Synthesis of novel heterobranched beta-cyclodextrins from 4(2)-O-beta-D-galactosyl-maltose and beta-cyclodextrin by the reverse action of pullulanase, and isolation and characterization of the products

From the mixture of 4(2)-O-beta-D-galactosyl-maltose (Gal-G2) and beta-cyclodextrin (betaCD), novel heterobranched betaCDs, (Gal-G2)-betaCD and (Gal-G2)2-betaCDs, were synthesized by the reverse action of debranching enzyme. The optimum conditions for the production of (Gal-G2)2-betaCDs were examined. A mixture of (Gal-G2)2-betaCDs was produced in about 4% yield when Aerobacter aerogenes pullulanase (64 units per 1 g of Gal-G2) was incubated with 1.6 M Gal-G2 and 0.16 M betaCD at 50 degrees C for 4 days. The reaction products, (Gal-G2)2-betaCDs, were separated into three peaks by HPLC analysis on a Hypercarb S column. Their structures were analyzed by fast atom bombardment mass spectroscopy and NMR spectroscopies, and confirmed by comparison of their hydrolyzates by beta-galactosidase with the authentic (G2)2 -betaCDs. The structures of (Gal-G2)-betaCD and three components of (Gal-G2)2-betaCDs were identified as 6-O-(GalG2)-betaCD, 6(1),6(2)-, 6(1),6(3)- and 6(1),6(4)-di-O-(Gal-G2)2-betaCD, respectively.     

Synthesis of novel heterobranched beta-cyclodextrins having beta-D-N-acetylglucosaminyl-maltotriose on the side chain

From a mixture of N-acetylglucosaminyl-beta-cyclodextrin (GlcNAc-betaCD) and lactose, beta-D-galactosyl-GlcNAc-betaCD (Gal-GlcNAc-betaCD) was synthesized by the transfer action of beta-galactosidase. GlcNAc-maltotriose (Glc3) and Gal-GlcNAc-Glc3 were produced with hydrolysis of GlcNAc-betaCD by cyclodextrin glycosyltransferase, and Gal-GlcNAc-betaCD by bacterial saccharifying alpha-amylase respectively. Finally, GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were synthesized in 5.2% and 3.5% yield when Klebsiella pneumoniae pullulanase was incubated with the mixture of GlcNAc-Glc(3) and betaCD, or Gal-GlcNAc-Glc3 and betaCD respectively. The structures of GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were analyzed by FAB-MS and NMR spectroscopy and identified as 6-O-alpha-(6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD, and 6-O-alpha-(4-O-beta-D-galactopyranosyl-6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD respectively.     

Isolation and characterization of di- and tri-mannosyl-cyclomaltoheptaoses (beta-cyclodextrins) produced by reverse action of alpha-mannosidase from jack bean

Di- and tri-mannosyl-cyclomaltoheptaoses (beta-cyclodextrins, beta CDs), which were synthesized together with monomannosyl-beta CD in a large-scale production by reverse action of alpha-mannosidase from jack bean, were isolated and purified by HPLC. The structures of seven isomers of di-mannosyl-beta CD and six isomers of tri-mannosyl-beta CD were elucidated by FABMS and NMR spectroscopy, and by enzymatic methods.     

Nanoparticles of beta-cyclodextrin esters obtained by self-assembling of biotransesterified beta-cyclodextrins

The synthesis of decanoate beta-cyclodextrin esters (beta-CDd) and hexanoate beta-cyclodextrin esters (beta-CDh) was biocatalyzed by thermolysin from native beta-cyclodextrin (beta-CD) and vinyl hexanoate or vinyl decanoate used as acyl donors. The products were chemically characterized by infrared, NMR, and mass spectrometry. Both beta-CDd and beta-CDh esters were identified as a mixture of beta-CD preferentially substituted on the C2 position by the corresponding acyl chain. The degree of substitution varied from 2 to 7 for beta-CDd and from 4 to 8 for beta-CDh. The ability of beta-CD esters to self-organize into nanoparticles was tested using a nanoprecipitation technique in various solvents. The mean size diameter and polydispersity measured by quasi-elastic light scattering were dramatically affected by the nature of solvent (acetone, ethanol, or tetrahydrofuran) used in the nanoprecipitation technique. When directly observed using cryo-transmission electron microscopy, beta-CDh appeared as uniformly dense nanospheres, whereas beta-CDd exhibited a multilamellar onion-like organization. A structural model was rationalized for the beta-CDd nanoparticles.     

Purification and characterization of a highly thermostable novel pullulanase from Clostridium thermohydrosulfuricum.

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.     

Purification and characterization of pullulanase from Lactococcus lactis

This paper describes a simple and efficient method of isolation of a plullulanase type I from amylolytic lactic acid bacteria (ALAB). Extracellular pullulanase type I was purified from a cell-free culture supernatant of Lactococcus lactis IBB 500 by using ammonium sulfate fractionation and dialysis (instead of ultrafiltration), and ion-exchange chromatography with CM Sepharose FF followed by gel filtration chromatography with Sephadex G-150 as the final step. A final purification factor of 14.36 was achieved. The molecular mass of the enzyme was estimated as 73.9 kD. The optimum temperature for the enzyme activity was 45°C and the optimum pH was 4.5. Pullulanase activity was increased by addition Co(2+) and completely inhibited by Hg(2+). The enzyme activity was specifically directed toward α-1,6 glycosidic linkages of pullulan giving maltotriose units. Enzymatic hydrolysis of starch and amylose produced a mixture of maltose and maltotriose.     

Moderately thermophilic Myxobacteria: novel potential for the production of natural products isolation and characterization

Novel moderately thermophilic Myxobacteria from both known suborders (Cystobacterineae and Sorangineae) were isolated from soil samples of semiarid and warm climates. The addition of the anthelmintic and amoebizidal agent levamisole was a new and crucial improvement for the fast isolation of overall 37 strains, which grew very fast at temperatures of 42-44 degrees C. When the 16S rDNAs were compared with GenBank data of common Myxobacteria, identities were 98-99%, thus not reflecting the physiological differences. Similar to the Myxobacteria described so far, the new isolates are multiresistant against a variety of antibiotics and are producers of typical myxobacterial secondary metabolites. Analysis of our previous strain collection isolated from soil samples taken worldwide revealed a more or less uniform distribution of strains which synthesize specific metabolites. Therefore these moderately thermophilic Myxobacteria, which grow 2-3 times faster, have the potential to replace the slow-growing isolates and provide a means for fast and cost-saving production of myxobacterial metabolites in the future.     

A novel lectin from the sponge Haliclona cratera: isolation,characterization and biological activity

A lectin from the Adriatic sponge Haliclona cratera was purified by ion-exchange and gel chromatography. The molecular mass of the lectin is approximately 29 kDa. Purified lectin is rich in hydrophobic and basic amino acids and has an isoelectric point at pH 8.6. H. cratera lectin is relatively heat- and pH-stable. It agglutinates native and trypsinized, papainized and neuraminidase-treated human A, B, O, AB and sheep erythrocytes, and the hemagglutinating activity is independent of Ca(2+), Mn(2+) and Mg(2+) ions; D-galactose and N-acetyl-D-galactosamine are found to be moderate inhibitors of the activity. H. cratera lectin displays cytotoxic effect on HeLa and FemX cells and weak mitogenic effect on human T-lymphocytes pretreated with phytohemagglutinin (PHA).     

Valosin: isolation and characterization of a novel peptide from porcine intestine

The isolation and primary structure of a novel gastrointestinal peptide, designated valosin, is described. The peptide was purified from porcine upper gut extracts using an HPLC and N-terminal sequence screening strategy which depends on chromatographic and structural characteristics as isolation criterion. The amino acid sequence of this peptide consists of 25 amino acid residues:     

A novel sucrose phosphorylase from the metagenomes of sucrose-rich environment: isolation and characterization

Sucrose phosphorylase, an important enzyme mainly involved in the generic starch and sucrose pathways, has now caught the attention of researchers due to its transglycosylation activity. A novel sucrose phosphorylase, unspase, has been isolated, and its transglycosylation properties were characterized. Compared with Bisp, the sucrose phosphorylase from Bifidobacterium adolescentis, unspase had two deleted regions in its C: -terminal. These deleted regions were probably equivalent to the important five-stranded anti-parallel β-sheet domain in sucrose phosphorylase. Unspase has a k(m) of 21.12?mM, a V(max) of 69.24?μmol?min(-1)?mg(-1) and a k(cat) of 31.19?s(-1) with sucrose as substrate. In 3-(N-morpholino) propanesulfonic acid (MOPS) buffer, unspase transferred the glycosyl moiety to L: -arabinose, D: -fructose and L: -sorbose. Much to our surprise, unspase can catalyze the transglycosylation in which a glycosyl moiety was transferred to L: -arabinose in the presence of phosphate, which is an interesting exception to the generally accepted fact that transglycosylation can only occur under the condition of phosphate absence. The final yield of the transglycosylation product (37.9?%) in phosphate buffer was even higher than that (5.8?%) in MOPS buffer. This is a novel phenomenon that a sucrose phosphorylase can catalyze a transglycosylation reaction in the presence of phosphate.     

A novel prothrombin activator from the venom of Micropechis ikaheka: isolation and characterization

A novel prothrombin activator, Mikarin, has been isolated from Micropechis ikaheka venom. It is a single polypeptide chain metalloproteinase with the apparent molecular weight of 47kDa. Mikarin exhibits Ca(2+)-independent prothrombin activation, but no effects on other blood coagulation factors, such as factor X and fibrinogen. Mikarin is the first member of group I prothrombin activators from elapid venom. Like other high-molecular-weight snake venom proteinases, it has three structural domains, metalloproteinase and disintegrin-like and Cys-rich domains, and belongs to the P-III class of snake venom metalloproteinases. The N-terminal of Mikarin exhibits 76% sequence identity with Cobrin, a metalloproteinase identified from Naja naja venom, but very lower identities were found when compared with those from viperid and crotalid venom. In addition, the presence of disintegrin-like and Cys-rich domains in snake venom metalloproteinases with diverse biological activities suggests that these domains may be important for their function.     

Cloning, sequencing, and characterization of a heat- and alkali-stable type I pullulanase from Anaerobranca gottschalkii

The gene encoding a type I pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii. In addition, the homologous gene was isolated from a gene library of Anaerobranca horikoshii and sequenced. The proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria Fervidobacterium pennivorans and Thermotoga maritima. The pullulanase gene from A. gottschalkii (encoding 865 amino acids with a predicted molecular mass of 98 kDa) was cloned and expressed in Escherichia coli strain BL21(DE3) so that the protein did not have the signal peptide. Accordingly, the molecular mass of the purified recombinant pullulanase (rPulAg) was 96 kDa. Pullulan hydrolysis activity was optimal at pH 8.0 and 70 degrees C, and under these physicochemical conditions the half-life of rPulAg was 22 h. By using an alternative expression strategy in E. coli Tuner(DE3)(pLysS), the pullulanase gene from A. gottschalkii, including its signal peptide-encoding sequence, was cloned. In this case, the purified recombinant enzyme was a truncated 70-kDa form (rPulAg'). The N-terminal sequence of purified rPulAg' was found 252 amino acids downstream from the start site, presumably indicating that there was alternative translation initiation or N-terminal protease cleavage by E. coli. Interestingly, most of the physicochemical properties of rPulAg' were identical to those of rPulAg. Both enzymes degraded pullulan via an endo-type mechanism, yielding maltotriose as the final product, and hydrolytic activity was also detected with amylopectin, starch, beta-limited dextrins, and glycogen but not with amylose. This substrate specificity is typical of type I pullulanases. rPulAg was inhibited by cyclodextrins, whereas addition of mono- or bivalent cations did not have a stimulating effect. In addition, rPulAg' was stable in the presence of 0.5% sodium dodecyl sulfate, 20% Tween, and 50% Triton X-100. The pullulanase from A. gottschalkii is the first thermoalkalistable type I pullulanase that has been described.     

嗜热地芽胞杆菌(Geobacillus kaustophilus)DY115普鲁兰酶基因的克隆、表达和酶学性质

采用基因工程方法对嗜热地芽胞杆菌(Geobacillus kaustophilus)DY115的普鲁兰酶基因pulA在大肠杆菌中进行了克隆表达。该基因ORF全长为2 157bp,编码718个氨基酸。重组PulA在大肠杆菌(Escherichia coli)BL21(DE3)中能够有效表达,经Ni-Sepharose亲和层析获得纯化的重组PulA蛋白。PulA最适作用温度为70℃,最适pH为8.0,在65℃和碱性条件下具有良好的热稳定性;K~+和Mn~(2+)对PulA活性有明显促进作用,Cu~(2+)和Zn~(2+)则强烈抑制PulA活性;PulA对普鲁兰糖水解能力最强,且其水解支链淀粉和糯米淀粉的能力明显高于直链淀粉;PulA可水解普鲁兰糖的α-(1,6)糖苷键生成麦芽三糖,属于I型普鲁兰酶。这是首次对来源于地芽胞杆菌属(Geobacillus)的高温碱性普鲁兰酶进行报道,由于PulA具有较好的水解淀粉支链的能力,因此其在淀粉加工业以及洗涤业上应用前景良好。     

Purification and characterization of a cold-adapted pullulanase from a psychrophilic bacterial isolate

There is a considerable potential of cold-active biocatalysts for versatile industrial applications. A psychrophilic bacterial strain, Shewanella arctica 40-3, has been isolated from arctic sea ice and was shown to exhibit pullulan-degrading activity. Purification of a monomeric, 150-kDa pullulanase was achieved using a five-step purification approach. The native enzyme was purified 50.0-fold to a final specific activity of 3.0 U/mg. The enzyme was active at a broad range of temperature (10–50 °C) and pH (5–9). Optimal activity was determined at 45 °C and pH 7. The presence of various metal ions is tolerated by the pullulanase, while detergents resulted in decreased activity. Complete conversion of pullulan to maltotriose as the sole product and N-terminal amino acid sequence indicated that the enzyme is a type-I pullulanase and belongs to rarely characterized pullulan-degrading enzymes from psychrophiles.     

Enzymatic nitration of the oleic acid derivatives: Synthesis,isolation and characterization

Unsaturated fatty acids are nitrated endogenously to produce nitrated lipids. Recent studies have shown that these nitrated lipids have high chemical reactivity and profound biological implications. We report an efficient, enzymatic synthesis of nitrated derivatives of the oleic acid. The methyl oleate could react with NO and horseradish peroxidase (HRP)/H₂O₂/NO based nitrating systems to give various nitration products which could be isolated by silica gel column and TLC fractionation, respectively. As reacting directly with NO, the obtained products contain (E)-methyl 9-nitrooctadec-9-enoate (1), (E)-methyl 10-nitrooctadec-9-enoate (2), (E)-methyl 9-nitrooctadec-10-enoate (3) and (E)-methyl 10-nitrooctadec-8-enoate (4), characterized by extensive IR, NMR and GC–MS analysis. Whereas the products of the reaction between the methyl oleate and NO with the presence of HRP/H₂O₂ were mainly composed of (E)-methyl 9-nitrooctadec-9-enoate and (E)-methyl 10-nitrooctadec-9-enoate. The improving selectivity of the products is attributed to the HRP catalysis system.     

Selective procedure for isolation of microorganisms producing pullulanase and isoamylase

Selective isolation of microorganisms producing pullulanase and isoamylase was accomplished using a two plate detection assay which distinguished both activities and excluded microorganisms producing other extracellular amylases. Over 115,000 colonies tested, 190 strains producing pullulanase and 57 strains producing isoamylase were isolated; extracellular activities of isolated strains were 0.2 to 4.2 and 0.5 to 2.1 nkat/ml of culture, respectively.This paper is dedicated to Prof. Dr. Raúl E. Trucco, in occasion of his 75th anniversary.     

Pathology, isolation, and preliminary molecular characterization of a novel iridovirus from tiger salamanders in Saskatchewan.

All iridovirus was confirmed to be the cause of an epizootic in larval and adult tiger salamanders (Ambystoma tigrinum diaboli) from four separate ponds in southern Saskatchewan (Canada) during the summer of 1997. This organism also is suspected, based on electron microscopic findings, to be the cause of mortality of larval tiger salamanders in a pond over 200 km to the north during the same year. Salamanders developed a generalized viremia which resulted in various lesions including: necrotizing, vesicular and ulcerative dermatitis; gastrointestinal ulceration; and necrosis of hepatic, splenic, renal, lymphoid, and hematopoietic tissues. In cells associated with these lesions, large lightly basophilic cytoplasmic inclusions and vacuolated nuclei with marginated chromatin were consistently found. Virus was isolated from tissue homogenates of infected salamanders following inoculation of epithelioma papilloma cyprini (EPC) cells. The virus, provisionally designated Regina ranavirus (RRV), was initially identified as an iridovirus by electron microscopy. Subsequent molecular characterization, including partial sequence analysis of the major capsid protein (MCP) gene, confirmed this assignment and established that RRV was a ranavirus distinct from frog virus 3 (FV3) and other members of the genus Ranavirus. Intraperitoneal inoculation of 5 x 10(6.23) TCID50 of the field isolate caused mortality in inoculated salamanders at 13 days post infection. Field, clinical, and molecular studies jointly suggest that the etiological agent of recent salamander mortalities is a highly infectious novel ranavirus.