Biochemical and molecular characterization of secreted α-xylosidase from Aspergillus niger

α-Linked xylose is a major component of xyloglucans in the cell walls of higher plants. An α-xylosidase (AxlA) was purified from a commercial enzyme preparation from Aspergillus niger, and the encoding gene was identified. The protein is a member of glycosyl hydrolase family 31. It was active on p-nitrophenyl-α-d-xyloside, isoprimeverose, xyloglucan heptasaccharide (XXXG), and tamarind xyloglucan. When expressed in Pichia pastoris, AxlA had activity comparable to the native enzyme on pNPαX and IP despite apparent hyperglycosylation. The pH optimum of AxlA was between 3.0 and 4.0. AxlA together with β-glucosidase depolymerized xyloglucan heptasaccharide. A combination of AxlA, β-glucosidase, xyloglucanase, and β-galactosidase in the optimal proportions of 51:5:19:25 or 59:5:11:25 could completely depolymerize tamarind XG to free Glc or Xyl, respectively. To the best of our knowledge, this is the first characterization of a secreted microbial α-xylosidase. Secreted α-xylosidases appear to be rare in nature, being absent from other tested commercial enzyme mixtures and from the genomes of most filamentous fungi.     

Biochemical and genetic characterization of 5-fluoro-2′-deoxyuridine-resistant mutants of Arabidopsis thaliana

Two independently isolated 5-fluoro-2-deoxyuridine (FUdR)-resistant mutant lines of Arabidopsis thaliana (L.) Heynh., FUD-1 and FUD-2, were identified by screening M2 populations of ethylmethane-sulfonatemutagenized seeds. The resistance was found to be due to single, recessive, nuclear gene mutations. Genetic complementation tests indicated that these two mutations were in the same gene locus, which was designated fur1, and mapped to linkage group four of Arabidopsis. Enzyme assays indicated that the mutants were not defective in thymidine-kinase activity. Greatly reduced concentrations of intracellular ³H were detected in fur1/fur1 plants compared with the wild type after incubation of wild-type and resistant plants in a medium with [³H]FUdR, indicating that either reduced uptake of FUdR or enhanced efflux of FUdR metabolites was the major reason for FUdR-resistance. fur1/fur1 plants also had significantly decreased uptake of thymidine and uridine compared with the wild type but no difference was found in the uptake of adenosine, guanosine, thymine, uracil or amino acids. It is suggested that the transport system affected in the fur1/fur1 mutants is one specific to pyrimidine nucleosides.Abbreviations BUdR 5-bromodeoxyuridine - FdUMP 5-fluoro-2-deoxyuridine monophosphate - FUdR 5-fluoro-2-deoxyuridine - FUR fluorouridine - TK thymidine kinase - TS thymidylate synthetase We thank Dr. George W. Haughn (Department of Biology, University of Saskatchewan) for providing Arabidopsis line W100 and Dr. George Mourad (Department of Biology, University of Saskatchewan) for help and advice. This work was supported by a Research Grant from the Natural Sciences and Engineering Research Council of Canada to J.K. K.W. is grateful for a University of Saskatchewan Graduate Scholarship.     

Selection and characterization of in vitro–induced mutants of Dendrobium ‘Earsakul’ resistant to black rot

In Vitro Cellular & Developmental Biology - Plant - The development of Dendrobium ‘Earsakul’ with improved resistance to black rot is essential for sustainable orchid production. In...     

Thiolutin resistant mutants of Escherichia coli are they RNA chain initiation mutants?

Summary Four mutants of Escherichia coli KL16 resistant to the antibiotic Thiolutin have been isolated. This drug was earlier reported to be an inhibitor of RNA chain elongation. The first mutant, TL^rI, is resistant only in rich or partially rich media: it can, however, grow in minimal medium containing the drug with a very long doubling time. The other mutants TL^rII, TL^rIIIa and TL^rIIIb are resistant in rich as well as minimal media. -galactosidase could not be induced in TL^rI and TL^rII in the presence of thiolutin whereas the enzyme is constitutively synthesised in TL^rIIIa and TL^rIIIb irrespective of the drug.The mutants do not support the development of phage T4 in presence of the drug, if the drug is added along with the phage, but escape the inhibition if phage development is allowed to proceed for some time before the addition of the drug. The time of this escape is characteristic of the mutant. Even in a sensitive strain, T7 growth escapes inhibition very soon after infection, around the time the phage-specific RNA polymerase is synthesised. In the parent strain the kinetics of inhibition of -galactosidase induction resembles more the inhibition caused by rifampicin than by streptolydigin. It is proposed that thiolutin could be an inhibitor of RNA chain initiation and resistance might be due to mutation in the subunit(s)/factor(s) involved in initiation.     

Purification and characterization of a β-glucosidase fromAspergillus niger

The high-molar mass from of β-glucosidase fromAspergillus niger strain NIAB280 was purified to homogeneity with a 46-fold increase in purification by a combination of ammonium sulfate precipitation, hydrophobic interaction, ion-exchange and gel-filtration chromatography. The native and subunit molar mass was 330 and 110 kDa, respectively. The pH and temperature optima were 4.6–5.3 and 70°C, respectively. TheK _m andk _cat for 4-nitrophenyl β-d-glucopyranoside at 40°C and pH 5 were 1.11 mmol/L and 4000/min, respectively. The enzyme was activated by low and inhibited by high concentrations of NaCl. Ammonium sulfate inhibited the enzyme. Thermolysin periodically inhibited and activated the enzyme during the course of reaction and after 150 min of proteinase treatment only 10% activity was lost with concomitant degradation of the enzyme into ten low-molar-mass active bands. When subjected to 0–9 mol/L transverse urea-gradient-PAGE for 105 min at 12°C, the nonpurified β-glucosidase showed two major bands which denatured at 4 and 8 mol/L urea, respectively, with half-lives of 73 min.     

Biochemical characterization of the β-chain variant haptoglobin Marburg

Summary -chains were isolated from two individuals heterozygous for the -chain mutant haptoglobin Marburg. Total amino acid composition and tryptic peptides were compared with -chains from common haptoglobin types. ^Mb chain preparations are characterized by the presence of -chains with an atypical electrophoretic migration rate and by at least three, possibly four additional peptides in their tryptic digests. It is probable that haptoglobin Marburg is the result of an mutational event other than a single base substitution.Supported by US-PHS grant AM 11796 and by US-PHS grant HD 03321 and aided by a grant from the Deutsche Forschungsgemeinschaft.     

Properties of β-glucosidase purified from Aspergillus niger mutants USDB 0827 and USDB 0828

Summary Two extracellular -glucosidases (EC 3.2.1.21) were isolated from Aspergillus niger USDB 0827 and A. niger USDB 0828, and their physical and kinetic properties studied. Both enzymes were very similar in terms of molecular size (230000 Da), pH optimum (pH 4.6), temperature optimum (65° C), stability at high temperatures and substrate preferences. They were capable of hydrolysing -linked disaccharides, phenyl -d-glucoside, p-nitrophenyl -d-glucoside (PNPG), o-nitrophenyl -d-glucoside, salicin and methyl -d-glucoside but lacked activity towards -linked disaccharides, a range of p-nitrophenyl monoglycosides and p-nitrophenyl diglycosides. Both -glucosidases were better at hydrolysing cellobiose than cellotriose, cellotetraose or cellopentaose. For both enzymes, glucose showed competitive inhibition with PNPG as substrate but had no effect with cellobiose. However, the two -glucosidases differed in inhibition by glucono-1,5-lactone and affinity for cellobiose. -Glucosidase from A. niger USDB 0827 also gave lower specific activity, and was more susceptible to metal ions (Ag⁺, Fe²⁺ and Fe³⁺) inhibition than that of A. niger USDB 0828. Correspondence to: Y. K. Hoh     

Biochemical and biophysical characterization of unique switch pocket inhibitors of p38α

Herein we describe the identification and characterization of a class of molecules that are believed to extend into a region of p38 known as the ‘switch pocket’. Although these molecules lack a canonical hinge binding motif, they show K_i values as low as 100 nM against p38. We show that molecules that interact with this region of the protein demonstrate different binding kinetics than a canonical ATP mimetic, as well as a wide range of kinome profiles. Thus, the switch pocket presents new opportunities for kinome selectivity which could result in unique biochemical responses and offer new opportunities in the field of kinase drug discovery.     

Isolation and characterization of deletion mutants affected in early genes of bacteriophage ?80

Summary When Escherichia coli cells that had been irradiated with ultraviolet light were infected with bacteriophage 80, five major (pE, pB, pA, pC and pD) and two minor (pU and pV) proteins were found to be synthesized during early stages of infection. The genss coding for the five major proteins were mapped on the 80 chromosome using various deletion mutants which lacked the capacity to synthesize some or all the major proteins. The size and positions of all the deletions were determined by gel electrophoresis of EcoRI digests of phage DNA and by electron microscopy of heteroduplexes between DNAs of the deletion and wild-type phage. The five major proteins designated pE(25K), pB(40K), pA(45K), pC(34K) and pD(31K) were shown to be encoded in this order presumably by a single operon that was located at 60.2–67.4% on the 80 genome. These proteins were found to be involved in phage recombination. The absence of pE or pB resulted in a Red^– phenotype and the absence of three proteins (pE, pB and pA) resulted in a Fec^– phenotype. The exact positions of the genes for the minor proteins pU(29K) and pV(26K) have not been determined.     

Replication mutants of the F-plasmid of Escherichia coli K-12: I. Isolation and characterization of temperature-sensitive replication mutants of F′-gal+

Eighteen mutants that are temperature-sensitive for vegetative replication (rep) were isolated from two F′-gal⁺ plasmids (F8 and F8-4) after N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis. Some of the mutants also have reduced transfer ability at both permissive and nonpermissive temperatures. Plasmid-plasmid P1 transduction has revealed that in some instances, the altered transfer ability is located in the transfer operon and is distinct from the rep mutation. However, in other cases, the replication and transfer defects have not been separated by P1 transduction. The implications of these results for the relationship between vegetative DNA replication and DNA replication during conjugation are discussed. In vivo recombinational results suggested that the temperature-sensitive mutations were not located in the same regions of the two F′-plasmids. We confirmed that no inversion, secondary deletion, or translocation of DNA had occurred in either F8 or F8-4, and suggest that the apparent difference is due to a recombination anomaly.     

Biochemical characterization of mIgM− variants of the murine B-cell lymphoma,WEHI 279.1

The membrane immunoglobulin M (m1gM) of a B lymphocyte serves as a receptor for its cognate antigen. Our aim is to elucidate the structure and function of this membrane-bound receptor. The first step is to determine the requirements for proper membrane placement of IgM. We have used mIgM-positive B lymphocyte tumors from which we isolated mIgM negative variants by immunoselection. We report here the initial characterization of mIgM-variants isolated by repeated cycles of selection of the murine B lymphoma, WEHI 279.1, with goat anti-mouse immunoglobulin (GMIg) and complement. These particular variants were chosen from a pool of more than 150 variants originally isolated because they resulted from several selection schemes and clearly had different origins. By analysis of their proteins, we have found three major phenotypes that do not produce mIgM: reduced _m, _s and L chain levels within cells, loss of _m and _s but retention of L chain synthesis, and loss of _m but retention of reduced amounts of _s and L chain. The defects underlying these phenotypes produce complex changes in the synthesis, turnover, and secretion of the or L chains involved. We performed experiments comparing the effects of the glycosylation inhibitor tunicamycin on variants with reduced p and L levels with its effects on variants with L but no chains. These experiments suggested that and L chain synthesis are controlled coordinately at the level of protein synthesis. We have not yet isolated any variants lacking L chain synthesis or any appearing to have gross structural defects in the _s protein. This analysis is the first phase of the detailed characterization of the requirements for proper synthesis, processing, tetramer formation, and membrane display of mIgM on B lymphoma tumors in mice.     

Biochemical characterization of Magnaporthe oryzae β-glucosidases for efficient β-glucan hydrolysis

β-Glucosidases designated MoCel3A and MoCel3B were successfully overexpressed in Magnaporthe oryzae. MoCel3A and MoCel3B showed optimal activity at 50 °C and pH 5.0–5.5. MoCel3A exhibited higher activity on higher degree of polymerization (DP) oligosaccharides and on β-1,3-linked oligosaccharides than on β-1,4-linked oligosaccharides. Furthermore, MoCel3A could liberate glucose from polysaccharides such as laminarin, 1,3-1,4-β-glucan, phosphoric acid-swollen cellulose, and pustulan, of which laminarin was the most suitable substrate. Conversely, MoCel3B preferentially hydrolyzed lower DP oligosaccharides such as cellobiose, cellotriose, and laminaribiose. Furthermore, the synergistic effects of combining enzymes including MoCel3A and MoCel3B were investigated. Depolymerization of 1,3-1,4-β-glucan by M. oryzae cellobiohydrolase (MoCel6A) enhanced the production of glucose by the actions of MoCel3A and MoCel3B. In these reactions, MoCel3A hydrolyzed higher DP oligosaccharides, resulting in the release of glucose and cellobiose, and MoCel3B preferentially hydrolyzed lower DP oligosaccharides including cellobiose. On the other hand, MoCel3A alone produced glucose from laminarin at levels equivalent to 80% of maximal hydrolysis obtained by the combined action of MoCel3A, MoCel3B, and endo-1,3-β-glucanase. Therefore, MoCel3A and MoCel3B activities yield glucose from not only cellulosic materials but also hemicellulosic polysaccharides.     

Application of RAPD markers for characterization of γ-ray-induced rose mutants and assessment of genetic diversity

Six parent and their 12 gamma ray-induced somatic flower colour mutants of garden rose were characterized to discriminate the mutants from their respective parents and understanding the genetic diversity using Random amplification of polymorphic DNA (RAPD) markers. Out of 20 primers screened, 14 primers yielded completely identical fragments patterns. The other 7 primers gave highly polymorphic banding patterns among the radiomutants. All the cultivars were identified by using only 7 primers. Moreover, individual mutants were also distinguished by unique RAPD marker bands. Based on the presence or absence of the 48 polymorphic bands, the genetic variations within and among the 18 cultivars were measured. Genetic distance between all 18 cultivars varied from 0.40 to 0.91, as revealed by Jaccard’s coefficient matrix. A dendrogram was constructed based on the similarity matrix using the Neighbor Joining Tree method showed three main clusters. The present RAPD analysis can be used not only for estimating genetic diversity present in gamma ray-induced mutants but also for correct identification of mutant/new varieties for their legal protection under plant variety rights.     

Biochemical characterization of Silene alba α4-fucosyltransferase and Lewis a products

1,4-Fucosylation has been recently detected in Arabidopsis thaliana [Léonard et al. (2002), Glycobiology 12: 299–306], and corresponding enzymes have also been characterized in Beta vulgaris [Bakker et al. (2001), FEBS Lett, 507: 307–312], and Lycopersicum aesculentum [Wilson (2001), Glycoconjugate J., 18: 439–447]. Here we demonstrated fucosyltransferase activity (FucT) in Silene alba cells and tissues. The Fuc linkage to GlcNAc residues of the lactosamine moiety of the Type I acceptor was confirmed by mass spectrometry experiments. Le^a-glycoconjugates are found in the Golgi apparatus and plasma membrane of plant cells. In planta, the highest levels of activity were detected in seedlings, young roots and male flowers. The enzyme was stable up to 45C and the optimum pH of reaction was 8.0. The enzyme required Mg²⁺ or Mn²⁺ for activity and was inhibited by Zn²⁺ and ethylenediaminetetraacetic acid. Chemical modification of the enzyme with group-selective reagents revealed that selective modifications of arginine and lysine residues had no effect on enzyme activity. However the enzyme contains histidine and tryptophan residues that are essential for its activity. In contrast to human FUT3, the S. alba 4-FucT was insensitive to N-ethylmaleimide (NEM) treatment. Measurement of enzyme activity in S. alba cell fractions indicated that the enzyme is bound to microsomal membranes, furthermore a soluble isoform of the protein may be present. Published in 2005.     

Biochemical characterization of two novel β-glucosidase genes by metagenome expression cloning

Two novel β-glucosidase genes designated as bgl1D and bgl1E, which encode 172- and 151-aa peptides, respectively, were cloned by function-based screening of a metagenomic library from uncultured soil microorganisms. Sequence analyses indicated that Bgl1D and Bgl1E exhibited lower similarities with some putative β-glucosidases. Functional characterization through high-performance liquid chromatography demonstrated that purified recombinant Bgl1D and Bgl1E proteins hydrolyzed D-glucosyl-β-(1-4)-D-glucose to glucose. Using p-nitrophenyl-β-D-glucoside as substrate, K(m) was 0.54 and 2.11 mM, and k(cat)/K(m) was 1489 and 787 mM(-1) min(-1) for Bgl1D and Bgl1E, respectively. The optimum pH and temperature for Bgl1D was pH 10.0 and 30°C, while the optimum values for Bgl1E were pH 10.0 and 25°C. Bgl1D exhibited habitat-specific characteristics, including higher activity in lower temperature and at high concentrations of AlCl(3) and LiCl. Bgl1D also displayed remarkable activity across a broad pH range (5.5-10.5), making it a potential candidate for industrial applications.     

Biochemical characterization of recombinant β-glucosyltransferase and analysis of global 5-hydroxymethylcytosine in unique genomes

5-Hydroxymethylcytosine (5-hmC) is an enzymatic oxidative product of 5-methylcytosine (5-mC). The Ten Eleven Translocation (TET) family of enzymes catalyze the conversion of 5-mC to 5-hmC. Phage-encoded glucosyltransferases are known to glucosylate 5-hmC, which can be utilized to detect and analyze the 5-hmC as an epigenetic mark in the mammalian epigenome. Here we have performed a detailed biochemical characterization and steady-state kinetic parameter analysis of T4 phage β-glucosyltransferase (β-GT). Recombinant β-GT glucosylates 5-hmC DNA in a nonprocessive manner, and binding to either 5-hmC DNA or uridine diphosphoglucose (UDP-glucose) substrates is random, with both binary complexes being catalytically competent. Product inhibition studies with β-GT demonstrated that UDP is a competitive inhibitor with respect to UDP-glucose and a mixed inhibitor with respect to 5-hmC DNA. Similarly, the glucosylated-5-hmC (5-ghmC) DNA is a competitive inhibitor with respect to 5-hmC DNA and mixed inhibitor with respect to UDP-glucose. 5-hmC DNA binds ~10 fold stronger to the β-GT enzyme when compared to its glucosylated product. The numbers of 5-hmC on target sequences influenced the turnover numbers for recombinant β-GT. Furthermore, we have utilized recombinant β-GT to estimate global 5-hmC content in a variety of genomic DNAs. Most of the genomic DNAs derived from vertebrate tissue and cell lines contained 5-hmC. DNA from mouse, human, and bovine brains displayed 0.5-0.9% of the total nucleotides as 5-hmC, which was higher compared to the levels found in other tissues. A comparison between cancer and healthy tissue genomes suggested a lower percentage of 5-hmC in cancer, which may reflect the global hypomethylation of 5-mC observed during oncogenesis.     

Biochemical analysis of related,independently arising histocompatibility mutants: bm17 and KB-98 enlarge the “bg series” of H-2Kb mutants

The “bg” series of MHC mutations is the most prevalent type of mutations of K^b in C57BL/6 mice screened by reciprocal tail skin grafting. The basis for identification of this series of mutations is the incompatibility of grafts between the parental B6 and the mutant. This series takes the longest to reciprocally reject the skin grafts. The series can be subdivided into “bg 1” and “bg 2” groups based on K^b-restricted recognition of virus-infected mutant target cells. The biochemical basis for these mutations are amino acid substitutions at residues 116 and 121 of the K^b transplantation antigen. These substitutions do not alter monoclonal antibody binding sites. The structural basis of MAb binding and the genetic basis of the mutation are discussed. This study was supported in part by USPHS Grants AI-07289, AI-10702, NCI P30-CA-13330, American Cancer Society Grant IM-236, and American Cancer Society Fellowship PF-2126. Stanley G. Nathenson is a member of the Irvington House Institute for Medical Research.     

Biochemical confirmation and characterization of the family-57-like α-amylase ofMethanococcus jannaschii

The gene encoding a family-57-like α-amylase in the hyperthermophilic archaeonMethanococcus jannaschii, has been cloned intoEscherichia coli. Extremely thermoactive α-amylase was confirmed in partially purified enzyme solution of the recombinant culture. This enzyme activity had a temperature optimum of 120°C and a pH optimum 5.0–8.0. The amylase activity is extremely stable against denaturants. Hydrolysis of large sugar polymers with α-1–6 and α-1–4 linkages yields products including glucose polymers of 1–7 units. Highest activity is exhibited on amylose. The catalyst exhibited a half-life of 50 h at 100°C, among the highest reported thermostabilities of natural amylases.