Characterization of a Thermostable Endo-β-1,4-D-galactanase from the Hyperthermophile Thermotoga maritima

A putative endo-β-1,4-D-galactanase gene of Thermotoga maritima was cloned and overexpressed in Escherichia coli. The recombinant enzyme hydrolyzed pectic galactans and produced D-galactose, β-1,4-D-galactobiose, β-1,4-D-galactotriose, and β-1,4-D-galactotetraose. The enzyme displayed optimum activity at 90 °C and pH 7.0. It was slowly inactivated above pH 8.0 and below pH 5.0 and stable at temperatures up to 80 °C.     

Cloning and Characterization of the Gene Encoding Endo-β-1,3-glucanase from Arthrobacter sp. NHB-10

The gluA gene, encoding an endo-β-1,3-glucanase from Arthrobacter sp. (strain NHB-10), was cloned and analyzed. The deduced endo-β-1,3-glucanase amino acid sequence was 750 amino acids long and contained a 42 amino acid signal peptide with a mature protein of 708 amino acids. There was no similarity to known endo-β-1,3-glucanases, but GluA was partially similar to two fungal exo-β-1,3-glucanases in glycoside hydrolase (GH) family 55. Of five possible residues for catalysis and two motifs in two β-helix heads of GH family 55, three residues and one motif were conserved in GluA, suggesting that GluA is the first bacterial endo-β-1,3-glucanase in GH family 55. Significant similarity was also found to two proteins of unknown function from Streptomyces coelicolor A3(2) and S. avermitilis.     

Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca, Ampullaria crossean

Two novel endo-β-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50℃ and 60 ℃; for EG45 it was 50 ℃. The analysis on the stability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 ℃, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 ℃ for 24 h. However, less than 10% residual activity of EG45 was detected at 50 ℃. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan （from oat spelt） and Sigmacell 101, and they were inactive to p-nitrophenyl-β-D-cellobioside, salicin and starch.     

Non-lysosomal Degradation of Singly Phosphorylated Oligosaccharides Initiated by the Action of a Cytosolic Endo-β-N-acetylglucosaminidase

Phosphorylated oligosaccharides (POSs) are produced by the degradation of dolichol-linked oligosaccharides (DLOs) by an unclarified mechanism in mammalian cells. Although POSs are exclusively found in the cytosol, their intracellular fates remain unclear. Our findings indicate that POSs are catabolized via a non-lysosomal glycan degradation pathway that involves a cytosolic endo-β-N-acetylglucosaminidase (ENGase). Quantitative and structural analyses of POSs revealed that ablation of the ENGase results in the significant accumulation of POSs with a hexasaccharide structure composed of Manα1,2Manα1,3(Manα1,6)Manβ1,4GlcNAcβ1,4GlcNAc. In vitro ENGase assays revealed that the presence of an α1,2-linked mannose residue facilitates the hydrolysis of POSs by the ENGase. Liquid chromatography-mass spectrometric analyses and fluorescent labeling experiments show that such POSs contain one phosphate group at the reducing end. These results indicate that ENGase efficiently hydrolyzes POSs that are larger than Man₄GlcNAc₂-P, generating GlcNAc-1-P and neutral Gn1-type free oligosaccharides. These results provide insight into important aspects of the generation and degradation of POSs.     

Isolation and Characterization of Two β-d-Glucosidases from Bifidobacterium breve 203

Two β-d-glucosidases were purified to homogeneity from Bifidobacterium breve 203: one ( β-d-glucosidase I; molecular weight, 96,000) showed reactivity toward p-nitrophenyl (p-NP) β-d-fucoside, 74% of that to p-NP β-d-glucoside, and the other ( β-dglucosidase II; molecular weight, 450,000) did not. They also differed in their thermal and pH stabilities. Laminaribiose, cellobiose and gentiobiose were hydrolyzed by β-d-glucosidase I, with 53%, 34% and 3% of the reactivity in the case of p-NP β-d-glucoside, and by β-dglucosidase II, with 53%, 6% and 107% of the reactivity. The reaction of β-dglucosidase I with p-NP β-dfucoside was enhanced by the addition of glucose and other monosaccharides to the reaction mixture, whereas that with p-NP β-dglucoside was not affected. The activity of β-dglucosidase II with p-NP β-dglucoside was inhibited by glucose.     

Isolation and Identification of l-Menthyl-β-d-glucoside from Shubi

Quinoxalines derived from d-galactose with o-phenylenediamine (OPD) in acidic media under reflux were studied by using GLC and NMR measurements. Four quinoxaline derivatives were obtained from the reaction mixture, and were identical with those derived from d-glucose. The yields of 2-(D-lyxo-tetrahydroxybutyl)quinoxaline (GA-III), and the stereoisomeric derivative of GA-III, i.e., 2-(D-arabino-tetrahydroxybutyl)quinoxaline (ATBQ), were 13.2 and 5.3–, respectively. The ratio of GA-III to ATBQ derived from d-galactose was reciprocally coincident with that from d-glucose. Some proposals are made on the relationship between the isomerization of these sugars and the formation of quinoxaline derivatives.     

Characterization of Cryptopygus antarcticus Endo-β-1,4-Glucanase from Bombyx mori Expression Systems

Endo-β-1,4-glucanase (CaCel) from Antarctic springtail, Cryptopygus antarcticus, a cellulase with high activity at low temperature, shows potential industrial use. To obtain sufficient active cellulase for characterization, CaCel gene was expressed in Bombyx mori-baculovirus expression systems. Recombinant CaCel (rCaCel) has been expressed in Escherichia coli (Ec-CaCel) at temperatures below 10 °C, but the expression yield was low. Here, rCaCel with a silkworm secretion signal (Bm-CaCel) was successfully expressed and secreted into pupal hemolymph and purified to near 90 % purity by Ni-affinity chromatography. The yield and specific activity of rCaCel purified from B. mori were estimated at 31 mg/l and 43.2 U/mg, respectively, which is significantly higher than the CaCel yield obtained from E. coli (0.46 mg/l and 35.8 U/mg). The optimal pH and temperature for the rCaCels purified from E. coli and B. mori were 3.5 and 50 °C. Both rCaCels were active at a broad range of pH values and temperatures, and retained more than 30 % of their maximal activity at 0 °C. Oligosaccharide structural analysis revealed that Bm-CaCel contains elaborated N- and O-linked glycans, whereas Ec-CaCel contains putative O-linked glycans. Thermostability of Bm-CaCel from B. mori at 60 °C was higher than that from E. coli, probably due to glycosylation.     

Isolation and Characterization of β-1,4- Endoxylanase from Trichoderma pseudokonigi

13-1,4-endoxylanase from Triehoderma pseudokonigi Rifai has been purified by anion-exchange chromatography on DEAE-Sephadex A50, DEAE-Sepharose CL-6B and mono Q. The endoxylanase was shown to be homogeneous by Native-PAGE and SDS-PAGE. This endoxylanase is a single-peptide chain protein with a molecular weight estimated as 66 kD. The endoxylanase was purified by 10-fold with a specific activity of 15.87 U·mg－1 Optimum endoxylanase activity was obtained when the enzyme was incubated at pH 4.5, 55 ℃ with a Km of 20 mg/mL and Vmax of 3.3 μmol·min－1·mg－1. Hg2 + and Cu2 + have a strong inhibition while Fe2 + and Mn2 + have a increasing effect on the enzymatic reaction rate.     

Isolation and Characterization of Novel β-Cyanoalanine Synthase and Cysteine Synthase Genes from Cassava

Cassava is an important staple food crop, feeding 600 million people worldwide, which produce cyanogenic glycosides. Cyanogenic glycosides in cassava are known to act as a deterrent for herbivores as well as serve as a mobile source of reduced nitrogen. Cassava is also equipped with a cyanide detoxification pathway, mediated by β-cyanoalanine synthase (β-CAS) which converts cyanide into asparagine. β-CAS, belonging to the Bsas family of enzymes, is multi functional and shares sequence homology with cysteine synthase (CS). Using rapid amplification of cDNA end-polymerase chain reaction (RACE-PCR), two cDNA sequences were isolated from cassava. The two clones named MANes;BsasA (accession no. EU350583) and MANes;BsasB (accession no. HQ257219), showed high homology to known β-CAS enzymes (80% and 75% amino acid similarity to Arabidopsis and 76% and 82% similarity to spinach, respectively). The kinetic properties of the two clones were characterized in a Escherichia coli NK3 mutant strain which lacks activity for any of the Bsas proteins. Kinetic studies showed that MANes;BsasB is a β-CAS with a CAS/CS activity ratio of 72 while MANes;BsasA is a CS showing bifunctional capabilities and with a CAS/CS activity ratio of 11. The isolation of cassava β-CAS and CS genes reported here paves the way for their utilization in genetically enhancing the cyanide detoxification potential of cassava and/or increase of the essential amino acid cysteine, which has been found to be low in nutritionally compromised individuals.     

Isolation and Characterization of Mutations in the β-Tubulin Gene of SACCHAROMYCES CEREVISIAE

Of 173 mutants of Saccharomyces cerevisiae resistant to the antimitotic drug benomyl (BenR), six also conferred cold-sensitivity for growth and three others conferred temperature-sensitivity for growth in the absence of benomyl. All of the benR mutations tested, including the nine conditional-lethal mutations, were shown to be in the same gene. This gene, TUB2, has previously been molecularly cloned and identified as the yeast structural gene encoding beta-tubulin. Four of the conditional-lethal alleles of TUB2 were mapped to particular restriction fragments within the gene. One of these mutations was cloned and sequenced, revealing a single amino acid change, from arginine to histidine at amino acid position 241, which is responsible for both the BenR and the cold-sensitive lethal phenotypes. The terminal arrest morphology of conditional-lethal alleles of TUB2 at their restrictive temperature showed a characteristic cell-division-cycle defect, suggesting a requirement for tubulin function primarily in mitosis during the vegetative growth cycle. The TUB2 gene was genetically mapped to the distal left arm of chromosome VI, very near the actin gene, ACT1; no CDC (cell-division-cycle) loci have been mapped previously to this location. TUB2 is thus the first cell-division-cycle gene known to encode a cytoskeletal protein that has been identified in S. cerevisiae.     

Isolation and Characterization of a β-Glucosidase from a Clavispora Strain with Potential Applications in Bioethanol Production from Cellulosic Materials

We previously reported on a new yeast strain of Clavispora sp. NRRL Y-50464 that is capable of utilizing cellobiose as sole source of carbon and energy by producing sufficient native β-glucosidase enzyme activity without further enzyme supplementation for cellulosic ethanol production using simultaneous saccharification and fermentation. Eliminating the addition of external β-glucosidase reduces the cost of cellulosic ethanol production. In this study, we present results on the isolation and identification of a β-glucosidase protein from strain Y-50464. Using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and blast search of the NCBInr database (National Center for Biotechnology Information nonredundant), the protein from Y-50464 was identified as a β-glucosidase (BGL1) with a molecular weight of 93.3 kDa. The BGL1 protein was purified through multiple chromatographic steps to a 26-fold purity (K _m?=?0.355 mM [pNPG]; K _i?=?15.2 mM [glucose]), which has a specific activity of 18.4 U/mg of protein with an optimal performance temperature at 45 °C and pH of 6.0. This protein appears to be intracellular although other forms of the enzyme may exist. The fast growth rate of Y-50464 and its capability to produce sufficient β-glucosidase activity for ethanol conversion from cellobiose provide a promising means for low-cost cellulosic ethanol production through a consolidated bioprocessing development.     

Isolation and Characterization of Polycyclic Aromatic Hydrocarbon–Degrading Mycobacterium Isolates from Soil

Bioremediation of soils contaminated with wood preservatives containing polycyclic aromatic hydrocarbons (PAHs) is desired because of their toxic, mutagenic, and carcinogenic properties. Creosote wood preservative–contaminated soils at the Champion International Superfund Site in Libby, Montana currently undergo bioremediation in a prepared-bed land treatment unit (LTU) process. Microbes isolated from these LTU soils rapidly mineralized the ¹⁴C-labeled PAH pyrene in the LTU soil. Gram staining, electron microscopy, and 16S rDNA-sequencing revealed that three of these bacteria, JLS, KMS, and MCS, were Mycobacterium strains. The phylogeny of the 16S rDNA showed that they were distinct from other Mycobacterium isolates with PAH-degrading activities. Catalase and superoxide dismutase (SOD) isozyme profiles confirmed that each isolate was distinct from each other and from the PAH-degrading mycobacterium, Mycobacterium vanbaalenii sp. nov, isolated from a petroleum-contaminated soil. We find that dioxygenase genes nidA and nidB are present in each of the Libby Mycobacterium isolates and are adjacent to each other in the sequence nidB-nidA, an order that is unique to the PAH-degrading mycobacteria.This revised version was published online in November 2004 with corrections to Volume 48.     

Isolation and Characterization of Photosystem Ⅱ of Porphyra yezoensis Ueda

The thylakoid membranes were isolated and purified from gametophyte of Porphyra yezoensis Ueda (P. yezoensis) by sucrose density gradient ultracentrifugation. After P. yezoensis gametophyte thylakoid membranes were solubilized with SDS, the photosystem Ⅱ (PSⅡ) particles were isolated and purified. The activity of PSⅡ     

Synthesis of Novel Heterobranched β-Cyclodextrins from α-D-Mannosyl- Maltotriose and β-Cyclodextrin by the Reverse Action of Pullulanase,and Isolation and Characterization of the Products

α-D-Mannosyl-maltotriose (Man-G3) were synthesized from methyl α-mannoside and maltotriose by the transfer action of α-mannosidase. (Man-G3)-βCD and (Man-G3)₂-βCD were produced in about 20% and 4% yield, respectively when Aerobacter aerogenes pullulanase (160 units per 1 g of Man-G3) was incubated with the mixture of 1.6 M Man-G3 and 0.16 M βCD at 50°C for 4 days. The reaction products, (Man-G3)-βCD were separated to three peaks by HPLC analysis on a YMC-PACK A-323-3 column and (Man-G3)₂-βCD were separated to several peaks by HPLC analysis on a Daisopak ODS column. The major product of (Man-G3)-βCDs was identified as 6-O-α-(6³-O-α-D-mannosyl-maltotriosyl)-βCD by FAB-MS and NMR spectroscopies. The structures of (Man-G3)₂-βCDs were analyzed by TOF-MS and NMR spectroscopies, and confirmed by comparison of elution profiles of their hydrolyzates by α-mannosidase and glucoamylase on a graphitized carbon column with those of the authentic di-glucosyl-βCDs. The structures of three main components of (Man-G3)₂-βCDs were identified as 6¹,6²-, 6¹,6³- and 6¹,6⁴-di-O-(6³-O-α-D-mannosyl-maltotriosyl)-βCD.     

Characterization of α-gliadin genes from diploid wheats and the comparative analysis with those from polyploid wheats

To carry out comparative analysis of the α-gliadin genes on A genomes of diploid and polyploid wheats, 8 full-length α-gliadin genes, including 3 functional genes and 5 pseudogenes, were obtained from diploid wheats, among which 2, 2 and 4 α-gliadin genes were isolated from T. urartu, T. monococcum, and T. boeoticum, respectively. The results indicated that higher number of α-gliadin pseudogenes have been present in diploid wheats before the formation of polyploid wheats. Amino acid sequence comparative analysis among 26 α-gliadin genes, including 16 functional genes and 10 pseudogenes, from diploid and polyploid wheats was conducted. The results indicated that all α-gliadins contained four coeliac toxic peptide sequences (i.e., PSQQ, QQQP, QQPY, and QPYP). The polyglutamine domains are highly variable, and the second polyglutamine stretch is usually disrupted by the lysine or arginine residue at the fourth position. The unique domain I is the most conserved domain. There are four and two conserved cysteine residues in the unique domains I and II, respectively. Comparative analysis indicated that the functional α-gliadin genes from A genome are highly conserved, whereas the identity of pseudogenes in diploid wheats are higher than those in hexaploid wheats. Phylogenetic analysis indicated that all the analyzed functional α-gliadin genes could be clustered into two major groups, among which one group could be further divided into 5 subgroups. The origin of α-gliadin pseudogene and functional genes were also discussed. The text was submitted by the authors in English.     

Isolation and characterization of 17β-hydroxy steroid dehydrogenase from human erythrocytes

1. The 17beta-hydroxy steroid dehydrogenase was solubilized during haemolysis of erythrocytes and was isolated from the membrane-free haemolysate. Membrane preparations isolated in different ways did not contain 17beta-hydroxy steroid dehydrogenase activity. The 17beta-hydroxy steroid dehydrogenase activity in the haemolysate was concentrated by repeated ammonium sulphate precipitation and gel filtration on Sephadex G-150. The 17beta-hydroxy steroid dehydrogenase activity of the purified preparation per unit weight of protein was 350-3000 times higher than the activity of the crude erythrocyte haemolysate. The 20alpha-hydroxy steroid dehydrogenase activity was lost during this purification procedure. 2. The 17beta-hydroxy steroid dehydrogenase was NADP-dependent and had a pH optimum for conversion of testosterone between 8.5 and 10. For the molecular weight of the enzyme a value of 64000 was calculated from Sephadex chromatography results. 3. p-Chloromercuribenzoate inhibited the enzymic activity. The oxidative activity of the enzyme for the 17beta-hydroxyl group was only partly inhibited when a large excess of 17-oxo steroids was added. The catalysing activity of the enzyme was influenced by the NADP(+)/NADPH ratio. The oxidation of the 17beta-hydroxyl group in the presence of NADP(+) proceeded faster than the reduction of the 17-oxo group with NADPH. When both reduced and oxidized cofactors were present the oxidation of the 17beta-hydroxyl group was inhibited to a considerable extent. 4. The enzyme had a broad substrate specificity and not only catalysed the conversion of androstanes with a 17beta-hydroxyl group, or 17-oxo group, but also the conversion oestradiolleft arrow over right arrowoestrone. In addition the steroid conjugates dehydroepiandrosterone sulphate and oestrone sulphate were also converted. There were no indications that more than one 17beta-hydroxy steroid dehydrogenase was present in the partially purified preparation.     

Purification and Some properties of Endo-β-1,6-Glucanase from Acinetobacter sp.

A kind of endo-β-1, 6-glucanase has been purified from the culture filtrate of Acinetobacter sp. grown in the medium containing baker’s yeast cells as a carbon source. A 100-fold purified preparation was obtained by DEAE-Sephadex A–50 column chromatography. The enzyme hydrolyzed pustulan giving a series of gentio-oligosaccharides and glucose. Gentiotriose and gentiotetraose were hydrolyzed by this enzyme yielding glucose and gentiobiose, and glucose, gentiobiose and gentiotriose, respectively. Gentiobiose was not hydrolyzed. Baker’s yeast glucans obtained from the isolated cell walls were also hydrolyzed by this enzyme giving a series of oligosaccharides and glucose. From the action patterns on these carbohydrates, we concluded the present enzyme being endo-β-1, 6-glucanase.     

Purification and Some Properties of an Endo-β,1,6-glucanase from Neurospora crassa

An endo-β-1,6-glucanase (E.C. 3.2.1.75) was purified from the culture filtrate of Neurospora crassa IFO-6O68 by chromatographies on CM-cellulofine, Con-A Sepharose 4B, and Sepharose Cl-6B followed by preparative affinity gel electrophoresis. The purified enzyme had an apparent molecular weight of 47,000. The pH and temperature optima for the activity were 5.0 and 50°C. The enzyme acted on β-1,6-glucan (Pustulan) and yielded a series of gentio-oligosaccharides with endo- type action, and finally, glucose and gentiobiose were produced. The enzyme was also able to act on N. crassa cell wall β-glucan, and a small amount of hydrolysis fragments were liberated without apparent change of the cell wall glucan molecules.