Process development for scale-up production of a therapeutic L-asparaginase by Streptomyces brollosae NEAE-115 from shake flasks to bioreactor

Abstract

L-asparaginase is a promising enzyme that has a wide range of significant applications including cancer therapy and starchy food industries. The statistical design of Plackett-Burman and face centered central composite design were employed to optimize L-asparaginase production by Streptomyces brollosae NEAE-115. As a result, a medium of the following formula is the optimum for producing L-asparaginase in the culture filtrate of Streptomyces brollosae NEAE-115: Dextrose 2 g, starch 20 g, L-asparagine 10 g, KNO₃ 1 g, K₂HPO₄ 1 g, MgSO₄.7H₂O 0.5 g, NaCl 0.1 g, pH 7, fermentation period 7 days, temperature 30 °C, inoculum size 4%, v/v, agitation speed 150 rpm and inoculum age 48 h. The kinetics of cell growth, carbohydrates consumption and L- asparaginase production were studied in 7-L stirred tank bioreactor under different cultivation conditions. A significant increase in both cell growth and carbohydrate consumption was observed as the stirring speed increased from 200 to 600 rpm under uncontrolled pH. The highest L- asparaginase activity of 108.46 U/mL was obtained after 96 h at 400 rpm. On the other hand, the specific enzyme production (Y_p/x) under uncontrolled pH reached its maximal value of about 20.3 U/mg cells. Further improvement of enzyme production was attained by controlling pH at 7 using the selected stirring speed of 400 rpm. Enzyme production of 162.11 U/mL obtained from the controlled pH cultures exceeded this value gained from uncontrolled pH (108.46 U/mL) by about 50%.

Figure 1

L-asparaginase activity of Streptomyces brollosae NEAE-115 detected by plate assay (A) production of the enzyme after two days; (B) control plate was prepared without dye.

Figure 2

Streptomyces brollosae NEAE-115 growth in large spherical pellets during L-asparaginase production in shake flasks.

Figure 3

(A) The main effects of different variables on L-asparaginase production according to the Packett–Burman experimental results; (B) Pareto chart illustrates the order of significance of each variable.

Figure 4

(A) The normal probability plot of residuals for L-asparaginase production as determined by the first-order polynomial equation, (B) correlation between predicted against observed values and (C) Plot of residuals against predicted values.

Figure 5

(A–C) Three-dimensional response surface plots showing the effect of incubation time (X₂), starch (X₈) and L-asparagine (X₉) and their interactions effects on the production of L-asparaginase by Streptomyces brollosae NEAE-115.

Figure 6

Time-course profile of (A) cell growth; (B) carbohydrates consumption; (C) L-asparaginase production; (D) specific activity; (E) dissolved oxygen and (F) final pH during batch cultivation of Streptomyces brollosae NEAE-115 in 7 L stirred tank bioreactor under different stirring speeds and uncontrolled pH.

Figure 7

Time profile of yield coefficients: (A) (Yp/x) unit of L-asparaginase per mg biomass; (B) (Yp/s) unit of L-asparaginase per mg carbohydrates; (C) (Yx/s) g cells per g carbohydrates in batch cultivation under different stirring speeds in 7-L stirred tank bioreactor.

Figure 8

Time-course profile of cell growth; L-asparaginase production; carbohydrates consumption; specific activity and dissolved oxygen during batch cultivation of Streptomyces brollosae NEAE-115 in 7-L stirred tank bioreactor at stirring speed of 400 rpm and controlled pH 7.

Figure 9

Microscopic observation of Streptomyces brollosae NEAE-115 cells at (A) early fermentation time showing mycelia clump, (B) dispersed mycelia at middle fermentation time and (C) freely dispersed mycelia and spores at late fermentation time of batch cultivations in 7-L stirred tank bioreactor.