family 72, and are believed to be involved in 1,3-beta-glucan elongation. Ecm33p belongs to a ubiquitous fungal protein family with an unknown but crucial function in cell wall integrity. Pwp1p is an abundant protein with an unknown but probably non-enzymatic function. All four CWPs were present in HF-pyridine extracts, indicating that they are linked via a phosphodiester bridge to the glucan network. Psu1p is a homologue of the Saccharomyces cerevisiae Sun family, whereas Asl1p has no homologues in S. cerevisiae but is related to Aspergillus fumigatus and Ustilago maydis proteins. Finally, although the protein content of Sz. pombe cell walls is only slightly less than in S. cerevisiae and Candida albicans, the amount of carbohydrate added to the proteins was found to be two- to three-fold decreased, consistent with earlier reported differences in outer chain N-glycosylation.

Alkalies↗
PubMed2000

A chromogenic substrate for a beta-xylosidase-coupled assay of alpha-glucuronidase.

4-Nitrophenyl 2-(4-O-methyl-alpha-d-glucopyranuronosyl)-beta-d-xylopyranoside obtained on deesterification of 4-nitrophenyl 2-O-(methyl 4-O-methyl-alpha-d-glucopyranosyluronate)-beta-d-xylopyranoside (Hirsch et al., Carbohydr. Res. 310, 145-149, 1998) was found to be an excellent substrate for the measurement of hemicellulolytic alpha-glucuronidase activity. A new precise alpha-glucuronidase assay was developed by coupling the alpha-glucuronidase-catalyzed formation of 4-nitrophenyl beta-d-xylopyranoside with its efficient hydrolysis by beta-xylosidase. A recombinant strain of Saccharomyces cerevisiae, harboring and expressing the beta-xylosidase gene xlnD of Aspergillus niger under control of the alcohol dehydrogenase II promoter on a multicopy plasmid, was used as a source of beta-xylosidase. The activity values of beta-xylosidase in the assay required to achieve a steady-state rate of 4-nitrophenol formation shortly after starting the alpha-glucuronidase reaction were obtained both experimentally and by calculation using the kinetics of coupled enzyme reactions.

Aspergillus niger↗