International Journal of Biological Macromolecules 75 (2015) 73–80
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Aspergillus oryzae S2 alpha-amylase production under solid state fermentation: Optimization of culture conditions Mouna Sahnoun, Mouna Kriaa, Fatma Elgharbi, Dorra-Zouari Ayadi, Samir Bejar ∗ , Radhouane Kammoun Laboratory of Microorganisms and Biomolecules (LMB), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia
a r t i c l e
i n f o
Article history: Received 27 January 2014 Received in revised form 12 January 2015 Accepted 13 January 2015 Available online 21 January 2015 Keywords: Alpha-amylase Form Cost-effective Soyabean meal Central composite design
a b s t r a c t Aspergillus oryzae S2 was assayed for alpha-amylase production under solid state fermentation (SSF). In addition to AmyA and AmyB already produced in monitored submerged culture, the strain was noted to produce new AmyB oligomeric forms, in particular a dominant tetrameric form named AmyC. The latter was purified to homogeneity through fractional acetone precipitation and size exclusion chromatography. SDS-PAGE and native PAGE analyses revealed that, purified AmyC was an approximately 172 kDa tetramer of four 42 kDa subunits. AmyC was also noted to display the same NH2 -terminal amino acid sequence residues and approximately the same physico-chemical properties of AmyA and AmyB, to exhibit maximum activity at pH 5.6 and 60 ◦ C, and to produce maltose and maltotriose as major starch hydrolysis end-products. Soyabean meal was the best substitute to yeast extract compared to fish powder waste and wheat gluten waste. AmyC production was optimized under SSF using statistical design methodology. Moisture content of 76.25%, C/N substrate ratio of 0.62, and inoculum size of 106.87 spores allowed maximum activity of 22118.34 U/g of dried substrate, which was 33 times higher than the one obtained before the application of the central composite design (CCD). © 2015 Elsevier B.V. All rights reserved.
1. Introduction Alpha amylases (endo-1,4-␣-d-glucan glucanohydrolase EC 3.2.1.1) are extracellular endo-enzymes that randomly cleave the ␣ (1,4) – linkages between adjacent glucose units of starch polymers and ultimately generate short oligosaccharide units. This family of enzymes has been widely applied in several industries, including bread making, brewing, textile, paper, and pharmaceutical industries. Alpha amylases production essentially depends on the cultivation system (liquid or solid state fermentation), bioreactor design, and medium composition. Solid state fermentation (SSF) has attracted growing attention in recent years as a promising technique for the efficient utilization of agro-industrial residues, raw materials, and solid wastes to produce value-added products of commercial interest [1]. This fermentation process refers to the microorganism cultivations on non-soluble materials, called solid substrates, which act both as physical support and nutrient source [2]. SSF is widely recognized as a relatively simple, low-cost, and
∗ Corresponding author. Tel.: +216 74 870 451; fax: +216 74 870 451. E-mail address: [email protected] (S. Bejar). http://dx.doi.org/10.1016/j.ijbiomac.2015.01.026 0141-8130/© 2015 Elsevier B.V. All rights reserved.
eco-friendly technology for use in various industrial processes, including the value-added bioconversion of biomass, the production of compost, ensiling, and animal feed from solid wastes [3]. This technique has been used for thousands of years in the production of traditional food, such as “miso”, “shoyu”, and “tempeh” in oriental countries and bread, cheese and yogurt in western countries. SSF offers distinct advantages over the conventional submerged fermentation (SmF) system, including ease of operation, lower operation costs, superior yields, shorter time, and enhanced product recovery [3]. The selection of an appropriate solid substrate is a key aspect of SSF. It depends on several factors, including cost, availability, particle size, and moisture content [4]. This selection may, therefore, involve the screening of several by-products, with the substrate of choice being the one that not only serves as the best nutrient source but also acts as the best support for cell growth. In fact, several substrates have been tested for ␣-amylase production, including wheat bran [5], spent brewing grains [6], barley and non-waxy rice [7], and corn gluten meal [8]. Low substrate moisture content makes such a fermentation process limited to a number of microorganisms, mainly yeasts and fungi, although some bacteria have also been used [4]. Filamentous fungi are considered as suitable microorganisms for amylase production under SSF, particularly
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because their morphology. They can also overcome the inevitable limitation of oxygen diffusion in the substrate surface layer by forming aerial mycelia [9]. They can even penetrate the hardest solid substrates, by the presence of a turgor pressure to the tip of the mycelium [10]. Alpha-amylase production by SSF has also been reported to depend on several other interrelated environmental factors, such as oxygen content, moisture level, additional nutrients, pH, and temperature. The monitoring of temperature has, for instance, been described to relate to heat conductivity, substrate hydrolysis, solid layer particles, and water content [2]. The authors have previously reported on the production, purification, and characterization of Aspergillus oryzae strain S2 ␣-amylase in submerged culture [11]. The present study aims to investigate the potential optimization of the SSF culture conditions for this ␣-amylase using a statistical experimental design. It also reports on the detection of new AmyB oligomeric forms, and a characterization of a dominant tetrameric form (AmyC). 2. Materials and methods 2.1. Microorganism The strain used for ␣-amylase production under SSF in this work was the A. oryzae strain S2, which was chosen for its efficiency and non-toxicity in amylase production [11] and for its easy cultivation as compared to other microorganisms [12]. The strain was propagated on PDA (Potato Dextrose Agar) (Fluka, France) medium plates at 30 ◦ C and stored at 4 ◦ C. 2.2. Substrates origins and characterization Unless otherwise specified, all substrates, chemicals, and reagents were of the analytical grade or highest commercially available purity, and were purchased from Sigma Chemical Co. (St. Louis, MO). Three agro-industrial waste residues were chosen as organic nitrogen sources to substitute yeast extract: tuna fish powder waste was obtained from a local tuna processing factory, prepared as previously described elsewhere [13], and consisted of 90.3% protein, 8.4% sugar, 0.237% lipid, 1.2% ash, and 1.3% water content (dry matter = 98.7%); wheat gluten waste was obtained from a local factory and contained 72.1% protein, 11.36% sugar, 0.034% lipid, and 1.2% ash, with 87.6% dry matter; soyabean meal was obtained from a local factory and contained about 75.01% protein, 11.15% sugar, 0.139% lipid, and 7.2% ash, with 91% dry matter. The economical carbon sources used in this study were: gruel, which obtained from a local semolina factory processing durum wheat, contained starch (60%), other carbohydrates (5%), cellulose (1.5%), gluten (12%), and total nitrogen (2.1%) [14]; and powder whey, which contained 13% protein, 70% sugar, 0.1% lipid, 9% ash, and 7.9% water content (dry matter = 92.1%). Total carbohydrates were determined after total acid hydrolysis [15]. Protein content was evaluated by Kjeldahl method [16]. Lipid content was determined gravimetrically after Soxhlet extraction using hexane solvent (Riedel-De Haën, Germany) [17]. Dry matter was determined by oven drying at 105 ◦ C to constant weight, and ash content was determined by the combustion in a muffle furnace at 550 ◦ C for 12 h. All contents were expressed as matter weight/weight. 2.3. Medium All preliminary and optimization assays of amylase production used a standard MS inoculum medium including: starch (BioChemika, Sigma–Aldrich Chemie GmbH, Switzerland) 10 g, yeast extract (Bio-Rad, France) 1 g, urea 0.5 g, KH2 PO4 0.2 g, NaNO3 5 g, MgSO4 ·7H2 O 0.03 g, CaCl2 ,·2H2 O 0.03 g, tween 80 1 ml, and trace
elements (1 ml) [composed of (g/L): MgSO4 1.6, ZnSO4 1.4, FeSO4 5, CaCl2 2]. 2.4. Optimization of process conditions All constituents of the MS medium were placed in a 1LErlenmeyer flask under static conditions at 30 ◦ C. At the end of fermentation, the enzyme was extracted by 0.1 M acetate buffer (pH 5.6) at a 1:3 (w/v) ratio. A preliminary investigation was performed on the effects of various culture parameters on ␣-amylase production, including initial moisture content (85.65%, 69.55%, 65.45%, 61.80%, 58.54%, 52.96%, w/w), time period (24, 36, 48, 72, 96 and 120 h), and aeration (250 ml, 500 ml and 1000 ml Erlenmeyer flasks). Standard conditions included 12 days of 1L-flask incubation under 60% of moisture level. Such condition was taken into account during all the preliminary studies on the effects of various SSF culture parameters. Agro-industrial waste nitrogen sources (tuna fish powder waste, wheat gluten waste, and soyabean meal) were tested as substitutes to yeast extract (Bio-Rad, France) in MS medium. Powder whey and gruel were assayed as economical carbon sources. 2.5. Experimental design and statistical analysis A central composite design (CCD) was employed to optimize the crude enzyme activity. C/N ratio, initial moisture content, inoculum size were screened as variables at five levels (−1.63, −1, 0, 1, 1.633) with a total number of 20 experiments. The latter included 23 full factorial design experiments (runs 1–8), six axial points (runs 13–18), and six replicates in the domain center (runs 9, 10, 11, 12, 19, 20) to estimate the variability of the experimental data. The data were analyzed using the Statistical Package for Social Sciences (SPSS, Version 11.0.1, 2001, LEAD Technologies, Inc., USA) software, and the response surface was generated using the Microsoft Excel (Version 2003, Microsoft office, Inc., USA) program. The regression model was constructed based on the SPSS procedure, which initially considers all the factors involved and then applies a step-by-step elimination of the non-significant ones. The response values of ␣amylase activity (U) used in each trial represented the average of triplicates. A quadratic model was adjusted to the responses of the design using the least-square method, which evaluated the effect of each independent variable on the response. This model can be represented by the following polynomial equation: Y = b0 + b1 X1 + b2 X2 + b3 X3 + b11 X21 + b22 X22 + b33 X23 + b12 X1 X2 + b13 X1 X3 + b23 X2 X3 where X1 , X2 , and X3 refer to the coded factors studied (Table 1), b0 to the intercept, b1 , b2 , b3 to linear coefficients, b11 , b22 , b33 to squared coefficients, and b12 , b13 , b23 to interaction coefficients. The model coefficients were estimated using multilinear regression. The significance of the coefficients was evaluated by a multiple regression analysis based on the F-test with unequal variance (P < 0.05). To check the compatibility of the proposed model with the experimental data obtained, an analysis of variance was performed. 2.6. Analytical methods 2.6.1. ˛-Amylase assay An appropriately extracted and diluted enzyme was added to 0.5 ml of 1% (w/v) starch that was solubilized in 0.1 M acetate buffer (pH 5.6). The reaction mixture was incubated for 30 min at 50 ◦ C, and the liberated reducing sugars (glucose equivalent) were measured using the 3,5-dinitrosalicylic acid method [18]. A separate blank was set up for each sample to correct the non-enzymatic
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Table 1 The CCD plan in actual and coded values along with experimental response for amylase production optimization under SSF. Run no.
Point type
Moisture (%)
C/N ratio
Inoculum size (spores)
Actual value
Coded value
Actual value
Coded value
−1
0.55
−1
1
0.55
−1
−1
0.71
1
1
0.71
1
−1
0.55
−1
1
0.55
−1
Actual value
Activity (U)
Coded value
106.5 106.5
−1
414.41 ± 0.18
106.5 106.5
−1
12,654.34 ± 0.23
−1
12,429.81 ± 0.12
−1
33,990.36 ± 0.19
1
Factorial
65
2
Factorial
85.04
3
Factorial
65
4
Factorial
85.04
5
Factorial
65
6
Factorial
85.04
7
Factorial
65
−1
0.71
1
9718.3 ± 0.10
Factorial
85.04
1
0.71
1
107.5 107.5
1
8
1
32,760.11 ± 0.10
9 10 11 12
Center Center Center Center
75.00 75.00 75.00 75.00
0 0 0 0
0.63 0.63 0.63 0.63
0 0 0 0
107 107 107 107
0 0 0 0
161,953.82 150,557.30 170,073.63 179,066.13
± ± ± ±
0.10 0.10 0.10 0.04
13 14 15 16
Axial Axial Axial Axial
58.67 91.33 75.00 75.00
0 0 0 0
12,515.26 71,726.45 58,265.83 21,021.78
± ± ± ±
0.10 0.05 0.11 0.12
17
Axial
75.00
0
0.63
0
107 107 107 107 105.77
−1.633
35,110.54 ± 0.05
18
Axial
75.00
0
0.63
0
107.73
1.633
13,351.45 ± 0.15
19 20
Center Center
75.00 75.00
0 0
0.63 0.63
0 0
107 107
−1.633 1.633 0 0
0.63 0.63 0.49 0.75
release of sugars. One unit (U) of ␣-amylase was defined as the amount of enzyme releasing 1 mol glucose/min under the assay conditions described. At least three replicates were performed for each analysis. The results are presented as x ± SD, where x refers to the mean of at least three replications and SD to the standard deviation. Student’s t-test was used to determine the significance of differences between means. The data were analyzed by SPSS for windows (Version 11.0.1, 2001, LEAD Technologies, Inc., USA). 2.6.2. Influence of temperature and pH The effect of temperature on enzyme activity was investigated using 100 mM sodium acetate buffer (pH 5.6) at temperatures ranging from 40 to 80 ◦ C. To evaluate thermal stability, AmyC purified solution was pre-incubated at temperatures ranging from 40 to 70 ◦ C, and residual activity was determined at regular time intervals. The optimum pH of activity and the effect of pH on enzymatic stability after incubation at 4 ◦ C for 48 h were studied using the following buffer solutions at 0.1 M: sodium acetate, pH 3.5–6; sodium phosphate, pH 6.0–8; Tris–HCl, pH 8–9; and glycine NaOH, pH 9–12. 2.6.3. Determination of kinetic parameters The hydrolysis of starch was performed in 100 mM sodium acetate buffer pH 5.6 at 60 ◦ C. Initial substrate concentrations ranging from 2 to 12 mg/ml of starch were used. The generated reducing sugars were determined at regular time intervals and expressed in equivalent micromoles of glucose by ml of purified AmyC. The Vmax and Km values were estimated from a Lineweaver and Burk plot using the hyper-32 program. 2.6.4. Analysis of reaction products A kinetic reaction mixture containing 100 U of the purified AmyC and 1% of soluble starch was prepared at 40 ◦ C. The reaction mixture was stopped by boiling for 10 min. The resulting products were analyzed by HPLC using an Aminex HPX-42A column. The products were eluted with water at a flow speed of 0.4 ml/min and detected with a Smartline Refractive Index Detector (RI2300). The solution of standards consisted of 8 g/L glucose and 5 g/L of each maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose.
0 0 −1.633 1.633
107.5 107.5
1
15,690.48 ± 0.12
1
23,182.79 ± 0.15
0 0
159,051.11 ± 0.09 160,710.36 ± 0.03
2.6.5. Protein quantification, electrophoresis, zymogram and purification Protein concentration was determined using the method of Bradford using bovine serum albumin as a standard [19] with three replicates for each analysis. The purified protein was migrated in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli [20]. Protein bands were visualized by Coomassie brilliant blue R-250 (Bio-Rad Laboratories, Hercules, CA) staining. The zymogram for amylase activity was performed using the same conditions of PAGE, (polyacrylamide gel did not contain SDS). Amylolytic activity was detected by placing the native gel onto an agarose gel containing (1%) soluble starch prior to incubation for 30 min at 50 ◦ C. The agarose gel was then stained with iodine reagent (2% iodine in 0.2% potassium iodine), and amylase activities were visualized as transparent bands on a dark blue background. The optimized SSF culture supernatant collected after 12 days of 1L-flask incubation was extracted as previously described. A preliminary concentration by evaporation was performed under vacuum at 40 ◦ C followed by acetone fractional precipitation (3%, v/v). The precipitate was applied to a size exclusion chromatography using BiosilSec 125 (300 mm × 7.8 mm) column equilibrated with 50 mM sodium acetate buffer (pH = 5.6) at a flow rate of 0.8 ml/min. 3. Results and discussion 3.1. Amylase production on solid state fermentation A. oryzae S2 was previously reported to produce two ␣amylases, namely AmyA and AmyB in submerged culture. AmyB was a derivative isoform of AmyA, sharing practically the same biochemical properties and resulting from proteolytic hydrolysis [11]. To evaluate the different amylase form productions during SSF cultivation, a zymogram of amylase activities was performed as described in Section 2. A zymogram of amylase activities under liquid culture was also developed for comparison (Fig. 1A). The findings revealed that, unlike submerged culture, SSF was marked by the formation of a new amylase form, called AmyC, which appeared since the beginning and persisted till the end of the SSF (Fig. 1B). In addition a new intermittent amylase form called AmyD was also
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Fig. 1. Zymogram of ␣-amylase activities during the incubation period. (A) Amylases produced in the culture medium from crude extracts of 1, 6, and 12 days under submerged culture incubation. (B) Amylases produced in culture medium from crude extracts of, 4, 6, 8, and 12 days under SSF.
monitored until the eight days of SSF. Contrary to the submerged culture, no reduction was observed for AmyA activity in the zymogram throughout the time of SSF (Fig. 1B). 3.2. Purification and characterization of the AmyC ˛-amylase form AmyC was purified 15 folds with a 56% recovery of initial amylase activity (Table 2). The SDS-PAGE analysis and native PAGE of this purified fraction showed that the AmyC form was a macromolecule of about 172 kDa with four 42 kDa subunits (Fig. 2). These results, therefore, indicated that AmyC was a tetrameric holoenzyme. Its molecular weight was higher than the ones already described for A. oryzae alpha amylase (52–53 kDa) and other Aspergillus alpha amylases (52–68 kDa) [21]. The starch hydrolysis product of AmyC was analyzed using HPLC. The profiles obtained after 24 h included maltose, maltotriose, and other maltoligosaccharides of higher polymerization degrees (Fig. 3). AmyC was noted to efficiently hydrolyze all the tested substrates (starch, amylose, amylopectine or glucogen) except pullulan (data not shown). Based on its mode of action, this amylase form can be classified as (␣-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1). In contrast to Aspergillus niger which produced two glucoamylase isoforms, in addition to an ␣-amylase produced in the beginning of SSF [12], A. oryzae strain S2 was noted to produce two isoforms and oligomeric derivatives ␣-amylases. This result is congruent with previous reports stipulating that A. oryzae strains are good producer of ␣-amylases [12,22]. The Km and Vmax Kinetic values calculated from the Lineweaver–Burk plot were 7.7 mg/ml and 874.4 U mg−1 , respectively (Table 3). The subsequent analysis of the kinetic parameters showed that the catalytic efficiency (kcat Km −1 ) exhibited by AmyC (353.39 ml mg−1 s−1 ) was lower than those of the two other isoforms, AmyA and AmyB, but within the kinetic parameter interval described for A. oryzae [21]. The effects of temperature and pH on AmyC activity and stability were evaluated as described in Section 2. AmyC was noted to exhibit pH-dependent activity and stability, which were close to those displayed by the AmyB and AmyA isoforms (Figs. 4A and 5A). AmyC was noted to reach maximum activity at pH 5.6 and to have an optimal temperature of 60 ◦ C, with a slightly higher thermal stability than the two other isoforms (Figs. 4B and 5B). These
Fig. 2. (A) Analysis of purified AmyC on non-denaturing PAGE and zymogram study. (A1) Zymogram of the purified AmyC. (A2) Non-denaturing PAGE analysis of purified AmyC. Lane 1, purified AmyC; Lane M, protein markers (molecular masses [in kilodaltons] are indicated on the left, Amersham, high molecular weight). (B) Coomassie brilliant blue-stained SDS-PAGE gel. Lane 1, purified Amy A (10 g); Lane 2, purified AmyC (10 g), Lane 3; 12 days of SSF extract crude; Lane 4, purified AmyB (20 g); Lane 5, protein markers (molecular masses [in kilodaltons] are indicated on the left, Amersham, low molecular weight).
values of optimal pH and temperature are consistent with the data previously reported for other A. oryzae ␣-amylases [21]. The effects of metal ions on AmyC activity were similar to those observed for AmyA and AmyB (data not shown). The NH2 -terminal sequence determined for native AmyC showed perfect identity with AmyA and B. Given that AmyC displayed the same biochemical properties of Amy B, this new amylase form could presumably represent a new Amy B complex molecule form, structured as a tetramer during SSF. The intermittent AmyD has a 126 kDa of molecular weight which equals to three times that of Amy B and could be most likely an Amy B trimeric form. Therefore, one can conclude that at high AmyB concentration, and at elevated viscosity, achieved in SSF conditions, AmyB tends to form its trimeric and tetrameric polymer and probably others oligomeric derivatives. This type of cultivation could presumably facilitate protein interaction by improving salt and hydrophobic amino-acid bridges through a mechanical force resulting from environmental parameters. In this field, several inter-relationships among amylase forms and various extracellular parameters, such as salt concentration [23], protease actions [11,24–26], and deglycosylation [27], were reported.
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Table 2 Summary of the purification steps of AmyC from crude extract collected after 12 days of SSF culture incubation. Data expressed as means ± SD. n = 3. Purification step SSF crude extract Evaporation Acetone concentrate Gel filtration
Total activity (U) 265.200 257.460 217.766 148.703
± ± ± ±
0.346 0.228 0.020 0.005
Total protein (mg) 4.826 1.556 0.440 0.180
± ± ± ±
0.037 0.015 0.000 0.000
specific activity (U mg−1 ) 54.947 165.401 494.550 824.608
± ± ± ±
0.476 1.482 0.662 2.603
Purification (fold)
Yield (%)
1 3.010 ± 0.053 9.000 ± 0.066 15.007 ± 0.085
100 97.081 ± 0.097 82.114 ± 0.099 56.072 ± 0.074
Fig. 3. HPLC of starch hydrolysate produced by the purified AmyC after incubation for 24 h (red). St: standard profile (blue) composed of 8 g/L glucose (16.483 min) and 5 g/L of each of maltose (14.417 min), maltotriose (12.883 min), maltotetraose (11.600 min), maltopentaose (10.517 min), maltohexaose (9.617 min), and maltoheptaose (9.017 min). (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)
Fig. 4. The effects of pH and temperature on the activity of the purified AmyA, AmyB and AmyC. (A) The effect of pH on the activity of purified AmyA (), AmyB () and AmyC (×); the relative activities were determined according to the standard assay with buffer solutions at 0.1 M as follows: sodium acetate, pH 3.5–6; sodium phosphate, pH 6.0–8; Tris–HCl, pH 8–9; and glycine NaOH, pH 9–12. (B) The effect of temperature on the activity of purified AmyA () AmyB () and AmyC (×); activities were determined according to the standard assay at temperatures varying from 37 to 80 ◦ C.
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Fig. 5. The effects of temperature and pH on the stability of the purified AmyA (), AmyB () and AmyC (×). (A) The effect of pH on the stability; residual activities were determined after the incubation of the enzymes at various pH values with the buffer solutions described above at 0.1 M (ranging between pH 3 and 12) at 4 ◦ C for 48 h. (B) The effect of temperature on the stability of purified AmyC (×) at 40 ◦ C (), 50 ◦ C (), 60 ◦ C (), and 70 ◦ C (×).
3.3. Preliminary study of the effects of incubation time, economical nitrogen source substitution, aeration, trace elements, and moisture content on amylase production by SSF The incubation time required to achieve maximum enzyme production yields was noted to depend on a number of culture parameters. The A. oryzae S2 strain produced high titers of enzyme (3595.5 U) within 12 days of SSF incubation in MS medium. To improve enzyme production and minimize production costs, cheap nitrogen sources from industrial by-products were assayed as potential substitutes for the expensive nitrogen source (yeast extract) in MS medium. Tuna powder waste, wheat gluten waste, and soyabean meal were tested using the one-factor-at-a-time method, and yeast extract was considered as control for a 12-day observation of SSF. The findings revealed that the highest levels of enzyme activity were obtained when soyabean meal was used as a nitrogen source (4447.14 U), which was equivalent to 494.13 U/g of dried substrate, followed by wheat gluten waste (3190.125 U), and tuna fish powder waste (2851.725 U). Soyabean meal could, therefore, be considered as a potential promising substitute for yeast extract which is described as the best inducer [5,8]. This result is also consistent with other reports [6] demonstrating that soybean meal positively affects the ␣-amylase production by A. oryzae NRRL 6270 in SSF. High levels of enzyme activity were also obtained when starch (BioChemika, Sigma–Aldrich Chemie GmbH, Switzerland) was used as a carbon source (3595.5 U). A decrease in enzyme activity
Table 3 Kinetic constants of the purified AmyC from Aspergillus oryzae strain S2 according to the representation of Lineweaver–Burk. The essay was performed in the optimal conditions using starch as a substrate. Enzyme
Km (mg/ml)
Vmax (U mg−1 )
kcat (s−1 )
kcat Km −1 (ml mg−1 s−1 )
AmyC
7.77
874.4
2721.11
353.39
was, however, noted when gruel (2635.8 U) and whey powder (2235.8 U) were used as carbon sources. This result could be explained mainly by the starch inducer role already described for Aspergillus amylase productions [28–30]. Accordingly, and given the fact that the starch is a relatively cheap and renewable carbon source [31], it has been selected in subsequent assays. In order to evaluate the effect of aeration on amylase under SSF, inoculum size of 106.5 spores of A. oryzae S2 strain was inoculated at a fixed culture volume (50 ml) and in various experimental flask volumes. Considering the constancy of medium volume, it would induce a change in the oxygen amount available for the mycelium. The findings revealed that appropriate aeration related to the ratio of flask volume, which in turn induced maximum ␣-amylase activity [1 L (3595.5 U) > 500 ml (3400 U) > 250 ml (1560 U)]. This result is in agreement with previous reports [9,32], which confirmed the direct relationship between O2 supply and A. oryzae ␣-amylase production by SSF. Moreover, the results demonstrated that the supplementation of trace elements in MS medium improved the production of ␣-amylase by 1.6 fold. This result is in agreement with previous reports confirming the ability of these metallic ions to promote Bacillus ␣-amylases activities [33]. The findings also indicated that high moisture content (80%) enhanced maximum enzyme production by 1.1, 1.31, and 1.43 folds as compared to the 50, 60, and 70% observed for their control counterparts, respectively (data not shown). The decrease of enzyme production at low initial moisture content may be attributed to a limitation in water uptake by the mycelium during the growth course and to hydrolysis reactions [2]. 3.4. Optimization of ˛-amylase production under SSF The experimental design methodology was used to determine the optimal values of the selected variables and enhance the ␣-amylase production yield. The central composite design was applied, and the experimental responses are presented in Table 1. The F-test (ANOVA) was used to identify the statistical significance of the second-order model (Table 4).
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Table 4 ANOVA analysis for ␣-amylase production in central composite design experiments. Source of variations
Sum of squares
Degree of freedom
Regression Residual
73,629,451,560.6778 12,342,033,693.4165
6 13
Total
85,971,485,254.0943
19
Mean square 12,271,575,260.113 949,387,207.185
F-value
Significance
12.927
8.10797E−05
Fig. 6. Response surface plots of amylase activity showing the interactions among the C/N ratio and log (inoculum size (spores number)) (A), moisture (%) and log (inoculum size (spores number)) (B), and C/N ratio and moisture (%) (C) by the central composite design.
The regression model for ˛-amylase production was highly significant (P < 0.01), with a satisfactory value of determination coefficient (R2 = 0. 92544), indicating that 92. 54% of the variability in the response could be explained by a second order polynomial Y = −11,694,594.3437526 + 73,852.125007941*X1 + equation: 9,676,437.00901642*X2 + 1,743,674.54536802*X3 − 484.240004680133*X1 *X1 − 7,683,999.84728185*X2 *X2 − 126,872.865469237*X3 *X3 , where Y refers to activity (U), X1 to the moisture (%), X2 to the C/N ratio, and X3 to log (inoculum size (spores number)). The analyses of the quadratic model showed that the variables with the largest effects were the linear and quadratic terms of the moisture, C/N ratio, and the inoculum size. The interaction between C/N ratio and inoculum size, C/N ratio and moisture, moisture and inoculum size could be better visualized by plotting the 3D response surface graphs. Accordingly, the response surfaces were obtained by varying the two factors while the third factor was maintained constant at its intermediate level (Fig. 6A–C). The elliptical contour plots of response surfaces showed that the interactions between the related variables are not significant. Under the optimized culture conditions, the quadratic model showed that the maximum ␣-amylase production yield of 199065.1 U, which is equivalent to 22118.34 U/g of dried substrate, could be attained when the strain was grown with a moisture content of 76. 25%, a C/N substrate ratio of 0.62, and an inoculum size of 106.87 spores.
3.5. Validation of optimized ˛-amylase production under SSF To validate the predicted results, fermentation experiments were performed in two tests. The production yield was 33 times better than the one obtained before optimization. In fact, this production rate was much higher than the ones previously described for A. oryzae [32,34], other Aspergillus species [35–37], Bacillus [5,8,38] and other fungi [39,40] under SSF. 4. Conclusions The present study is the first to report on the purification and characterization of a new high molecular weight ␣-amylase form, called AmyC, from the SSF culture supernatant of A. oryzae strain S2. Except for its relatively higher thermostability, AmyC was a macromolecule of about 172 kDa with four 42 kDa subunits displays the same biochemical proprieties of AmyB, and AmyA, presumably suggesting that it is an AmyB tetrameric derivative form. Statistical design methodology was used to optimize AmyC production under SSF, demonstrating the usefulness of SSF for the production of high ␣-amylase yields and the valorization of soyabean meal as a nitrogen source. The optimum conditions for achieving maximum enzyme activity (corresponding to 22118.34 U/g of dried substrate) consisted of a moisture content of 76.25%, a C/N substrate ratio of 0.62, and an inoculum size of 106.87 spores. This activity was about 33 times higher than the one obtained before the application of the
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central composite design. The promising properties and attributes reported for AmyC require that further studies be performed to better understand the structure–function relationships of ␣-amylases and their value for carbohydrate enzyme engineering. Acknowledgements This work was in part supported by a grant from the Tunisian government MESRS-TIC contract program CBS-LMB/code: LR10CBS04 2010-2013. We extend our thanks to Dr. Bassem Jaouadi (LMB-CBS) for his constructive discussions and suggestions. The authors would like to express their sincere gratitude to Mr. Anouar Smaoui and Mrs. Hanen Ben Salem from the English Language Unit at the Sfax Faculty of Science, Tunisia for their constructive proofreading and language polishing services. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
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[14] S. Bejar, R. Ghorbel, R. Ben Amar, R. Kammoun, E. Ben Messaoud, K. Belguith, R. Gargouri, R. Ellouz, Procédé de fabrication de sirop de glucose. INNORPITUNISIE, 1999, Brevet d’invention No. 17235. [15] C.J. Israilides, G.A. Grant, Y.W. Han, Appl. Environ. Microbiol. 36 (1978) 43–46. [16] D. Pearson, The Chemical Analysis of Foods, seventh ed., Churchhill Livingstone, Edinburgh, 1970, pp. 6–25. [17] AOAC, Official Methods for Analysis of Association of Official Analytical Chemists, fourteenth ed., 1984, pp. 1141, Arlington, VA. [18] G.L. Miller, Anal. Chem. 31 (1959) 426–428. [19] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254. [20] U.K. Laemmli, Nature 227 (1970) 680–685. [21] R. Gupta, P. Gigras, K.V. Goswami, B. Chauhan, J. Biochem. Tokyo 38 (2003) 1599–1616. [22] A. Pandey, C.R. Soccol, C. Larroche, Current Developments in Solid State Fermentations, Springer, New Delhi, 2008. [23] B. Prakash, M. Vidyasagar, M.S. Madhukumar, G. Muralikrishna, K. Sreeramulu, Process Biochem. 44 (2009) 210–215. [24] S. Murakami, K. Nagasaki, H. Nishimoto, R. Shigematu, J. Umesaki, S. Takenaka, Enzym. Microb. Technol. 43 (2008) 321–328. [25] A.K. Dubey, C. Suresh, R. Kavitha, N.G. Karanth, S. Umesh-Kumar, FEBS Lett. 471 (2000) 251–255. [26] K. Ravi-Kumar, K.S. Venkatesh, S. Umesh-Kumar, Appl. Microbiol. Biotechnol. 74 (2007) 1011–1015. [27] B.A. Gopal, S.A. Singh, G. Muralikrishna, Int. J. Biol. Macromol. 43 (2008) 100–105. [28] S. Djekrif-Dakhmouche, Z. Gheribi-Aoulmi, Z. Meraihi, L. Bennamoun, J. Food Eng. 73 (2006) 190–197. [29] R.R. Ray, Acta Microbiol. Pol. 50 (3–4) (2001) 305–309. [30] S. Tani, T. Kawaguchi, M. Kato, T. Kobayashi, N. Tsukagoshi, Mol. Gen. Genet. 263 (2) (2000) 232–238. [31] V. Kumar, V. Sahai, V.S. Bisariaa, Biocatal. Agric. Biotechnol. 1 (2012) 124–128. [32] Y.S.P. Rahardjo, F.J. Weber, S. Haemers, J. Tramper, A. Rinzema, Enzym. Microb. Technol. 36 (2005) 900–902. [33] R.A.K. Srivastava, J.N. Baruah, Appl. Environ. Microbiol. 52 (1986) 179–184. [34] R.J.S. de Castro, H.H. Sato, Ind. Crops Prod. 49 (2013) 813–821. [35] S. Negi, R. Benerjee, Food Technol. Biotechnol. 44 (2006) 257–261. [36] P. Selvakumar, L. Ashakumary, A. Pandey, Bioresour. Technol. 65 (1998) 83–85. [37] R. Suganthi, J.F. Benazir, R. Santhi, K.V. Ramesh, H. Anjana, N. Meenakshi, K.A. Nidhiya, G. Kavitha, R. Lakshmi, Int. J. Eng. Sci. Technol. (3) (2011) 1756–1763. [38] H. Karatas, F. Uyar, V. Tolan, Z. Baysal, Ann. Microbiol. 63 (2013) 45–52. [39] A. Kunamneni, K. Permaul, S. Singh, J. Biosci. Bioeng. 100 (2) (2005) 168–171. [40] P. Vijayaraghavan, C.S. Remya, S.G.P. Vincent, Res. J. Microbiol. 6 (4) (2011) 366–375.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Aspergillus oryzae S2 alpha-amylase production under solid state fermentation: Optimization of culture conditions Mouna Sahnoun, Mouna Kriaa, Fatma Elgharbi, Dorra-Zouari Ayadi, Samir Bejar ∗ , Radhouane Kammoun Laboratory of Microorganisms and Biomolecules (LMB), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia
a r t i c l e
i n f o
Article history: Received 27 January 2014 Received in revised form 12 January 2015 Accepted 13 January 2015 Available online 21 January 2015 Keywords: Alpha-amylase Form Cost-effective Soyabean meal Central composite design
a b s t r a c t Aspergillus oryzae S2 was assayed for alpha-amylase production under solid state fermentation (SSF). In addition to AmyA and AmyB already produced in monitored submerged culture, the strain was noted to produce new AmyB oligomeric forms, in particular a dominant tetrameric form named AmyC. The latter was purified to homogeneity through fractional acetone precipitation and size exclusion chromatography. SDS-PAGE and native PAGE analyses revealed that, purified AmyC was an approximately 172 kDa tetramer of four 42 kDa subunits. AmyC was also noted to display the same NH2 -terminal amino acid sequence residues and approximately the same physico-chemical properties of AmyA and AmyB, to exhibit maximum activity at pH 5.6 and 60 ◦ C, and to produce maltose and maltotriose as major starch hydrolysis end-products. Soyabean meal was the best substitute to yeast extract compared to fish powder waste and wheat gluten waste. AmyC production was optimized under SSF using statistical design methodology. Moisture content of 76.25%, C/N substrate ratio of 0.62, and inoculum size of 106.87 spores allowed maximum activity of 22118.34 U/g of dried substrate, which was 33 times higher than the one obtained before the application of the central composite design (CCD). © 2015 Elsevier B.V. All rights reserved.
1. Introduction Alpha amylases (endo-1,4-␣-d-glucan glucanohydrolase EC 3.2.1.1) are extracellular endo-enzymes that randomly cleave the ␣ (1,4) – linkages between adjacent glucose units of starch polymers and ultimately generate short oligosaccharide units. This family of enzymes has been widely applied in several industries, including bread making, brewing, textile, paper, and pharmaceutical industries. Alpha amylases production essentially depends on the cultivation system (liquid or solid state fermentation), bioreactor design, and medium composition. Solid state fermentation (SSF) has attracted growing attention in recent years as a promising technique for the efficient utilization of agro-industrial residues, raw materials, and solid wastes to produce value-added products of commercial interest [1]. This fermentation process refers to the microorganism cultivations on non-soluble materials, called solid substrates, which act both as physical support and nutrient source [2]. SSF is widely recognized as a relatively simple, low-cost, and
∗ Corresponding author. Tel.: +216 74 870 451; fax: +216 74 870 451. E-mail address: [email protected] (S. Bejar). http://dx.doi.org/10.1016/j.ijbiomac.2015.01.026 0141-8130/© 2015 Elsevier B.V. All rights reserved.
eco-friendly technology for use in various industrial processes, including the value-added bioconversion of biomass, the production of compost, ensiling, and animal feed from solid wastes [3]. This technique has been used for thousands of years in the production of traditional food, such as “miso”, “shoyu”, and “tempeh” in oriental countries and bread, cheese and yogurt in western countries. SSF offers distinct advantages over the conventional submerged fermentation (SmF) system, including ease of operation, lower operation costs, superior yields, shorter time, and enhanced product recovery [3]. The selection of an appropriate solid substrate is a key aspect of SSF. It depends on several factors, including cost, availability, particle size, and moisture content [4]. This selection may, therefore, involve the screening of several by-products, with the substrate of choice being the one that not only serves as the best nutrient source but also acts as the best support for cell growth. In fact, several substrates have been tested for ␣-amylase production, including wheat bran [5], spent brewing grains [6], barley and non-waxy rice [7], and corn gluten meal [8]. Low substrate moisture content makes such a fermentation process limited to a number of microorganisms, mainly yeasts and fungi, although some bacteria have also been used [4]. Filamentous fungi are considered as suitable microorganisms for amylase production under SSF, particularly
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because their morphology. They can also overcome the inevitable limitation of oxygen diffusion in the substrate surface layer by forming aerial mycelia [9]. They can even penetrate the hardest solid substrates, by the presence of a turgor pressure to the tip of the mycelium [10]. Alpha-amylase production by SSF has also been reported to depend on several other interrelated environmental factors, such as oxygen content, moisture level, additional nutrients, pH, and temperature. The monitoring of temperature has, for instance, been described to relate to heat conductivity, substrate hydrolysis, solid layer particles, and water content [2]. The authors have previously reported on the production, purification, and characterization of Aspergillus oryzae strain S2 ␣-amylase in submerged culture [11]. The present study aims to investigate the potential optimization of the SSF culture conditions for this ␣-amylase using a statistical experimental design. It also reports on the detection of new AmyB oligomeric forms, and a characterization of a dominant tetrameric form (AmyC). 2. Materials and methods 2.1. Microorganism The strain used for ␣-amylase production under SSF in this work was the A. oryzae strain S2, which was chosen for its efficiency and non-toxicity in amylase production [11] and for its easy cultivation as compared to other microorganisms [12]. The strain was propagated on PDA (Potato Dextrose Agar) (Fluka, France) medium plates at 30 ◦ C and stored at 4 ◦ C. 2.2. Substrates origins and characterization Unless otherwise specified, all substrates, chemicals, and reagents were of the analytical grade or highest commercially available purity, and were purchased from Sigma Chemical Co. (St. Louis, MO). Three agro-industrial waste residues were chosen as organic nitrogen sources to substitute yeast extract: tuna fish powder waste was obtained from a local tuna processing factory, prepared as previously described elsewhere [13], and consisted of 90.3% protein, 8.4% sugar, 0.237% lipid, 1.2% ash, and 1.3% water content (dry matter = 98.7%); wheat gluten waste was obtained from a local factory and contained 72.1% protein, 11.36% sugar, 0.034% lipid, and 1.2% ash, with 87.6% dry matter; soyabean meal was obtained from a local factory and contained about 75.01% protein, 11.15% sugar, 0.139% lipid, and 7.2% ash, with 91% dry matter. The economical carbon sources used in this study were: gruel, which obtained from a local semolina factory processing durum wheat, contained starch (60%), other carbohydrates (5%), cellulose (1.5%), gluten (12%), and total nitrogen (2.1%) [14]; and powder whey, which contained 13% protein, 70% sugar, 0.1% lipid, 9% ash, and 7.9% water content (dry matter = 92.1%). Total carbohydrates were determined after total acid hydrolysis [15]. Protein content was evaluated by Kjeldahl method [16]. Lipid content was determined gravimetrically after Soxhlet extraction using hexane solvent (Riedel-De Haën, Germany) [17]. Dry matter was determined by oven drying at 105 ◦ C to constant weight, and ash content was determined by the combustion in a muffle furnace at 550 ◦ C for 12 h. All contents were expressed as matter weight/weight. 2.3. Medium All preliminary and optimization assays of amylase production used a standard MS inoculum medium including: starch (BioChemika, Sigma–Aldrich Chemie GmbH, Switzerland) 10 g, yeast extract (Bio-Rad, France) 1 g, urea 0.5 g, KH2 PO4 0.2 g, NaNO3 5 g, MgSO4 ·7H2 O 0.03 g, CaCl2 ,·2H2 O 0.03 g, tween 80 1 ml, and trace
elements (1 ml) [composed of (g/L): MgSO4 1.6, ZnSO4 1.4, FeSO4 5, CaCl2 2]. 2.4. Optimization of process conditions All constituents of the MS medium were placed in a 1LErlenmeyer flask under static conditions at 30 ◦ C. At the end of fermentation, the enzyme was extracted by 0.1 M acetate buffer (pH 5.6) at a 1:3 (w/v) ratio. A preliminary investigation was performed on the effects of various culture parameters on ␣-amylase production, including initial moisture content (85.65%, 69.55%, 65.45%, 61.80%, 58.54%, 52.96%, w/w), time period (24, 36, 48, 72, 96 and 120 h), and aeration (250 ml, 500 ml and 1000 ml Erlenmeyer flasks). Standard conditions included 12 days of 1L-flask incubation under 60% of moisture level. Such condition was taken into account during all the preliminary studies on the effects of various SSF culture parameters. Agro-industrial waste nitrogen sources (tuna fish powder waste, wheat gluten waste, and soyabean meal) were tested as substitutes to yeast extract (Bio-Rad, France) in MS medium. Powder whey and gruel were assayed as economical carbon sources. 2.5. Experimental design and statistical analysis A central composite design (CCD) was employed to optimize the crude enzyme activity. C/N ratio, initial moisture content, inoculum size were screened as variables at five levels (−1.63, −1, 0, 1, 1.633) with a total number of 20 experiments. The latter included 23 full factorial design experiments (runs 1–8), six axial points (runs 13–18), and six replicates in the domain center (runs 9, 10, 11, 12, 19, 20) to estimate the variability of the experimental data. The data were analyzed using the Statistical Package for Social Sciences (SPSS, Version 11.0.1, 2001, LEAD Technologies, Inc., USA) software, and the response surface was generated using the Microsoft Excel (Version 2003, Microsoft office, Inc., USA) program. The regression model was constructed based on the SPSS procedure, which initially considers all the factors involved and then applies a step-by-step elimination of the non-significant ones. The response values of ␣amylase activity (U) used in each trial represented the average of triplicates. A quadratic model was adjusted to the responses of the design using the least-square method, which evaluated the effect of each independent variable on the response. This model can be represented by the following polynomial equation: Y = b0 + b1 X1 + b2 X2 + b3 X3 + b11 X21 + b22 X22 + b33 X23 + b12 X1 X2 + b13 X1 X3 + b23 X2 X3 where X1 , X2 , and X3 refer to the coded factors studied (Table 1), b0 to the intercept, b1 , b2 , b3 to linear coefficients, b11 , b22 , b33 to squared coefficients, and b12 , b13 , b23 to interaction coefficients. The model coefficients were estimated using multilinear regression. The significance of the coefficients was evaluated by a multiple regression analysis based on the F-test with unequal variance (P < 0.05). To check the compatibility of the proposed model with the experimental data obtained, an analysis of variance was performed. 2.6. Analytical methods 2.6.1. ˛-Amylase assay An appropriately extracted and diluted enzyme was added to 0.5 ml of 1% (w/v) starch that was solubilized in 0.1 M acetate buffer (pH 5.6). The reaction mixture was incubated for 30 min at 50 ◦ C, and the liberated reducing sugars (glucose equivalent) were measured using the 3,5-dinitrosalicylic acid method [18]. A separate blank was set up for each sample to correct the non-enzymatic
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Table 1 The CCD plan in actual and coded values along with experimental response for amylase production optimization under SSF. Run no.
Point type
Moisture (%)
C/N ratio
Inoculum size (spores)
Actual value
Coded value
Actual value
Coded value
−1
0.55
−1
1
0.55
−1
−1
0.71
1
1
0.71
1
−1
0.55
−1
1
0.55
−1
Actual value
Activity (U)
Coded value
106.5 106.5
−1
414.41 ± 0.18
106.5 106.5
−1
12,654.34 ± 0.23
−1
12,429.81 ± 0.12
−1
33,990.36 ± 0.19
1
Factorial
65
2
Factorial
85.04
3
Factorial
65
4
Factorial
85.04
5
Factorial
65
6
Factorial
85.04
7
Factorial
65
−1
0.71
1
9718.3 ± 0.10
Factorial
85.04
1
0.71
1
107.5 107.5
1
8
1
32,760.11 ± 0.10
9 10 11 12
Center Center Center Center
75.00 75.00 75.00 75.00
0 0 0 0
0.63 0.63 0.63 0.63
0 0 0 0
107 107 107 107
0 0 0 0
161,953.82 150,557.30 170,073.63 179,066.13
± ± ± ±
0.10 0.10 0.10 0.04
13 14 15 16
Axial Axial Axial Axial
58.67 91.33 75.00 75.00
0 0 0 0
12,515.26 71,726.45 58,265.83 21,021.78
± ± ± ±
0.10 0.05 0.11 0.12
17
Axial
75.00
0
0.63
0
107 107 107 107 105.77
−1.633
35,110.54 ± 0.05
18
Axial
75.00
0
0.63
0
107.73
1.633
13,351.45 ± 0.15
19 20
Center Center
75.00 75.00
0 0
0.63 0.63
0 0
107 107
−1.633 1.633 0 0
0.63 0.63 0.49 0.75
release of sugars. One unit (U) of ␣-amylase was defined as the amount of enzyme releasing 1 mol glucose/min under the assay conditions described. At least three replicates were performed for each analysis. The results are presented as x ± SD, where x refers to the mean of at least three replications and SD to the standard deviation. Student’s t-test was used to determine the significance of differences between means. The data were analyzed by SPSS for windows (Version 11.0.1, 2001, LEAD Technologies, Inc., USA). 2.6.2. Influence of temperature and pH The effect of temperature on enzyme activity was investigated using 100 mM sodium acetate buffer (pH 5.6) at temperatures ranging from 40 to 80 ◦ C. To evaluate thermal stability, AmyC purified solution was pre-incubated at temperatures ranging from 40 to 70 ◦ C, and residual activity was determined at regular time intervals. The optimum pH of activity and the effect of pH on enzymatic stability after incubation at 4 ◦ C for 48 h were studied using the following buffer solutions at 0.1 M: sodium acetate, pH 3.5–6; sodium phosphate, pH 6.0–8; Tris–HCl, pH 8–9; and glycine NaOH, pH 9–12. 2.6.3. Determination of kinetic parameters The hydrolysis of starch was performed in 100 mM sodium acetate buffer pH 5.6 at 60 ◦ C. Initial substrate concentrations ranging from 2 to 12 mg/ml of starch were used. The generated reducing sugars were determined at regular time intervals and expressed in equivalent micromoles of glucose by ml of purified AmyC. The Vmax and Km values were estimated from a Lineweaver and Burk plot using the hyper-32 program. 2.6.4. Analysis of reaction products A kinetic reaction mixture containing 100 U of the purified AmyC and 1% of soluble starch was prepared at 40 ◦ C. The reaction mixture was stopped by boiling for 10 min. The resulting products were analyzed by HPLC using an Aminex HPX-42A column. The products were eluted with water at a flow speed of 0.4 ml/min and detected with a Smartline Refractive Index Detector (RI2300). The solution of standards consisted of 8 g/L glucose and 5 g/L of each maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose.
0 0 −1.633 1.633
107.5 107.5
1
15,690.48 ± 0.12
1
23,182.79 ± 0.15
0 0
159,051.11 ± 0.09 160,710.36 ± 0.03
2.6.5. Protein quantification, electrophoresis, zymogram and purification Protein concentration was determined using the method of Bradford using bovine serum albumin as a standard [19] with three replicates for each analysis. The purified protein was migrated in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli [20]. Protein bands were visualized by Coomassie brilliant blue R-250 (Bio-Rad Laboratories, Hercules, CA) staining. The zymogram for amylase activity was performed using the same conditions of PAGE, (polyacrylamide gel did not contain SDS). Amylolytic activity was detected by placing the native gel onto an agarose gel containing (1%) soluble starch prior to incubation for 30 min at 50 ◦ C. The agarose gel was then stained with iodine reagent (2% iodine in 0.2% potassium iodine), and amylase activities were visualized as transparent bands on a dark blue background. The optimized SSF culture supernatant collected after 12 days of 1L-flask incubation was extracted as previously described. A preliminary concentration by evaporation was performed under vacuum at 40 ◦ C followed by acetone fractional precipitation (3%, v/v). The precipitate was applied to a size exclusion chromatography using BiosilSec 125 (300 mm × 7.8 mm) column equilibrated with 50 mM sodium acetate buffer (pH = 5.6) at a flow rate of 0.8 ml/min. 3. Results and discussion 3.1. Amylase production on solid state fermentation A. oryzae S2 was previously reported to produce two ␣amylases, namely AmyA and AmyB in submerged culture. AmyB was a derivative isoform of AmyA, sharing practically the same biochemical properties and resulting from proteolytic hydrolysis [11]. To evaluate the different amylase form productions during SSF cultivation, a zymogram of amylase activities was performed as described in Section 2. A zymogram of amylase activities under liquid culture was also developed for comparison (Fig. 1A). The findings revealed that, unlike submerged culture, SSF was marked by the formation of a new amylase form, called AmyC, which appeared since the beginning and persisted till the end of the SSF (Fig. 1B). In addition a new intermittent amylase form called AmyD was also
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Fig. 1. Zymogram of ␣-amylase activities during the incubation period. (A) Amylases produced in the culture medium from crude extracts of 1, 6, and 12 days under submerged culture incubation. (B) Amylases produced in culture medium from crude extracts of, 4, 6, 8, and 12 days under SSF.
monitored until the eight days of SSF. Contrary to the submerged culture, no reduction was observed for AmyA activity in the zymogram throughout the time of SSF (Fig. 1B). 3.2. Purification and characterization of the AmyC ˛-amylase form AmyC was purified 15 folds with a 56% recovery of initial amylase activity (Table 2). The SDS-PAGE analysis and native PAGE of this purified fraction showed that the AmyC form was a macromolecule of about 172 kDa with four 42 kDa subunits (Fig. 2). These results, therefore, indicated that AmyC was a tetrameric holoenzyme. Its molecular weight was higher than the ones already described for A. oryzae alpha amylase (52–53 kDa) and other Aspergillus alpha amylases (52–68 kDa) [21]. The starch hydrolysis product of AmyC was analyzed using HPLC. The profiles obtained after 24 h included maltose, maltotriose, and other maltoligosaccharides of higher polymerization degrees (Fig. 3). AmyC was noted to efficiently hydrolyze all the tested substrates (starch, amylose, amylopectine or glucogen) except pullulan (data not shown). Based on its mode of action, this amylase form can be classified as (␣-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1). In contrast to Aspergillus niger which produced two glucoamylase isoforms, in addition to an ␣-amylase produced in the beginning of SSF [12], A. oryzae strain S2 was noted to produce two isoforms and oligomeric derivatives ␣-amylases. This result is congruent with previous reports stipulating that A. oryzae strains are good producer of ␣-amylases [12,22]. The Km and Vmax Kinetic values calculated from the Lineweaver–Burk plot were 7.7 mg/ml and 874.4 U mg−1 , respectively (Table 3). The subsequent analysis of the kinetic parameters showed that the catalytic efficiency (kcat Km −1 ) exhibited by AmyC (353.39 ml mg−1 s−1 ) was lower than those of the two other isoforms, AmyA and AmyB, but within the kinetic parameter interval described for A. oryzae [21]. The effects of temperature and pH on AmyC activity and stability were evaluated as described in Section 2. AmyC was noted to exhibit pH-dependent activity and stability, which were close to those displayed by the AmyB and AmyA isoforms (Figs. 4A and 5A). AmyC was noted to reach maximum activity at pH 5.6 and to have an optimal temperature of 60 ◦ C, with a slightly higher thermal stability than the two other isoforms (Figs. 4B and 5B). These
Fig. 2. (A) Analysis of purified AmyC on non-denaturing PAGE and zymogram study. (A1) Zymogram of the purified AmyC. (A2) Non-denaturing PAGE analysis of purified AmyC. Lane 1, purified AmyC; Lane M, protein markers (molecular masses [in kilodaltons] are indicated on the left, Amersham, high molecular weight). (B) Coomassie brilliant blue-stained SDS-PAGE gel. Lane 1, purified Amy A (10 g); Lane 2, purified AmyC (10 g), Lane 3; 12 days of SSF extract crude; Lane 4, purified AmyB (20 g); Lane 5, protein markers (molecular masses [in kilodaltons] are indicated on the left, Amersham, low molecular weight).
values of optimal pH and temperature are consistent with the data previously reported for other A. oryzae ␣-amylases [21]. The effects of metal ions on AmyC activity were similar to those observed for AmyA and AmyB (data not shown). The NH2 -terminal sequence determined for native AmyC showed perfect identity with AmyA and B. Given that AmyC displayed the same biochemical properties of Amy B, this new amylase form could presumably represent a new Amy B complex molecule form, structured as a tetramer during SSF. The intermittent AmyD has a 126 kDa of molecular weight which equals to three times that of Amy B and could be most likely an Amy B trimeric form. Therefore, one can conclude that at high AmyB concentration, and at elevated viscosity, achieved in SSF conditions, AmyB tends to form its trimeric and tetrameric polymer and probably others oligomeric derivatives. This type of cultivation could presumably facilitate protein interaction by improving salt and hydrophobic amino-acid bridges through a mechanical force resulting from environmental parameters. In this field, several inter-relationships among amylase forms and various extracellular parameters, such as salt concentration [23], protease actions [11,24–26], and deglycosylation [27], were reported.
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Table 2 Summary of the purification steps of AmyC from crude extract collected after 12 days of SSF culture incubation. Data expressed as means ± SD. n = 3. Purification step SSF crude extract Evaporation Acetone concentrate Gel filtration
Total activity (U) 265.200 257.460 217.766 148.703
± ± ± ±
0.346 0.228 0.020 0.005
Total protein (mg) 4.826 1.556 0.440 0.180
± ± ± ±
0.037 0.015 0.000 0.000
specific activity (U mg−1 ) 54.947 165.401 494.550 824.608
± ± ± ±
0.476 1.482 0.662 2.603
Purification (fold)
Yield (%)
1 3.010 ± 0.053 9.000 ± 0.066 15.007 ± 0.085
100 97.081 ± 0.097 82.114 ± 0.099 56.072 ± 0.074
Fig. 3. HPLC of starch hydrolysate produced by the purified AmyC after incubation for 24 h (red). St: standard profile (blue) composed of 8 g/L glucose (16.483 min) and 5 g/L of each of maltose (14.417 min), maltotriose (12.883 min), maltotetraose (11.600 min), maltopentaose (10.517 min), maltohexaose (9.617 min), and maltoheptaose (9.017 min). (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)
Fig. 4. The effects of pH and temperature on the activity of the purified AmyA, AmyB and AmyC. (A) The effect of pH on the activity of purified AmyA (), AmyB () and AmyC (×); the relative activities were determined according to the standard assay with buffer solutions at 0.1 M as follows: sodium acetate, pH 3.5–6; sodium phosphate, pH 6.0–8; Tris–HCl, pH 8–9; and glycine NaOH, pH 9–12. (B) The effect of temperature on the activity of purified AmyA () AmyB () and AmyC (×); activities were determined according to the standard assay at temperatures varying from 37 to 80 ◦ C.
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Fig. 5. The effects of temperature and pH on the stability of the purified AmyA (), AmyB () and AmyC (×). (A) The effect of pH on the stability; residual activities were determined after the incubation of the enzymes at various pH values with the buffer solutions described above at 0.1 M (ranging between pH 3 and 12) at 4 ◦ C for 48 h. (B) The effect of temperature on the stability of purified AmyC (×) at 40 ◦ C (), 50 ◦ C (), 60 ◦ C (), and 70 ◦ C (×).
3.3. Preliminary study of the effects of incubation time, economical nitrogen source substitution, aeration, trace elements, and moisture content on amylase production by SSF The incubation time required to achieve maximum enzyme production yields was noted to depend on a number of culture parameters. The A. oryzae S2 strain produced high titers of enzyme (3595.5 U) within 12 days of SSF incubation in MS medium. To improve enzyme production and minimize production costs, cheap nitrogen sources from industrial by-products were assayed as potential substitutes for the expensive nitrogen source (yeast extract) in MS medium. Tuna powder waste, wheat gluten waste, and soyabean meal were tested using the one-factor-at-a-time method, and yeast extract was considered as control for a 12-day observation of SSF. The findings revealed that the highest levels of enzyme activity were obtained when soyabean meal was used as a nitrogen source (4447.14 U), which was equivalent to 494.13 U/g of dried substrate, followed by wheat gluten waste (3190.125 U), and tuna fish powder waste (2851.725 U). Soyabean meal could, therefore, be considered as a potential promising substitute for yeast extract which is described as the best inducer [5,8]. This result is also consistent with other reports [6] demonstrating that soybean meal positively affects the ␣-amylase production by A. oryzae NRRL 6270 in SSF. High levels of enzyme activity were also obtained when starch (BioChemika, Sigma–Aldrich Chemie GmbH, Switzerland) was used as a carbon source (3595.5 U). A decrease in enzyme activity
Table 3 Kinetic constants of the purified AmyC from Aspergillus oryzae strain S2 according to the representation of Lineweaver–Burk. The essay was performed in the optimal conditions using starch as a substrate. Enzyme
Km (mg/ml)
Vmax (U mg−1 )
kcat (s−1 )
kcat Km −1 (ml mg−1 s−1 )
AmyC
7.77
874.4
2721.11
353.39
was, however, noted when gruel (2635.8 U) and whey powder (2235.8 U) were used as carbon sources. This result could be explained mainly by the starch inducer role already described for Aspergillus amylase productions [28–30]. Accordingly, and given the fact that the starch is a relatively cheap and renewable carbon source [31], it has been selected in subsequent assays. In order to evaluate the effect of aeration on amylase under SSF, inoculum size of 106.5 spores of A. oryzae S2 strain was inoculated at a fixed culture volume (50 ml) and in various experimental flask volumes. Considering the constancy of medium volume, it would induce a change in the oxygen amount available for the mycelium. The findings revealed that appropriate aeration related to the ratio of flask volume, which in turn induced maximum ␣-amylase activity [1 L (3595.5 U) > 500 ml (3400 U) > 250 ml (1560 U)]. This result is in agreement with previous reports [9,32], which confirmed the direct relationship between O2 supply and A. oryzae ␣-amylase production by SSF. Moreover, the results demonstrated that the supplementation of trace elements in MS medium improved the production of ␣-amylase by 1.6 fold. This result is in agreement with previous reports confirming the ability of these metallic ions to promote Bacillus ␣-amylases activities [33]. The findings also indicated that high moisture content (80%) enhanced maximum enzyme production by 1.1, 1.31, and 1.43 folds as compared to the 50, 60, and 70% observed for their control counterparts, respectively (data not shown). The decrease of enzyme production at low initial moisture content may be attributed to a limitation in water uptake by the mycelium during the growth course and to hydrolysis reactions [2]. 3.4. Optimization of ˛-amylase production under SSF The experimental design methodology was used to determine the optimal values of the selected variables and enhance the ␣-amylase production yield. The central composite design was applied, and the experimental responses are presented in Table 1. The F-test (ANOVA) was used to identify the statistical significance of the second-order model (Table 4).
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Table 4 ANOVA analysis for ␣-amylase production in central composite design experiments. Source of variations
Sum of squares
Degree of freedom
Regression Residual
73,629,451,560.6778 12,342,033,693.4165
6 13
Total
85,971,485,254.0943
19
Mean square 12,271,575,260.113 949,387,207.185
F-value
Significance
12.927
8.10797E−05
Fig. 6. Response surface plots of amylase activity showing the interactions among the C/N ratio and log (inoculum size (spores number)) (A), moisture (%) and log (inoculum size (spores number)) (B), and C/N ratio and moisture (%) (C) by the central composite design.
The regression model for ˛-amylase production was highly significant (P < 0.01), with a satisfactory value of determination coefficient (R2 = 0. 92544), indicating that 92. 54% of the variability in the response could be explained by a second order polynomial Y = −11,694,594.3437526 + 73,852.125007941*X1 + equation: 9,676,437.00901642*X2 + 1,743,674.54536802*X3 − 484.240004680133*X1 *X1 − 7,683,999.84728185*X2 *X2 − 126,872.865469237*X3 *X3 , where Y refers to activity (U), X1 to the moisture (%), X2 to the C/N ratio, and X3 to log (inoculum size (spores number)). The analyses of the quadratic model showed that the variables with the largest effects were the linear and quadratic terms of the moisture, C/N ratio, and the inoculum size. The interaction between C/N ratio and inoculum size, C/N ratio and moisture, moisture and inoculum size could be better visualized by plotting the 3D response surface graphs. Accordingly, the response surfaces were obtained by varying the two factors while the third factor was maintained constant at its intermediate level (Fig. 6A–C). The elliptical contour plots of response surfaces showed that the interactions between the related variables are not significant. Under the optimized culture conditions, the quadratic model showed that the maximum ␣-amylase production yield of 199065.1 U, which is equivalent to 22118.34 U/g of dried substrate, could be attained when the strain was grown with a moisture content of 76. 25%, a C/N substrate ratio of 0.62, and an inoculum size of 106.87 spores.
3.5. Validation of optimized ˛-amylase production under SSF To validate the predicted results, fermentation experiments were performed in two tests. The production yield was 33 times better than the one obtained before optimization. In fact, this production rate was much higher than the ones previously described for A. oryzae [32,34], other Aspergillus species [35–37], Bacillus [5,8,38] and other fungi [39,40] under SSF. 4. Conclusions The present study is the first to report on the purification and characterization of a new high molecular weight ␣-amylase form, called AmyC, from the SSF culture supernatant of A. oryzae strain S2. Except for its relatively higher thermostability, AmyC was a macromolecule of about 172 kDa with four 42 kDa subunits displays the same biochemical proprieties of AmyB, and AmyA, presumably suggesting that it is an AmyB tetrameric derivative form. Statistical design methodology was used to optimize AmyC production under SSF, demonstrating the usefulness of SSF for the production of high ␣-amylase yields and the valorization of soyabean meal as a nitrogen source. The optimum conditions for achieving maximum enzyme activity (corresponding to 22118.34 U/g of dried substrate) consisted of a moisture content of 76.25%, a C/N substrate ratio of 0.62, and an inoculum size of 106.87 spores. This activity was about 33 times higher than the one obtained before the application of the
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central composite design. The promising properties and attributes reported for AmyC require that further studies be performed to better understand the structure–function relationships of ␣-amylases and their value for carbohydrate enzyme engineering. Acknowledgements This work was in part supported by a grant from the Tunisian government MESRS-TIC contract program CBS-LMB/code: LR10CBS04 2010-2013. We extend our thanks to Dr. Bassem Jaouadi (LMB-CBS) for his constructive discussions and suggestions. The authors would like to express their sincere gratitude to Mr. Anouar Smaoui and Mrs. Hanen Ben Salem from the English Language Unit at the Sfax Faculty of Science, Tunisia for their constructive proofreading and language polishing services. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
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