Optimization of growth and extracellular glucoamylase production by Candida famata isolate

Candida famata was isolated from traditional Moroccan sourdough. It exhibited high glucoamylase and biomass production. Starch induces high glucoamylase production C. famata with maximum glucoamylase activity at 5 g/L. Glucose stimulates good production in biomass but strongly inhibits glucoamylase production. Among the sources of nitrogen tested, yeast extract and the (NH 4 ) 2 HPO 4 gave maximum glucoamylase and biomass after 72 h of incubation in liquid medium at 30°C, pH 5 and 105 rpm.

Different culture conditions greatly affect the production of amylase.So, it becomes necessary to investigate different factors involved in maximum production of amylase.Yeast amylases production have been reported *Corresponding author.E-mail: lagzoulimohamed@yahoo.fr. at different pH and temperatures of incubation (Spenser-Martins and Van Uden, 1979;Oteng-Gyang et al., 1981).
In the present study, screening was carried out with Candida famata, which was isolated from traditional Moroccan sourdough.Studies were also conducted to determine the optimum culture conditions and factors involved in maximum production of glucoamylase.

Microorganism
C. famata was isolated from traditional Moroccan sourdough using the following medium (g/L): soluble starch 5, KH2PO4 3; (NH4)2SO4 1; MgSO4 0.5; and yeast extract 4. pH was adjusted to 5 with 0.1 M HCl.Medium was solidified by the addition of 1.5% agar, and autoclaved at 121°C for 15 min.Liquid medium was incubated at 30°C in a flask on rotary shaker set at 105 rpm for 72 h.

Isolation and screening of the isolate
1 g of traditional Moroccan sourdough were suspended in 10 ml of sterile physiological water and plated on medium using pour plate method and then incubated at 30°C for 72 h.around microbial colonies after addition of iodine solution.Colonies with the largest halo-forming zone were isolated for further investigation.Evaluation of the clear zones was estimated as diameter (mm) of the clear zone.Yeast colonies producing large clear zones were picked up and purified three times by streaking on Potato Dextrose Agar (PDA).Pure cultures were maintained on PDA, stored in a refrigerator and sub-cultured at 3 months interval.

Measurement of enzyme activity
The fermented broth was taken after 72 h and centrifuged at 7000 rpm for 10 min, and then substrate-free supernatant was used for estimation of enzyme activity.Amylase activity was determined by measuring the reducing sugar formed by the enzymatic hydrolysis of starch using the method of Somogyi and Nelson (Nelson et al., 1944).0.25 ml soluble starch (1%), 0.15 ml phosphate buffer (0.1 M) and 0.1 ml enzyme solution were mixed and incubated at 40°C in water bath for 30 min.The reaction was stopped with 2 ml of Somogyi reactive, and 1.5 ml of distilled water, followed by boiling for 15 min to develop blue color.The absorbance was measured at 540 nm with a spectrophotometer.The blue color was measured against the control in which no enzyme was added.A calibration curve of absorbance and concentration of glucose was established with known amount of glucose.
One unit (µmol/L/min) of glucoamylase was defined as the amount of µmol of reducing sugar per liter of enzymes per min, measured as glucose under the conditions of assay.

Biomass yield
It was determined by measuring the absorbance of the suspension at 600 nm.

Effect of incubation time, temperature and medium pH
To ascertain the effect of culture conditions the present study was carried out at different incubation periods (24, 48, 72 and 96 h), temperatures (20, 25, 33 and 40°C), and medium pH (3.0, 5.0, 6.0, 7.0 and 8.0).Their effects on biomass yield and glucoamylase production were recorded.

Effect of carbon and nitrogen sources
The production of extracellular amylases under different carbon and nitrogen availability were studied in liquid medium.Seven carbon sources (starch, sucrose, lactose, maltose, galactose, fructose and glucose), and nitrogen sources: organic (3 g/L) yeast extract, peptone, tryptone, meat extract and inorganic: CH4N2O; NaNO3; (NH4)2HPO4; SO4(NH4)2 were added at the same concentration of nitrogen (0.318 g/L N equivalent of 1 g of SO4(NH4)2) and initial medium pH 5; Effects of carbon and nitrogen sources on production of glucoamylase and biomass yield was recorded.Fermentation experiments were carried out for 72 h.

Batch fermentation in a laboratory bioreactor
C. famata was grown in a (2 liter Setric Set 002 M Bioreactor) containing 1 L of the production medium.The bioreactor was operated at 30°C, and 105 rpm.The initial pH of the medium was adjusted to 5.5 with 0.1 M of KH2PO4 before sterilization; inoculum was prepared in Erlenmeyer flasks in a volume corresponding to 10% of fermentation broth medium and incubated at 30°C at 150 rpm.Samples were withdrawn at 1 h intervals, and centrifuged at 6000 rpm for 10 min after the recording biomass; cellfree supernatant was used for the assay of glucoamylase.

RESULTS AND DISCUSSION
Of the sixteen yeasts isolated, four were considered to be the best amylase producing strains.One of them identified as C. famata using API gallery ID 32 C (Biomérieux REF 32 200) was selected for the present work.

Effect of incubation time
C. famata showed different glucoamylase activities at different incubation period; it was found that the isolate produced maximum activity after 72 h of incubation (Figure 1) but highest biomass yield was recorded after 48 h of incubation time.The final pH of the supernatant was found to range from 6 to 7.5.Production of amylase after 3 days of incubation by Aspergillus sp. was also reported by Rahman et al. (1993) and Cherry et al. (2004).

Effect of temperature on biomasse and glucoamylase activity
The influence of temperature on glucoamylase activity of the crude enzyme showed that enzyme activity increased progressively with increase in temperature from 25°C reaching a maximum at 30°C (Figure 2).Above 35°C, there was a reduction in the glucoamylase activity.Similar results were also reported by Rene and Hubert (1985) with Filobasidium capsuligenum.Other researchers (Bertrand et al., 2005) also reported that maximum glucoamylase production occurred at the same temperature.Temperature for optimal production of amylolytic enzymes by Yarrowia lipolytica was 28°C (Cheon et al., 1997).These results disagree with results found by Chandra et al. (1980).This organism did not produce α-amylase at 30°C although it grew very well at this temperature.

Effect of initial pH of the medium
Medium pH also plays an important role on the production of microbial enzymes.In the present study, maximum production was achieved at medium with initial pH 5 (Figure 3); the pH of the culture filtrates after 72 h were ranged from 6 to 7.5.Similarly, Quang et al. (2000) found that optimum pH of fermentation medium of Thermomyces lanuginosus ATCC 34626 was found to be 4.9.In the same way Reiser and Gasperik (1995) reported that optimum activity of Saccharomycopsis fibuligera glucoamylase was observed at pH 5.5.
Comparable results were found by Rene and Hubert (1985) with F. capsuligenum at pH ranging from 5 to 5.6.Others found that the optimum pH for a better production of amylolytic enzymes by Lipomyces kononenkoae is 5.5 (Isabel, 1982).Also, Taylor et al. (1978) reported that Humicola lanuginosa produces the two shapes of amylolytic enzymes, with optimum of pH of 4.9 and 6.6, whereas for other groups of bacteria, optimum pH of growth and production of the enzyme by Bacillus sp. is 7 (Carlos and Souza, 2001).

Effect of starch concentration
Amylase became more active in relation to the increase in starch concentration from 2.5 g/L to 5 g/L.Beyond 5 g/L, there was a decline in amylase activity (Figure 4).Similar starch concentration (5 g/L) was used for the production of amylase by F. capsuligenum (Rene and Hubert, 1985), Clostridium thermosulfurogenes (Hyun and Zeikus, 1985) and Clostridium sp.(Madi et al., 1987).On the contrary, other investigators reported that maximum amylase production was produced at 10 g/L, with Lipomyces kononenkoae, (Isabel,982), Schwanniomyces alluvius (Jeffrey and Michael, 1982) and 15 g/L with Chaetomium thermophilum (Jing et al., 2005).Also, growth of the studied yeast increases according to the concentration of the starch to reach a maximum value at 10 g/L.

Effect of yeast extracts concentration
Figure 5 shows that maximum of glucoamylase activity is obtained with the concentration 2 g/L of yeast extract.Production of -amylase by Aspergillus fumigatus was obtained at 2 g/L (Cherry et al., 2004), Pichia subpelliculosa ABWF-64 (Sanjeev-Kumar and Satyanarayana, 2001), Lactobacillus amylovorus at 1 g/L (Oda et al., 2002), and 1.5 g/L with Penicillium sp.(Capuccino and Sherman, 2001).Also, Suzuki and Chishiro (1983) reported that Endomycopsis fibuliger gives maximum growth and glucoamylase activity at 3 g/L of yeast extract.
These results disagree with the maximum enzyme production obtained with yeast extract by T. lanuginosus (Nguyen et al., 2000) and Aspergillus niger (Djekrif-Dakhmouche et al., 2006).

Effect of nitrogen sources on glucoamylase production
Yeast extract was replaced in separate medium with 0.3% of different organic and equivalent of 1 g/L of sodium sulphate of inorganic compounds as nitrogen source keeping the rest of the medium composition the same.Among the nitrogen sources, yeast extract was the best organic one followed by meat extract, peptone and tryptone (Table 1).On the other hand, urea was the best inorganic nitrogen source followed by (NH 4 ) 2 PO 4 .
Yeast extract has been reported to significantly influence enzyme production (Hamilton et al., 1999;Nguyen et al., 2000).Similarly, Han et al. (2005) reported that Basidiomycete Ganoderma lucidum gave the highest starch degradation with yeast extract.This is in agreement with the observation of Cherry et al. (2004) who reported that the fungus A. fumigatus produces high amylase activity with yeast extract.
The effects of combination of organic and inorganic compounds on amylase production were also studied.We observed that the combination of urea and yeast extract gives highest amylase yields (Table 1), followed by combination of beef extract and urea.

Batch fermentation in laboratory bioreactor
Optimum conditions of growth and of production of enzymatic activity are limited in Erlenmeyer because of the consumption of the substrates and the accumulation of cellular metabolites.To control various parameters of culture, the kinetics of growth and enzymatic production of C. famata were carried out by bioreactor under the optimum conditions with the culture previously established.
Results reported in Figure 6 show that enzymatic activity was related to the cell multiplication.Glucoamylase activity increased gradually in the medium after 8 h of culture time and reached a maximum value of 2926.19 µmol/L/min at the end of the exponential phase.The initial pH of the nutrient solution was adjusted to 5. The results showed that pH had an important effect on glucoamylase production.pH increase from 5 at 0 h of culture to 7.8 at 62 h was conversely proportional to starch degradation.In parallel starch is gradually consumed in the medium during the growth.Its hydrolysis is noticeable during logarithmic phase.Starch degradation starts from 7 h to draw near at its maximum after 38 h of the fermentation period.

Conclusion
Yeast extract, soluble starch and urea would be beneficial for glucoamylase production; the enzyme activity was increased by urea.The yeast grown on glucose as well as on all tested simple sugars showed considerable growth production but strongly inhibited glucoamylase activity.Based on these results, the proposed compositions of the fermentation medium (w/v) are soluble starch 5 g/L; yeast extract 3 g/L, urea 0.77 g/L, KH 2 PO 4 3 g/L and MgSO 4 0.5 g/L.The pH should be adjusted to 5, and cultivation should be at 30°C under 105 rpm agitation.To reach maximum glucoamylase activity, 72 h fermentation time is needed.

Figure 1 .
Figure 1.Effect of incubation time on biomass production and glucoamylase activity by Candida famata.

Figure 2 .
Figure 2. Effect of temperature on biomass production and glucoamylase activity by Candida famata.

Figure 3 .Figure 4 .
Figure 3.Effect of initial pH of medium on biomass production and glucoamylase activity by Candida famata.

Figure 5 .
Figure 5.Effect of yeast extract concentration on biomass production and glucoamylase activity by Candida famata.

Table 1 .
Effect of nitrogen sources on biomass production and glucoamylase activity by Candida famata.