Enzymatic synthesis of two novel non-reducing oligosaccharides using transfructosylation activity with β-fructofuranosidase from Arthrobacter globiformis

Two novel non-reducing oligosaccharides together with tri- and tetra-saccharides were synthesized by transfructosylation activity from sucrose as a donor and cellobiose or cellotriose as an acceptor with a purified beta-fructofuranosidase from Arthrobacter globiformis IFO 3062, and these oligosaccharides were identified as O-beta-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranosyl-(1-->2)-alpha,beta-D-fructofuranoside and O-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranosyl-(1-->2)-alpha,beta-D-fructofuranoside by spectrometric analyses. Both oligosaccharides were stable under condition at 100 degrees C for 30 min, and showed no degradation at pH 2.     

Cloning,expression and functional validation of a β-fructofuranosidase from Lactobacillus plantarum

Fructooligosaccharides (FOS) are prebiotics that selectively stimulate the growth and activity of lactobacilli and bifidobacteria. These strains metabolize FOS with endogenous β-fructofuranosidase. In this study, a β-fructofuranosidase gene from Lactobacillus plantarum ST-III designated sacA was cloned into Escherichia coli, and the properties of the recombinant protein (SacA) were examined. The sacA gene encodes a peptide of 501 amino acids with a predicted molecular weight of 56.7 kDa. Sequence alignment revealed the presence of three highly conserved motifs, NDPNG, RDP and EC, indicating that the enzyme belongs to glycoside hydrolase family 32. The predicted three-dimensional structure of the SacA enzyme was similar to β-fructofuranosidases of bifidobacteria, such that it contained a five-blade β-propeller module and a β-sandwich domain with one additional N-terminal α-helix. The optimal reaction temperature and pH of the enzyme were 37 °C and 6.0, respectively. Substrate hydrolysis and kinetic parameters demonstrated that β-fructofuranosidase from L. plantarum ST-III liberated fructosyl residues from the non-reducing terminus of fructans, such as sucrose, FOS, levan or inulin, and FOS was the preferred substrate. The expression of the sacA gene in a non-FOS-fermenting strain, Lactobacillus rhamnosus GG, enabled the recombinant strain to metabolize FOS and sucrose.     

Immobilization of β-glucosidase in calcium alginate gel using genipin as a new type of cross-linking reagent of natural origin

Summary -Glucosidase was immobilized in calcium alginate gel using genipin as the cross-linking reagent. The activity of this immobilized enzyme with genipin cross-linking was higher than that with other cross-linking reagents and no decrease in activity was observed even after using the gel 12 times.     

Design,synthesis and in vitro cytotoxicity studies of novel β-carbolinium bromides

A series of novel β-carbolinium bromides has been synthesized from easily accessible β-carbolines and 1-aryl-2-bromoethanones. The newly synthesized compounds were evaluated for their in vitro anticancer activity. Among the synthesized derivatives, compounds 16l, 16o and 16s exhibited potent anticancer activity with IC₅₀ values of <10 μM against tested cancer cell lines. The most potent analogue 16l was broadly active against all the tested cancer cell lines (IC₅₀ = 3.16–7.93 μM). In order to test the mechanism of cell death, we exposed castration resistant prostate cancer cell line (C4-2) to compounds 16l and 16s, which resulted in increased levels of cleaved PARP1 and AO/EB staining, indicating that β-carbolinium salts induce apoptosis in these cells. Additionally, the most potent β-carbolines 16l and 16s were found to inhibit tubulin polymerization.     

Screening,identification, and characterization of a novel saccharide-stimulated β-glycosidase from a soil metagenomic library

Applied Microbiology and Biotechnology - MeBglD2, a β-glycosidase that is highly activated in the presence of various monosaccharides and disaccharides, was isolated from a soil metagenomic...     

Evolution, expression and effectiveness in a cluster of novel bovine β-defensins

The anti-microbial peptides β-defensins constitute a large family of innate immune effector molecules, conserved across a wide species range. In this paper, we describe a systematic search of the sequenced bovine genome to characterise this extensive gene family in Bos taurus, providing an insight into the pattern of conservation of β-defensin genes between species. We have sequenced a sub-set of these newly discovered bovine β-defensin genes and also report expression data for these genes across a range of tissues. We have synthesised the peptide product of one of these genes, bovine β-defensin 123, and found it to be a potent inhibitor of several pathogenic microbes, particularly Escherichia coli and Listeria monocytogenes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phytochrome-mediated synthesis of novel growth inhibitors,A-2α and β, and dwarfism in peas

Variations in the content of A-2α and β, novel endogenous growth inhibitors, andcis,trans- andtrans, trans-xanthoxins were determined in 6- or 7-d-old, dark-grown seedlings of peas (Pisum sativum L. cvs. Progress No. 9, dwarf, and Alaska, tall) under various treatments with red light (R), and compared with R-induced growth inhibition. After transfer of the plants to continuous R the contents of the A-2s in cv. Progress increased after a 20-min lag, and reached plateaus after 12 h, whereas they remained almost unchanged in darkness. Both the rates of increase of the A-2s and the plateau levels were proportional to the logarithm of the irradiance applied. After a 10-min R pulse, the contents of both A-2α and β increased with the same rapidity to reach peaks after 6 h, and then gradually decreased to the initial levels after about 24 h. The effect of R was shown to be phytochrome-dependent, being nullified by far-red light. The level of neithercis,trans- nortrans,trans-xanthoxin showed such a close correlation with growth inhibition, although both xanthoxins increased as a result of phytochrome action. It is highly suggestive that the A-2s, rather than the xanthoxins, are responsible for phytochrome-dependent growth inhibition in cv. Progress. In cv. Alaska, in contrast, R-induced increase of the A-2s was rapid but slight, and could not explain the transient growth inhibition, which was found to be as large as that in cv. Progress shortly after the onset of R. The large content of the A-2s in cv. Progress in the steady state under continuous R, compared with that in cv. Alaska, may explain the dwarfism of cv. Progress. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Identification and characterization of a novel antibacterial peptide,avian β-defensin 2 from ducks

In this study, a novel avian β-defensin (AvBD) was isolated from duck pancreas. The complete nucleotide sequence of the gene contained an 195 bp open reading frame encoding 64 amino acids. Homology, characterization and comparison of the gene with AvBD from other avian species confirmed that it was duck AvBD2. The mRNA expression of the gene was analyzed in 17 tissues from 21-day-old ducks. AvBD2 was highly expressed in the trachea, crop, heart, bone marrow, and pancreas; moderately expressed in the muscular stomach, small intestine, kidney, spleen, thymus, and bursa of Fabricius; and weakly expressed in skin. We produced and purified recombinant AvBD2 by expressing the gene in Escherichia coli. As expected, the recombinant peptide exhibited strong bactericidal properties against Bacillus cereus, Staphylococcus aureus, and Pasteurella multocida, and weak bactericidal properties against E. coli and Salmonella choleraesuis. In addition, the recombinant protein retained antimicrobial activity against S. aureus under different temperatures (range, −20°C to 100°C) and pH values (range, 3 to 12)     

Purification and characterization of a novel β-agarase, AgaA34, from Agarivorans albus YKW-34

An extracellular β-agarase (AgaA34) was purified from a newly isolated marine bacterium, Agarivorans albus YKW-34 from the gut of a turban shell. AgaA34 was purified to homogeneity by ion exchange and gel filtration chromatographies with a recovery of 30% and a fold of ten. AgaA34 was composed of a single polypeptide chain with the molecular mass of 50 kDa. N-terminal amino acid sequencing revealed a sequence of ASLVTSFEEA, which exhibited a high similarity (90%) with those of agarases from glycoside hydrolase family 50. The pH and temperature optima of AgaA34 were pH 8.0 and 40°C, respectively. It was stable over pH 6.0–11.0 and at temperature up to 50°C. Hydrolysis of agarose by AgaA34 produced neoagarobiose (75 mol%) and neoagarotetraose (25 mol%), whose structures were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy and ¹³C NMR. AgaA34 cleaved both neoagarohexaose and neoagarotetraose into neoagarobiose. The k _cat/K _m values for hydrolysis agarose and neoagarotetraose were 4.04 × 10³ and 8.1 × 10² s⁻¹ M⁻¹, respectively. AgaA34 was resistant to denaturing reagents (sodium dodecyl sulfate and urea). Metal ions were not required for its activity, while reducing reagents (β-Me and dithiothreitol, DTT) increased its activity by 30%.     

The synthesis of novel sulfamides derived from β-benzylphenethylamines as acetylcholinesterase,butyrylcholinesterase and carbonic anhydrase enzymes inhibitors

In this study, a series of novel β-benzylphenethylamines and their sulfamide derivatives were synthesized starting from (Z)-2,3-diphenylacrylonitriles. Pd-C catalysed hydrogenation of diphenylacrylonitriles, reduction of propanenitriles with LiAlH₄ in the presence of AlCl₃ followed by addition of conc. HCl afforded β-benzylphenethylamine hydrochloride salts. The reactions of these amine hydrochloride salts with chlorosulfonyl isocyanate (CSI) in the presence of tert-BuOH and excess Et₃N gave sulfamoylcarbamates. Removing of Boc group from the synthesized sulfamoylcarbamates with trifluoroacetic acid (TFA) yielded novel sulfamides in good yields. These novel sulfamides derived from β-benzylphenethylamines were effective inhibitors of the cytosolic carbonic anhydrase I and II isoenzymes (hCA I and II), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with K_i values in the range of 0.278–2.260 nM for hCA I, 0.187–1.478 nM for hCA II, 0.127–2.452 nM for AChE and 0.494–1.790 nM for BChE. The inhibitory effects of the synthesized novel sulfamides derived from β-benzylphenethylamines were compared to those of acetazolamide and dorzolamide as clinical hCA I and II isoenzymes inhibitors and tacrine as a clinical AChE and BChE enzymes inhibitors. In addition to in vitro tests, molecular modeling approaches are implemented not only for prediction of the binding affinities of the compounds but also to study their inhibition mechanisms in atomic level at the catalytic domains.     

Studies on the synthesis of β-keto esters derived from dipeptides: search for a low-epimerizing method

-Keto esters derived from dipeptides areprepared by application of common methodologiesemployed for the synthesis of amino acid-derived-keto esters; however, epimerization of theC-terminal residue occurred to different extentsdepending on the method. In imidazolide activateddipeptides, this epimerization is due to the CDIactivation step and to the configurational instabilityof the intermediate imidazolides in different reactionmedia. Regarding yield and diastereomeric purity, themethod of choice proved to be the reaction ofdipeptide-derived imidazolide with the potassium saltof malonic half esters in the presence of MgCl₂.     

Studies on the synthesis of β-keto esters derived from dipeptides: search for a low-epimerizing method

Summary β-Keto esters derived from dipeptides are prepared by application of common methodologies employed for the synthesis of amino acid-derived β-keto esters; however, epimerization of the C-terminal residue occurred to different extents depending on the method. In imidazolide activated dipeptides, this epimerization is due to the CDI activation step and to the configurational instability of the intermediate imidazolides in different reaction media. Regarding yield and diastereomeric purity, the method of choice proved to be the reaction of dipeptide-derived imidazolide with the potassium salt of malonic half esters in the presence of MgCl₂.     

Design,synthesis and biological evaluation of novel tripeptidyl epoxyketone derivatives constructed from β-amino acid as proteasome inhibitors

A series of novel tripeptidyl epoxyketone derivatives constructed from β-amino acid were designed, synthesized and evaluated as proteasome inhibitors. All target compounds were tested for their proteasome inhibitory activities and selected compounds were tested for their anti-proliferation activities against two multiple myeloma (MM) cell lines RPMI 8226 and NCI-H929. Among them, eleven compounds exhibited proteasome inhibitory rates of more than 50% at the concentration of 1 μg/mL and nine compounds showed anti-proliferation activities with IC₅₀ values at low micromolar level. Compound 20h displayed the most potent proteasome inhibitory activities (IC₅₀: 0.11 ± 0.01 μM) and anti-proliferation activities with IC₅₀ values at 0.23 ± 0.01 and 0.17 ± 0.02 μM against two tested cell lines. Additionally, the poly-ubiquitin accumulation in the western blot analysis supported that proteasome inhibition in a cellular system was induced by compound 20h. All these experimental results confirmed that β-amino acid can be introduced as a building block for the development of proteasome inhibitors.     

Characterization and identification of SFDC-1, a novel AmpC-type β-lactamase in Serratia fonticola

The clinical and environmental infections caused by AmpC β-lactamases have been increasingly reported recently. In this study, we characterize the novel chromosome-encoded AmpC β-lactamase SFDC-1 identified in Serratia fonticola strain R28, which was isolated from a rabbit raised on a farm in southern China. SFDC-1 shared the highest amino acid identity of 79.6% with the functionally characterized AmpC β-lactamase gene bla_YRC-1, although it had highly homologous functionally uncharacterized relatives in the same species from different sources, including some of the clinical significance. The cloned bla_SFDC-1 exhibited resistance to a broad spectrum of β-lactam antibiotics, including most cephalosporins with the highest resistance to ampicillin, cefazolin and ceftazidime, with increased MIC levels ≥128-fold compared with the control strains. The purified SFDC-1 showed catalytic activities against β-lactams with the highest catalytic activity to cefazolin. The genetic context of bla_SFDC-1 and its relatives was conserved in the chromosome, and no mobile genetic elements were found surrounding them.     

A substitution of cytosine for thymine in codon 110 of the human β-globin gene is a novel cause of β-thalassemia phenotypes

Summary We have described a novel human globin gene mutation that produced in a Japanese family the -thalassemia phenotype through a post-translational mechanism. Substitution of proline for leucine at position 110 in the G-helix of the -globin chain greatly reduced the molecular stability of the -globin subunit, leading to total destruction of the variant globin chains by proteolysis and hence to the -thalassemia phenotype. The mutation could be identified after MspI digestion. This detection of the mutation on the gene level is valuable for diagnostic purposes.