Optimization of asparaginase production from Zymomonas mobilis by continuous fermentation Francieli

Asparaginase is an enzyme used in clinical treatments as a chemotherapeutic agent and in food technology to prevent acrylamide formation in fried and baked foods. Asparaginase is industrially produced by microorganisms, mainly gram-negative bacteria. Zymomonas mobilis is a Gram-negative bacterium that utilizes glucose, fructose and sucrose as carbon source and has been known for its efficiency in producing ethanol, sorbitol, levan, gluconic acid and has recently aroused interest for asparaginase production. Current assay optimizes the production of Z. mobilis asparaginase by continuous fermentation using response surface experimental design and methodology. The studied variables comprised sucrose, yeast extract and asparagine. Optimized condition obtained 117.45 IU L with dilution rate 0.20 h, yeast extract 0.5 g L, sucrose 20 g L and asparagine 1.3 g L. Moreover, carbon:nitrogen ratio (1:0.025) strongly affected the response of asparaginase activity. The use of Z. mobilis by continuous fermentation has proved to be a promising alternative for the biotechnological production of asparaginase.

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).
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.

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.3.5 Sucrose a 10.0, 20.0 and 30.0 Asparagine a 0.1, 0.5 and 0.9 Yeast extract a 0.5 , 1.0 and 1.5 a According to factorial design (Table 2).

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 adjustablespeed 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.
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: Y 1 represents the expected asparaginase activity; X 1 is the concentration of yeast extract; X 2 is sucrose concentration; X 3 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 (117.45IU 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).

Figure 1 .
Figure 1.Response surface showing the effect of asparagine and sucrose on asparaginase activity.Source: Authors.

Table 1 .
Composition of the fermentation medium.

Table 2 )
. Statistica 7.0 was used for statistical analysis.

Table 2 .
Box-Behnken design with the variation levels of tested variables

Table 3 .
Matrix containing numbered assays, decoded variables and results obtained for asparaginase.

Table 4 .
Decoded variables and obtained results.

Table 5 .
Values obtained in optimized culture of Z. mobilis at different dilution rates.