Introduction

Asparaginase (asparagine amidohydrolase EC 3.5.1.1) hydrolyzes the amino acid asparagine into aspartic acid and ammonia. Asparaginase is an intracellular enzyme produced by several different microorganisms, including Gram-negative bacteria, mycobacteria, yeasts and fungi, and may also be extracted from plants and from the plasma of certain vertebrates. It was the first enzyme with antitumor activity to be studied extensively in humans (Maladkar, Singh, & Naik, 1993). Although the bacterium E. coli produces antitumor asparaginase (Müller, & Boos, 1998; Shanmugaprakash et al., 2015; Olu et al., 2008; Song et al., 2015), it causes toxic side effects when used continuously (Prakasham, Rao, Rao, Lakshmi, & Sarma, 2007; Kotzia & Labrou, 2005), coupled to high production costs.

Besides its use in antitumor treatments, asparaginase is also employed in food technology to reduce the formation of acrylamide (a toxic carcinogenic compound) in fried and baked food (Kornbrust, Stringer, Lange, & Hendriksen, 2009; Zuo, Zhang, Jiang, & Mu, 2015; Kumar, Shimray, Indrani, & Manonmani, 2014; Batool, Makky, Jalal, & Yusoff, 2016).

Interestingly, L-asparaginases from Aspergillus niger and Aspergillus oryzae have been commercially used to decrease the formation of acrylamide (Hendriksen, Kornbrust, Ostergaard, & Stringer, 2009). Asparaginase has been focused in the search for other microorganisms that produce the enzyme with low toxicity and low-cost raw materials in the fermentation process.

Zymomonas mobilis is one of several microorganisms that have been studied extensively in recent years and have aroused interest in the field of biotechnology. Besides its use in the production of ethanol, it provides asparaginase, levan, sorbitol (Ernandes, Boscolo, & Cruz, 2010) and gluconic acid (Silveira et al., 1999). Further, current research group has also studied the utilization of commercial sugar and sugarcane juice for the production of asparaginase by Z. mobilis and Erwinia herbicola by submerged fermentation. Results revealed that Z. mobilis has a great potential for the production of asparaginase (Wietchorek, Buzato, Menegat, & Celligoi, 2013).

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Zymomonas mobilis is a Gram-negative bacterium that uses glucose, fructose and sucrose as energy sources (Swings & Ley, 1977). Carbohydrate catabolism and ethanol production occur through the Entner-Doudoroff pathway (Sprenger, 1996). Since continuous fermentation in fermentation processes achieves highest production and yield rates, it may be an interesting approach for the production of asparaginase from Z. mobilis.

Thus, the objective of this work was to optimize the production of asparaginase from Z. mobilis through continuous fermentation, using the response surface experimental design and methodology, and testing the following variables: sucrose, yeast extract and asparagine. Results show that continuous fermentation is a useful method to produce asparaginase by Z. mobilis. To our knowledge, this is one of the few studies to describe the production of asparaginase by Z. mobilis using continuous fermentation.

Material and methods

Microorganism and growth media

Zymomonas mobilis ATCC 35001 was used in a slant culture containing glucose (10 g L-1), yeast extract (5 g L-1) and agar (15 g L-1), and stored in a refrigerator (2 to 8°C). Table 1 shows the composition of the fermentation medium. The components were solubilized in distilled water and sterilized separately in groups (sulfates, phosphates, chlorides, sucrose, asparagine and yeast extract) and mixed aseptically.

Continuous fermentation

Continuous culture was performed in a 0.5-L reactor and a 0.3-L working volume. Temperature was set at 30°C ± 1, with constant agitation. The culture was fed continuously with a new autoclaved medium (from a reservoir) using an adjustable-speed peristaltic pump. Initially, fermentations were carried out in batch mode for approximately 8 hours until the culture reached the exponential growth phase; the culture was afterwards fed continuously. Dilution ratio was set at 0.20 h-1 during the phase in which the variables were tested. During the volumetric production stage and medium optimized, the dilution rates 0.20, 0.25 and 0.30 h-1 were tested.

Analytical Determinations

Determination of asparaginase activity followed Peterson and Ciegler (1969). Briefly, 0.1 mL of enzymatic extract was added to a solution containing asparagine (0.04 M) and incubated at 37ºC. After 30 minutes, the reaction was stopped and 15% of trichloroacetic acid were added. After centrifugation, the supernatant was diluted in distilled water and treated with 0.1 ml of Nessler’s reagent. The optical density was measured at 400 nm. One international unit (IU) of L-asparaginase is defined as the amount of enzyme required to release 1 µmol of ammonia per minute at 37°C under pH 8.6 (buffer Tris-HCl 0.05 M). Asparaginase activity was expressed as IU L-1 of the fermented broth. Biomass was estimated by spectrophotometry at a wavelength of 610 nm, and the corresponding dry mass was obtained by a calibration curve.

Experimental design

The evaluation of the different conditions for asparaginase production from Z. mobilis was evaluated by incomplete 3³ factorial design, with 3 replications in the central. Variables comprised concentration of yeast extract (X1), concentration of sucrose (X2), and concentration of asparagine (X3), (Table 2). Statistica 7.0 was used for statistical analysis.

Results and discussion

The factorial experimental design has been used by several authors in studies on asparaginase production from Zymomonas mobilis (Camilios-Neto, Buzato, & Borsato, 2006; Casotti et al., 2007) and other microorganisms (Prakasham et al., 2007; Hymavathi, Sathish, Subba, & Prakasham, 2009; Kenari, Alemzadeh, & Maghsodi, 2011; Babu, Ramagopal, & Rami-Reddy, 2010; Agarwal, Kumar, & Veeranki, 2011; Usha, Mala, Venil, & Palaniswamy, 2011; Gurunathan, & Sahadevan, 2012; Singh & Srivastav, 2013).

Table 3 presents the design matrix used in this work, along with the experimental values obtained for asparaginase activity. It may be observed that assay 7, with 0.5, 20 and 0.9 g L-1 concentrations, respectively for yeast extract, sucrose and asparagine, demonstrated the highest enzyme activity, namely, 99.09 IU L-1. High concentration of asparagine was a factor that improved the rates of asparaginase activity. The presence of amino acid asparagine, related to asparaginase production, was demonstrated by the pioneer works by Galani, Drainas, and Typas (1985) with submerged fermentation from Z. mobilis in growth medium, with and without asparagine.

Assay 11 revealed the weakest performance of Z. mobilis for enzyme activity and biomass activity. The combination of variables tested showed it to be a low-yield growth medium with unsatisfactory fermentation. Assays 8 and 12, with progressive performance (asparaginase activity of 26.37 and 58.00 IU L-1, respectively), indicated that the ideal ratio between sucrose and yeast extract would feature ever-decreasing amounts of yeast extract. Assay 7 demonstrated that the ideal ratio was 1:0.025 (sucrose : yeast extract) among the four culture variations analyzed. Another observation is that the greater the enzyme activity, the higher the biomass value obtained.

Table 4 shows the four results obtained in the assays with asparagine concentration in the upper limit of the tested condition (0.9 g L-1). Amounts of yeast extract and sucrose used, as well as the biomass and ratio between yeast extract and sucrose.

Previous research have shown that the carbon source:yeast extract ratio had a strong effect on enzyme ativity response. Research by Camilios-Neto et al. (2006) yielded a maximum rate (16.55 IU L-1) of asparaginase from Z. mobilis, even though the above authors used different concentrations of molasses and yeast extract as variables. Lower asparaginase rates seem to be influenced by the high concentration of total reducing sugars in the molasses. Lee, Tribe, and Rogers (1979) reported that glucose at high concentrations produced incomplete fermentations since the sugar was not fully metabolized. Moreover, the minerals in the molasses could have inhibited growth, particularly magnesium salts in the substrate. Casotti et al. (2007) used sugarcane juice and yeast extract at high concentrations as variables, and asparagine at 1.0 g L-1. The authors obtained 9.75 IU L-1 as the best asparaginase activity which proved that, although sugarcane juice and yeast extract were rich substrates and represented an excellent medium for the growth of microorganisms, they did not favor asparaginase production, but merely stimulated alcohol fermentation (90% sugar intake) and biomass production.

The results of the activity were analyzed by response surface methodology (Figure 1). The equation below fitted the model using the incomplete 3³ factorial design:

where: Y1 represents the expected asparaginase activity; X1 is the concentration of yeast extract; X2 is sucrose concentration; X3 is the concentration of asparagine.

The surface response graph indicates that, to increase asparaginase activity, the concentration of sucrose and yeast extract must remain at low levels, while the amount of asparagine must increase.

So that the volumetric production stage could be evaluated, an assay was performed with an asparagine concentration of 1.3 g L-1 at different dilution rates: 0.20, 0.25 and 0.30 h-1. Table 5 shows the obtained results.

Activity and yield values were 15.6% higher for dilution rate 0.20 h-1 in an equal volume of produced fermented broth as that obtained from assay 7, with less asparagine. Results confirm that increase in asparagine stimulates asparaginase activity.

Nevertheless, activity, biomass and yield rates decrease as dilution rate increases. Dilution rates that exceeded maximum growth rate of the microorganism produced lower biomass rates. These data characterize cell wash since the microorganism cannot reproduce at the same speed as growth medium renovation.

Conclusion

Among the evaluated conditions, the best asparaginase production (117.45 IU L-1) occurred when the medium under optimum conditions contained 0.5 g L-1 yeast extract, 20 g L-1 sucrose and 1.3 g L-1 asparagine. Dilution rate over 0.20 h-1 resulted in decreased production parameters. In fact, the carbon:nitrogen ratio greatly affected asparaginase activity response, with the best ratio (1:0.025).

Acknowledgements

The authors would like to thank Indústria Farmacêutica Prati and Donaduzzi Cia Ltda for providing the laboratory facilities to conduct the experiments. Thanks are also due to the Universidade Paranaense, at Toledo, for providing the laboratory facilities for the determination of enzyme activity.

References

Agarwal, A., Kumar, S., & Veeranki, V. D. (2011). Effect of chemical and physical parameters on the production of L-asparaginase from a newly isolated Serratia marcescens SK-07. Letters in Applied Microbiology, 52(4), 307-313.

babu, U. K., Ramagopal, N., & Rami-Reddy, D. S. (2010). Optimization of L-asparaginase from isolated Aspergillus niger by using state fermentation on sesame cake via application of genetic algorithm, and artificial neural network-based design model. Journal of Biotechnology, 150(11), 538-539.

Batool, T., Makky, E. A., Jalal. M., & Yusoff, M. M. (2016). A comprehensive review on L-Asparaginase and Its Applications. Applied Biochemical and Biotechnology, 178(5), 900-923.

Camilios-Neto, D., Buzato, J. B., & Borsato, D. (2006). L-asparaginase production by Zymomonas mobilis during molasses fermentation: optimization of culture conditions using factorial design. Acta Scientiarum.Technology, 28(2) 151-153.

Casotti, J. F., Camilios-Neto, D., Bruzon, G., Machado, R. A. M., Buzato, J. B., & Celligoi, M. A. P. C. (2007). Utilization of raw materials from agroindustry – sugar cane juice and yeast extract – for asparaginase production by Zymomonas mobilis CP4. Semina: Ciências Agrárias, 28(4), 653-658.

Ernandes, F. M. P. G., Boscolo, M., & Cruz, C. H. G. (2010). Influência da composição do meio para a produção de etanol por Zymomomas mobilis. Acta Scientiarum. Technology, 32(1), 21-26.

Galani, I., Drainas, C., & Typas, M. A. (1985). Growth requirements and establishment of a chemically defined minimal medium in Zymomonas mobilis. Biotechnology Letters, 7(9), 673-679.

Gurunathan, B., & Sahadevan, R. (2012). Optimization of culture conditions and bench-scale production of L-asparaginase by submerged fermentation of Aspergillus terreus MTCC 1782. Journal of Microbiology and Biotechnology, 22(7), 923-929.

Hendriksen, H. V., Kornbrust, B. A., Ostergaard, P. R., & Stringer, M. A. (2009). Evaluating the potential for enzymatic acrylamide mitigation in a range of food products using an asparaginase from Aspergillus oryzae. Journal of Agricultural and Food Chemistry, 57(10), 4168-4176.

Hymavathi, M., Sathish, T., Subba, R. C. H., & Prakasham, R. S. (2009). Enhancement of L-asparaginase production by isolated Bacillus circulans (MTCC 8574) using response surface methodology. Applied Biochemical Biotechnology, 159(1), 191-198.

Kenari, S. L. D., Alemzadeh, I., & Maghsodi, V. (2011). Production of L-asparaginase from Escherichia coli ATCC 11303: optimization by response surface methodology. Food and Bioproducts Processing, 89(4), 315-321.

Kornbrust, B. A., Stringer, M. A., Lange, N. E. K., & Hendriksen, H. V. (2009). Asparaginase- an enzyme for acrylamide reduction in food products. In R. J. Whitehurst, M. V. Oort (Ed.). Enzymes in food technology (p. 59-87). Oxford-UK; Hoboken-NJ: Wiley-Blackwell.

Kotzia, G. A., & Labrou, N. E., (2005). Cloning, expression and characterization of Erwinia carotovora asparaginase. Journal of Biotechnology, 119(4), 309-323.

Kumar, N. M., Shimray, C. A., Indrani, D., & Manonmani, H. (2014). Reduction of acrylamide formation in sweet bread with L-asparaginase treatment. Food Bioprocess Technology, 7(3), 741-748.

Lee, K. J., Tribe, D. E., & Rogers, P. L. (1979). Ethanol production by Zymomonas mobilis in continuous culture at high glucose concentration. Biotechnology Letters, 10(1), 421-426.

Maladkar, N. K., Singh, V. K., & Naik, S. R. (1993). Fermentative production and isolation of L-asparaginase from Erwinia carotovora, EC-113. Hindustan Antibiotics Bulletin, 35(1-2), 77-86.

Müller, H. J., & Boos, J. (1998). Use of L-asparaginase in childhood ALL. Critical Reviews in Oncology: Hematology, 28(2), 97-113.

Olu, A., Podobed, O. V., Borisova, A. A., Sidoruk, K. V., Aleksandrova, S. S., Omel’ianiuk, N. M., & Sokolov, N. N. (2008). Antitumor activity of L-asparaginase from Yersinia pseudotuberculosis. Biomed Khim, 54(6), 712-719.

Peterson, R. E., & Ciegler, A. (1969). L-Asparaginase production by Erwinia aroideae. Applied Microbiology, 18(1), 64-67.

Prakasham, R. S., Rao, C. H. S., Rao R. S., Lakshmi, G. S., & Sarma, P. N. (2007). L-asparaginase production by isolated Staphylococcus sp. - 6A: design of experiment considering interaction effect for process parameter optimization. Journal of Applied Microbiology, 102(5), 1382-1391.

Shanmugaprakash, M., Jayashree, C., Vinothkumar, V., Senthilkumar, S. N. S., Siddiqui, S., Rawat, V., & Arshad, M. (2015). Biochemical characterization and antitumor activity of three phase partitioned L-asparaginase from Capsicum annuum. Separation and Purification Technology, 142(4), 258-267.

Silveira, M. M., Wisberck, E., Lemmel, C., Erzinger, G., Costa, J. P. L., & Bertasso, M. (1999). Bioconversion of glucose and fructose to sorbitol and gluconic acid by untreated cells of Zymomonas mobilis. Journal of Biotechnology, 75(2-3), 99-103.

Singh, Y., & Srivastav, S. K. (2013). Statistical and evolutionary optimization for enhanced production of an antileukemic enzyme, L-asparaginase, in a protease-deficient Bacillus aryabhattai ITBHU02 isolated from the soil contaminated with hospital waste. Indian Journal of Experimental Biology, 51(4), 322-335.

Song, P., Ye, L., Fan, J., Li, Y., Zeng, X., Wang, Z., & Ju, D. (2015). Asparaginase induces apoptosis and cytoprotective autophagy in chronic myeloid leukemia cells. Oncotarget, 28(6), 3861-3873.

Sprenger, G. A., (1996). Carbohydrate metabolism in Zymomonas mobilis: a catabolic highway with some scenic routes. FEMS Microbiology letters, 145(3), 301-307.

Swings, J., & Ley, J. (1977). The biology of Zymomonas. Bacteriology Review, 41(1), 1-46.

Usha, R., Mala, K. K., Venil, C. K., & Palaniswamy, M. (2011). Screening of Actinomycetes from mangrove ecosystem for L-asparaginase activity and optimization by response surface methodology. Polish Journal of Microbiology, 60(3), 213-221.

Wietchorek, P. J. A. G., Buzato, J. B., Menegat, F. B., & Celligoi, M. A. P. C. (2013). Assessment of Zymomonas mobilis and Erwinia herbicola for the anti leukaemia asparaginase production. BBR - Biochemistry and Biotechnology Reports, 2(1), 31-34.

Zuo, S., Zhang, T., Jiang, B., & Mu, W. (2015). Reduction of acrylamide level through blanching with treatment by an extremely thermostable L-asparaginase during French fries processing. Applied Biochemical Biotechnology, 19(4), 841-851.

Author notes

buzato@uel.br