Resumo A α-amilase secretada, com atividade dominante, foi purificada a partir do extrato bruto de Mon Thong durian através de passos de precipitação utilizando sulfato de amônia e cromatografia de afinidade. A α-amilase purificada (DzAmy1) tem uma massa molecular de aproximadamente 44 kDa. O pH e temperatura ótimos para a atividade foram 7.0 e 50°C, respectivamente. A enzima ficou estável no pH entre 6 a 10 e temperatura entre 30 a 60°C. Vários íons metais não afetaram a atividade amilásica. A clonagem do gene DzAmy1 foi feita e confirmou que esse gene consiste em 1.254 bp de região codificante, na qual se codificam 23 aminoácidos de peptídeo sinal e 395 aminoácidos da proteína madura com massa molecular calculada de 43.7 kDa. O ponto isoelétrico da enzima é de 5.78. A DzAmy1 pertence a subfamília das α-amilases de planta, de acordo com dados de filogenia. A caracterização estrutural através de modelagem por homologia sugeriu que a DzAmyl consiste em 3 domínios, com sua tríade catalítica presente no domínio A. A DzAmy1 recombinante (rDzAmy1) foi expressa em Escherichia coli com sucesso e teve sua atividade de hidrólise de amido e etilideno-pNP-G7, as quais claramente confirmaram a autenticidade da DzAmy1 como uma α-amilase funcional.

Abstract The secreted α-amylase with dominant activity was purified from the crude extract of Mon Thong durian by steps of ammonium sulphate precipitation and the affinity column chromatography. The purified α-amylase (DzAmy1) had a molecular mass of approximately 44 kDa. Its optimum pH and temperature for activity were 7.0 and 50°C, respectively. The enzyme was stable from pH 6 to 10 and from 30 to 60°C. Many metal ions did not affect amylase activity. The gene cloning of DzAmy1 was carried out and it was confirmed that DzAmy1 gene consisted of 1,254 bp open reading frame, which encoded 23 amino acids of the signal peptide and 395 amino acids of mature protein with a calculated molecular mass of 43.7 kDa. The isoelectric point of the enzyme was 5.78. DzAmy1 was shown to belong to sub-family one of the plant α-amylases based on phylogenetic tree analysis. Structural characterization by homology modelling suggested that it consisted of 3 domains with a catalytic triad in domain A. Recombinant DzAmy1 (rDzAmy1) was successfully expressed in Escherichia coli and had hydrolysis activity for starch and ethylidene-pNP-G7, which was clearly confirmed the authenticity of DzAmy1 as a functional α-amylase.

Abstract Background: The domestic cat serves as an animal model for assisted reproductive studies of endangered felid species. To date, there are no data on the protein alterations following cryopreservation of oocytes in felid family. Methods: Immature (germinal vesicle) domestic cat oocytes were vitrified in the vitrification solution containing 35% ethylene glycol, 20% DMSO and 0.5 mM sucrose. The vitrified-warmed oocytes were matured (metaphase II) in vitro and subjected to proteomic analysis using 1DE SDS-PAGE prefractionation combined with LC-MS/MS. Results: A total of 1712 proteins were identified in in vitro matured oocytes. Of the 1712 proteins, 1454 proteins were found in both groups, whereas, 258 proteins were differentially expressed between control and vitrified-warmed groups. In vitrified-warmed oocytes, the missing proteins were membrane and nuclear proteins; whereas, apoptosis and DNA repair proteins were overrepresented. Conclusions: The identified missing and overexpressed proteins in vitrified-warmed oocytes represent potential markers of cryoinjuries and the developmental pathways of oocytes. The findings of differential expressed proteins may contribute to effective ways of proteome analysis of oocyte/embryo quality in order to assess safety of cryopreservation in felid species.

Abstract Background: Wasp venom is a complex mixture containing proteins, enzymes and small molecules, including some of the most dangerous allergens. The greater banded wasp (Vespa tropica) is well-known for its lethal venom, whose one of the major components is a hyaluronidase (HAase). It is believed that the high protein proportion and activity of this enzyme is responsible for the venom potency. Methods: In the present study, cDNA cloning, sequencing and 3D-structure of Vespa tropica venom HAase were described. Anti-native HAase antibody was used for neutralization assay. Results: Two isoforms, VesT2a and VesT2b, were classified as members of the glycosidase hydrolase 56 family with high similarity (42–97 %) to the allergen venom HAase. VesT2a gene contained 1486 nucleotide residues encoding 357 amino acids whereas the VesT2b isoform consisted of 1411 residues encoding 356 amino acids. The mature VesT2a and VesT2b are similar in mass and pI after prediction. They are 39119.73 Da/pI 8.91 and 39571.5 Da/pI 9.38, respectively. Two catalytic residues in VesT2a, Asp107 and Glu109 were substituted in VesT2b by Asn, thus impeding enzymatic activity. The 3D-structure of the VesT2s isoform consisted of a central core (α/β)7 barrel and two disulfide bridges. The five putative glycosylation sites (Asn79, Asn99, Asn127, Asn187 and Asn325) of VesT2a and the three glycosylation sites (Asn1, Asn66 and Asn81) in VesT2b were predicted. An allergenic property significantly depends on the number of putative N-glycosylation sites. The anti-native HAase serum specifically recognized to venom HAase was able to neutralize toxicity of V. tropica venom. The ratio of venom antiserum was 1:12. Conclusions: The wasp venom allergy is known to cause life-threatening and fatal IgE-mediated anaphylactic reactions in allergic individuals. Structural analysis was a helpful tool for prediction of allergenic properties including their cross reactivity among the vespid HAase.