بهینه ‏سازی تولید آلفا آمیلاز از گونه تثبیت شده باسیلوس آمیلوفاسینس ATCC 23350

Introduction

Some microorganisms use secreted enzymes such as α-amylase to convert starch to glycosyl residues in order to generate sufficient energy for their survival (Table 1).

Species of Bacillus is well known for their α-amylase production (5 & 6) and in recent years product optimization has received great attention (7 & 8). It has been demonstrated that physicochemical properties of the medium have crucial roles in cell density and α-amylase production (9 & 10). Bacterial immobilization as cell factory/reactor has been considered useful for the production of industrially-important enzymes (11). The microbial cell and enzyme entrapment in polymer matrices such as agar, alginate, carrageenan, cellulose and their derivatives has been used as an efficient immobilization technique. Amongst which, alginate gel proved to be more popular due to its simple manipulation steps being set at milder conditionsand non-toxic character (12). Accordingly, we have examined secreted α-amylase production from Bacillus amyloliquefaciens in free and immobilized conditions and optimized the culture conditions with low cost substrates via implementing statistical analyses.

Materials and methods

Fermentation medium: Homemade starch from rice, wheat, corn, green bean and potato was prepared from 500g of each plant. The plant samples were washed, peeled, smashed, suspended in 1l of distilled water and cooked at 100 ^○C for 30 min. The juice was filtered through muslin cloth under vacuum. Starch content of filtered juice was measured according to Thivend et al's method (13) and 60 g/l was used in fermentation medium. Protein content was obtained according to Lowry's method (14) and adjusted with either of the peptone, urea, free fat cotton seed and soy bean powder to have 30 g/l protein within the fermentation medium. Therefore, fermentation medium contained 60 g/l carbon sources (glucose, rice starch, wheat starch, corn starch, green bean starch and potato starch, Merck starch), 30 g/l protein sources (peptone, urea, free fat cotton seed and soy bean powder), 1 g/l KH₂PO₄,0.1 g/l CaCl₂, 0.7 g/l NaCl and 0.3 g/l MgSO₄ (15).

Table 1- α-Amylase production of Bacillus in various media with or without starch supplement by submerge fermentation.

Microorganisms and seed culture: Bacillus amyloliquefaciensATCC 23350 was obtained from Persian Type Culture Collection (Tehran, Iran). The seeding medium contained 15 g/l glucose, 2 g/l of each of meat extract, yeast extract and 0.5 g/l NaCl (15). The medium was seeded with a single Bacillus amyloliquefaciens cells and incubated at 37°C for 48 h with a constant shake at 180 rpm. Cell suspension culture from seeding medium (5% v/v) was transferred into fermentation media with three replicates and incubated for 18 h at 37°C (15).

Bacillus amyloliquefaciens Immobilization: The cells of the Bacillus amyloliquefaciens were inoculated into 50 ml of theseed culture media. The polyvinyl alcohol (PVA) borate cell beads (100, 300 and 500 beads) were formed according to Zhao et al (16) and the beads were transferred into 50 ml of the fermentation media.

α-Amylase Assay: The supernatant were collected after incubation in fermentation medium. The cultures were centrifuged at 7656 ×g for 20 min and the supernatant was used for enzyme production assay according to Bernfeld (17). A reaction mixture containing 520μl of 4% (w/v) soluble starch in 0.02 M phosphate buffer (pH = 5), 80μl of sample or crude enzyme (for calibration via standard curve) and 400 μl of the 0.15 M phosphate citrate buffer (pH =5) was incubated for 10min at 44^○C. The reaction was incubated for 10 min at 90 C. The amount of liberated reducing sugar was determined by the dinitrosalicylic (DNS) acid method (18). 2 mL of DNSA (3, 5-dinitrosalicylic acid) reagent was added to the mixture, and then kept for 5 min in boiling water bath. Amount of reduced suger released from starch degradation was estimated by measuring OD at 540 nm.

The assay was followed by “one factor at a time” method considering number of beads (100, 300 and 500), protein and carbon sources as stated above. For continuation of our study, the demonstrated maximum enzyme production at each specific condition (bead number, protein content and starch source) was considered.

Optimization of medium components by RSM: RSM using Composite Central Design (CCD) was used to establish the concentration of starch and protein sources for the maximum production of α-Amylase. The statistical software package Design-Expert 6.0 (Stat- Ease Inc, Minneapolis, USA) was used to produce a regression model for predicting the result of combined variables on α-amylase production.

Results

Effects of number of beads, nitrogen and carbon sources on the α-amylase production by "one factor at a time" method: Analysis of the number of bead (100, 300, and 500) showed that the highest α-amylase production was achieved at 300 beads in the following condition: 37°C, pH = 7.0 and 18 h of incubation in green bean starch (60 g/l) as the carbon source and cotton seed powder (30 g/l) as a nitrogen source on a rotary shaker. Conversely, the lowest production was achieved in free cell culture (Fig. 1).

Fig 1- Effect of number of bed on α-amylase production

By defining the optimum number of beads (i.e. 300), contribution of nitrogen sources was evaluated. The results showed that the lowest (33 U/ml) and highest (197 U/ml) activities were achieved in the presence of peptone in free and immobilized cells, respectively. In contrast, urea demonstrated to act in the opposite direction to the peptone (Fig. 2). At this stage, peptone (30 g/l) was chosen amongst tested nitrogen sources.

Fig 2- α-amylase production of the free and the immobilized cell on difference protein sources

As the final step carbon sources were analyzed for α-amylase production in the availability of 300 beads and 30 g/l peptone. Free and immobilized cells responded differently to glucose and rice starch. In free cell condition, glucose demonstrated the highest activity (136 U/ml). Whereas rice starch showed to be the most efficient carbon source (170 U/ml), once the cells were immobilized (Fig. 3).

Fig 3- α-amylase production of the free and the immobilized cell on difference carbon resources

Optimization by RSM: By defining the suitable carbon and nitrogen sources and number of beads, RSM was used to define the optimum concentrations of rice starch and peptone and their interactions. Accordingly, the high and low values of selected nitrogen and carbon sources were defined for Design-Expert 6.0. The software package introduced more coded values (Table 2) and RSM analysis was performed to generate an equation describing the α-amylase production, considering ranges of rice starch and peptone.

RSM followed by multivariate regression analysis, resulted the following equation:

Y₁=+124.47901– 0.40919 × C– 1.51742 × N+0.030473 × C × N+3.24755E^-003× C²+9.54545E-003 × N²– 1.31590E^-004× C²× N (R² = 0.990371, CV = 4.06, P < 0.05)

Where, Y₁ is the presented α-amylase production, C and N correspond to starch and peptone respectively. Further analysis revealed that the fitted model well explain the α-amylase production. In this regression model, the interaction between carbon and nitrogen sources (C × N) and N² were not statistically significant (P < 0.05, Table 3).

Table 2- Results of Face Centered Central Composite Design (FCCCD) using two variables demonstrating observed and predicted responses

Table 3- Model coefficients assessed by multivariate regression and significance of regression coefficient for α-amylase.