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An innovative simultaneous glucoamylase extraction and recovery using colloidal gas aphrons
Separation and Purification Technology 67 (2009) 8–13
Contents lists available at ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
An innovative simultaneous glucoamylase extraction and recovery using colloidal gas aphrons A. Zad Zidehsaraei, M. Moshkelani, M.C. Amiri ∗ Department of Chemical Engineering, Isfahan University of Technology, Isfahan, Iran
a r t i c l e
i n f o
Article history: Received 15 November 2008 Received in revised form 25 February 2009 Accepted 25 February 2009 Keywords: Glucoamylase Colloidal gas aphron Recovery Extraction
a b s t r a c t Recovery and purification of biological products generally involve complicated processes that cost even up to 70% of final product price. Because of many commercial applications of glucoamylase, its innovative purification methods are still desired. In this paper, colloidal gas aphrons, generated with cationic and anionic surfactants at their critical micelle concentration (CMC), have been used to recover and purify glucoamylase enzyme from actual biomass produced by solid state fermentation. Three diverse separation methods (mixing, integrated and combined) were examined. In these tests, actual biomass samples, composed of a mixture of various proteins and enzymes, were used. Using aphron flotation, it was found that recovery of glucoamylase in a simultaneous glucoamylase extraction and recovery mode can be enhanced by using a cationic surfactant and a flocculation agent. The experimental results revealed that this innovative procedure is the most effective method in recovery and purifying glucoamylase. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Glucoamylase (EC 3.2.1.3, 1,4-a-d-glucan glucohydrolase) is an important industrial enzyme that catalyzes the breakdown of malto-oligosaccharides to glucose and is used to produce high fructose corn syrup (HFCS) and alcohol by hydrolysis of starch and grains [1]. It is produced from various microorganism cultures, especially those in the Aspergillus and Rhizopus genus. The glucoamylase preparation from Aspergillus Niger is produced on a commercial scale and the preparation has been widely utilized for industrial production of crystalline d-glucose, high fructose corn syrups, wine, ethanol, beer, and in other fermentation processes [2]. Glucoamylase purification can be done by different methods such as affinity chromatography [3,4], affinity precipitation [5–10], fast protein liquid chromatography [11,12], and aqueous two-phase extraction [13–16]. Moreover, protein recovery using colloidal gas aphrons (CGAs) has been an attractive area for researchers since the 1990s [17–25]. However, only Wallis et al. [26] and Save et al. [16] have applied colloidal gas aphrons for glucoamylase recovery in order to intensify mass transfer while using aqueous two-phase method. Unfortunately there has been no report in the scientific literatures on the recovery and purification of glucoamylase from a real fermented biomass (a mixture of various proteins, enzymes and spores) directly using colloidal gas aphrons.
∗ Corresponding author. Tel.: +98 311 3915615; fax: +98 311 3912677. E-mail addresses: [email protected] (A.Z. Zidehsaraei), [email protected] (M. Moshkelani), [email protected] (M.C. Amiri). 1383-5866/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2009.02.019
Solid state fermentation holds tremendous potential for the production of glucoamylase in comparison to submerged fermentation because of its high volumetric productivity, relatively higher concentration of the products, less effluent generation and simple fermentation equipments. However, recovery and purification of produced glucoamylase remains a challenging step, since various enzymes and components like protease, cellulose, xylose derivatives, arabitol, erythrose and glyceraldehydes are produced during the fermentation process and inhibit the activity of produced glucoamylase. Thus recovery of glucoamylase from fermented matter is an important factor that affects the cost-effectiveness of the overall process. In this work, glucoamylase has been produced through a solid state fermentation method. Then three different techniques using aphron flotation have been applied for recovery and purification. Glucoamylase separation parameters for various techniques have been investigated, in the presence and absence of a flocculating agent. Finally, an innovative simultaneous glucoamylase extraction, recovery, and purification method was developed. 2. Theory CGAs are microbubbles (typically 25–100 m in diameter) with colloidal properties, first introduced by Sebba [27]. The drainage of CGA dispersion and the ionisation of chemical groups at the surface (when using ionic surfactant molecules) are two important factors in aphron flotation. Due to the low density of CGA dispersion, it undergoes drainage process during which the dispersion separates into a clear solution region at the bottom and froth phase at the
A.Z. Zidehsaraei et al. / Separation and Purification Technology 67 (2009) 8–13
9
Fig. 1. Details of various methods and steps in testing procedure.
top [28]. The drainage rate depends on environmental conditions such as solution ionic strength, pH and additives. Basically an individual CGA and its most closely associated ions can move through the solution as a unit. The pH of solution and the electrical conductivity affects the zeta potential of each unit. Since CGA dispersion possesses a size distribution that changes with progress of drainage process, they can act similarly to a charged microfilter bed with non-uniform size distribution. Therefore, they can collect particles based on their sizes as well as their charges. On the basis of the theoretical analysis developed by Jameson et al. [29] the efficiency of collision (Ec ) between a particle (diameter of dp ) and a bubble (diameter of db ) is given by Eq. (1) in which m appears to have a value of 1–2 and k a value of 3.5–4. Ec =
dpm
(1)
dbk
Equation (1) shows that the efficiency generally increases as dp /db increase. Since glucoamylase and other molecules are submicron size particles, a flocculating agent can be used to enlarge their sizes. Obviously adding a flocculating agent not only results in improving collection efficiency but also increases impurities; therefore, there should be an optimized dose of flocculating agent. It seems that CGA dispersion is able to differentiate a mixture of particles and high molecular weight molecules if experimental conditions are carefully monitored. In order to quantify the recovery and purity of produced glucoamylase, the following parameters have been used to determine the effectiveness of the separation process: 1. Total amount of enzyme activity (U) = activity (U/ml) × volume (ml) activity (U/ml) 2. specific activity (U/mg) = protein content (mg/ml) 3. recovery (yield)% =
× 100
specific activity of enzyme after purification step specific activity before purification step
5. enrichment ratio =
3. Materials and methods 3.1. Materials Sodium dodecyl sulfate (SDS) (FSA, England), an anionic surfactant, and tetradecyl trimethyl ammonium bromide (TTAB) (Merk), a cationic surfactant were used to produce CGAs at CMC concentrations. Citric acid and sodium citrate (citrate buffer) were utilized as a buffer solution for pH adjustment in range of 3–6.2. Aluminum sulfate (Alum) was used as a flocculating agent. In order to determine the glucoamylase activity, 2-hydroxy-3,5-dinitrobenzoic acid (DNS), glucose and starch solution were applied. Bovine serum albumin, copper (II) sulfate, potassium sodium tartrate and FolinCiocalteu reagent were used for total solution protein content determination. All chemicals except surfactants were supplied by Sigma–Aldrich Co. 3.2. Biomass and glucoamylase production
total amount of enzyme activity after purification step total amount of activity enzyme before purification step
4. purification factor =
operations involve separately CGA generation with distilled water according to the Sebba method [27]. In the batch process, a definite ratio of CGA dispersion is mixed with protein or enzyme and the mixture is stirred for a certain time. But in continues mode, the generated CGA is pumped into a column of protein or enzyme feed. The optimum recovery can be achieved by controlling the residence time in the column. In both operations, the top foamy phase and drained liquid are tested to measure separation parameters. However, Amiri undertook a different approach by producing CGA directly from whey [23]. In this study, batch and continuous modes as well as a totally innovative extraction and recovery combination method for recovery and purification of glucoamylase have been examined.
glucoamylase activity in aphron phase glucoamylase activity in mixture of aphron and glucoamylse
Almost all researchers have utilized batch or continues operations in their protein or enzyme recovery studies [17–25]. Both
Glucoamylase was produced by solid state fermentation at the Isfahan Research Center for Agricultural and Natural Resources. The reported value of isoelectric point for glucoamylase was pI 3.5. Aspergillus Niger (CCUG 33991, Sweden) with Mw = 90,000 = 90 kDa, was added to 50% wet bran on a tray and left for 40 h at 35 ◦ C to complete the fermentation process. The produced biomass was left to decrease its humidity to less than 5% at 40 ◦ C. The purified enzyme was reported to have a molar mass of 90 kDa with an isoelectric point at pH 3.5. In order to extract glucoamylase from dry biomass,
10
A.Z. Zidehsaraei et al. / Separation and Purification Technology 67 (2009) 8–13
Fig. 2. Outline of experimental design: (A) mixing method, (B) integrated method and (C) combined method.
the sample was diluted 10-fold with distilled water and shaken for 70 min at 30 ◦ C and speed of 120 rounds per minutes. Glucoamylase was finally obtained by filtration.
experimental design. All tests for each method were double checked to generate reliable results. 3.5. Glucoamylase activity determination
3.3. Colloidal gas aphron generation CGAs dispersions were generated using anionic and cationic surfactants both at their CMC through method proposed by Sebba [27]. Stirring rate and time were 7000 rpm and 5 min, respectively for all tests. For each production method, different feeds were used. Distilled water, glucoamylase solution, and solid biomass solution were chosen to generate CGAs for the mixing, integrated, and combined methods, respectively. 3.4. Testing procedures Fig. 1 demonstrates the details of various steps in different testing procedures. Three different approaches were undertaken in batch wise mode. In the mixing method, enzyme to CGA ratios of 1, 2, or 3 were mixed together with a magnetic stirrer for 5 min at pH values of 3.5, 4, 4.3, or 4.8. As for integrated tests, CGA was produced directly from glucoamylase dispersion and pH was adjusted at 3.5, 4.3 and 4.8. The sample was transferred to a cylinder for differentiated sampling. In these series of tests, the effect of flocculating agent was also investigated by adding 100 or 50 ppm of alum to the glucoamylase solution before CGA generation. In the case of the combined method, CGA was generated by adding 1000 and 750 ml of distilled water to solid biomass and alum concentrations of 40, 30, 20, 15, 10, and 0 ppm to check the maximum recovery. The details of differentiated sampling during the drainage process in the measuring cylinder were reported elsewhere [28]. Fig. 2 shows the
In order to determine the glucoamylase activity, a specified amount of enzyme sample and 1 ml of distilled water were added to 1 ml of starch substrate solution (pH 4.5–4.6). The mixture was kept at 60 ◦ C for 15 min to produce glucose monomers. It was then put in to boiling water to deactivate the enzyme. Following the calorimetric assay, the absorption intensity of the final solution was then measured at wave length of 540 nm for determining the amount of released glucose, which is directly related to enzyme activity. The spectrophotometer equipment was calibrated using distilled water for all tests. To assure precise results, each test was repeated twice. 3.6. Total protein content determination Total content of protein in recovered samples were determined according to the Lowry protein assay [30]. 4. Results and discussion 4.1. Mixing method Table 1 shows the effect of pH on separation factors for a CGA/glucoamylase mixing ratio of one. The best separation result is obtained for TTAB at pH 3.5 with the highest recovery and enrichment factor. Comparing SDS with TTAB, cationic surfactant (TTAB) was more successful in recovering glucoamylase because of higher enrichment and recovery factors. Because the positively
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11
Table 1 Effect of pH for SDS and TTAB for CGA/glucoamylase mixing ratio of one. pH
Maximum enzyme activity%
Recovery%
Enrichment ratio
Time of maximum enrichment
SDS 3.5 4 4.3 4.8
72.5 78.8 82.2 91.5
96 101 95.5 93.2
1.05 1.01 1.03 1.08
End of macroscopic drainage Start and end of macroscopic drainage End of macroscopic drainage End of macroscopic drainage
TTAB 3.5 4 4.3 4.8
89 84.4 87 83
117 107 83.7 104
1.18 1.11 1.03 1.06
End of microscopic drainage End of microscopic drainage End of microscopic drainage End of microscopic drainage
Table 2 Separation factors for various mixing ratio of CGA/glucoamylase for SDS and TTAB, pH 3.5. Surfactant
CGA/glucoamylase ratio
Recovery%
Enrichment ratio
SDS
1/1 1/2 1/3
96 112 101
1.05 1.25 1.04
TTAB
1/1 1/2
117 92
1.18 1.05
charged hydrophobic alkyl group in TTAB does not interact with H+ ions in solution and facilitate quick and easy dispersion of CGAs consequently [16]. Moreover, it seems that glucoamylase should accumulate at foam phase; therefore maximum enrichment factor can be achieved during microscopic drainage [28], which was well observed for TTAB. Additionally in the TTAB case for total pH range (except pH 4.3) and for SDS at pH 4, total enzyme activity after the separation procedure is more than of the initial glucoamylase solution. Since no glucoamylase is being produced through the separation process, it is concluded that the mixing method has been successful in removing solid impurities like pigments and spores (confirmed observationally) as well as some proteins and other enzymes that decrease glucoamylase activity. However, maximum glucoamylase activity of less than 100, and an enrichment factor of approximately 1 for all tests reveals that mixing method is not the efficient approach in recovering glucoamylase. Results also showed that TTAB should be considered for further experimental work (see Table 2). 4.2. Integrated method Table 3 shows effect of pH on performance of aphron flotation in integrated method. Best recovery was obtained at pH 3.5. Based on analysis developed in the theoretical section, according to Eq. (1), efficiency should be improved by using a flocculating agent [29,31]. Moreover, total protein content of each sample was also measured to probe the effect of a flocculating agent on the integrated method.
Table 3 Effect of pH on performance of aphron flotation in integrated method. pH
Maximum enzyme activity%
Recovery%
Time of maximum enrichment
3.5 4.3 4.8
110 77.4 62.5
102 71 62
End of microscopic drainage End of microscopic drainage End of microscopic drainage
Table 4 shows that high concentration of flocculating agent reduces the recovery of glucoamylase and leads to the waste of more protein and enzyme. However, an extremely clear retentate was drained due to efficient removal of solid impurities. Thus finding an optimum concentration of flocculating agent is a key point in any successful aphron flotation. 4.3. Combined (simultaneous extraction and recovery) method In this novel method, simultaneous CGA generation and enzyme extraction from biomass has been done. The extracted glucoamylase then was recovered during the drainage process. Table 5 compares the efficiency of the combined method for 1 l of such dispersion in presence and absence of alum. It was found that the maximum enzyme activity and recovery is highly improved even in comparison with the results obtained by Shin et al. [32]. They obtained 65% recovery in a mixture of only two different proteins. Adding alum has positive effects in reducing the amount of wasted protein and glucoamylase. Approaching maximum specific activity of nearly 10 shows a clearly successful selective recovery of glucoamylase. To improve the effectiveness of combined method, tests were done for 750 ml of feed solution. Purification factor and recovery is illustrated for different alum concentrations in Fig. 3 which shows that double purification factor and the highest glucoamylase efficiency were obtained at alum dosage of 15 ppm. As is shown in Table 6, at lower concentrations of alum, less protein was wasted and also glucoamylase waste was not detectable. To double check the correctness of measurements, total protein content in the remaining sludge after separation was analysed.
Table 4 Effect of flocculating agent (alum) on glucoamylase recovery by integrated method. Alum concentrations (ppm) 100 50
Maximum enzyme activity%
Recovery%
Purification factor
Waste protein%
Waste enzyme%
58.3 77.3
48 56.7
0.95 0.89
47.6 24
52 43.3
Table 5 Effect of flocculating agent on 1000 ml of enzyme/CGA in simultaneous extraction and recovery method. Alum concentration (ppm)
Maximum enzyme activity%
Recovery%
Maximum specific enzyme activity
Purification factor
Waste protein%
Waste enzyme%
0 30
107 107.2
84.2 91.1
9.9 9.7
1.13 1.13
30.4 7.8
16.2 8.9
12
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Fig. 3. Effect of alum concentration on efficacy of produced glucoamylase: (A) recovery% and maximum GA activity% and (B) purification factor and maximum specific activity.
Table 6 Effect of flocculating agent on 750 ml of enzyme/CGA in simultaneous extraction and recovery method. Alum concentrations (ppm)
Maximum enzyme activity%
Recovery%
Maximum specific enzyme activity
Purification factor
Waste protein%
Waste enzyme%
40 20 15 10
167.4 196.6 210.4 206.5
134.1 147.5 152.7 137.4
6.3 6.7 6.9 5.9
1.86 1.99 2.05 1.76
25 22.4 22.5 17.1
0 0 0 0
Fig. 4. Recovery during drainage, alum conc. = 15 ppm: (A) glucoamylase ratio activity and (B) protein ratio.
The results showed high amount of protein with negligible glucoamylase activity, that is, extremely lean content of glucoamylase in wasted protein. It can be concluded that glucoamylase has been selectively recovered from a mixture of several proteins mixture by aphron flotation as illustrated in Fig. 4. Although CGA generation with 750 ml of feed resulted in a uniquely high separation parameters, extra reduction of the feed volume did not lead to improvement due to volume restriction in CGA generation. Fig. 5 compares the efficiency of the mixing, integrated and combined methods. These results show clearly that the combined
method is the most successful approach in recovering and purifying glucoamylase. 5. Conclusion Recovery and purification of glucoamylase, produced from an actual solid state fermented biomass, was studied in this work. This is the first reported experimental study on the actual, and not synthetic, samples, composed of a mixture of various proteins, enzymes and solid impurities. Three diverse separation procedures (mixing, integrated and combined) based on aphron flotation were tested to investigate the most effective separation method. It was found that a novel combined method (simultaneous extraction and recovery of glucoamylase) is a straightforward and suitable technique, which not only eliminates the extraction step, but also results in a relatively pure glucoamylase. Experimental results also showed that, according to the proposed theory, there is an optimized dose of flocculating agent. Carefully controlling the amount of flocculent agent resulted in enhancing the efficiency of aphron flotation. Acknowledgements
Fig. 5. Comparison among performance of various methods.
Isfahan University of Technology is gratefully acknowledged for financial support of this project as well as Dr. Khanahmadi from Isfahan Research Center for Agricultural and Natural Resources for supplying glucoamylase biomass.
A.Z. Zidehsaraei et al. / Separation and Purification Technology 67 (2009) 8–13
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[16] S.V. Save, V.G. Pangarkar, S.V. Kumar, Intensification of mass transfer in aqueous two-phase systems, Biotechnol. Bioeng. 41 (1992) 72–78. [17] P. Jauregi, J. Varley, Lysozyme separation by colloidal gas aphrons, Colloid Polym. Sci. 100 (1996) 362–367. [18] P. Jauregi, S. Gilmour, J. Varley, Characterization of colloidal gas aphrons for subsequent use for protein recovery, Chem. Eng. J. 65 (1997) 1–11. [19] M. Nobel, A. Brown, P. Jauregi, A. Kaul, J. Varley, Protein recovery using gas–liquid dispersion, J. Chromatogr. B 711 (1998) 31–43. [20] P.J. Oconnell, J. Varley, Immobilization of Candida rugosa lipase on colloidal gas aphrons, Biotechnol. Bioeng. 74 (2001) 264–269. [21] S. Fernandes, R. Hatti-Kaul, B. Mattiasson, Selective recovery of lactate dehydrogenase using affinity foam, Biotechnol. Bioeng. 79 (2002) 72–480. [22] S. Jarudilokkul, K. Rungphetcharat, V. Boonamnuayvitaya, Protein separation by colloidal gas aphrons using non-ionic surfactant, Sep. Purif. Technol. 35 (2004) 23–29. [23] M.C. Amiri, K.T. Valsaraj, Effect of gas transfer on separation of whey protein with aphron flotation, Sep. Purif. Technol. 35 (2004) 161–167. [24] E. Fuda, D. Bhatia, D.L. Pyle, P. Jauregi, Selective separation of ␥-lactoglobulin from sweet whey using CGAs generated from the cationic surfactant CTAB, Biotechnol. Bioeng. 90 (2005) 532–542. [25] E. Fuda, P. Jauregi, D.L. Pyle, Recovery of lactoferrin and lactoperoxidase from sweet whey using CGAs generated from anionic surfactant AOT, Biotechnol. Prog. 20 (2004) 514–525. [26] D.A. Wallis, D.L. Michelsen, F. Sebba, J.K. Carpenter, D. Houle, Application of aphron technology to biological separations, Biotechnol. Bioeng. Symp. (1986) 399–408. [27] F. Sebba, An unexpected colloid system, J. Colloid. Interface Sci. 35 (1971) 643–646. [28] M. Moshkelani, M.C. Amiri, Electrical conductivity as a novel technique for characterization of colloidal gas aphrons (CGA), Colloids Surf. A: Physicochem. Eng. Aspects 317 (2008) 262–269. [29] G.J. Jameson, S. Nam, M.M. Young, Physical factors affecting recovery rates in flotation, Miner. Sci. Eng. 9 (1977) 103–118. [30] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin reagent, J. Biol. Chem. 193 (1951) 265–275. [31] E.H.A. Mansur, Y. Wang, Y. Dai, Removal of suspensions of fine particles from water by colloidal gas aphrons (CGAs), Sep. Purif. Technol. 48 (2006) 71–77. [32] Y.O. Shin, D. Wahnon, M.E. Weber, J.H. Vera, Selective precipitation and recovery of xylanase using surfactant and organic solvent, Biotechnol. Bioeng. 86 (2004) 698–705.
Contents lists available at ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
An innovative simultaneous glucoamylase extraction and recovery using colloidal gas aphrons A. Zad Zidehsaraei, M. Moshkelani, M.C. Amiri ∗ Department of Chemical Engineering, Isfahan University of Technology, Isfahan, Iran
a r t i c l e
i n f o
Article history: Received 15 November 2008 Received in revised form 25 February 2009 Accepted 25 February 2009 Keywords: Glucoamylase Colloidal gas aphron Recovery Extraction
a b s t r a c t Recovery and purification of biological products generally involve complicated processes that cost even up to 70% of final product price. Because of many commercial applications of glucoamylase, its innovative purification methods are still desired. In this paper, colloidal gas aphrons, generated with cationic and anionic surfactants at their critical micelle concentration (CMC), have been used to recover and purify glucoamylase enzyme from actual biomass produced by solid state fermentation. Three diverse separation methods (mixing, integrated and combined) were examined. In these tests, actual biomass samples, composed of a mixture of various proteins and enzymes, were used. Using aphron flotation, it was found that recovery of glucoamylase in a simultaneous glucoamylase extraction and recovery mode can be enhanced by using a cationic surfactant and a flocculation agent. The experimental results revealed that this innovative procedure is the most effective method in recovery and purifying glucoamylase. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Glucoamylase (EC 3.2.1.3, 1,4-a-d-glucan glucohydrolase) is an important industrial enzyme that catalyzes the breakdown of malto-oligosaccharides to glucose and is used to produce high fructose corn syrup (HFCS) and alcohol by hydrolysis of starch and grains [1]. It is produced from various microorganism cultures, especially those in the Aspergillus and Rhizopus genus. The glucoamylase preparation from Aspergillus Niger is produced on a commercial scale and the preparation has been widely utilized for industrial production of crystalline d-glucose, high fructose corn syrups, wine, ethanol, beer, and in other fermentation processes [2]. Glucoamylase purification can be done by different methods such as affinity chromatography [3,4], affinity precipitation [5–10], fast protein liquid chromatography [11,12], and aqueous two-phase extraction [13–16]. Moreover, protein recovery using colloidal gas aphrons (CGAs) has been an attractive area for researchers since the 1990s [17–25]. However, only Wallis et al. [26] and Save et al. [16] have applied colloidal gas aphrons for glucoamylase recovery in order to intensify mass transfer while using aqueous two-phase method. Unfortunately there has been no report in the scientific literatures on the recovery and purification of glucoamylase from a real fermented biomass (a mixture of various proteins, enzymes and spores) directly using colloidal gas aphrons.
∗ Corresponding author. Tel.: +98 311 3915615; fax: +98 311 3912677. E-mail addresses: [email protected] (A.Z. Zidehsaraei), [email protected] (M. Moshkelani), [email protected] (M.C. Amiri). 1383-5866/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2009.02.019
Solid state fermentation holds tremendous potential for the production of glucoamylase in comparison to submerged fermentation because of its high volumetric productivity, relatively higher concentration of the products, less effluent generation and simple fermentation equipments. However, recovery and purification of produced glucoamylase remains a challenging step, since various enzymes and components like protease, cellulose, xylose derivatives, arabitol, erythrose and glyceraldehydes are produced during the fermentation process and inhibit the activity of produced glucoamylase. Thus recovery of glucoamylase from fermented matter is an important factor that affects the cost-effectiveness of the overall process. In this work, glucoamylase has been produced through a solid state fermentation method. Then three different techniques using aphron flotation have been applied for recovery and purification. Glucoamylase separation parameters for various techniques have been investigated, in the presence and absence of a flocculating agent. Finally, an innovative simultaneous glucoamylase extraction, recovery, and purification method was developed. 2. Theory CGAs are microbubbles (typically 25–100 m in diameter) with colloidal properties, first introduced by Sebba [27]. The drainage of CGA dispersion and the ionisation of chemical groups at the surface (when using ionic surfactant molecules) are two important factors in aphron flotation. Due to the low density of CGA dispersion, it undergoes drainage process during which the dispersion separates into a clear solution region at the bottom and froth phase at the
A.Z. Zidehsaraei et al. / Separation and Purification Technology 67 (2009) 8–13
9
Fig. 1. Details of various methods and steps in testing procedure.
top [28]. The drainage rate depends on environmental conditions such as solution ionic strength, pH and additives. Basically an individual CGA and its most closely associated ions can move through the solution as a unit. The pH of solution and the electrical conductivity affects the zeta potential of each unit. Since CGA dispersion possesses a size distribution that changes with progress of drainage process, they can act similarly to a charged microfilter bed with non-uniform size distribution. Therefore, they can collect particles based on their sizes as well as their charges. On the basis of the theoretical analysis developed by Jameson et al. [29] the efficiency of collision (Ec ) between a particle (diameter of dp ) and a bubble (diameter of db ) is given by Eq. (1) in which m appears to have a value of 1–2 and k a value of 3.5–4. Ec =
dpm
(1)
dbk
Equation (1) shows that the efficiency generally increases as dp /db increase. Since glucoamylase and other molecules are submicron size particles, a flocculating agent can be used to enlarge their sizes. Obviously adding a flocculating agent not only results in improving collection efficiency but also increases impurities; therefore, there should be an optimized dose of flocculating agent. It seems that CGA dispersion is able to differentiate a mixture of particles and high molecular weight molecules if experimental conditions are carefully monitored. In order to quantify the recovery and purity of produced glucoamylase, the following parameters have been used to determine the effectiveness of the separation process: 1. Total amount of enzyme activity (U) = activity (U/ml) × volume (ml) activity (U/ml) 2. specific activity (U/mg) = protein content (mg/ml) 3. recovery (yield)% =
× 100
specific activity of enzyme after purification step specific activity before purification step
5. enrichment ratio =
3. Materials and methods 3.1. Materials Sodium dodecyl sulfate (SDS) (FSA, England), an anionic surfactant, and tetradecyl trimethyl ammonium bromide (TTAB) (Merk), a cationic surfactant were used to produce CGAs at CMC concentrations. Citric acid and sodium citrate (citrate buffer) were utilized as a buffer solution for pH adjustment in range of 3–6.2. Aluminum sulfate (Alum) was used as a flocculating agent. In order to determine the glucoamylase activity, 2-hydroxy-3,5-dinitrobenzoic acid (DNS), glucose and starch solution were applied. Bovine serum albumin, copper (II) sulfate, potassium sodium tartrate and FolinCiocalteu reagent were used for total solution protein content determination. All chemicals except surfactants were supplied by Sigma–Aldrich Co. 3.2. Biomass and glucoamylase production
total amount of enzyme activity after purification step total amount of activity enzyme before purification step
4. purification factor =
operations involve separately CGA generation with distilled water according to the Sebba method [27]. In the batch process, a definite ratio of CGA dispersion is mixed with protein or enzyme and the mixture is stirred for a certain time. But in continues mode, the generated CGA is pumped into a column of protein or enzyme feed. The optimum recovery can be achieved by controlling the residence time in the column. In both operations, the top foamy phase and drained liquid are tested to measure separation parameters. However, Amiri undertook a different approach by producing CGA directly from whey [23]. In this study, batch and continuous modes as well as a totally innovative extraction and recovery combination method for recovery and purification of glucoamylase have been examined.
glucoamylase activity in aphron phase glucoamylase activity in mixture of aphron and glucoamylse
Almost all researchers have utilized batch or continues operations in their protein or enzyme recovery studies [17–25]. Both
Glucoamylase was produced by solid state fermentation at the Isfahan Research Center for Agricultural and Natural Resources. The reported value of isoelectric point for glucoamylase was pI 3.5. Aspergillus Niger (CCUG 33991, Sweden) with Mw = 90,000 = 90 kDa, was added to 50% wet bran on a tray and left for 40 h at 35 ◦ C to complete the fermentation process. The produced biomass was left to decrease its humidity to less than 5% at 40 ◦ C. The purified enzyme was reported to have a molar mass of 90 kDa with an isoelectric point at pH 3.5. In order to extract glucoamylase from dry biomass,
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Fig. 2. Outline of experimental design: (A) mixing method, (B) integrated method and (C) combined method.
the sample was diluted 10-fold with distilled water and shaken for 70 min at 30 ◦ C and speed of 120 rounds per minutes. Glucoamylase was finally obtained by filtration.
experimental design. All tests for each method were double checked to generate reliable results. 3.5. Glucoamylase activity determination
3.3. Colloidal gas aphron generation CGAs dispersions were generated using anionic and cationic surfactants both at their CMC through method proposed by Sebba [27]. Stirring rate and time were 7000 rpm and 5 min, respectively for all tests. For each production method, different feeds were used. Distilled water, glucoamylase solution, and solid biomass solution were chosen to generate CGAs for the mixing, integrated, and combined methods, respectively. 3.4. Testing procedures Fig. 1 demonstrates the details of various steps in different testing procedures. Three different approaches were undertaken in batch wise mode. In the mixing method, enzyme to CGA ratios of 1, 2, or 3 were mixed together with a magnetic stirrer for 5 min at pH values of 3.5, 4, 4.3, or 4.8. As for integrated tests, CGA was produced directly from glucoamylase dispersion and pH was adjusted at 3.5, 4.3 and 4.8. The sample was transferred to a cylinder for differentiated sampling. In these series of tests, the effect of flocculating agent was also investigated by adding 100 or 50 ppm of alum to the glucoamylase solution before CGA generation. In the case of the combined method, CGA was generated by adding 1000 and 750 ml of distilled water to solid biomass and alum concentrations of 40, 30, 20, 15, 10, and 0 ppm to check the maximum recovery. The details of differentiated sampling during the drainage process in the measuring cylinder were reported elsewhere [28]. Fig. 2 shows the
In order to determine the glucoamylase activity, a specified amount of enzyme sample and 1 ml of distilled water were added to 1 ml of starch substrate solution (pH 4.5–4.6). The mixture was kept at 60 ◦ C for 15 min to produce glucose monomers. It was then put in to boiling water to deactivate the enzyme. Following the calorimetric assay, the absorption intensity of the final solution was then measured at wave length of 540 nm for determining the amount of released glucose, which is directly related to enzyme activity. The spectrophotometer equipment was calibrated using distilled water for all tests. To assure precise results, each test was repeated twice. 3.6. Total protein content determination Total content of protein in recovered samples were determined according to the Lowry protein assay [30]. 4. Results and discussion 4.1. Mixing method Table 1 shows the effect of pH on separation factors for a CGA/glucoamylase mixing ratio of one. The best separation result is obtained for TTAB at pH 3.5 with the highest recovery and enrichment factor. Comparing SDS with TTAB, cationic surfactant (TTAB) was more successful in recovering glucoamylase because of higher enrichment and recovery factors. Because the positively
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Table 1 Effect of pH for SDS and TTAB for CGA/glucoamylase mixing ratio of one. pH
Maximum enzyme activity%
Recovery%
Enrichment ratio
Time of maximum enrichment
SDS 3.5 4 4.3 4.8
72.5 78.8 82.2 91.5
96 101 95.5 93.2
1.05 1.01 1.03 1.08
End of macroscopic drainage Start and end of macroscopic drainage End of macroscopic drainage End of macroscopic drainage
TTAB 3.5 4 4.3 4.8
89 84.4 87 83
117 107 83.7 104
1.18 1.11 1.03 1.06
End of microscopic drainage End of microscopic drainage End of microscopic drainage End of microscopic drainage
Table 2 Separation factors for various mixing ratio of CGA/glucoamylase for SDS and TTAB, pH 3.5. Surfactant
CGA/glucoamylase ratio
Recovery%
Enrichment ratio
SDS
1/1 1/2 1/3
96 112 101
1.05 1.25 1.04
TTAB
1/1 1/2
117 92
1.18 1.05
charged hydrophobic alkyl group in TTAB does not interact with H+ ions in solution and facilitate quick and easy dispersion of CGAs consequently [16]. Moreover, it seems that glucoamylase should accumulate at foam phase; therefore maximum enrichment factor can be achieved during microscopic drainage [28], which was well observed for TTAB. Additionally in the TTAB case for total pH range (except pH 4.3) and for SDS at pH 4, total enzyme activity after the separation procedure is more than of the initial glucoamylase solution. Since no glucoamylase is being produced through the separation process, it is concluded that the mixing method has been successful in removing solid impurities like pigments and spores (confirmed observationally) as well as some proteins and other enzymes that decrease glucoamylase activity. However, maximum glucoamylase activity of less than 100, and an enrichment factor of approximately 1 for all tests reveals that mixing method is not the efficient approach in recovering glucoamylase. Results also showed that TTAB should be considered for further experimental work (see Table 2). 4.2. Integrated method Table 3 shows effect of pH on performance of aphron flotation in integrated method. Best recovery was obtained at pH 3.5. Based on analysis developed in the theoretical section, according to Eq. (1), efficiency should be improved by using a flocculating agent [29,31]. Moreover, total protein content of each sample was also measured to probe the effect of a flocculating agent on the integrated method.
Table 3 Effect of pH on performance of aphron flotation in integrated method. pH
Maximum enzyme activity%
Recovery%
Time of maximum enrichment
3.5 4.3 4.8
110 77.4 62.5
102 71 62
End of microscopic drainage End of microscopic drainage End of microscopic drainage
Table 4 shows that high concentration of flocculating agent reduces the recovery of glucoamylase and leads to the waste of more protein and enzyme. However, an extremely clear retentate was drained due to efficient removal of solid impurities. Thus finding an optimum concentration of flocculating agent is a key point in any successful aphron flotation. 4.3. Combined (simultaneous extraction and recovery) method In this novel method, simultaneous CGA generation and enzyme extraction from biomass has been done. The extracted glucoamylase then was recovered during the drainage process. Table 5 compares the efficiency of the combined method for 1 l of such dispersion in presence and absence of alum. It was found that the maximum enzyme activity and recovery is highly improved even in comparison with the results obtained by Shin et al. [32]. They obtained 65% recovery in a mixture of only two different proteins. Adding alum has positive effects in reducing the amount of wasted protein and glucoamylase. Approaching maximum specific activity of nearly 10 shows a clearly successful selective recovery of glucoamylase. To improve the effectiveness of combined method, tests were done for 750 ml of feed solution. Purification factor and recovery is illustrated for different alum concentrations in Fig. 3 which shows that double purification factor and the highest glucoamylase efficiency were obtained at alum dosage of 15 ppm. As is shown in Table 6, at lower concentrations of alum, less protein was wasted and also glucoamylase waste was not detectable. To double check the correctness of measurements, total protein content in the remaining sludge after separation was analysed.
Table 4 Effect of flocculating agent (alum) on glucoamylase recovery by integrated method. Alum concentrations (ppm) 100 50
Maximum enzyme activity%
Recovery%
Purification factor
Waste protein%
Waste enzyme%
58.3 77.3
48 56.7
0.95 0.89
47.6 24
52 43.3
Table 5 Effect of flocculating agent on 1000 ml of enzyme/CGA in simultaneous extraction and recovery method. Alum concentration (ppm)
Maximum enzyme activity%
Recovery%
Maximum specific enzyme activity
Purification factor
Waste protein%
Waste enzyme%
0 30
107 107.2
84.2 91.1
9.9 9.7
1.13 1.13
30.4 7.8
16.2 8.9
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Fig. 3. Effect of alum concentration on efficacy of produced glucoamylase: (A) recovery% and maximum GA activity% and (B) purification factor and maximum specific activity.
Table 6 Effect of flocculating agent on 750 ml of enzyme/CGA in simultaneous extraction and recovery method. Alum concentrations (ppm)
Maximum enzyme activity%
Recovery%
Maximum specific enzyme activity
Purification factor
Waste protein%
Waste enzyme%
40 20 15 10
167.4 196.6 210.4 206.5
134.1 147.5 152.7 137.4
6.3 6.7 6.9 5.9
1.86 1.99 2.05 1.76
25 22.4 22.5 17.1
0 0 0 0
Fig. 4. Recovery during drainage, alum conc. = 15 ppm: (A) glucoamylase ratio activity and (B) protein ratio.
The results showed high amount of protein with negligible glucoamylase activity, that is, extremely lean content of glucoamylase in wasted protein. It can be concluded that glucoamylase has been selectively recovered from a mixture of several proteins mixture by aphron flotation as illustrated in Fig. 4. Although CGA generation with 750 ml of feed resulted in a uniquely high separation parameters, extra reduction of the feed volume did not lead to improvement due to volume restriction in CGA generation. Fig. 5 compares the efficiency of the mixing, integrated and combined methods. These results show clearly that the combined
method is the most successful approach in recovering and purifying glucoamylase. 5. Conclusion Recovery and purification of glucoamylase, produced from an actual solid state fermented biomass, was studied in this work. This is the first reported experimental study on the actual, and not synthetic, samples, composed of a mixture of various proteins, enzymes and solid impurities. Three diverse separation procedures (mixing, integrated and combined) based on aphron flotation were tested to investigate the most effective separation method. It was found that a novel combined method (simultaneous extraction and recovery of glucoamylase) is a straightforward and suitable technique, which not only eliminates the extraction step, but also results in a relatively pure glucoamylase. Experimental results also showed that, according to the proposed theory, there is an optimized dose of flocculating agent. Carefully controlling the amount of flocculent agent resulted in enhancing the efficiency of aphron flotation. Acknowledgements
Fig. 5. Comparison among performance of various methods.
Isfahan University of Technology is gratefully acknowledged for financial support of this project as well as Dr. Khanahmadi from Isfahan Research Center for Agricultural and Natural Resources for supplying glucoamylase biomass.
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