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1.
Chinese Journal of Biotechnology ; (12): 1293-1301, 2010.
Artículo en Chino | WPRIM | ID: wpr-351593

RESUMEN

In vitro evolution methods are often used to modify protein with improved characteristics. We developed a directed evolution protocol to enhance the thermostability of the beta-1,3-1,4-glucanase. The thermostability of the enzyme was significantly improved after two rounds of directed evolution. Three variants with higher thermostability were obtained. The mutant enzymes were further analyzed by their melting temperature, halftime and kinetic parameters. Comparing to intact enzyme, the T50 of mutant enzymes 2-JF-01, 2-JF-02 and 2-JF-03 were increased by 2.2 degrees C, 5.5 degrees C and 3.5 degrees C, respectively, the halftime (t1/2, 60 degrees C) of mutant enzymes 2-JF-01, 2-JF-02 and 2-JF-03 were shortened by 4,13 and 17 min, respectively, the V(max) of mutant enzymes were decreased by 8.3%, 2.6% and 10.6%, respectively, while K(m) of mutant enzymes were nearly unchanged. Sequence analysis revealed seven single amino acid mutant happened among three mutant enzymes, such as 2-JF-01 (N36S, G213R), 2-JF-02 (C86R, S115I, N150G) and 2-JF-03 (E156V, K105R). Homology-modeling showed that five of seven substituted amino acids were located on the surface of or in hole of protein. 42.8% of substituted amino acids were arginine, which indicated that arginine may play a role in the improvement of the thermostability of the beta-1,3-1,4-glucanase.This study provide some intresting results of the structural basis of the thermostability of beta-1,3-1,4-glucanase,and provide some new point of view in modifying enzyme for future industrial use.


Asunto(s)
Secuencia de Aminoácidos , Bacillus , Evolución Molecular Dirigida , Endo-1,3(4)-beta-Glucanasa , Química , Genética , Estabilidad de Enzimas , Calor , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión , Química , Genética
2.
Chinese Journal of Biotechnology ; (12): 542-548, 2009.
Artículo en Chino | WPRIM | ID: wpr-286676

RESUMEN

To compare of performance of beta-1,3-1,4-glucanase gene (bgl) in different expression systems, the beta-1,3-1,4-glucanase gene (GenBank Accession No. EU623974) was amplified from Bacillus amyloliquefaciens BS5582 by PCR and was cloned into three vectors pEGX-4T-1, pET20b(+) and pET28a(+) to construct pEGX-4T-1-bgl, pET20b(+)-bgl and pET28a(+)-bgl recombinant plasmids. The pEGX-4T-1-bgl was transformed into three different Escherichia coli host strains. The pET20b (+)-bgl and pET28a (+)-bgl were transformed into E. coli BL21 (DE3) respectively. Recombinant beta-glucanase was expressed by IPTG inducement in these recombinants. E. coli BL21 (DE3)-pET28a (+)-bgl had the highest enzyme activity. In Luria-Bertani medium, the total enzyme activity was (322.0 +/- 8.8) U/mL, which was 40.1% of original strain in optimal shaking flask condition. This recombinant's performance was studied in Terrific Broth medium under inducement of IPTG and lactose at the same time., and the highest total enzyme activity could reach (1883.3 +/- 45.8) U/mL (818.8% of the original), which indicate that the recombinant strain has a good value in industry application.


Asunto(s)
Bacillus , Genética , Clonación Molecular , Endo-1,3(4)-beta-Glucanasa , Genética , Metabolismo , Escherichia coli , Genética , Metabolismo , Vectores Genéticos , Genética , Datos de Secuencia Molecular , Proteínas Recombinantes , Genética , Metabolismo
3.
Chinese Journal of Biotechnology ; (12): 1420-1427, 2008.
Artículo en Chino | WPRIM | ID: wpr-275368

RESUMEN

The ECM25 deletion mutant of industrial brewing yeast, G03/a, was constructed by replacing the ECM25 gene with the kanMX gene. The transformant was verified to be genetically stable. The PCR analysis showed that ECM25 gene in the G-03/a was deleted. Under aerobic conditions of ll degrees C and 28 degrees C, compared with the host strain G-03, the excretive glutathione concentration of G-03/a increased by 21.4% and 14.7%, respectively. Strains G-03 and G-03/a were inoculated in flasks and cultivated continuously for 4 generations. Compared with the host strain G-03, the glutathione concentration in the main fermentation broth and final beer of strain G-03/a increased by 32.1% and 13.8%, the stability index (SI) increased by 7.7% and 5.3%, respectively, and the flavor resistance staling value (RSV value) in final beer increased by 45.0%. During EBC fermentation, the glutathione concentration in the main fermentation broth of strain G-03/a increased by 34.0%, compared with the host strain G-03. Furthermore, no significant difference in routine fermentation parameters was found. The strain G-03/a is proved to be an excellent anti-staling brewing yeast to improve beer flavor stability.


Asunto(s)
Cerveza , Microbiología , Fermentación , Técnicas de Inactivación de Genes , Glutatión , Secreciones Corporales , Recombinación Genética , Saccharomyces , Genética
4.
Journal of Third Military Medical University ; (24)1984.
Artículo en Chino | WPRIM | ID: wpr-546696

RESUMEN

Twenty-five hybrid dogs were injected intravenously with oleic acid of the dose 0.3 ml/kg of body weight to prepare a model of respiratory distress syndrome (RDS) . The animals were killed 24 hours after injection. PaO2, PaCO2 and pH of the arterial and mixed venous blood were determined before and immediately, 0.5, 1?2, 4, 6, 22 and 24 hours after injection. The average pulmonary arterial pressure was measured hourly. The chest x-ray films were taken 2,4, 6 and 24 hours after injection. The electrolytes T3, T4, the hematocrit and RBC count, and the serum corticosteroid level were measured before and 24 hours after injection.25 dogs were divided into two groups; the control group consisted of 8 dogs and the therapeutic group consisted of 17 dogs, among which nine were treated with hyosine hydrobromide and 8 with dexamethasone. The histologic specimens of the animals of the control group and hyosine hydrobromide treated group were examined with both light and electron microscopes but the specimens of the animals of dexamethasone treated group were examined with light microscope only.It was found that dexamethasone is effective in the treatment of RDS produced with oleic acid injection while hyosine hydrobromide is of no value.

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