A study of the optimal conditions for glucoamylases obtained from Aspergillus niger using amylopectin from cassava starch as carbon source

A fourteen day pilot study carried out showed that high glucoamylase activities were obtained on the 4 and 11 th day of fermentation and the enzymes were harvested on the respective days giving the codes GluAgCSV4 and GluAgCSV11. The optimal pH and optimal temperatures for enzyme activities GluAgCSV4 and GluAgCSV11 were in a range of 6 to 7 and 50 to 55, using cassava, guinea corn and tiger nut starch as substrates, respectively. The enzyme activity (GluAgCSV4) was enhanced by Ca 2+ , Mn 2+ , Fe 2+ and Zn 2+ . Co 2+ had inhibitory effect on the enzyme while Pb 2+ completely inactivated the enzyme. The enzyme activity (GluAgCSV11) was enhanced by Ca 2+ and Co 2+ . Zn 2+ , Fe 2+ Mn 2+ and Pb 2+

Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License industry to make sweets, drinks, juices and for fermentation into products like citric acid, ethanol, bakers' 'yeast, as well as in paper and textile.The original method of starch hydrolysis known as acid hydrolysis (Kolusheva and Marinova, 2007) requires the use of corrosion resistant materials, gives rise to high colour and salt ash content (after neutralisation), needs more energy for heating and is relatively difficult to control.As a result of these problems, enzymatic hydrolysis becomes a better method for starch hydrolysis since the above mentioned constraints are not peculiar.Starch hydrolysis in industry most times requires a combination of enzymes for complete hydrolysis.There is the need therefore to optimize the necessary conditions for glucoamylase activity to achieve complete starch hydrolysis without necessarily involving multiple enzymes.Starch is a major storage product of economically important crops like cassava, guinea corn and tiger nut.These plants have the potentials to become the principle feedstock for fuel, alcohol and glucose syrup market.This research is aimed at production of glucoamylases from Aspergillus niger in a submerged fermentation using amylopectin fractionated from cassava starch as the only carbon source and possibly optimize the conditions for the applications in hydrolysis of cassava, guinea corn and tiger nut starch.

Collection of plant samples
The plant materials (cassava tuber, guinea corn, and tiger nut) were obtained at Orba main market, Udenu Local Government, Enugu State, Nigeria.

Processing of cassava and cassava starch
Cassava starch was processed using the method described by Corbishley and Miller (1984) with the following modifications.Freshly harvested cassava tubers were peeled washed clean and grated.The grated cassava (1.2 kg) was soaked in 4 L of distilled water for 1 h after which it was sieved (3 times) with muslin cloth.This was allowed to stand for 4 h and the supernatant decanted.The isolated wet starch was sun dried and packaged in plastic air tight container, labelled and kept in a cool, dry place.

Processing of cereal starch
The starch from cereals was processed using the method described by Agboola et al. (1990) with the following modifications.The seeds were sun dried and ground to fine flour.300 g of the flours were suspended in 3 L of distilled water for 24 h.The suspended flour was sieved using muslin cloth.The extracted starch was allowed to sediment for 4 h at room temperature.The supernatant was decanted off and the starch washed with 3 L of distilled water twice and finally allowed to stand for 4 h.The supernatant was then decanted and the resulting wet starch was sun dried and then packaged in an air tight container and stored at room temperature.

Fractionation of starch into amylose and amylopectin
Fractionation of amylose and amylopectin was carried out by following the general procedure of (Sobukola and Aboderin, 2012).This consists of heating and stirring starch dispersion (0.8%, w/v in water) in water bath at 100°C until starch is gelatinized.Starch solutions were filtered using filter paper to remove insoluble residues, and the pH adjusted to 6.3 using phosphate buffer.The solution was stirred in a boiling water bath for 2 h to disperse the starch molecules.Thereafter, n-Butyl alcohol was added (20%, v/v), and the solution was stirred at 100°C for 1 hr, followed by cooling to room temperature over a period of 24-36 h.Amylose butyl alcohol complex crystals was formed and precipitated during cooling, and was separated by filtration.The amylopectin remaining in the supernatant was recovered by adding excess methyl alcohol.

Isolation of glucoamylase producing fungi
Glucoamylase producing fungi were isolated by adopting the method of Martin et al. (2004) as modified by Okoye et al. (2013).Tiger nut starch was fractionated into amylose and amylopectin.The wet amylopectin was left open on shelve to allow microorganisms to grow on it.A loop of each organism was streaked onto potato dextrose agar PDA under the flame of Bunsen burner.Streaks were made from each side of the plate, marking an initial point, with sterilization of the wire loop after each side has been completed.The plates were thereafter incubated at 35°C till visible colonies were observed.All morphological contrasting colonies were purified by repeated streaking and sub-culturing on separate plates.This process was continued till pure fungal culture was obtained.Pure fungal isolates were maintained on potato dextrose agar (PDA) slopes or slants as stock cultures.PDA media were prepared according to the manufacture's description.In the description, 3.9 g of PDA powder was weighed and added in small volume of distilled water and made up to 100ml.The medium was autoclaved at 121°C, 15 psi for 15 min.It was allowed to cool to about 45°C and then poured into petri dishes and allowed to gel.The plates were then incubated in a B and T Trimline incubator at 37°C for 24 h to check for sterility.Three day old pure cultures were examined.The colour, texture, nature of mycelia or spores and growth patterns were also observed.The three day old pure cultures were used in preparing microscopic slides.A little bit of the mycelia was dropped on the slide and a drop of lactophenol blue was added to it.A cover slip was placed over it and examination was performed under the light microscope at X400 magnification.Identification was carried out by relating features and the micrographs to "Atlas of mycology" by Barnett and Hunter (1972).

Glucoamylase production
Glucoamylase was produced by adopting the method described by Bagheri et al. (2014) with the following modifications.A 250 ml Erlenmeyer flask containing 100 ml of sterile cultivation medium optimized for glucoamylase with 0.3% ammonium sulphate (NH4)2SO3), potassium dihydrogen phosphate (0.6% KH2PO4), magnesium sulphate hepta hydrate 0.1% (MgSO4.7H2O),0.01% ferrous sulphate hepta hydrate (FeSO4.7H2O),0.1% calcium chloride (CaCl2) and 1%, amylopectin.The flask was stoppered with aluminium foil and autoclaved at 121°C, 15 psi for 15 min.From the PDA slants, fresh plates were prepared and three day old cultures were used to inoculate the flasks.In every sterile flask, two discs of the respective fungal isolates were added using a cork borer of diameter 10 mm and then plugged properly.The culture was incubated for 7 days at room temperature (30°C).At each day of harvest, flasks were selected from the respective groups and mycelia biomass separated by filtration using filter paper.Each day, the filtrate was analyzed for glucoamylase activity and extracellular protein concentration till the 14 th day of fermentation.After the 14 days pilot SmF studies, 4 and 11 th day of fermentation were chosen for mass production.

Glucoamylase assay
Glucoamylase activity was assayed by the method of Parbat and Singhal (2011) with the following modifications.0.5ml of the enzyme was added into a clean test tube followed by 0.5 ml 1% soluble starch solution in 50 mM acetate buffer (pH 5.5) at 50°C and was allowed to stand for 20 min. 1 ml of 3, 5-dinitrosalicyclic acid (DNSA) reagent was added and boiled for 10 min to stop the reaction.1 ml of sodium potassium tartarate was added to stabilize the red colour produced.The mixture was then allowed to cool and the glucose released was measured using a JENWAY 6405 UV/VIS spectrophotometer (Beckman/Instruments, Inc., Huston Texas) at 540 nm.Absorbance values were converted to glucose concentrations by extrapolation from the glucose standard curve.Glucoamylase activity unit (U) was expressed as the amount of enzyme releasing one μ mole of glucose equivalent per minute per ml.

α-amylase assay
The α-amylase activity was assayed by adopted the method of Bernfield (1955).The reaction mixture contains 0.5 ml of the enzyme preparation and 0.5 ml of (1% w/v) starch solution in 20mM sodium phosphate buffer (pH 7.0).The reaction mixture was incubated at 55°C for 60 min after which the reaction was stopped by addiction of 1 ml 3, 5-dinitrosalicyclic acid (DNSA) reagent and boiling for 10 min. 1 ml of sodium potassium tartarate was added to stabilize the red colour produced.The mixture was then allowed to cool and the glucose released was measured using a JENWAY 6405 UV/VIS spectrophotometer (Beckman/Instruments, Inc., Huston Texas) at 600 nm.α-amylase activity unit (U) was expressed as the amount of enzyme releasing one μ mole of reducing end groups (maltose) per minute under assay conditions.

Protein determination
Protein content of the enzyme was determined by the method of Lowry et al. (1951), using Bovine Serum Albumin as standard.

Partial purification
The crude enzyme preparation was made up to 20 and 70% ammonium sulphate saturation with solid (NH4)2SO4 for both GluAgCSV4 and GluAgCSV11, respectively.This was kept at 4°C for 30 h; thereafter it was centrifuged with Cole-palmer VS-13000 micro centrifuge at 4000 rmp for 30 min.The precipitate was collected and re-dissolved in 20 mM acetate buffer pH 5.5.The glucoamylase activity and protein were determined as described above.

Gel filtration chromatography
A volume (20 ml) of the precipitated enzyme was introduced into a (50 × 2.5 cm) gel chromatographic column and subjected to gel filtration using sephadex G-100 pre-equilibrated with 0.02 M sodium acetate buffer pH 5.5.Fractions were collected at a flow rate of 5 ml/20 min.The protein concentration of each fraction was monitored using a JENWAY 6405 UV/VIS spectrophotometer (Beckman/Instruments, Inc.Huston Texas) at 280 nm.The glucoamylase activity of each fraction was assayed as earlier discussed with the active fractions pooled together and stored at -10°C Optimizing glucoamylase activity with respect to pH, temperature, metal ion concentration and substrate concentration

Optimum pH
The optimum pH for enzyme activity was determined using 0.02 M sodium acetate buffer pH 3.5 to 5.5, phosphate buffer pH 6.0 to 7.5 and Tris-HCl buffer pH 8.0 to 10.0 at intervals of 0.5.0.5 ml of gelatinized starch solution (1%) was equilibrated with 1 ml of the buffers (20 mM) of respective pHs for 5 min at 37°C.0.5 ml of the enzyme was added and the reaction mixture was mixed properly and allowed to stand for 20 min at 50°C.The glucoamylase activity was assayed as described above using starch as substrate.

Metal ions concentration
The concentrations, 20, 30, 40 and 50 mM of metal salts (ZnCL2, CoCl2, MnCl2, FeCl2, PbCl2 and CaCl2) were prepared in 20 mM sodium acetate.Each of the reaction mixtures contains 0.5 ml of enzyme solution, 0.5 ml of starch solution (1%) and 1 ml of metal ion solutions (Ca 2+ , Mg 2+ , Mn 2+ , Fe 2+ , Co 2+ and Zn 2+ ).The mixtures were incubated for 20 min at the predetermined optimal pHs and temperatures.To study the effect of metal ions on glucoamylase activity/stability, the reaction was carried out with and without metal ions.In all the above experiments, the enzyme activity was calculated as the average of three independent sets of experiments and the standard deviation in all cases was negligible.

Optimum temperature
The optimum temperature for glucoamylase activity was determined by incubating the enzyme with gelatinized starch solution (1%) at 30 to 80°C for 20 min at respective predetermined optimal pHs.Glucoamylase activity was assayed as described above using starch as substrate.

Substrate concentration
The effect of substrate concentration on glucoamylase activity was determined by incubating 0.5 ml of enzyme with 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg/ml of starch solution for 20 min at the respective predetermined optimal pHs and temperatures.The glucoamylase activity was assayed as described above using starch from cassava, guinea corn and tiger nut starch as substrate.

RESULTS AND DISCUSSION
In all the above experiments, the results were calculated as the average of three independent sets of experiments and the standard deviation in all cases was negligible.A fourteen day pilot study was carried out to determine the day of highest protein production, α-amylase and glucoamylase activity in submerged fermentation using amylopectin obtained from cassava starch as carbon source.Enzyme activities were assayed using cassava,  guinea corn and tiger nut starch as substrate (Figures 1,  2 and 3).Two major peaks were obtained on day 4 and 11 with activities 299.56 and 298.47 µmol/min, respectively, using cassava starch was used as substrate.Also, using guinea corn starch as substrate, a major peak was obtained on day 4 with glucoamylase activity 236.15 µmol/min, while day 4 exhibited the highest peak with glucoamylase activity (304.30µmol/min) using tiger nut starch as substrate.Therefore, day 4 and 11 were chosen for mass production of the enzyme.The high increase in glucoamylase activity observed in the study could be due to de-branching activity exhibited by glucoamylase in addition to its α-1, 4glucosidic activity.Glucoamylase first hydrolyses the amorphous regions before attacking the crystalline regions and both amylose and amylopectin are hydrolysed simultaneously to smaller molecular sizes (Wang and Wang, 2001).The decrease in glucoamylase activity could be due to depletion in the level of branch points in the carbon sources, product inhibition as well as depletion in growth supplement in the broth (Nahar et al., 2008).Alva et al. (2007) reported that the productivity of glucoamylase production is generally subjected to catabolite repression by glucose and other readily  Ammonium sulphate saturation (20 and 70%) were found suitable to precipitate proteins with highest glucoamylase activity in GluAgCSV4 and GluAgCSV4.After ammonium sulphate precipitation and gel filtration, the specific activities were found to increase from 83.43 to 188.08 U/mg protein with purification fold 2.3 and 156.83 to 245.22 U/mg protein with purification fold 1.56 for GluAgCSV4 and GluAgCSV11, respectively (Table 1).The increase in specific activity could be as a result of exclusion of impurities contributed by ammonium sulphate salt that may have inhibited the enzyme activity.
The optimal pH for GluAgCSV4 were 7.5, 7.0 and 6.0 using cassava, guinea corn and tiger nut starch as substrates, respectively while that for GluAgCSV11 were 7.0, 5.0 and 6.0 using cassava, guinea corn and tiger nut starch as substrates (Figures 4 and 5).This suggests that at optimum pH range of 5.0-7.5, cassava, guinea corn and tiger nut starch hydrolysis by GluAgCSV4 and GluAgCSV11 could be very much enhanced.Parbat and Singhal, (2011) reported optimum pH of 5.0 for glucoamylase from Aspergillus oryzae using agro industrial products.Similarly, Puri et al. (2013) reported optimum pH of 5.0 for glucoamylase from A. oryzae.Rangabhashiyam et al. (2012) reported optimum pH of 5.2 for glucoamylase obtained from A. niger using agro waste as carbon source.Nahid et al. (2012) reported acidic pH range of 4.5 to 4.7 for glucoamylase from A. niger.Kumar et al. (2013) reported optimum pH 6.0 for glucoamylase from A. oryzae using wheat bran as carbon source.Koc and Metin (2010) also reported optimum pH of 6.0 for glucoamylase produced by Aspergillus flavus.Zambare (2010) reported optimum pH of 6.0 for glucoamylase from A. oryzae.The result of this study has similarity with that of aforementioned authors.The disparity could be as a result of difference substrates used for the enzyme assay.The optimum pH range suggests that, glucoamylases obtained in this study have    wide pH stability which is of industrial importance.Most of amylases are known to be metal ion-dependent enzymes (Deb et al., 2013).The effect of metal ion on glucoamylase activity showed that, GluAgCSV4 activity was enhanced by Ca 2+ , Mn 2+ , Fe 2+ and Zn 2+ while Co 2+ had inhibitory effect on the enzyme but Pb 2+ inactivated the enzyme (Figure 6).Also, GluAgCSV11 activity was enhanced by Ca  and Pb 2+ completely inactivated the enzyme (Figure 7).The optimum pH range obtained suggest that is more likely to readily form nucleophilic hydroxide ions with metals like Ca 2+ , Co 2+ , Fe 2+ , Mn 2+ and Zn 2+ by activating a bound water molecule, this stabilizes the negative charges that are formed at the active sites allowing more weak interactions to hold the substrates in proper orientation at the active site of the enzyme there by enhancing the activity.Also, it has been suggested that metal ions probably force amylases to adopt a compact structure, by salting out the hydrophobic residues of the enzyme, thereby inducing resistance to extreme pH and temperatures (Kareem et al., 2014) ions can make it more suitable for use in industrial starch bioconversion process.The inhibition on glucoamylase activity inhibition may be due to the complexes formed between metals and the enzyme which prevent it from binding to the substrate and form the product (Aziz and Ali, 2012).Koc and Metin, (2010)

Figure 1 .
Figure 1.Monitoring the day of highest glucoamylase, α-amylase activity and protein concentration in liquid broth using amylopetin from cassava starch as the only carbon source (cassava starch was used as substrate).

Figure 2 .
Figure 2. Monitoring the day of highest glucoamylase, α-amylase activity and protein concentration in liquid broth using amylopectin from cassava starch as the only carbon source (tiger nut starch was used as substrate).

Figure 3 .
Figure 3. Monitoring the day of highest glucoamylase, α-amylase activity and protein concentration in liquid broth using amylopectin from cassava starch as the only carbon source.

Figure 4 .
Figure 4. Effect of pH on glucoamylase activity obtained from Aspergillus niger on the 4 th day of fermentation using amylopectin from cassava starch as carbon source (GluAgCSV4).

Figure 5 .
Figure 5.Effect of pH on glucoamylase activity obtained from Aspergillus niger on the 11 th day of fermentation using amylopectin from cassava starch as carbon source (GluAgCSV11).

Figure 6 .
Figure 6.Effect of ion concentration on glucoamylase activity obtained from Aspergillus niger on the 4 th day of fermentation using amylopectin from cassava starch as carbon source (GluAgCSV4).

Figure 7 .
Figure7.Effect of ion concentration on glucoamylase activity from Aspergillus niger on the 11 th day of fermentation using amylopectin from cassava starch as carbon source (GluAgCSV11).
. Since glucoamylases are used together with α-amylase, which requires Ca