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The maximum protease production obtained experimentally was found to be 128.5 U/ml.
A 24 full factorial central composite design was employed to determine the maximum protease production.
Glucose, soybean meal and ammonium sulfate were resulted in maximum protease production at 644 U/ml, 720 U/ml, and 806 U/ml when screened for carbon, organic nitrogen and inorganic nitrogen sources, respectively, using optimized immobilization conditions.
Using this methodology, the quadratic regression model of producing organic solvent-stable protease was built and the optimal combinations of media constituents and culture conditions for maximum protease production were determined as glycerol 12.47 g/L, MgSO4·7H2O 0.73 g/L, and pH 8.25.
Some cultivation parameters were studied and the results showed that the optimum fermentation medium was composed of wheat bran, 2.0% (w/w) peptone and 2.0% (w/w) yeast extract, and the conditions for maximum protease production were an initial moisture content of 50.0%, an inoculum level of 107 spores g−1 and an incubation at 23 °C.
From these results was also possible to establish the use of at least three different culture media formulations with a maximum protease production above 260 U g−1, making it a versatile and efficient system for protease production and that can be easily applicable to other groups of enzymes.
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At 30°C, maximal protease production was observed at 48 h in the case of both cultures (p < 0.05).
Eleven carbon, 11 organic nitrogen and seven inorganic nitrogen sources were screened by two-level Plackett Burman design for maximum alkaline protease production by using optimized immobilized conditions.
The maximum alkaline protease production was 1520 ± 35 U ml−1at 42 h of incubation which showed about 1.5-fold increase in protease production over the central point and an overall 6-fold enhancement (1520 250 U ml−1) over the basal medium.
Regarding protease production, the maximum quantity of 39.2 U/g was nearly 1.25-fold higher in the transformed fungus compared to the parental strain after 6 days of fermentation (Fig. 4d).
The fermentation time course for protease production by Streptomyces flavogriseus HS1 (data not shown) indicates that the maximum protease activity (99 U/mL) was obtained after 96 h of cultivation, when cells were in the stationary phase, and its production was not growth associated.
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