Heterologous expression and enhanced production of ß-1, 4-glucanase of Bacillus halodurans C-125 in Escherichia coli

Background: Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-ß-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-ß-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results: A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+). On induction with isopropyl-b-D-1-thiogalactopyranoside, the enzyme expression reached upto ~20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced ß-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the ß-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion: Production of endo-1, 4 ß-glucanase enzymes from B. halodurans increased several folds when cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 ß-glucanases from B. halodurans.

Immunological analysis of allergenic cross-reactivity between Cheyletus malaccensis and Dermatophagoides farinae, Dermatophagoides pteronyssinus and Blomia tropicalis / Análise imunológica da reatividade cruzada alergênica entre Cheyletus malaccensis e Dermatophagoides farinae, Dermatophagoides pteronyssinus e Blomia tropicalis

Objective: The mite Cheyletus malaccensis is cited in the literature as a predator of other mite species. Little is known about its protein composition, and few studies have evaluated its ability to trigger atopic respiratory allergic reactions. The present study aims to investigate the protein profile fingerprint present in Cheyletus malaccensis extract and to evaluate its immunologic reactivity in the presence of specific immunoglobulins (IgE) from the serum of individuals diagnosed with allergy to the mites Dermatophagoides farinae, Dermatophagoides pteronyssinus and Blomia tropicalis. These three species carry proteins responsible for the most cases of atopic respiratory allergies, hence the interest in comparing them to Cheyletus malaccensis. Methods: Samples of aspirated dust containing Cheyletus malaccensis were collected from households in the city of Rio de Janeiro, Brazil. From the collected mass of this mite, extracts were prepared for analysis. Proteins present in the extracts were identified by electrophoresis under denaturing conditions. Results: Proteins with a molecular mass of 24 kDa, 26 kDa, 12 kDa, 45 kDa and 70 kDa were visualized. The immunoblotting assay showed positive cross-reactivity for proteins of molecular mass ranging from 20 kDa to 45 kDa. These results indicate that specific links were established between IgE present in the serum of individuals allergic to the comparator mite and proteins from Cheyletus malaccensis. Conclusions: These findings are relevant for their potential clinical and immunotherapeutic applications, as well as information base for further studies.

Objetivo: O ácaro Cheyletus malaccensis é referido na literatura como um predador de outras espécies de ácaro. Pouco se sabe sobre sua composição proteica, e poucos estudos avaliaram sua habilidade de desencadear reações alérgicas respiratórias atópicas. O objetivo do presente estudo é investigar a impressão digital do perfil proteico presente em um extrato de Cheyletus malaccensis e avaliar sua reatividade imunológica na presença de imunoglobulinas (IgE) específicas do soro de indivíduos diagnosticados com alergia aos ácaros Dermatophagoides farinae, Dermatophagoides pteronyssinus e Blomia tropicalis. Essas três espécies carregam proteínas responsáveis pela maioria dos casos de alergias respiratórias atópicas, o que justifica o interesse em compará-las ao Cheyletus malaccensis. Métodos: Amostras de poeira aspirada contendo Cheyletus malaccensis foram coletadas de domicílios na cidade do Rio de Janeiro, no Brasil. A partir da massa coletada desse ácaro, extratos foram preparados para análise. As proteínas presentes nos extratos foram identificadas por eletroforese sob condições desnaturantes. Resultados: Proteínas com massa molecular de 24 kDa, 26 kDa, 12 kDa, 45 kDa e 70 kDa foram visualizadas. O ensaio imunoenzimático mostrou reatividade cruzada positiva para proteínas de massa molecular variando de 20 kDa a 45 kDa. Esses resultados indicam que ligações específicas foram estabelecidas entre a IgE presente no soro de indivíduos alérgicos ao ácaro usado como comparador e proteínas de Cheyletus malaccensis. Conclusões: Os achados são relevantes por seu potencial clínico e aplicações imunoterapêuticas, bem como sua base de informações para futuros estudos.