Solid state fermentation for production of L – asparaginase in rice bran by Serratia marcescens SB08
C Venil, P Lakshmanaperumalsamy
l – asparaginase, rice bran, serratia marcescens sb08, solid state cultivation, yeast extract
C Venil, P Lakshmanaperumalsamy. Solid state fermentation for production of L – asparaginase in rice bran by Serratia marcescens SB08. The Internet Journal of Microbiology. 2008 Volume 7 Number 1.
L – asparaginase, produced by various genera under solid-state and submerged cultivation, is an important chemotherapeutic agent used for the treatment of a variety of lymphoproliferative disorders and lymphomas, acute lymphoblastic leukemia in particular. In this study, L – asparaginase production by
L – asparaginase is an important chemotherapeutic agent used for the treatment of a variety of lymphoproliferative disorders and lymphomas, acute lymphoblastic leukemia in particular. In recent years, the use of L-asparaginase in the treatment of leukemia and other lymphoproliferative disorders has expanded immensely. For these reasons L – asparaginase has established itself to be an indispensable component
Several microorganisms including
In recent years, the production of enzymes by solid state fermentation (SSF) has emerged. The SSF has numerous advantages over submerged fermentation, including superior productivity, simple technique, low capital investment, low energy requirement and less water output, better product recovery and reported to be the most appropriate process for developing countries (Carrizales and Jaffe, 1986). SSF holds tremendous potential for the production of secondary metabolites and has been increasingly applied in recent years (Sangeetha
In this work, the different media ingredients along with rice bran were tested under solid state conditions for L – asparaginase synthesis by
Material and Methods
Growth of organisms
Solid-state cultivation in flask
Rice bran was collected from a local market and grinded to obtain 0.5 mm size particle using a standard sieve and preserved at room temperature. 10 g (w/w) of this substrate was distributed in a 250 mL wide-mouth flask and then moisturized with 40 mL of L – asparagine broth medium. It had the following composition (g/L): Peptone: 0.5; Beef extract: 0.5; Yeast extract: 0.5; L – asparagine: 0.1. The initial pH of the medium was adjusted to 7.0 and sterilized at 121 oC for 15 min. The fermentation process was started by adding one mL (v/v) of inoculum (3 x 106 cells /mL) as prepared above, whole content mixed thoroughly and then incubated at 30 oC for 24 h in a stationary condition. A similar experiment was conducted for L - asparaginase production by replacing rice bran with 10 g (w/w) wheat bran. Every 6 hrs sample was withdrawn aseptically and used for determining the L - asparaginase activity. Moisture and biomass were measured by constant dry weight basis.
Enzyme production on different media supplements
The effect of different media supplements including carbon, nitrogen and other starchy materials along with rice bran was studied in this work by using L – asparagine broth medium. 1% (w/v) carbon source was separately tested in 40 mL of the above medium containing 10 g (w/w) rice bran for enzyme activity. For checking the effect of nitrogen sources on L - asparaginase production, 0.5% (w/v) organic nitrogen source and 0.3% (w/v) inorganic nitrogen source were separately added in 40 mL of the above medium. The influence of different media ingredients on L - asparaginase production was examined by replacing appropriate sources in the same medium with rice bran as a main substrate. 10 g of starchy material was used to replace the rice bran in the same minimal medium containing sucrose (1%) and yeast extract (0.5%). One mL of cells (3 x 106 cells/mL) was used as an inoculum to initiate fermentation and then all of the flasks were incubated at 30oC for 24 h in a stationary condition.
Solid-state cultivation in aluminum tray
One mL (v/v) of inoculum (3 x 106 cells/mL) was transferred to a 500 mL wide-mouth flask containing 25 g (w/w) rice bran moisturized with 100 mL L – asparagine broth medium and then sterilized at 121o C for 15 min. After 24 h incubation in stationary condition, substrate was thoroughly mixed with inoculum to obtain dough. In tray culture method, one Kg (w/w) pulverized (0.5 mm particle size) rice bran was distributed and uniformly layered on an aluminum tray (30 x 15 cm). Then, the same minimal medium was sprinkled on the surface of the rice bran until it acquired proper moisture content. 25 g (w/w) dough was spread on the surface of rice bran and dispersed thoroughly. This tray was closed invertically with another one tray by keeping a space between two trays with wooden strips and then incubated at 28oC for 48 h.
Extraction of enzyme
After 48 h incubation, 50 mM citrate buffer (pH-6.8) (1:10 ratio) was added to fermented dough and homogenized with a constant stirring. This suspension was filtered through Whatman filter paper number 1 and the filtrate was again centrifuged at 6000 rpm for 15 min. This solid free supernatant was used as enzyme source for assaying L – asparaginase activity.
L – asparaginase assay
L-asparaginase activity was determined by measuring the amount of ammonia formed by nesslerization (Wriston and Yellin, 1973). A 0.5 mL sample of crude enzyme, 1.0 mL of 0.1 M sodium borate buffer (pH 8.5) and 0.5 mL of 0.04 M L-asparagine solution were mixed and incubated for 10 min at 37oC. The reaction was then stopped by the addition of 0.5 mL of 15% trichloroacetic acid. The precipitated protein was removed by centrifugation and the liberated ammonia was determined by direct nesslerization. Suitable blanks of substrate and enzyme containing samples were included in all assays. The yellow color was read in a spectrophotometer (Hitachi – 3210 UV - Vis) at 500 nm. One unit (U) of L-asparaginase was the amount of enzyme which liberates 1 µmole of ammonia in 1 min at 37oC.
Results and Discussion
The cell free culture supernatant analyzed by paper chromatography using n-butanol: pyridine: distilled water as mobile phase (50:28:32 ratio) indicated that
Figure1 shows the effect of incubation time on L – asparaginase production by
The moisture content in rice bran tested for maximum L – asparaginase production indicated enhanced enzyme production at 50 % moisture content. The results are furnished in Figure 2.
Moisture content below and above 50% was significantly lowering the formation of L - asparaginase and the biomass yield of
In rice bran supplemented with yeast extract (0.5%),
Rice bran alone, without addition of minimal medium, served as a potential carbon source for the solid state cultivation of
Additional carbon supplements in rice bran, exclusively galactose, lactose and sucrose in L – asparagine broth medium, were found to induce maximum L – asparaginase activity whereas supplementation of xylose and fructose lowered L – asparaginase production drastically (Table 2). Anto
Addition of organic and inorganic nitrogen sources in L – asparagine broth medium, was found to produce varied L – asparaginase activity by
The concentration of organic and inorganic nitrogen sources were 0.5 % (w/v) and 0.025 % (w/v) respectively.
Additional nitrogen supplements, exclusively organic nitrogen in L – asparagine broth medium, were found to induce maximum L – asparaginase activity whereas supplementation of inorganic nitrogen sources lowered L – asparaginase production drastically (Table 3). The results indicated that the selective additions of nitrogen supplements favored L - asparaginase enhancement under solid state fermentation in rice bran.
Tray culture method of enzyme production attempted to compare with the results of solid state cultivation of
We are thankful to the authorities of Bharathiar University, Coimbatore, Tamil Nadu, India for providing necessary facilities to carry out this investigation.