Introduction

L-Asparaginase the enzyme which catalyses the hydrolysis of asparagine to form aspartic acid and ammonia, is an important natural product that possess a broad spectrum of anti-tumor activity. It has been successfully applied to the treatment of several diseases such as lymphocyte sarcoma and leukemia1-3. Literally called as L-Asparagine amino hydrolase E.C 3.5.1-1, the enzyme belongs to amide group has been successfully applied to various applications. Clinical studies have demonstrated higher recovery efficiency for leukaemia patients treated with L-Asparaginase than those with IL-24. L-Asparaginase is an important chemotherapeutic agent used for the treatment of a variety of lymphoproliferative disorders and lymphomas, acute lymphoblastic leukaemia (ALL) in particular. It has been a mainstay of combination chemotherapy protocols used in treatment of pediatric ALL for almost 30 years5. Based on this, it has also been included in most contemporary, multi-agent regimens for adult ALL6,7. L-Asparaginase as a drug has demonstrated effectiveness in induction and subsequent phases of various chemotherapeutic strategies.

The pioneer observation that turned out to be important for the development of L-Asparaginase as a potential antineoplastic agent was made by Clementi 8 in 1922, revealing the presence of high activity of L-Asparaginase in the serum of guinea pig. High L-Asparaginase activity was observed only in guinea pig serum, whereas other mammals were found devoid of this enzyme. In 1953, Kidd9  described the regression of transplanted lymphomas in mice and rats by the administration of guinea pig serum. This cytotoxic activity was not present in horse or rabbit serum. Neumann and McCoy10 extended these observations in 1956. They demonstrated that the growth of cell line derived from Walker carcinosarcoma required L-asparagine. Haley et al.11   in 1961 obtained similar results, using a mouse leukaemia cell line. It was Broome in 1961 while working in Kidd’s laboratory, who compared Kidd’s finding of growth inhibition with the earliest observation by Clementi, and succeeded in concluding that the anti lymphoma activity in guinea pig sera was due to L-Asparaginase12. Further investigations of the same author confirmed its therapeutic potential13. Yellin and Wriston in 1966, succeeded in partial purification of two isoforms of L- asparaginase from the serum of guinea pig14. Interestingly, only one isoform exhibited anti lymphoma activity in vivo15. Since the extraction of this enzyme from the guinea pig serum in sufficient amounts was difficult, other sources like microbes were looked into. Mashburn and Wriston, in 1964 and Campbell and Mashburn in 1969 reported the purification of E. coli L-Asparaginase, and demonstrated its tumoricidal activity similar to that of guinea pig sera16. These findings provided a practical base for large-scale production of enzyme for pre- clinical and clinical studies17.Tumor cells, more specifically lymphatic cells, require huge amount of asparagine to keep up with their rapid malignant growth. This means they use both asparagine from the diet (blood serum) as well as what they can make themselves (which is limited) to satisfy their large L-asparagine demand. L-Asparaginase as a drug exploits this unusually high requirement tumor cells have for the amino acid asparagine. L-Asparaginase catalyses the hydrolysis of L-asparagine to L- aspartic acid and ammonia (Fig. 1).

Figure 1: Schematic representation of mechanism of action of 1-asparaginase.   Click here to View figure

L-asparaginase have been produced by Escherichia coli, Serratia marcescens, Erwinia   carotovora, Pseudomonas acidivoras and P. geniculata under aerobic conditions15, 18,19. The discovery of new L-asparaginase serologically different but having similar therapeutic effects is highly desired. L-asparaginase was produced throughout the world by submerged fermentation (SF)20-22. The presence of the product at low concentration and the consequent handling, reduction and disposal of large volumes of water during down stream  processing  in  SF  are  known  as  cost  intensive, highly  problematic  and  poorly  understood  unit  operation23. Solid-state   fermentation   (SSF),   is   a   very effective  technique  as  the  yield  of  the  product  is  many times  higher  than  that  in  SF24  It  also  offers  many other advantages including resistance to contamination, ease of product extraction does not require complicated methods   of   treating   the   fermented   residue25.   In comparison with SF, SSF offers better opportunity for the biosynthesis of low-volume-high cost products26. Screening and evaluation of nutritional and environmental requirements of microorganism is an important step for bioprocess development. Our objective was to elaborate the  best  conditions  for  production  of  extracellular
L- asparaginase by isolated Aspergillus niger in SSF to  take  the  advantage  of  SSF  specially  in  resisting bacterial  contamination,  ease  of  purification  and  the use of cheap natural materials like Sesame cake. To the best   of   our   knowledge,   this is the first report of production of L-asparaginase in SSF using sesame cake.  SSF  was  carried out through a stepwise optimization strategy including, elucidation of medium and environmental  components that affect enzyme production significantly using a One-factor-at-a-time approach, This method consists of selecting a starting point, or baseline set of levels, for each factors, then successively varying each factor over its range with the other factors held constant at the baseline level. Maximum preliminary studied are conducted using this type of strategy of experimentation.

Material and Methods

Microorganism

The fungal culture used in this study was isolated from soil sample and identified as Aspergillus niger.

Raw material

Local Sesame cake, after extracting the oil from sesame, was collected grounded as coarse powder. The ground material was collected after passing through a sieve, to attain 0.2cm particle size.

Cultivation conditions and crude enzyme extraction

After isolation of the pure culture of Aspergillus niger, a stock culture of Aspergillus niger was maintained on Potato dextrose agar slants, and were stored at 4°C. Cultivation was achieved by solid-state fermentation as reported previously27. The Fungal strain was grown for 7days  at  30°C  in  Czapek Dox  broth,  from this culture conidial suspension was prepared with concentrations of 102-103 conidial spores ml-1, 1   ml   of   the Fungal  conidial suspension   was   used   to   inoculate   10 gms of Sesame cake   dispensed  in  250  ml  Erlenmeyer flasks. The fermentation medium has the following composition in 250  ml  flask,  Sesame cake,  10  g  and  5  ml  of  0.1  M sodium  phosphate  buffer  (pH  7.6).  Unless  otherwise indicated,  the  flasks  were  autoclaved  for  15  min  at 121oC  and  inoculated with 1 ml  of  the  previously prepared  Fungal suspension. The culture  flasks  were then  incubated at  30°C for 4 days  without  shaking.
The extracellular enzyme was prepared at the end of the fermentation period by the addition of 90 ml of 0.01 M sodium phosphate  buffer (pH 7.0) to the medium followed  by centrifugation at 8000 rpm  for 20 min. The cell-free supernatant was used as  crude enzyme preparation.

Protein determination

The protein content was determined according to the modified Lowry’s method28.

Enzyme assay

L-asparaginase was assayed colorimetrically29. A standard curve was prepared with ammonium sulfate. One L-asparaginase unit (IU) is defined as that amount of enzyme which liberates 1 m mole of ammonia per min under the optimal assay conditions.

Results and Discussion

Optimization of Production conditions

The effect of pH on L-asparaginase activity was determined. The pH ranges from 5.5 to 8 was employed. pH 6.5was found to be optimum for the L-asparaginase production (Fig.2). The influence of Fermentation time was shown in Fig.3.
The maximum Activity and specific activity of  L-Asparaginase was observed as 75.25 IU at 108 hours. To determine the incubation temperature, flasks were incubated at different temperatures ranging from 24 to 44°C. At 32°c incubation temperature was found to be optimum with 75.5 IU of enzyme activity (Fig.4).

Figure 2: Effect of pH on L-asparaginase activity.   Click here to View figure

Figure 3: Effect of incubation time on L-asparaginase activity.   Click here to View figure

Figure 4: Effect of temperature on L-asparaginase activity.   Click here to View figure

Effect of different supplements was studied by the addition of carbon nitrogen, growth promoter and different mineral salts were added and checked for the L-Asparaginase enzyme activity. Some supplements like glucose, ammonium sulphate, L-Asparagine, Magnesium sulphate and sodium chloride have shown the positive effect, some are negative effect and some have shown the neutral effect. Addition of those positively influenced supplements improves the enzymatic activity from 75.5 IU to 121.7 IU. Further each ingredient in the production medium was optimized. The L-Asparaginase activities were considerably increased with the change of each ingredient concentration. Table.1 clearly indicated that the L-Asparaginase activity and specific activity can be increased from 121.7 IU and 13.003 IU/mg when the basal medium added with the supplements, to 191.2 IU and 19.877 IU/mg with optimization of all supplements with the medium.

Analysis of variance showed that there was significant improvement in the L-Asparaginase activity when the sesame cake was supplemented with the different carbon, nitrogen and growth promoters. Glucose, fructose, and sucrose were suitable carbon sources for the L-Asparaginase activity with the 172.5, 191.2 and 165.6 IU respectively (Table 2). Five different Nitrogen sources (Ammonium Sulphate, Ammonium Phosphate, Ammonium Chloride, Urea and Yeast extract) and five different Growth promoters
(L-Asparagine, Tryptophan, Phenyl alanine, Thiamine and Folic acid) were used to supplement the Sesame cake for the L-Asparaginase activity. Ammonium sulphate (191.2 IU), Yeast extract (173.6 IU), L-Asparagine (191.2 IU) and Tryptophan (176.4 IU) have significantly influenced the
L-Asparaginase activity with Sesame cake as a sole substrate. Maximal  enzyme  activity  (191.2  IU)  has  been detected when medium was supplemented with 3%w/w Fructose, 3%w/w Ammonium sulphate, 0.1%w/w L-Asparagine, 0.01% w/w Magnesium Sulphate,01% w/w sodium chloride and inoculum size of 1.5ml (1.6 X 103Spores/ml) which is nearly three folds the activity in basal medium (Table 1)

Table 1: Optimization of environmental and additional nutrient factors in Sesame cake for L-Asparaginase activity with isolated Aspergillus Species.

Table 2: Effect of supplementary Additions in Sesame.

Conclusion

In screening the factors affecting   production of certain  secondary  metabolites  it  is  very  important  to test as  many  factors  as  possible  and  to  identify  the significance  of  each  of  these. One-factor-at-a-time approach design offers a good procedure for computing the significance of individual factors and maintains convincing  information on each component. Although, other  interactions  are  not  included  in  this design,  it is the  first  priority  in  the  screening program  to  examine  the  effect of individual factors, both environmental factors like Particle size of the solid medium, Moisture content, Incubation pH, Time and temperature and nutritional factors on the accumulation of Extracellular enzyme, L-asparaginase.  Of  these,  only  the  most  effective factors  with  positive  significance  would  be  selected  for further optimization, in which different alternative sources were screened for the better accumulation of enzyme.

Our preliminary studies had shown that, a better activity of enzyme was achieved at a pH of 6.5 and temperature of 32°C after 108 hours of incubation with a particle size of 0.67 cm by adding moisture content of 1:1 (Media: buffer). Various nutritional factors like Carbon, Nitrogen, growth factors, and mineral sources had been screened among which carbon, nitrogen and growth factors shown positive influence on the enzyme activity where as other factors like mineral sources had no influence. The present work was significant as it paves a new era for the production of L-Asparaginase using a cheap medium like Sesame cake from fungal strains. It is useful to advise  microbial industry sponsors to adopt such process to maintain high efficiency and economic bioprocesses There is lot of scope for future work in many aspects like examination of  the  interaction  between  a  large number  of  variables by applying the statistical methods, which  converts  the  bioprocess  factor  correlations  into  a mathematical model that predicts where the optimum is likely  to  be  located.

Acknowledgements

We are thankful to Bapatla Education Society, Bapatla for providing the necessary lab facilities to carry out the research work.

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