Abstract
The present paper describes optimization of fermentation conditions in shaken flasks and scale-up of fermentor production up to 115 L. The response surface methodology (RSM) has been successfully applied in standardization of mutanase production by Trichoderma harzianum CCM F-340. The model was very well fitted to the experimental data and explained more than 96% of the whole variation of the response (adjusted R2 = 0.962). In order to confirm the adequacy of the regression model based on the experimental data, validation cultures were grown in conditions created through optimization. The highest enzyme activity (0.747 U/mL) was reached in shaken flask cultures on Mandels’ medium in a volume of 140 mL modified in terms of carbon (cell wall preparation from the polypore fungus Laetiporus sulphureus 8.08 g/L) and nitrogen (soybean peptone 1.38 g/L) sources, under culture conditions 30°C, pH 5.3, agitation 270 rpm. The scale-up of the culture in the bioreactors with a working volume of 5 and 115 L resulted in a slight decrease in the mutanase activity (0.734 and 0.682 U/mL, respectively). The validation experiment showed a 70.6% increase in the production of mutanase compared with the culture before optimization. The results proved that the cultures could be scaled-up successfully from shaken flasks to the bioreactor scale. Our results indicate that in optimal conditions, T. harzianum could be a highly effective extracellular mutanase source. This report is the first to deal with optimization of mutanase biosynthesis using a mathematical model and scale-up of enzyme production in controlled fermentors with a view to facilitate application thereof in industry. Key words: Mutanase, Trichoderma harzianum, response surface methodology (RSM), bioreactors, submerged culture.
Highlights
Mutanases (α-(1→3)-glucan 3-glucanohydrolases) hydrolyze the α-(1→3)-glycosidic bonds of streptococcal mutan- water-insoluble, alkali-soluble α-glucan found in oral biofilms
The present paper describes production of mutanase from T. harzianum CCM F-340 using a sequential study of the factorial Plackett–Burman design followed by central composite design (CCD)
The cell wall preparation from L. sulphureus (CWP) is rich in α-(1→3)-glucans; it effectively induces mutanase activity and fully substitutes other inducers when used as a sole carbon source in the culture medium (Wiater et al, 2008)
Summary
Mutanases (α-(1→3)-glucan 3-glucanohydrolases) hydrolyze the α-(1→3)-glycosidic bonds of streptococcal mutan- water-insoluble, alkali-soluble α-glucan found in oral biofilms. Mutanases (α-(1→3)-glucan 3-glucanohydrolases) hydrolyze the α-(1→3)-glycosidic bonds of streptococcal mutan. Mutanases could be used as an active additive in preparations intended for oral hygiene, such as mouthwashes, toothpastes, and dental gels, and for washing and storage of prosthesis and prosthetic devices for removal of denture plaque located on their acrylic surfaces. Mutanases could become useful supplements to mechanical cleaning of teeth and dentures with a toothbrush, dental sticks, and dental floss. In addition to their potential usefulness in dentistry as oral therapeutic agents, α-(1→3)-glucanases might be applicable in investigations of α-(1→3)-glycosidic linkages found in microbial cell-wall structures and glucans of certain higher plants. Various preparations of mutanases have been successfully used for obtaining fungal protoplasts (Balasubramanian et al, 2003)
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