Farid, Mohamed Abdel-Fattah Mohamed
(Natural and Microbial Products Department, National Research Centre)
,
Kamel, Zinat
(Faculty of Science, Cairo University)
,
Elsayed, Elsayed Ahmed
(Natural and Microbial Products Department, National Research Centre, Advanced Chair for Proteomics and Cytomics Research, Faculty of Science, King Saud University)
,
El-Deen, Azza Mohamed Noor
(Natural and Microbial Products Department, National Research Centre)
Fructooligosaccharides have been mainly produced by microbial fructosyltransferases (FTase) enzymes. The present work focuses on the optimization of medium composition and cultivation parameters affecting FTase produced by Penicillium aurantiogriseum AUMC 5605 in shake flask cultivation. FTase produ...
Fructooligosaccharides have been mainly produced by microbial fructosyltransferases (FTase) enzymes. The present work focuses on the optimization of medium composition and cultivation parameters affecting FTase produced by Penicillium aurantiogriseum AUMC 5605 in shake flask cultivation. FTase production was optimized in two steps using DeMeo's fractional factorial design. A 1.46-fold increase in FTase production (105.4 U/mL) was achieved using the optimized culture medium consisting of (g/L): sucrose, 600; yeast extract, 10; $K_2HPO_4$, 5; $MgSO_4{\cdot}7H_2O$, 0.5; $(NH_4)_2SO_4$, 1.0 and KCl, 0.5. The obtained results showed that the maximum FTase enzyme activity was produced at initial cultivation pH values ranging from 6.0-6.5, at agitation speed of 200 rpm and using vegetative fungal cells as inoculum. Moreover, results showed that optimization of medium composition and some cultivation parameters resulted in an increase of about 93.7% in the enzyme activity than the nonoptimized cultivation conditions after 96 h of cultivation. Additionally, maximum production and specific production rates recorded 2340 U/L/h and 102 U/L/h/g cells, respectively.
Fructooligosaccharides have been mainly produced by microbial fructosyltransferases (FTase) enzymes. The present work focuses on the optimization of medium composition and cultivation parameters affecting FTase produced by Penicillium aurantiogriseum AUMC 5605 in shake flask cultivation. FTase production was optimized in two steps using DeMeo's fractional factorial design. A 1.46-fold increase in FTase production (105.4 U/mL) was achieved using the optimized culture medium consisting of (g/L): sucrose, 600; yeast extract, 10; $K_2HPO_4$, 5; $MgSO_4{\cdot}7H_2O$, 0.5; $(NH_4)_2SO_4$, 1.0 and KCl, 0.5. The obtained results showed that the maximum FTase enzyme activity was produced at initial cultivation pH values ranging from 6.0-6.5, at agitation speed of 200 rpm and using vegetative fungal cells as inoculum. Moreover, results showed that optimization of medium composition and some cultivation parameters resulted in an increase of about 93.7% in the enzyme activity than the nonoptimized cultivation conditions after 96 h of cultivation. Additionally, maximum production and specific production rates recorded 2340 U/L/h and 102 U/L/h/g cells, respectively.
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문제 정의
However, there is no report on the transfructosylating enzyme activity of Penicillium aurantiogriseum. The aim of this study is to optimize the media components and cultivation parameters affecting the production of FTase by a locally isolated fungus, Penicillium aurantiogriseum AUMC 5605, in shake-flask cultivation.
가설 설정
4 mL of 50% (W/V) sucrose solution. The reaction mixture was incubated at 55 ℃ for one h. At the end of the incubation time, the reaction was stopped by adding copper reagent. The amount of reducing sugar liberated was determined spectrophotometrically at 520 nm using glucose as a standard according to Somogyi (1945).
Here, Aj means either H or L level in experimental run i. If a calculated coefficient has a positive value, it means that the particular constituent has positive effect at its high level. P refers to FTase activity, X to biomass dry weight and S to specific activity of FTase.
제안 방법
De Meo’s fractional factorial design is a valuable tool for rapid evaluation of the effects of different media constituents. This design is a preliminary technique, which tests only two levels of each medium component, but cannot determine the exact quantity of each constituent required for best performance in the medium. However, the method suggests the importance of each constituent.
대상 데이터
In the present study, seven different components namely: sucrose (A), yeast extract (B), K 2HPO4 (C), MgSO4 ·7H2O (D), NaNO3 (E), (NH4)2SO4 (F), and KCl (G) were taken into consideration in eight experiments.
데이터처리
Data analysis. Treatment effects were analyzed and the mean comparison was performed by ANOVA One-Way analysis of variance using computer software Minitab 16 and the average values were reported. Significant differences among the replicates have been presented at the 95% confidence level (p ≤0.
이론/모형
Spores of the producing strain from 6–7 day old agar slants (approximately 1×106 spore/mL) were used to inoculate 50 mL of the vegetative growth medium and then incubated at 28±2 ℃ in an incubator shaker at 200 rpm for 24 hours (approximately 1×104 cells/mL). Cells count was measured by the dilution plate count method (Parkinson et al., 1971).
Determination of extracellular protein. The protein content of the crude enzyme source was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.
성능/효과
FTase activity, specific activity and enzyme yield from the fermentation trails are given in Table 3. The calculated coefficient values for FTase activity (C p ), biomass dry weight (C x ) and specific activity (C s) are also shown in Table 4. In all the eight runs, FTase activity varied from 70.26 to101 U/mL with a mean of 86.7 U/mL while its specific activity ranged between 9.7 and 13.7 U/mg proteins with a mean of about 11.4 U/mg proteins. Similarly, there were wide variations in the amount of the soluble protein (7.
4) reveal that highest FTase level was obtained when the culture was inoculated with vegetative cells (10 mL/50 mL culture) of 24 h old. Furthermore, the results showed that increasing the inoculums size more than 10 mL/50 mL culture, resulted in a gradual decrease in the enzyme level. However, it was noticed that increasing the inoculum size from 15–25 mL/50 mL culture was accompanied with increase in cell dry mass.
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