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Fibrinolytic enzymes from a newly isolated marine bacterium Bacillus subtilis A26: characterization and statistical media optimization

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A fibrinolytic enzyme producing bacterium was isolated and identified as Bacillus subtilis A26 on the basis of the 16S rRNA gene sequence. The fibrin zymography analysis reveals the presence of at least three fibrinolytic enzymes. The crude enzyme exhibited maximal activity at 60 °C and pH 8.0. Medium composition and culture conditions for the enzyme production by B. subtilis A26 were optimized using two statistical methods. The Plackett-Burman statistical design was applied to find the key ingredients and conditions for the best yield of enzyme production. Five significant variables (hulled grain of wheat, casein peptone, NaCl, CaCl2, and initial pH) were selected for the optimization studies. The response surface methodological approach was used to determine the optimal concentrations and conditions. The optimized medium contained 40.0 g·L-1 hulled grain of wheat, 3.53 g·L-1 casein peptone, 4.0 g·L-1 CaCl2, 3.99 g·L-1 NaCl, 0.01 g·L-1 MgSO4, and 0.01 g·L-1 KH2PO4, pH 7.78. The medium optimization resulted in a 4.2-fold increased level of fibrinolytic production (269.36 U·mL-1) compared with that obtained with the initial medium (63.45 U·mL-1). A successful and significant improvement in the production of protease by the A26 strain was accomplished using inexpensive carbon substrate (hulled grain of wheat), allowing a significant reduction in the cost of medium constituents.

Une souche bactérienne productrice d’enzymes fibrinolytique a été isolée et identifiée sur la base de la séquence du gène codant pour l’ARN ribosomal 16S comme étant Bacillus subtilis A26. L’analyse par zymogramme sur fibrine révèle la présence d’au moins trois enzymes fibrinolytiques. L’extrait enzymatique présente un optimum d’activité à 60 °C et à pH 8,0. La composition du milieu et les conditions de cultures pour la production d’enzymes fibrinolytiques par B. subtilis A26 ont été optimisées par la méthodologie des plans d’expériences. La matrice de Plackett-Burman a été employée pour cribler les facteurs ayant un effet significatif sur la production d’enzymes fibrinolytiques. Cinq facteurs significatifs (gruau, peptone de caséine, NaCl, CaCl2 et pH initial) ont été sélectionnés pour l’optimisation par la méthodologie des surfaces de réponses. Le milieu optimisé est composé de 40,0 g·L-1 gruau, 3,53 g·L-1 peptone de caséine, 4,0 g·L-1 CaCl, 3,99 g·L-1 NaCl, 0,01 g·L-1 MgSO4 et 0,01 g·L-1 KH2PO4, pH 7,78. L’optimisation a permis d’améliorer le niveau de production d’enzymes fibrinolytiques d’un facteur de 4,2 (269,36 U·mL-1) par rapport au milieu initial (63,45 U·mL-1). Une amélioration significative du niveau de production de protéases fibrinolytiques par la souche A26 est obtenue en utilisant un substrat carboné bon marché (gruau), ce qui permet une réduction intéressante du coût des constituants du milieu de culture et donc de la production.

Document Type: Research Article

Publication date: 2009-09-01

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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