Characterization of a Hexameric Exo-Acting GH51 alpha-l-Arabinofuranosidase from the Mesophilic Bacillus subtilis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural basis for branching-enzyme activity of glycoside hydrolase family 57: Structure and stability studies of a novel branching enzyme from the hyperthermophilic archaeon Thermococcus Kodakaraensis KOD1

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aspergillus niger beta-Glucosidase Has a Cellulase-like Tadpole Molecular Shape INSIGHTS INTO GLYCOSIDE HYDROLASE FAMILY 3 (GH3) beta-GLUCOSIDASE STRUCTURE AND FUNCTION

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Understanding the Function of Conserved Variations in the Catalytic Loops of Fungal Glycoside Hydrolase Family 12

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

cDNA cloning and 1.75 angstrom crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 +/- 15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed beta(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-beta-D-glucopyranose units in chitin. The full-lengthamino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 angstrom resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (beta alpha)(8) barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182.

cDNA cloning and 1.75 Å crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 ± 15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed β(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-β-d-glucopyranose units in chitin. The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 Å resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (βα) 8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182. © 2006 The Authors.

Development and Biotechnological Application of a Novel Endoxylanase Family GH10 Identified from Sugarcane Soil Metagenome

Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45°C. The crystal structure was solved at 2.75 Å resolution, revealing the classical (β/α)8-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production. © 2013 Alvarez et al.

CYTOGENETIC SURVEY OF SOME BRAZILIAN CERAMBYCIDAE (COLEOPTERA, POLYPHAGA, CHRYSOMELIDAE)

Meioformula was determined in 16 species of the family Cerambycidae, seven of them of the subfamily Cerambycinae, eight of Lamiinae and one of Prioninae. Only two species of Cerambycinae and one of Lamiinae showed the basic Polypliaga karyotype (9 + Xy(p)) and the remaining ones showed occurrence of centric fusions and fissions that have modified the basic karyotypes. These results are in accordance with previous studies on other species of this family.

Hyaluronidase from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae): Cloning, structural modeling, purification, and immunological analysis

In this study, we describe the cDNA cloning, sequencing, and 3-D structure of the allergen hyaluronidase from Polybia paulista venom (Pp-Hyal). Using a proteomic approach, the native form of Pp-Hyal was purified to homogeneity and used to produce a Pp-specific polyclonal antibody. The results revealed that Pp-Hyal can be classified as a glycosyl hydrolase and that the full-length Pp-Hyal cDNA (1315 bp; GI: 302201582) is similar (80-90%) to hyaluronidase from the venoms of endemic Northern wasp species. The isolated mature protein is comprised of 338 amino acids, with a theoretical pI of 8.77 and a molecular mass of 39,648.8 Da versus a pI of 8.13 and 43,277.0 Da indicated by MS. The Pp-Hyal 3D-structural model revealed a central core (α/β)7 barrel, two sulfide bonds (Cys 19-308 and Cys 185-197), and three putative glycosylation sites (Asn79, Asn187, and Asn325), two of which are also found in the rVes v 2 protein. Based on the model, residues Ser299, Asp107, and Glu109 interact with the substrate and potential epitopes (five conformational and seven linear) located at surface-exposed regions of the structure. Purified native Pp-Hyal showed high similarity (97%) with hyaluronidase from Polistes annularis venom (Q9U6V9). Immunoblotting analysis confirmed the specificity of the Pp-Hyal-specific antibody as it recognized the Pp-Hyal protein in both the purified fraction and P. paulista crude venom. No reaction was observed with the venoms of Apis mellifera, Solenopsis invicta, Agelaia pallipes pallipes, and Polistes lanio lanio, with the exception of immune cross-reactivity with venoms of the genus Polybia (sericea and ignobilis). Our results demonstrate cross-reactivity only between wasp venoms from the genus Polybia. The absence of cross-reactivity between the venoms of wasps and bees observed here is important because it allows identification of the insect responsible for sensitization, or at least of the phylogenetically closest insect, in order to facilitate effective immunotherapy in allergic patients. © 2013 Elsevier Ltd.

Estudo do potencial enzimático hidrolítico e oxidativo do microorganismo Myceliophthora thermophila M.7.7

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

PRP8 intein in Ajellomycetaceae family pathogens: Sequence analysis, splicing evaluation and homing endonuclease activity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chromosome banding and 18S rDNA in situ hybridization analysis of seven species of the family Achiridae (Teleostei : Pleuronectiformes)

Achiridae is an important family of the order Pleuronectiformes widely distributed in North, Central, and South America with freshwater and marine species. In the present study cytogenetic analyses comprising conventional and molecular techniques were carried out in seven species of this family. The following diploid numbers (2n) and fundamental numbers (FN) were obtained: Achirus declivis 2n = 34, FN = 52; Achirus lineatus 2n = 40, FN = 66; Catathyridium jenynsi 2n = 40 and FN = 50; Gymnachirus nudus 2n = 36 and FN = 50; Hypoclinemus mentalis 2n = 38 and FN = 54; Trinectes paulistanus 2n = 42 and FN = 52; and Trinectes sp. 2n = 38 and FN = 54. All species presented a single nucleolar organizer region (NOR) bearing chromosome pair and C-band positive segments mainly distributed at the pericentromeric position. The wide variation observed in chromosome number and FN suggests the occurrence of larger chromosome rearrangements in the family Achiridae if compared with other families of the same order.

Isolation and characterization of a satellite DNA family in Achirus lineatus (Teleostei : Pleuronectiformes : Achiridae)

Agarose gels stained with Ethidium bromide and Southern blot experiments of HindIII-digested genomic DNA of Achirus lineatus evidenced the presence of monomers and multimers of a DNA segment of about 200 bp, named here Al-HindIII sequence. No signals were observed in Southern blot experiments with genomic DNA of other flatfish species. The DNA sequencing of four recombinant clones showed that Al-HindIII sequences had 204 bp and were 63.72% AT-rich. FISH experiments using a Al-HindIII sequence as probe showed bright signals in the centromeric position of all chromosomes of A. lineatus.

Molecular phylogeny of the armored catfish family Callichthyidae (Ostariophysi, Siluriformes)

The family Callichthyidae comprises eight genera of fishes widely distributed across the Neotropical region. In the present study, sequences of the mitochondrial genes 12S rRNA, 16S rRNA, ND4, tRNA(His), and tRNA(Scr) were obtained from 28 callichthyid specimens. The sample included 12 species of Corydoras, three species of Aspidoras, two species of Brochis, Dianema, Lepthoplosternum, and Megalechis, and two local populations of Callichthys and Hoplosternum. Sequences of Nematogenys inermis (Nematogenyidae), Trichomycterus areolatus, and Henonemus punctatus (Trichomycteridae), Astroblepus sp. (Astroblepidae), and Neopleeostomus paranensis, Delturus parahybae, and Hemipsilichthys nimius (Loricariidae) were included as the outgroup. Phylogenetic analyses were performed by using the methods of maximum parsimony and maximum likelihood. The results of almost all analyses were very similar. The family Callichthyidae is monophyletic and comprises two natural groups: the subfamilies Corydoradinae (Aspidoras, Brochis, and Corydoras) and Callichthyinae (Callichthys, Dianema, Hoplosternum, Lepthoplosternum, and Megalechis), as previously demonstrated by morphological studies. The relationships observed within these subfamilies are in several ways different from those previously proposed on the basis of morphological data. Molecular results were compared with the morphologic and cytogenetic data available on the family. (C) 2003 Elsevier B.V. All rights reserved.

Karyotype characterization of Mugil incilis Hancock, 1830 (Mugiliformes: Mugilidae), including a description of an unusual co-localization of major and minor ribosomal genes in the family

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)