Solid-State Fermentation for the Production of L-Asparaginase by Aspergillus Sp

 

V Sreenivasulu1*, KN Jayaveera2 and P Mallikarjuna Rao3

1Department of Biotechnology, KLR Pharmacy College, Paloncha- 507115, Khammam (District), A.P, India.

2 Oil Technological Research Institute, JNT University, Anantapur–15001, A.P, India.

3 International Medical University, Kualalumpur, Malaysia-57000.

 

ABSTRACT

Production of L-asparaginase employing Aspergillus sp. VEM-9 under solid-state fermentation was optimized. Different substrates like rice bran, green gram bran, wheat rawa, wheat bran, Bombay rawa, black gram bran, barley, saw dust, jowar flour, rice flour, castor oil cake, ground nut oil cake, coconut oil cake, sesame oil cake were studied to optimize the best substrate. Groundnut oil cake showed the highest enzyme yield. Different physical fermentation factors were optimized. The maximum productivity of L-asparaginase (60 U/gds) was achieved by employing groundnut oil cake and optimized process parameters including incubation period of 5 days, initial moisture content of solid substrate 90%, 1: 10 (v/w) ratio of salt solution to weight of groundnut oil cake, inoculum level 20%(v/w), incubation temperature at 30 oC and initial pH 6.5.

 

Keywords: L-asparaginase,  Aspergillus sp. VEM-9,  groundnut oil cake,  optimization, Solid-state fermentation.

 

INTRODUCTION

L-Asparaginase (L-asparagine amidohydrolase, E.C. 3.5.1.1) catalyses the hydrolysis   of L-asparagine into L-aspartate and ammonia. This enzyme acquired some clinical importance in 1961, when the antitumour effect of guinea pig serum, originally discovered by Kidd, 19531 was traced by Broome, 19612 to the presence of this enzyme.  A wide variety of microbial strains also produce L-asparaginase 3,4,5. The enzyme from Escherichia coli and Erwinia carotovora was clinically used to treat patients suffering from asparagines- dependent leukemias and lymphomas6. A number of undesirable side effects, ascribed to the acute lymphoblastic leukemia, presence of contaminating bacterial endotoxins in the enzyme preparations (from bacterial source) were observed7. The search for the other asparaginase sources like eukaryotes can lead to an enzyme with less adverse effects.  It has been observed that eukaryote microorganisms like yeast and filamentous fungi have a potential for asparaginase production8,9. Hence, we attempted to prepare this enzyme from different other microbial source like fungi.  L-asparaginase is also used for the treatment of pancreatic carcinoma10 and bovine lymphomosarcoma11. Moreover, L-asparaginase preparation from food grade Aspergillus niger is used as a processing aid during food production to covert asparagine to aspartic acid in order to reduce cancer causing acrylamide formation 12.  Very little work has been carried out on L-asparaginase from fungal source.

 

L-asparaginase is produced throughout the world by submerged fermentation (SmF). This technique has many disadvantages, such as the low concentration production, consequent handling, reduction and disposal of large volumes of water during the downstream processing. Therefore, the SmF technique is a cost effect intensive, highly problematic and poorly understood unit operation13.  Solid-state fermentation (SSF) is a very effective technique as the yield of the product is many times higher when compared to that in SmF14 and it also offers many other advantages15.

 

 

In the present work, it is aimed to investigate production of L-asparaginase by Aspergillus sp. VEM-9 under SSF conditions. In this paper we have reported the physical factors that influence maximization of L-asparaginase production through SSF.

 

MATERIALS AND METHODS:

Micro organism:

A mutant strain of Aspergillus sp. VEM-9 was used in the present study. It was isolated from soil samples by using pH and dye based method 5 and was maintained on Czapeck Dox agar medium slants at 4˚C and subcultured at every 4 weeks.

 

Seed inoculum:

Inoculum was prepared by transferring 5ml of spore suspension prepared from 7 days old slant culture, into 250-ml Erlenmeyer flasks containing 45 ml sterile inoculum medium. The composition of the inoculum medium: Czapeck Dox medium 16 was (gl-1): Glucose, 2.0; L- Asparagine, 10.0; KH2PO4, 1.52; KCl, 0.52; MgSO4.7H2O, 0.52; CuNO3.3H2O, trace; ZnSO4.7H2O, trace; FeSO4.7H2O.trace; pH 6.2. The flasks were incubated on a rotary shaker at 150 rpm at 30˚C for 2 days. 

 

The composition of salt solution was (g/l):  KH2PO4 0.5, MgSO4.7 H2O 0.5, FeSO4.7H2O 0.01 and NaCl, 0.5.

 

Optimization of fermentation process under SSF:

Factors including selection of solid substrate, incubation time, initial moisture content, level of salt solution, inoculum level, incubation temperature, initial pH affecting the secretion of L-asparaginase enzyme by Aspergillus sp. VEM-9 under SSF were optimized by adopting a search technique 17 and varying parameters one at a time. Static experiments were conducted in 250ml Erlenmeyer flasks containing 10 g of groundnut oil cake, distilled water was added to adjust the final substrate moisture content to 70%.  After sterilization by autoclaving, flasks were cooled and inoculated with a 10% inoculum level (4x107spores/ml) and incubated at 30 ˚C for 4 days under appropriate experimental conditions. 

 

Effect of various substrates:

Agro-industrial residues of rice bran, green gram bran, wheat rawa, wheat bran, Bombay rawa, black gram bran, barley, saw dust, jowar flour, rice flour, castor oil cake, ground nut oil cake, coconut oil cake, sesame oil cake were procured from  local market and used as  solid substrate. They were assessed to study their effect on production of L-asparaginase. The best solid substrate achieved by this step was fixed for subsequent experiments.

 

Effect of incubation period:

 Different incubation periods (2, 3, 4, 5, 6, 7, 8 and 9 days) were employed to study their effect on L-asparaginase production. The fermentation was carried out at 30°C and other experimental conditions were 10% inoculum level, 70% moisture content. The optimum incubation period achieved by this step was fixed for subsequent experiments.

 

Effect of initial moisture content:

To investigate the influence of the initial total moisture content (before autoclaving) of the substrate, the fermentation was carried out under various initial moisture contents (50, 60, 70, 80, 90, 100 and 110 %) of groundnut oil cake, which was adjusted with distilled water. The other conditions were 10% inoculum level. The fermentation was carried out for 5 days at 30°C. The optimum initial moisture content of solid substrate achieved by this step was fixed for subsequent experiments.

 

Effect of level of salt solution:

Various concentrations of the standard salt solution were studied by changing the ratio of volume of salt solution to the weight of the substrate. Different volumes of standard salt solution to weight of the substrate (v/w-0.5: 10, 1.0:10, 1.5:10, and 2.0:10) in different flasks were assessed. Fermentation was carried out at 30°C and other experimental conditions were 10% inoculum level. The optimum volume of salt solution to solid substrate achieved by this step was fixed for subsequent experiments.

 

Effect of level of inoculum:

Various inoculum levels (5, 10, 20, 30, 40 and 50%) were tried to study their effect on enzyme production. The study was carried out at 30°C keeping other conditions at their optimum levels. The optimum inoculum level achieved by this step was fixed for subsequent experiments.

 

Effect of incubation temperature:

The fermentation was carried out at various temperatures such as 20, 25, 30, 35, 40, 45 and 50°C and to study their effects on Enzyme production, keeping other conditions at their optimum levels. The optimum incubation temperature achieved by this step was fixed for subsequent experiments.

 

Effect of initial pH:

While optimizing the initial pH of basal medium, the pH of aqueous solution was carried from 4.0 to 9.0 with 1N HCl or 1N NaOH.  The fermentation was carried out at 30°C to study their effect on Enzyme production, keeping all other conditions at their optimum level. The optimum initial pH of the solid substrate achieved by this step was fixed for subsequent experiments.

 

Analytical methods:

Enzyme extraction:

At the end of fermentation, the biomass was treated with 50 ml of sterile deionized water and agitated thoroughly on the rotary shaker for 1 h at 150 rpm and kept in the refrigerator at 4°C for soaking (5-6 hrs).  The whole content was filtered through Whatman No.1 filter paper. The residue was again treated with another 50ml of sterile deionised water in the same way and filtered. The filtrates were pooled together and the clear filtrate was evaluated for the enzyme content.

 

Estimation of L-asparaginase:

L-asparaginase assayed according to Nesselerisation method 3. The reaction mixture, containing 0.5ml of L-asparagine (0.04 M), 0.5ml of phosphate buffer 0.1M (pH 8.0), 0.8 ml of deionised water and 0.2ml enzyme solution was incubated at 37 ˚C for 30 minutes.  The reaction was stopped by addition of 0.5 ml of 1.5 M trichloroacetic acid. Then to 3.4 ml deionized water, 0.1 ml of the above mixture and 0.5ml of Nesselers reagent were added and colour developed was read at 450 nm in Elico double beam UV –visible spectrophotometer (SL-164).  Enzyme blank was used as control. A standard curve was prepared with ammonium sulphate. Enzyme activity expressed in Units per gram dry fermented substrate (U/gds).  One unit of L-asparaginase activity was defined as that amount of enzyme which catalyses the formation of 1mmol of ammonia per minute under optimal assay conditions.  All the experiments were carried out in triplicate and the mean of the three was presented.

 

RESULTS AND DISCUSSION:

The SSF process has been observed to be less sensitive to contamination than SmF14, 15.   In SSF, the selection of a suitable  solid substrate for  fermentation process is critical factor and thus involves screening of a number of agro-industrial material for microbial growth and product formation in the present study.  Fourteen substrates, viz.  rice bran, green gram bran, wheat rawa, wheat bran, Bombay rawa, black gram bran, barley, saw dust, jowar flour, rice flour, castor oil cake, ground nut oil cake, coconut oil cake, sesame oil cake were  used  for growth  and L-asparaginase  production by  Aspergillus sp. VEM-9.  The results are shown in fig.1. All the substrates supported growth and enzyme formation by the culture, while groundnut oil cake has proved superior to other substrates. A high titre of L-asparaginase (21.7 U/gds) was obtained in medium containing groundnut oil cake alone as the substrate followed by castor oil cake. The order of substrate suitability was groundnut oil cake> castor oil cake> barley>wheat rawa> coconut oil cake>wheat bran>rice bran > sesame oil cake>Bombay rawa> black gram bran> riceflour>green gram bran > saw dust> jowar flour.  Hence, groundnut oil cake was selected and used for subsequent studies.

 

The effect of incubation periods on L-asparaginase production was studied and results are shown in fig.2.  The results indicated that enzyme yield was increased gradually and high titres (29 U/gds) were attained at 5 days incubation, further incubation resulted in gradual decrease in L-asparaginase production.   

 

High enzyme titre  (37 U/gds) was attained when the initial moisture level was 90% in comparison with that at low or high moisture levels (fig.3). The critical importance of moisture level in SSF media and influence on the biosynthesis and secretion of enzymes can be attributed to the interference of moisture in the physical properties of the solid particles.  Increased moisture level is believed to reduce the porosity of the groundnut oil cake, thus limiting oxygen transfer 18,19. Low moisture content causes reduction in the solubility of nutrients of the substrates and low degree of swelling 20.

 

The effect  of concentration of standard salt solution on enzyme production was studied by altering the ratio of volume of salt solution to the weight of the substrate (0.5:10, 1:10, 1.5:10 and 2:10 v/w).  Maximum enzyme production (41 U/gds) was obtained at salt solution concentration to ground nut oil cake weight ratio of 1.0: 10, while an increasing salt concentration reduced the Enzyme yield. The results are presented in fig.4.  

 

Inoculum level was also an important factor for the production of L-asparaginase.  High inoculum levels are inhibitory in nature.  Various inoculums levels (5, 10, 20, 30, 40 and 50%) were tried to study their effect on L-asparaginase production.  The higher enzyme production (50. 2 U/gds) was obtained at 20%v/w inoculum levels as compared low or high inoculum levels.  The results are presented in fig. 5.  It is important to provide an optimum inoculum level in fermentation process. Lower inoculum density may give insufficient biomass causing reduced product formation, where as a higher inoculum may produce too much biomass leading to the poor product formation 21.

 

The maximum enzyme production (50. 4 U/gds) was attained at 30°C.  Results are presented in fig. 6. Decrease in the yield of L-asparaginase was observed when the incubation temperature was higher or lower than the optimum incubation temperature. Higher temperature had some adverse effect on the metabolic activities of the microorganisms and it has been reported by various scientists that the metabolic activities of the microorganisms become slow at lower temperature. Hence, incubation temperature and its control in SSF process is crucial as the heat evolved during SSF process is accumulated in the medium due to poor heat dessipation in  solid medium. This results in reduced microbial activity, there by decreasing the product yield 22, 23. The same incubation temperature was repoted on the production of L-asparaginase from Aspergillus niger grown on bran of pulses in SSF 24.

 

The effect of initial pH on L-asparaginase production was studied and the results presented in fig.7. The results indicated that a gradual increase in L-asparaginase production was observed from pH 5.0 to 6.5 followed by a gradual decrease of enzyme yield beyond pH 6.5. Higher or lower than this pH (6.5) resulted in lower yield of L-asparaginase. The optimum quantity of L-asparaginase production  (60 U/gds) was obtained at pH 6.5.  The same pH was observed on the production of L-asparaginase from Aspergillus niger in SSF, the nearest physiological pH (6.5) makes this enzyme superior to that of bacterial origin as a chemotherapeutic agent in treatment of   leukemia 24.

 

 

CONCLUSION:

The optimum productivity of L-asparaginase (60 U/gds) was achieved by employing ground nut oil cake with optimized process parameters such as   incubation period of 5 days, moisture content of solid substrate 90%, 1.0:10(v/w) ratio of salt solution to weight of ground nut oil cake, inoculum level at 20%, incubation temperature at 30°C and initial pH 6.5.

 

Before optimization studies, the yield was only 21.7 U/gds. The above optimization studies finally resulted an improved yield of 60 U/gds. This accounts enhanced yield, 2.76 times over the control, which is significant achievement.

 

In this conclusion, the results of the present study indicate scope for exploring soil fungi as a source for L-asparaginase, an enzyme that has gained industrial and pharmaceutical significance recently.

 

ACKNOWDGEMENT:

The authors are grateful to KLR Pharmacy College, Paloncha, Khammam  (Dist.), for providing necessary facilities to carry out the research work. The authors are also grateful to Department of Pharmaceutical Biotechnology, University college of Pharmaceutical Sciences, Andhra University, Visakhapatnam for providing valuable suggestions and constant encouragement.

 

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Received on 06.04.2009

Accepted on 22.05.2009     

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 1(1): July.-Aug. 2009, 21-25