Intensification of β-glucosidase enzyme production from Aspergillus niger using extractive fermentation with an aqueous two-phase system

2.1 Micro-organisms

Aspergillus niger NRRL 322 was obtained from the National Chemical Laboratory Pune, India. The fungus was maintained on potato dextrose agar (PDA) medium. Spore suspension was prepared by dislodging the spores from fully sporulated PDA slant into sterile 0.05% Tween 80 solution. Spore count was determined using a haemocytometer.

2.2 Substrate

Wheat bran was obtained from Surya Rice and Poha Mills, Thane (W), Mumbai (India). The substrate was dried at 105°C to constant weight and then ground to a 20 mesh size [6].

2.3 Chemicals

The substrate for β-glucosidase, i.e. p-nitrophenyl β-d-glucopyranoside (pNPG) was purchased from Sesco Reaserch Laboratory (SRL), Mumbai. All the chemicals, media components and solvents used in the medium were of reagent grade and were purchased from Himedia (India) and Sigma (USA).

2.4 Fermentation

Basal media composition used in g/l urea: 0.3, (NH₄)₂SO₄: 1.4, KH₂PO₄: 0.4, MgSO_4; 7H₂O: 0.3, peptone: 0.75, yeast extract: 0.25, FeSO₄; 7H₂O: 0.05, MnSO_4; 7H₂O: 0.01, ZnSO_4; 7H₂O: 0.01, and COCl₂: 0.01) as per the reported literature [5]. About 1% wheat bran was used as a carbon source. Fermentation was carried out in a 250-ml Erlenmeyer flask with 50 ml working volume. Different fermentation parameters, such as incubation period, inoculum size, and pH, were optimized by using One Factor at a Time approach (data not shown). The fungus expressed high enzyme production, i.e. 790 U/ml at initial pH 4, Inoculum size 2×10⁶ per ml after 96 h at 180 RPM.

2.5 Extractive fermentation with ATPS

ATPS was prepared by mixing aqueous dispersions of a PEG and Potassium phosphate salt (polymer-salt ATPS), the pH of which was adjusted to 4.0 with 10% H₂SO_4. This system was supplemented with 1% wheat bran as a main carbon source, and the same was used for fermentation. Total system made was 50% (w/w). The medium was autoclaved and inoculated with 0.5 ml spore suspension, i.e. 2×10⁶ spores in 50 ml of medium and incubated at 30°C for 96 h at 180 rpm. Finally, cells were separated by centrifugation, and the supernatant was collected in a separating funnel to separate the phases. Each phase was analyzed for enzyme activity.

2.6 Enzyme assay

β-glucosidase assay was performed by using pNPG as substrate (5 mm pNPG in 0.05 m acetate buffer, pH 4.8). About 0.1 ml of appropriately diluted enzyme sample was incubated with 1 ml of substrate solution at 50°C for 10 min. The reaction was stopped by adding 2 ml of 1 m Na₂CO₃. The activity of β-glucosidase was estimated spectro-photometrically by reading the absorbance of the liberated p-nitrophenol at 400 nm. A standard graph was prepared with varying concentrations of p-nitrophenol (0–500 μm) in 0.05 m acetate buffer. One unit of BGL activity was defined as the μM of p-nitrophenol released per ml of enzyme per minute under the standard assay conditions [7].

2.7 Protein assay

The protein content of the sample was determined at 595 nm according to Bradford [8]. A system without a sample was taken as a blank reading. Bovine serum albumin was used as a standard. The protein estimation was carried out in duplicate for each sample, and the mean value was used.

3.1 Enzyme production kinetic in a shake flask

As the primary metabolite of the organism incubation period, the enzyme is the most important parameter to be optimized in extractive fermentation with ATPS. The incubation period was studied in a biphasic system containing 12% (w/w) PEG and 14% (w/w) salt. As shown in Figure 1, enzyme activity continuously increased up to 96 h and then gradually decreased. This is because enzymes are primary metabolite proteins and their concentrations increase at the exponential phase of the growth. Maximum protein production of 0.59 mg/ml is observed at 96 h of growth. Here, the growth of A. niger occurred continuously up to 96 h; hence, enzyme activity was maximum, i.e. 946 U/ml (sum of enzyme activity in both phases). Further, in-growth decreases may be attributed to the depletion of substrate and utilization of enzyme as a substrate by micro-organism. Thus, all further fermentation experiments were performed till 96 h.

Figure 1:

Enzyme production kinetics of Aspergillus niger in a shake flask at pH 4.0 and temperature of 30°C.

3.2 Effect of PEG concentration on yield

The distribution of enzymes in ATPS depends upon the phase concentration, which has to be optimized for maximum enzyme production and purification. The influence of PEG concentration on the production and separation of β-glucosidase enzyme was studied at different PEG 4000 concentrations, while maintaining the constant salt concentration of 14% (w/w). Thus, enzyme activity was measured at different PEG concentrations ranging from 10% to 18% (w/w), as reported in Figure 2. It has been observed that maximum β-glucosidase production occurred at a PEG concentration of 12% (w/w) with a gradual increase from 10% to 12% (w/w) and then decreased slowly. The possible reason is explained as follows. PEG is a hydrophilic polymer and, at particular concentration, it forms a matrix network in which the maximum number of macromolecules become enclosed and further increase in concentration, leading to the formation of a dense matrix network that repels the molecule towards the opposite phase. Partitioning of the protein molecules is dependent on the type of protein when the polymer concentration is increased. Usually, an increased polymer concentration pushes the cells at the interface or into lower phase [9, 10]. Similarly, an increase in the molecular weight of a phase-forming polymer can cause a protein to partition more towards the opposite phase [3]. Here, the matrix network formed at 12% w/w was sufficient to enclose the maximum enzyme, hence producing the maximum yield (630 U/ml). A further increase in PEG concentration from 14% to 18% (w/w) repels enzyme molecules in the opposite phase due to the formation of dense PEG matrix, which reduces enzyme yield. The calculation of the fold yield was done by comparing the results with submerged fermentation under the same conditions of constant salt concentration, 14% (w/w) and at different PEG concentration. Maximum fold yield (i.e. 1.2) was obtained when the biphasic system has PEG and salt concentrations of 12% (w/w) and 14% (w/w) as expressed in Figure 3.

Figure 2:

β-Glucosidase production at different PEG concentrations.

pH 4.0 and temperature 30°C.

Figure 3:

Effects of PEG concentration on fold yield.

pH 4.0 and temperature 30°C.

3.3 Effect of salt concentration on yield

The influence of different salt concentrations with constant PEG concentration, 12% (w/w) was studied on the production as well as separation of β-glucosidase enzyme. The enzyme activity was measured at different salt concentrations ranging from 12% to 20% (w/w), and results are reported in Figure 4. As can be seen, enzyme activity was increased from salt concentration of 12% to 14% (w/w) and then decreased slowly up to 20% (w/w). This is attributed to the fact that the potassium phosphate salt forms multivalent ions in its aqueous dispersion; moreover, the phase-forming ability of multivalent salts with PEG is closely related to the lyotropic series of Hoffmeister [11]. An electrical potential is created between the two phases when a salt is included in the phase system. This can be attributed to the fact that different salts have different chemical affinities for the two phases. A potential difference can influence the partitioning of charged macromolecules and particles, especially those carrying a high number of surface electrical charges [3]. On the other hand, total proteins and β-glucosidase to the top and bottom phases, respectively, are partitioned based on their isoelectric points (pIs). If the pH of the medium is lower than the isoelectric point of the enzyme, then the enzyme will have a positive surface charge and then partitioned in a phase with a negative surface charged molecule and vice-versa. Here, the top phase of an aqueous biphasic system is composed of PEG, which carries a positive charge, whereas the bottom phase has phosphate salt, which carries a negative charge [12]. The pH of the medium was adjusted to 4.0, which is below the pI of β-glucosidase enzyme, i.e. pI~8.7 [13]; hence, the enzyme will have a positive charge and get partitioned in the salt phase, which has a negative charge.

Figure 4:

β-Glucosidase production at different salt concentrations.

pH 4.0 and temperature 30°C.

Here, maximum yield (i.e. 885 U/ml) was obtained at a salt concentration of 14% (w/w), indicating that this concentration was optimum for partitioning the maximum enzyme molecules and also favors the growth of A. niger. A further increase in salt concentration decreases the growth of micro-organisms, thus reducing the yield of the enzyme. As shown in Figure 5, the fold yield was calculated by comparing with that of the submerge fermentation under the same conditions at different salt concentrations, but with constant PEG concentration, 12% (w/w). Maximum fold yield (i.e. 1.2) was obtained when the biphasic system has PEG and salt concentrations of 12% (w/w) and 14% (w/w), respectively.

Figure 5:

Effects of salt concentrations on fold yield.

pH 4.0 and temperature 30°C.