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Improvement of catalytic properties of starch hydrolyzing fungal amyloglucosidase: Utilization of agar-agar as an organic matrix for immobilization
Carbohydrate Research 486 (2019) 107860
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Improvement of catalytic properties of starch hydrolyzing fungal amyloglucosidase: Utilization of agar-agar as an organic matrix for immobilization
T
Sidra Perveza, Muhammad Asif Nawazb,∗, Muhsin Jamalc, Tour Jand, Farhana Maqboola, Ismail Shahe, Afsheen Amanf, Shah Ali Ul Qaderg a
Department of Microbiology, Hazara University, Mansehra, Khyber Pakhtunkhwa, Pakistan Department of Biotechnology, Shaheed Benazir Bhutto University, Sheringal, Dir (Upper), Khyber Pakhtunkhwa, Pakistan Department of Microbiology, Abdul Wali Khan University, Garden Campus, Mardan, Pakistan d Department of Botany, University of Malakand, Chakdrara, Khyber Pakhtunkhwa, Pakistan e Department of Pharmacy, Abdul Wali Khan University, Garden Campus, Mardan, Pakistan f The Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi, 75270, Karachi, Pakistan g Department of Biochemistry, University of Karachi, Karachi, 75270, Pakistan b c
ARTICLE INFO
ABSTRACT
Keywords: Agar-agar Entrapment Immobilization Reusability Starch hydrolysis Bioprocessing
In this study, amyloglucosidase was immobilized within agar-agar through entrapment technique for the hydrolysis of soluble starch. Enzymatic activities of soluble and entrapped amyloglucosidase were compared using soluble starch as a substrate. Partially purified enzyme was immobilized and maximum immobilization yield (80%) was attained at 40 gL-1 of agar-agar. Enzyme catalysis reaction time shifted from 5.0 min to 10 min after immobilization. Similarly, a five-degree shift in temperature (60 °C–65 °C) and a 0.5 unit increase in pH (pH-5.0 to pH-5.5) were also observed. Substrate saturation kinetics revealed that Km of entrapped amyloglucosidase increased from 1.41 mg ml−1 (soluble enzyme) to 3.39 mg ml−1 (immobilized enzyme) whereas, Vmax decreased from 947 kU mg−1 (soluble enzyme) to 698 kU mg−1 (immobilized enzyme). Entrapped amyloglucosidase also exhibited significant catalytic performance during thermal and storage stability when compared with soluble enzyme. Reusability of entrapped amyloglucosidase for hydrolysis of soluble starch demonstrated its recycling efficiency up to six cycles which is an exceptional characteristic for continuous bioprocessing of soluble starch into glucose.
1. Introduction Starch debranching enzymes are among the most important industrial enzymes used for the production of wide variety of commercial products. They specifically act on O-glycosidic linkages of starch molecules and produce variety of monosaccharide and oligosaccharides. The hydrolyzed products obtained from starch are extensively used in various industrial processes. Hydrolysis of starch can be performed by enzymatic, alkaline or acidic treatments. Hui [1] suggested that starch hydrolysis can further be facilitated by performing other treatments like microwaving, heating and extrusion. During treatment, the glycosidic bonds in starch are fragmented and results in the formation of different oligomers (maltotriose, maltose) and monomers (glucose). Enzymatic cleavage of starch by means of hydrolases is most widely used because of the low energy consumption and high product formation. Whereas, ∗
as in case of acid hydrolysis, the product yield is lower along with unwanted color and bitter taste development [2,3]. Among different starch debranching enzymes, amyloglucosidase [E.C. 3.2.1.3] have more commercial interest due to complete conversion of starch and other related polysaccharides into D-glucose monomers. Free or soluble amyloglucosidase is widely used in starch saccharification, ethanol production, baking, food, brewing, pharmaceutical and textile industries [4,5]. However, there are some major difficulties associated in the use of soluble enzyme due to their instability under harsh industrial conditions like at various temperatures and pH [6,7]. Enzyme immobilization is a strategy to increase the stability and recycling efficiency of various industrial enzymes. Subsequently, various approaches have been used for immobilization of enzymes which are mainly classified as physical (adsorption and entrapment) and chemical (covalent binding and cross-linking) methods. Physical methods are simple and
Corresponding author. Department of Biotechnology, Shaheed Benazir Bhutto University, Sheringal, Dir Upper, KPK, Pakistan. E-mail address: [email protected] (M.A. Nawaz).
https://doi.org/10.1016/j.carres.2019.107860 Received 4 May 2019; Received in revised form 2 October 2019; Accepted 25 October 2019 Available online 01 November 2019 0008-6215/ © 2019 Elsevier Ltd. All rights reserved.
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cost effective but the interaction between the soluble enzyme and matrix is usually reversible and weak while in case of chemical methods, the interaction is strong and irreversible [8]. The method of entrapment involves occlusion and confinement of soluble enzyme in a porous fiber or a gel matrix through either covalent or non-covalent interactions. In this method, substrate and product can easily diffuse out through the polymeric network. However, because of the enzyme being larger in molecular size, it remains entrapped within the matrix [9]. Additionally, gel entrapment is a simple and effective approach for immobilization as it preserves the biological function of the biocatalyst which is entrapped [10]. Entrapment method also permits co-immobilization of multiple enzymes for efficient hydrolysis of complex substrates like starch. Agar-agar, agarose, cellulose triacetate, sodium alginate, gelatin, chitosan, diacetyl cellulose and polyacrylamide have been used as different matrices for the entrapment of various enzymes [11]. Agar-agar is natural polysaccharide obtained from red algae (Rhodophyta). It is composed of two components which are agarose (linear) and agaropectin (branched) linked together by α-(1 → 3) and β(1 → 4) linkages. Due to its chemical structure, non-reactivity to other biomolecules, resistance to acid and capability to form robust gel at low concentrations this biopolymer favors applications in various biotechnological industries [12]. Beside all these properties, agar-agar is highly porous which makes it an excellent matrix for entrapment of proteins. Previously this amyloglucosidase have been immobilized using different supports including alginate and agarose through entrapment method and onto chitosan through covalent binding [13,14]. However, data is very scarce for entrapment of amyloglucosidase within agar. Hence, this is the first study in which fungal amyloglucosidase is immobilized using agar-agar as a supporting matrix to improve its catalytic efficiency, thermal and storage stabilities.
allowed to solidify at room temperature. Solid agar gel which contain amyloglucosidase was cut into small beads (3.0 mm) with the help of metallic cork-borer. These beads were then undergo several washing steps with citrate buffer (50 mM; pH-5.0) to remove unbound or loosely bound enzyme molecules. For preparation of control beads, agar-agar solution was mixed with citrate buffer (50 mM; pH-5.0) only and same procedure was followed for the preparation of amyloglucosidase beads. Immobilized beads were stored at 4 °C for further analysis. 2.5. Analysis of amyloglucosidase and total protein Enzyme activity of amyloglucosidase was determined as described previously by Ghani et al. [16]. Concisely, 0.1 ml of desalted soluble amyloglucosidase and 0.25 gm of immobilized amyloglucosidase beads were mixed with 1.0 ml of starch solution prepared in 50 mM citrate buffer (pH: 5.0 for soluble and 5.5 for entrapped enzyme). The reaction mixture was kept at appropriate temperature (60 °C for soluble and 65 °C for immobilized) for specified time (5.0 min for soluble and 10.0 min for immobilized enzyme). Glucose liberated after reaction was then determined spectrophotometrically at 546 nm using GOD-PAP method [17,18] with glucose as standard. Total protein was estimated by Lowry's method using bovine serum albumin as standard [19]. 2.6. Immobilization yield Immobilization yield was calculated using following formula:
Immobilization Yield (%) =
Activity of immobilized enzyme Activity of soluble enzyme
× 100
2. Material and methods
2.7. Selection of reaction time, reaction pH and reaction temperature
2.1. Chemicals and reagents
Aspergillus fumigatus KIBGE-IB33 [GenBank Accession: KF905648] was cultivated in a previously optimized medium of pH: 7.0 for 4 days at 30 °C [15]. After fermentation, the crude soluble amyloglucosidase was separated from fungal biomass by centrifuging the medium at 40,000 ✕g for 15 min at 4 °C [15].
Enzyme performance of immobilized amyloglucosidase was investigated by incubating the enzyme with substrate for different time intervals (5.0–60.0 min). For the selection of optimum pH, amyloglucosidase activity was performed at different pH values ranging from 4.0 to 8.0 of same ionic strength (50.0 mM) using various buffers including acetate buffer (pH: 4.0–4.5), citrate buffer (pH: 5.0–6.0) and phosphate buffer (pH: 6.5–8.0). A wide range of temperature was studied to determine the maximum activity of soluble and entrapped amyloglucosidase (35–80 °C). Arrhenius plot was constructed for the determination of activation energy using the following equation:
2.3. Partial purification of amyloglucosidase
InA =
The ammonium sulphate precipitation method with 40% saturation was used for partial purification of crude amyloglucosidase. Further, this partially purified enzyme was desalted using PD-10 desalting column (GE Healthcare) as described previously [14].
2.8. Analysis of Vmax and Km
Agar-agar was purchased from Research Organics (USA). All other chemicals were of analytical grade. 2.2. Amyloglucosidase production
Ea × InB RT
The Vmax and Km was determined using Lineweaver-Burk double reciprocal plot. Enzymatic assay was performed with different concentration of potato starch (1.0–30.0 mg ml−1) at optimum assay conditions.
2.4. Immobilization of amyloglucosidase using agar-agar Amyloglucosidase was immobilized within agar-agar matrix by mixing desalted enzyme with agar-agar solution in 1:1 ratio. The effect of concentration of agar-agar on entrapment yield of amyloglucosidase was studied at different concentrations (20.0–50.0 gL-1) and most appropriate concentration selected was further used in subsequent studies. The agar-agar solution was prepared by dissolving 40 gL-1 of agaragar in citrate buffer (50 mM; pH-5.0) at 100 °C. After cooling the solution to 40 °C, amyloglucosidase was mixed thoroughly and immediately casted in a preassembled glass plate. This mixture was
2.9. Thermal stability To determine the thermal tolerance, soluble enzyme and agar-agar beads (with or without enzyme) were pre-incubated at wide range of temperatures (30 °C–80 °C) for 120 min. After every 20 min, aliquots were taken out and immediately placed on ice bath and enzyme activity was performed and calculated. 2
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2.10. Storage stability
enzymes [21]. Beyond this concentration, a decrease in enzymatic activity was noticed which could be due to either stearic hindrance of the enzyme due to the formation of multilayered protein complexes on charged matrix [22] or due to the mass transfer limitation of substrate [23]. Mass transfer limitation can be of two types including internal and external limitation [24,25]. In internal limitation, the diffusion of starch molecules (substrate) into the matrix (agar-agar) is constrained due to small or less pore size while, in case of external limitation the release of glucose molecules (product) from the internal environment of the matrix is restricted.
Soluble enzyme and enzyme immobilized beads were stored at different temperatures (4 °C and 37 °C) for 90 days in citrate buffer (50 mM; pH-5.0). The residual activity of amyloglucosidase was measured in percentage after every 10 days of storage. 2.11. Recycling studies For the determination of operational stability of immobilized amyloglucosidase, beads were incubated with 10 gL-1 starch prepared in citrate buffer (50 mM; pH-5.0) under optimal conditions. After the determination of glucose using the standard assay conditions, the beads were washed with activity buffer and experiment was repeated until no catalytic activity of amyloglucosidase was observed or it become constant.
3.1.2. Effect of reaction time To investigate the optimum reaction time required to hydrolyze starch into glucose by immobilized amyloglucosidase, the catalytic activity of enzyme was carried out at different time intervals. Fig. 2A demonstrates that the relative activity of immobilized amyloglucosidase increased with the increase in the reaction time and maximum starch hydrolysis was achieved after 10.0 min of reaction as compared to the soluble enzyme which exhibited maximum starch hydrolysis by amyloglucosidase after 5.0 min. This variation in the reaction time in both the cases supported the probability of resistance towards accessibility of high molecular weight substrate (starch) to diffuse into the agar-agar matrix and react with the enzyme to produce glucose molecules as a product. Therefore, starch requires relatively more time to reach the active site of the enzyme that is entrapped within the matrix. Mahajan et al. [26] and Sattar et al. [27] also noticed 8.0 min (soluble enzyme: 11.0 min; immobilized enzyme: 19.0 min) and 5.0 min increments (soluble enzyme: 15.0 min; immobilized enzyme: 20.0 min) in reaction time after immobilization of amylolytic and proteolytic enzyme on agar-agar beads, respectively.
2.12. Scanning electron microscopy (SEM) Cross-sectional topological structure of agar-agar beads (with and without amyloglucosidase) was investigated through SEM as described previously [18]. 3. Results and discussion Industrial scale utilization of enzyme involves some practical problems associated with the use of soluble enzyme. Being soluble in water, the recovery of enzymes in active form after completion of a reaction is very difficult. Furthermore, free enzymes possess very low operational stability (thermal and storage) that renders its successful application impossible. These limitations can be overcome by immobilizing the biocatalysts using suitable support system and appropriate methodology. In this study, entrapment method was used with agar-agar as a matrix to improve the catalytic efficiency of amyloglucosidase which hydrolyzes starch into glucose molecules.
3.1.3. Effect of reaction pH The influence of pH (Fig. 2B) on the catalytic activity of soluble and entrapped amyloglucosidase was studied and an approximate pH shift of 0.5 units was observed in case of immobilized biocatalyst (pH: 5.5) as compared to soluble enzyme (pH: 5.0). It has been suggested that the charge surface within the agar-agar beads and the entrapped enzyme can create a charged microenvironment, which could eventually affect the active site of an enzyme and thus can alters the pH of the entrapped enzyme [28]. Liu et al. [29] reported that cationic matrices containing enzymes usually shifts pH towards acidic side while, anionic shifts it towards alkaline region. Agar-agar is composed of two components in which agarose is neutral while, agro-pectin is negatively charged due to presence of sulphate. Hence in the current results, a 0.5 unit shift of pH towards alkaline side was observed. Soluble amyloglucosidase showed 30% relative activity at pH-8.0 while, entrapped enzyme retained approximately 50% of its initial activity at the same pH value (Fig. 2B). Similarly, a slight change (0.5 units) in optimum pH from 6.0 to 6.5 was observed in case of entrapment of α-amylase from A. oryzae on calcium agar beads [30]. Previously the pH of amyloglucosidase is also increased 1.0 unit (soluble enzyme: 5.0; immobilized enzyme: 6.0) when calcium alginate was used as a matrix for entrapment [15].
3.1. Immobilization of amyloglucosidase 3.1.1. Effect of agar-agar concentration The selection of optimum concentration of support is very important for immobilization as it is responsible for the penetration of substrate into the matrix as well as it is also accountable for the leaching of enzyme molecules. Additionally, it also governs the porosity of agar-agar beads because as the concentration increases, the pore size of the matrix will be decreased and hence, this situation will eventually render both the enzyme entrapment and substrate diffusion within the support. The porosity of matrix lattice is the most crucial aspect in entrapment method. In the case of small pore size, the entrapment of biocatalyst only occurs on the external surface of the matrix which could result in low enzyme loading while, in case of large pore size of the matrix, enzyme leakage can occur [20]. Similar phenomenon was noticed when amyloglucosidase was entrapped within agar-agar matrix. Maximum immobilization of approximately 80% was observed when 40.0 gL-1 concentration was used (Fig. 1). Beyond this concentration, the entrapment of amyloglucosidase declined and approximately 24% decrease in immobilization yield was noticed at 50.0 gL-1. This might be due to the outflow of enzyme from agar-agar beads because of its large pore size and after subsequent washing of beads the enzyme leached out. Minimum immobilization yield was detected at 20.0 gL-1 which exhibited 40% less entrapment as compared to the 40.0 gL-1 concentration. This could be because at this low concentration of agar-agar the beads were very delicate and fragile and were unable to sturdily entrap the enzyme. These results confirmed that agar-agar concentration directly affects the entrapment of enzyme and also inversely affects the pore size of the matrix. A similar concentration of agar-agar (40.0 gL-1) was also previously reported for entrapment of amylolytic
3.1.4. Effect of reaction temperature Kinetic behavior of soluble and entrapped amyloglucosidase was observed at different temperatures ranges. Maximum catalysis of substrate was observed at 60 °C when using soluble enzyme and at 65 °C when using entrapped amyloglucosidase (Fig. 2C). Increase in relative activity for immobilized enzyme at higher temperatures could be due to the unique structural nature of the matrix that protected the enzyme from denaturation [31]. Along with this, the energy of activation for soluble and entrapped amyloglucosidase was also assessed using Arrhenius plot and it was nearly 5.55 kcal mol−1 and 4.55 kcal mol−1, respectively (Fig. 2D). A decrease in activation energy for entrapped enzyme as compared to soluble enzyme could be due to an increase in optimum reaction temperature (65 °C) of entrapped enzyme (Fig. 2C). 3
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Fig. 1. Effect of agar-agar concentration on immobilization yield of amyloglucosidase (A); Agar-agar beads with and without entrapped amyloglucosidase (B).
Fig. 2. Effect of enzyme-substrate reaction time (A); pH (B); temperature (C) and construction of Arrhenius plot (D).
Kumar et al. [31] have suggested that the immobilization matrices are able to protect the enzymes from denaturation by absorbing surrounding heat and may also cause some conformational changes in the enzyme structure. In the present study, besides the change in the optimum reaction temperature, the temperature profile of immobilized amyloglucosidase became wider as compared to the soluble enzyme with a retention of 41% of relative enzyme activity even at 80 °C while, soluble enzyme only retained 20%. A five-degree shift in the optimum temperature of immobilized fungal α-amylase was previously reported when calcium agar gels and agarose were used, respectively [30]. Previously, immobilization of urease in agar-agar tablets showed extreme change in optimum temperature from 30 °C (free enzyme) to 60 °C (immobilized enzyme) [32]. The current results also advocate that agar-agar can act as a protective barrier which could absorb heat and protect enzymes from denaturation by stabilizing its structure within the microenvironment.
The results disclosed that after entrapment of amyloglucosidase, Km amplified by 1.97 mg ml−1 whereas, Vmax units were lowered by 249 kU mg−1. This increase in Km could be due to the low affinity of the enzyme towards soluble potato starch [33]. Other possible reasons in this case which resulted in decrease in Vmax value could be the confinement of amyloglucosidase in the microenvironment of agar-agar and because of the diffusional resistance or stearic hindrance that is presented during penetration of soluble potato starch into the matrix. Similar findings have been reported about the increased Km and declined Vmax values of the immobilized alpha amylase and urease within agar matrix [32]. However, Sharma et al. [34] found no change in affinity of amylase towards soluble starch after entrapment in calcium agar tablets whereas, a 37.5 fold increase in Vmax was observed. Previously, amyloglucosidase entrapped in alginate also showed higher Km and lower Vmax values [15]. 3.1.6. Stability studies Thermal stability data of immobilized enzyme showed that it retained additional catalytic activity at all temperatures as compared to soluble enzyme. Soluble enzyme lost approximately 30%, 40% and 58% of its initial residual activity at 40 °C, 50 °C and 60 °C after 120 min, respectively (Fig. 3A, B, 3C). However, at 70 °C and 80 °C a complete loss in enzyme activity was observed after 100 and 80 min, respectively (Fig. 3D and E). On the other hand, agar-agar entrapped amyloglucosidase retained about 70%, 40% and 27% of its relative activity at 60 °C, 70 °C and 80 °C even after 120 min, respectively. The unfolding of
3.1.5. Effect of substrate concentration Studies on kinetic parameters exhibited that Vmax and Km of amyloglucosidase were affected by immobilized entrapment method. It was observed that Km increased from 1.41 mg ml−1 (standard error: 0.054 mg ml−1) for soluble amyloglucosidase to 3.39 mg ml−1 (standard error: 0.396 mg ml−1) for agar-agar entrapped amyloglucosidase. While, a decrease in Vmax value was detected from 947 kU mg−1 (standard error: 40.1 kU mg−1) for soluble amyloglucosidase to 698 kU mg−1 (standard error: 29.2 kU mg−1) for agar-agar entrapped enzyme. 4
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Fig. 3. Stability studies of soluble and entrapped amyloglucosidase at various temperatures: 40 °C (A); 50 °C (B); 60 °C (C); 70 °C (D); 80 °C (E).
Fig. 4. Storage stability of soluble and immobilized amyloglucosidase at 37 °C (A) and 4 °C (B).
When comparing performance of immobilized biocatalysts intended for industrial use against soluble enzymes, the portrayal of their storage stabilities is also important. If storage stability is high then the practical application cost of the majority of bioprocesses could be reduced. Storage stability was examined for up to 90 days. As observed from Fig. 4A and B, agar-agar entrapped amyloglucosidase retained around 33% and 57% of its initial activity after 60 days of storage at 37 °C and 4 °C, respectively. Immobilized amyloglucosidase is more stable as compared to its soluble counterpart at these temperatures. These findings revealed that agar-agar provided a shielded effect to amyloglucosidase. Thus, it is suggested that the confined microenvironment of the agar-agar beads would able to prevent the biocatalyst from harsh industrial conditions. Similarly, 50% activity of agar-agar immobilized urease was observed at 4 °C even after 53 days [32].
Fig. 5. Recycling efficiency of immobilized amyloglucosidase.
3.1.7. Recycling studies The main objective of the current study was to design an immobilization method that can proficiently recover amyloglucosidase from the reaction mixture for reuse because for continuous utilization of enzymes, operational stability is among the most important factor. Therefore, reusability potential of entrapped amyloglucosidase was examined at 60 °C up to 10 cycles. Results of current findings showed that agar-agar entrapped amyloglucosidase possess considerable operational stability with a retention of 68% and 46% activity after the 4th and 6th recycles, respectively (Fig. 5). For incessant bioprocessing at industrial scale, such operational stabilities always provide benefit by
protein structure after exposure to higher temperatures which is above its enzyme kinetic maxima is one of the main phenomenon that is involved in deactivation of enzyme's catalytic performance. It is assumed that after immobilization, some conformational changes may have occurred that improves the stability of an enzyme by reducing the unfolding rate [34]. On a commercial scale, the thermostable amyloglucosidase is mostly required [35] and hence, it is suggested that this thermostable amyloglucosidase entrapped in agar-agar could be chosen for starch saccharification at higher temperatures. 5
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Fig. 6. Scanning electron micrograph of agar-agar beads without (A) and with (B) amyloglucosidase entrapment.
increasing the yield of the product through continuous recycling and in the case of starch saccharification, immobilized amyloglucosidase can continuously produce glucose molecules. After multiple cycles the enzyme efficiency was decreased which might be due to the outflow of the enzyme molecules from the beads during several washing steps. Sharma et al. [34] reported that α-amylase entrapped in agar beads lost 80% of its catalytic activity after the 6th cycle but when α-amylase was entrapped in calcium agar beads then it retained 80% of its activity.
Declaration of competing interest Authors declare no conflict of interest regarding publication of this manuscript. Acknowledgment Authors gratefully acknowledge the indigenous financial support from The Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi, Karachi, Pakistan.
3.1.8. Scanning electron microscopy Surface topology of agar-agar matrix beads in the presence and absence of amyloglucosidase was observed under scanning electron microscopy at different magnifications. Results of obtained micrographs exhibited that relatively plane surface was visualized (Fig. 6A) in the case of control beads (without enzyme) whereas, after entrapment of enzyme, heterogeneous spherical aggregates were observed on the surface of agar-agar beads (Fig. 6B). Sattar et al. [30] also observed globular particles when protease was entrapped in agar-agar beads. Previously, amyloglucosidase from Aspergillus fumigatus KIBGE-IB33 has been immobilized using derivative of agar i.e. agarose [13]. However, results indicated that immobilization of amyloglucosidase using agar has more catalytic efficiency as it showed higher activity at wide range of pH and temperatures as compared to agarose. Furthermore, the stability studies in terms of thermal and storage stability was also higher in case of agar entrapped amyloglucosidase as at higher temperatures i.e. at 70 °C and 80 °C agarose entrapped amyloglucosidase retained 25% and 14% activity after 120 min while agar entrapped amyloglucosidase retained 40% and 27% activity after same time at same temperatures, respectively. The stability study profile of agar entrapped amyloglucosidase is also higher as at 4 °C and 37 °C agarose entrapped amyloglucosidase retained 23% and 3% activity after 90 days while agar entrapped amyloglucosidase retained 37% and 13% activity after same time at same temperatures, respectively [13]. Therefore, it is suggested that agar is suitable support for immobilization of amyloglucosidase as compared to agarose.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.carres.2019.107860. References [1] Y.H. Hui, Food Biochemistry and Food Processing, first ed., Wiley-Blackwell, London, 2006. [2] B. Jensen, J. Olsen, Amylases and their industrial potential, in: B.N. Johri, T. Satyanarayana, J. Olsen (Eds.), Thermophilic Molds in Biotechnology, Netherland, Kluwer Academic Publishers, 1999, pp. 115–137. [3] A.G. Glazer, H. Nikaido, Microbial enzymes, in: A.G. Glazer, H. Nikaido (Eds.), Microbial Biotechnology: Fundamental of Applied Microbiology, W.H. Freeman and Company, New York, 1995, pp. 241–263. [4] S. Pervez, A. Aman, S. Iqbal, N.N. Siddiqui, S.A. Qader, Saccharification and liquefaction of cassava starch: an alternative source for the production of bioethanol using amylolytic enzymes by double fermentation process, BMC Biotechnol. 14 (2014) 49. [5] R. Gupta, P. Gigras, H. Mohapatra, V.K. Goswami, B. Chauhan, Microbial α-amylases: a biotechnological prospective, Process Biochem. 38 (2003) 1599–1616. [6] R. DiCosimo, J. McAuliffe, A.J. Poulose, G. Bohlmann, Industrial use of immobilized enzymes, Chem. Soc. Rev. 42 (2013) 6437–6474. [7] A. Liese, L. Hilterhaus, Evaluation of immobilized enzymes for industrial applications, Chem. Soc. Rev. 42 (2013) 6236–6249. [8] Y. Arıca, G. Bayramoğlu, Reversible immobilization of tyrosinase onto polyethyleneimine-grafted and Cu (II) chelated poly (HEMAco-GMA) reactive membranes, J. Mol. Catal. B Enzym. 27 (2004) 255–265. [9] K.F. O'Driscoll, Techniques of enzyme entrapment in gels, in: K. Mosbach (Ed.), Methods in Enzymology, Academic Press, New York, 1976, pp. 169–183. [10] L.E. Rodgers, R.B. Knott, P.J. Holden, K.J. Pike, J.V. Hanna, L.J.R. Foster, J.R. Bartlett, Structural evolution and stability of sol–gel biocatalysts, Phys. B Condens. Matter 385–386 (2006) 508–510. [11] L. Cao, Immobilized enzymes: science or art? Curr. Opin. Chem. Biol. 9 (2005) 217–226. [12] A.H. Clark, S.B. Ross-Murphy, Structural and Mechanical Properties of Biopolymer Gels. Biopolymers, Springer, Berlin, 1987. [13] S. Pervez, M.A. Nawaz, A. Aman, S. Qayyum, F. Nawaz, S.A. Qader, Agarose hydrogel beads: an effective approach to improve the catalytic activity, stability and reusability of fungal amyloglucosidase of GH15 family, Catal. Lett. 148 (2018) 2643–2653. [14] S. Pervez, A. Aman, S.A. Qader, Role of two polysaccharide matrices on activity, stability and recycling efficiency of immobilized fungal amyloglucosidase of GH15 family, Int. J. Biol. Macromol. 96 (2017) 70–77. [15] S. Pervez, N.N. Siddiqui, A. Ansari, A. Aman, S.A. Qader, Phenotypic and molecular characterization of Aspergillus species for the production of starch-saccharifying
4. Conclusions Results of the current findings suggested that immobilization of amyloglucosidase using agar-agar as an entrapment matrix improves the kinetic properties of the biocatalyst for that catalyzes hydrolysis of soluble potato starch into glucose molecules. The thermal and storage stability of amyloglucosidase was also considerably enhanced after entrapment. Thus, it is proposed that amyloglucosidase entrapped in agar-agar has a potential to be applied for saccharification of starch in starch processing industry. 6
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7
entrapping higher content of enzymes for commercial applications, Indian J. Pharm. Sci. 72 (2010) 223–228. H. Sattar, A. Aman, S.A. Qader, Agar-agar immobilization: an alternative approach for the entrapment of protease to improve the catalytic efficiency, thermal stability and recycling efficiency, Int. J. Biol. Macromol. 111 (2018) 917–922. D. Norouzian, Enzyme immobilization: the state of art in biotechnology, Iran. J. Biotechnol. 1 (2003) 197–206. Q. Liu, Y. Hua, X. Kong, C. Zhang, Y. Chen, Covalent immobilization of hydroperoxide lyase on chitosan hybrid hydrogels and production of C6 aldehydes by immobilized enzyme, J. Mol. Catal. B Enzym. 95 (2013) 89–98. M. Sharma, V. Sharma, D.K. Majumdar, Entrapment of α-amylase in agar beads for biocatalysis of macromolecular substrate, Int. Sch. Res. Not. (2014) 8 936129 http://dx.doi.org/10.1155/2014/936129. S. Kumar, A. Dwevedi, A.M. Kayastha, Immobilization of soybean (Glycine Max) urease on alginate and chitosan beads showing improved stability: analytical applications, J. Mol. Catal. B Enzym. 58 (2009) 138–145. S. Mulagalapalli, S. Kumar, R.C. Kalathur, A.M. Kayastha, Immobilization of urease from pigeonpea (Cajanus cajan) on agar tablets and its application in urea assay, Appl. Biochem. Biotechnol. 142 (2007) 291–297. B.L. Tee, G. Kaletunç, Immobilization of a thermostable a-amylase by covalent binding to an alginate matrix increases high temperature usability, Biotechnol. Prog. 25 (2009) 436–445. V.V. Mozhaev, Mechanism based strategies for protein thermostabilization, Trends Biotechnol. 11 (1993) 88–95. Y. Zheng, Y. Xue, Y. Zhang, C. Zhou, U. Schwaneberg, Y. Ma, Cloning, expression, and characterization of a thermostable glucoamylase from Thermoanaerobacter tengcongensis MB4 Appl, Microbiol. Biotechnol. 87 (2010) 225–233.
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Improvement of catalytic properties of starch hydrolyzing fungal amyloglucosidase: Utilization of agar-agar as an organic matrix for immobilization
T
Sidra Perveza, Muhammad Asif Nawazb,∗, Muhsin Jamalc, Tour Jand, Farhana Maqboola, Ismail Shahe, Afsheen Amanf, Shah Ali Ul Qaderg a
Department of Microbiology, Hazara University, Mansehra, Khyber Pakhtunkhwa, Pakistan Department of Biotechnology, Shaheed Benazir Bhutto University, Sheringal, Dir (Upper), Khyber Pakhtunkhwa, Pakistan Department of Microbiology, Abdul Wali Khan University, Garden Campus, Mardan, Pakistan d Department of Botany, University of Malakand, Chakdrara, Khyber Pakhtunkhwa, Pakistan e Department of Pharmacy, Abdul Wali Khan University, Garden Campus, Mardan, Pakistan f The Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi, 75270, Karachi, Pakistan g Department of Biochemistry, University of Karachi, Karachi, 75270, Pakistan b c
ARTICLE INFO
ABSTRACT
Keywords: Agar-agar Entrapment Immobilization Reusability Starch hydrolysis Bioprocessing
In this study, amyloglucosidase was immobilized within agar-agar through entrapment technique for the hydrolysis of soluble starch. Enzymatic activities of soluble and entrapped amyloglucosidase were compared using soluble starch as a substrate. Partially purified enzyme was immobilized and maximum immobilization yield (80%) was attained at 40 gL-1 of agar-agar. Enzyme catalysis reaction time shifted from 5.0 min to 10 min after immobilization. Similarly, a five-degree shift in temperature (60 °C–65 °C) and a 0.5 unit increase in pH (pH-5.0 to pH-5.5) were also observed. Substrate saturation kinetics revealed that Km of entrapped amyloglucosidase increased from 1.41 mg ml−1 (soluble enzyme) to 3.39 mg ml−1 (immobilized enzyme) whereas, Vmax decreased from 947 kU mg−1 (soluble enzyme) to 698 kU mg−1 (immobilized enzyme). Entrapped amyloglucosidase also exhibited significant catalytic performance during thermal and storage stability when compared with soluble enzyme. Reusability of entrapped amyloglucosidase for hydrolysis of soluble starch demonstrated its recycling efficiency up to six cycles which is an exceptional characteristic for continuous bioprocessing of soluble starch into glucose.
1. Introduction Starch debranching enzymes are among the most important industrial enzymes used for the production of wide variety of commercial products. They specifically act on O-glycosidic linkages of starch molecules and produce variety of monosaccharide and oligosaccharides. The hydrolyzed products obtained from starch are extensively used in various industrial processes. Hydrolysis of starch can be performed by enzymatic, alkaline or acidic treatments. Hui [1] suggested that starch hydrolysis can further be facilitated by performing other treatments like microwaving, heating and extrusion. During treatment, the glycosidic bonds in starch are fragmented and results in the formation of different oligomers (maltotriose, maltose) and monomers (glucose). Enzymatic cleavage of starch by means of hydrolases is most widely used because of the low energy consumption and high product formation. Whereas, ∗
as in case of acid hydrolysis, the product yield is lower along with unwanted color and bitter taste development [2,3]. Among different starch debranching enzymes, amyloglucosidase [E.C. 3.2.1.3] have more commercial interest due to complete conversion of starch and other related polysaccharides into D-glucose monomers. Free or soluble amyloglucosidase is widely used in starch saccharification, ethanol production, baking, food, brewing, pharmaceutical and textile industries [4,5]. However, there are some major difficulties associated in the use of soluble enzyme due to their instability under harsh industrial conditions like at various temperatures and pH [6,7]. Enzyme immobilization is a strategy to increase the stability and recycling efficiency of various industrial enzymes. Subsequently, various approaches have been used for immobilization of enzymes which are mainly classified as physical (adsorption and entrapment) and chemical (covalent binding and cross-linking) methods. Physical methods are simple and
Corresponding author. Department of Biotechnology, Shaheed Benazir Bhutto University, Sheringal, Dir Upper, KPK, Pakistan. E-mail address: [email protected] (M.A. Nawaz).
https://doi.org/10.1016/j.carres.2019.107860 Received 4 May 2019; Received in revised form 2 October 2019; Accepted 25 October 2019 Available online 01 November 2019 0008-6215/ © 2019 Elsevier Ltd. All rights reserved.
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cost effective but the interaction between the soluble enzyme and matrix is usually reversible and weak while in case of chemical methods, the interaction is strong and irreversible [8]. The method of entrapment involves occlusion and confinement of soluble enzyme in a porous fiber or a gel matrix through either covalent or non-covalent interactions. In this method, substrate and product can easily diffuse out through the polymeric network. However, because of the enzyme being larger in molecular size, it remains entrapped within the matrix [9]. Additionally, gel entrapment is a simple and effective approach for immobilization as it preserves the biological function of the biocatalyst which is entrapped [10]. Entrapment method also permits co-immobilization of multiple enzymes for efficient hydrolysis of complex substrates like starch. Agar-agar, agarose, cellulose triacetate, sodium alginate, gelatin, chitosan, diacetyl cellulose and polyacrylamide have been used as different matrices for the entrapment of various enzymes [11]. Agar-agar is natural polysaccharide obtained from red algae (Rhodophyta). It is composed of two components which are agarose (linear) and agaropectin (branched) linked together by α-(1 → 3) and β(1 → 4) linkages. Due to its chemical structure, non-reactivity to other biomolecules, resistance to acid and capability to form robust gel at low concentrations this biopolymer favors applications in various biotechnological industries [12]. Beside all these properties, agar-agar is highly porous which makes it an excellent matrix for entrapment of proteins. Previously this amyloglucosidase have been immobilized using different supports including alginate and agarose through entrapment method and onto chitosan through covalent binding [13,14]. However, data is very scarce for entrapment of amyloglucosidase within agar. Hence, this is the first study in which fungal amyloglucosidase is immobilized using agar-agar as a supporting matrix to improve its catalytic efficiency, thermal and storage stabilities.
allowed to solidify at room temperature. Solid agar gel which contain amyloglucosidase was cut into small beads (3.0 mm) with the help of metallic cork-borer. These beads were then undergo several washing steps with citrate buffer (50 mM; pH-5.0) to remove unbound or loosely bound enzyme molecules. For preparation of control beads, agar-agar solution was mixed with citrate buffer (50 mM; pH-5.0) only and same procedure was followed for the preparation of amyloglucosidase beads. Immobilized beads were stored at 4 °C for further analysis. 2.5. Analysis of amyloglucosidase and total protein Enzyme activity of amyloglucosidase was determined as described previously by Ghani et al. [16]. Concisely, 0.1 ml of desalted soluble amyloglucosidase and 0.25 gm of immobilized amyloglucosidase beads were mixed with 1.0 ml of starch solution prepared in 50 mM citrate buffer (pH: 5.0 for soluble and 5.5 for entrapped enzyme). The reaction mixture was kept at appropriate temperature (60 °C for soluble and 65 °C for immobilized) for specified time (5.0 min for soluble and 10.0 min for immobilized enzyme). Glucose liberated after reaction was then determined spectrophotometrically at 546 nm using GOD-PAP method [17,18] with glucose as standard. Total protein was estimated by Lowry's method using bovine serum albumin as standard [19]. 2.6. Immobilization yield Immobilization yield was calculated using following formula:
Immobilization Yield (%) =
Activity of immobilized enzyme Activity of soluble enzyme
× 100
2. Material and methods
2.7. Selection of reaction time, reaction pH and reaction temperature
2.1. Chemicals and reagents
Aspergillus fumigatus KIBGE-IB33 [GenBank Accession: KF905648] was cultivated in a previously optimized medium of pH: 7.0 for 4 days at 30 °C [15]. After fermentation, the crude soluble amyloglucosidase was separated from fungal biomass by centrifuging the medium at 40,000 ✕g for 15 min at 4 °C [15].
Enzyme performance of immobilized amyloglucosidase was investigated by incubating the enzyme with substrate for different time intervals (5.0–60.0 min). For the selection of optimum pH, amyloglucosidase activity was performed at different pH values ranging from 4.0 to 8.0 of same ionic strength (50.0 mM) using various buffers including acetate buffer (pH: 4.0–4.5), citrate buffer (pH: 5.0–6.0) and phosphate buffer (pH: 6.5–8.0). A wide range of temperature was studied to determine the maximum activity of soluble and entrapped amyloglucosidase (35–80 °C). Arrhenius plot was constructed for the determination of activation energy using the following equation:
2.3. Partial purification of amyloglucosidase
InA =
The ammonium sulphate precipitation method with 40% saturation was used for partial purification of crude amyloglucosidase. Further, this partially purified enzyme was desalted using PD-10 desalting column (GE Healthcare) as described previously [14].
2.8. Analysis of Vmax and Km
Agar-agar was purchased from Research Organics (USA). All other chemicals were of analytical grade. 2.2. Amyloglucosidase production
Ea × InB RT
The Vmax and Km was determined using Lineweaver-Burk double reciprocal plot. Enzymatic assay was performed with different concentration of potato starch (1.0–30.0 mg ml−1) at optimum assay conditions.
2.4. Immobilization of amyloglucosidase using agar-agar Amyloglucosidase was immobilized within agar-agar matrix by mixing desalted enzyme with agar-agar solution in 1:1 ratio. The effect of concentration of agar-agar on entrapment yield of amyloglucosidase was studied at different concentrations (20.0–50.0 gL-1) and most appropriate concentration selected was further used in subsequent studies. The agar-agar solution was prepared by dissolving 40 gL-1 of agaragar in citrate buffer (50 mM; pH-5.0) at 100 °C. After cooling the solution to 40 °C, amyloglucosidase was mixed thoroughly and immediately casted in a preassembled glass plate. This mixture was
2.9. Thermal stability To determine the thermal tolerance, soluble enzyme and agar-agar beads (with or without enzyme) were pre-incubated at wide range of temperatures (30 °C–80 °C) for 120 min. After every 20 min, aliquots were taken out and immediately placed on ice bath and enzyme activity was performed and calculated. 2
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2.10. Storage stability
enzymes [21]. Beyond this concentration, a decrease in enzymatic activity was noticed which could be due to either stearic hindrance of the enzyme due to the formation of multilayered protein complexes on charged matrix [22] or due to the mass transfer limitation of substrate [23]. Mass transfer limitation can be of two types including internal and external limitation [24,25]. In internal limitation, the diffusion of starch molecules (substrate) into the matrix (agar-agar) is constrained due to small or less pore size while, in case of external limitation the release of glucose molecules (product) from the internal environment of the matrix is restricted.
Soluble enzyme and enzyme immobilized beads were stored at different temperatures (4 °C and 37 °C) for 90 days in citrate buffer (50 mM; pH-5.0). The residual activity of amyloglucosidase was measured in percentage after every 10 days of storage. 2.11. Recycling studies For the determination of operational stability of immobilized amyloglucosidase, beads were incubated with 10 gL-1 starch prepared in citrate buffer (50 mM; pH-5.0) under optimal conditions. After the determination of glucose using the standard assay conditions, the beads were washed with activity buffer and experiment was repeated until no catalytic activity of amyloglucosidase was observed or it become constant.
3.1.2. Effect of reaction time To investigate the optimum reaction time required to hydrolyze starch into glucose by immobilized amyloglucosidase, the catalytic activity of enzyme was carried out at different time intervals. Fig. 2A demonstrates that the relative activity of immobilized amyloglucosidase increased with the increase in the reaction time and maximum starch hydrolysis was achieved after 10.0 min of reaction as compared to the soluble enzyme which exhibited maximum starch hydrolysis by amyloglucosidase after 5.0 min. This variation in the reaction time in both the cases supported the probability of resistance towards accessibility of high molecular weight substrate (starch) to diffuse into the agar-agar matrix and react with the enzyme to produce glucose molecules as a product. Therefore, starch requires relatively more time to reach the active site of the enzyme that is entrapped within the matrix. Mahajan et al. [26] and Sattar et al. [27] also noticed 8.0 min (soluble enzyme: 11.0 min; immobilized enzyme: 19.0 min) and 5.0 min increments (soluble enzyme: 15.0 min; immobilized enzyme: 20.0 min) in reaction time after immobilization of amylolytic and proteolytic enzyme on agar-agar beads, respectively.
2.12. Scanning electron microscopy (SEM) Cross-sectional topological structure of agar-agar beads (with and without amyloglucosidase) was investigated through SEM as described previously [18]. 3. Results and discussion Industrial scale utilization of enzyme involves some practical problems associated with the use of soluble enzyme. Being soluble in water, the recovery of enzymes in active form after completion of a reaction is very difficult. Furthermore, free enzymes possess very low operational stability (thermal and storage) that renders its successful application impossible. These limitations can be overcome by immobilizing the biocatalysts using suitable support system and appropriate methodology. In this study, entrapment method was used with agar-agar as a matrix to improve the catalytic efficiency of amyloglucosidase which hydrolyzes starch into glucose molecules.
3.1.3. Effect of reaction pH The influence of pH (Fig. 2B) on the catalytic activity of soluble and entrapped amyloglucosidase was studied and an approximate pH shift of 0.5 units was observed in case of immobilized biocatalyst (pH: 5.5) as compared to soluble enzyme (pH: 5.0). It has been suggested that the charge surface within the agar-agar beads and the entrapped enzyme can create a charged microenvironment, which could eventually affect the active site of an enzyme and thus can alters the pH of the entrapped enzyme [28]. Liu et al. [29] reported that cationic matrices containing enzymes usually shifts pH towards acidic side while, anionic shifts it towards alkaline region. Agar-agar is composed of two components in which agarose is neutral while, agro-pectin is negatively charged due to presence of sulphate. Hence in the current results, a 0.5 unit shift of pH towards alkaline side was observed. Soluble amyloglucosidase showed 30% relative activity at pH-8.0 while, entrapped enzyme retained approximately 50% of its initial activity at the same pH value (Fig. 2B). Similarly, a slight change (0.5 units) in optimum pH from 6.0 to 6.5 was observed in case of entrapment of α-amylase from A. oryzae on calcium agar beads [30]. Previously the pH of amyloglucosidase is also increased 1.0 unit (soluble enzyme: 5.0; immobilized enzyme: 6.0) when calcium alginate was used as a matrix for entrapment [15].
3.1. Immobilization of amyloglucosidase 3.1.1. Effect of agar-agar concentration The selection of optimum concentration of support is very important for immobilization as it is responsible for the penetration of substrate into the matrix as well as it is also accountable for the leaching of enzyme molecules. Additionally, it also governs the porosity of agar-agar beads because as the concentration increases, the pore size of the matrix will be decreased and hence, this situation will eventually render both the enzyme entrapment and substrate diffusion within the support. The porosity of matrix lattice is the most crucial aspect in entrapment method. In the case of small pore size, the entrapment of biocatalyst only occurs on the external surface of the matrix which could result in low enzyme loading while, in case of large pore size of the matrix, enzyme leakage can occur [20]. Similar phenomenon was noticed when amyloglucosidase was entrapped within agar-agar matrix. Maximum immobilization of approximately 80% was observed when 40.0 gL-1 concentration was used (Fig. 1). Beyond this concentration, the entrapment of amyloglucosidase declined and approximately 24% decrease in immobilization yield was noticed at 50.0 gL-1. This might be due to the outflow of enzyme from agar-agar beads because of its large pore size and after subsequent washing of beads the enzyme leached out. Minimum immobilization yield was detected at 20.0 gL-1 which exhibited 40% less entrapment as compared to the 40.0 gL-1 concentration. This could be because at this low concentration of agar-agar the beads were very delicate and fragile and were unable to sturdily entrap the enzyme. These results confirmed that agar-agar concentration directly affects the entrapment of enzyme and also inversely affects the pore size of the matrix. A similar concentration of agar-agar (40.0 gL-1) was also previously reported for entrapment of amylolytic
3.1.4. Effect of reaction temperature Kinetic behavior of soluble and entrapped amyloglucosidase was observed at different temperatures ranges. Maximum catalysis of substrate was observed at 60 °C when using soluble enzyme and at 65 °C when using entrapped amyloglucosidase (Fig. 2C). Increase in relative activity for immobilized enzyme at higher temperatures could be due to the unique structural nature of the matrix that protected the enzyme from denaturation [31]. Along with this, the energy of activation for soluble and entrapped amyloglucosidase was also assessed using Arrhenius plot and it was nearly 5.55 kcal mol−1 and 4.55 kcal mol−1, respectively (Fig. 2D). A decrease in activation energy for entrapped enzyme as compared to soluble enzyme could be due to an increase in optimum reaction temperature (65 °C) of entrapped enzyme (Fig. 2C). 3
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Fig. 1. Effect of agar-agar concentration on immobilization yield of amyloglucosidase (A); Agar-agar beads with and without entrapped amyloglucosidase (B).
Fig. 2. Effect of enzyme-substrate reaction time (A); pH (B); temperature (C) and construction of Arrhenius plot (D).
Kumar et al. [31] have suggested that the immobilization matrices are able to protect the enzymes from denaturation by absorbing surrounding heat and may also cause some conformational changes in the enzyme structure. In the present study, besides the change in the optimum reaction temperature, the temperature profile of immobilized amyloglucosidase became wider as compared to the soluble enzyme with a retention of 41% of relative enzyme activity even at 80 °C while, soluble enzyme only retained 20%. A five-degree shift in the optimum temperature of immobilized fungal α-amylase was previously reported when calcium agar gels and agarose were used, respectively [30]. Previously, immobilization of urease in agar-agar tablets showed extreme change in optimum temperature from 30 °C (free enzyme) to 60 °C (immobilized enzyme) [32]. The current results also advocate that agar-agar can act as a protective barrier which could absorb heat and protect enzymes from denaturation by stabilizing its structure within the microenvironment.
The results disclosed that after entrapment of amyloglucosidase, Km amplified by 1.97 mg ml−1 whereas, Vmax units were lowered by 249 kU mg−1. This increase in Km could be due to the low affinity of the enzyme towards soluble potato starch [33]. Other possible reasons in this case which resulted in decrease in Vmax value could be the confinement of amyloglucosidase in the microenvironment of agar-agar and because of the diffusional resistance or stearic hindrance that is presented during penetration of soluble potato starch into the matrix. Similar findings have been reported about the increased Km and declined Vmax values of the immobilized alpha amylase and urease within agar matrix [32]. However, Sharma et al. [34] found no change in affinity of amylase towards soluble starch after entrapment in calcium agar tablets whereas, a 37.5 fold increase in Vmax was observed. Previously, amyloglucosidase entrapped in alginate also showed higher Km and lower Vmax values [15]. 3.1.6. Stability studies Thermal stability data of immobilized enzyme showed that it retained additional catalytic activity at all temperatures as compared to soluble enzyme. Soluble enzyme lost approximately 30%, 40% and 58% of its initial residual activity at 40 °C, 50 °C and 60 °C after 120 min, respectively (Fig. 3A, B, 3C). However, at 70 °C and 80 °C a complete loss in enzyme activity was observed after 100 and 80 min, respectively (Fig. 3D and E). On the other hand, agar-agar entrapped amyloglucosidase retained about 70%, 40% and 27% of its relative activity at 60 °C, 70 °C and 80 °C even after 120 min, respectively. The unfolding of
3.1.5. Effect of substrate concentration Studies on kinetic parameters exhibited that Vmax and Km of amyloglucosidase were affected by immobilized entrapment method. It was observed that Km increased from 1.41 mg ml−1 (standard error: 0.054 mg ml−1) for soluble amyloglucosidase to 3.39 mg ml−1 (standard error: 0.396 mg ml−1) for agar-agar entrapped amyloglucosidase. While, a decrease in Vmax value was detected from 947 kU mg−1 (standard error: 40.1 kU mg−1) for soluble amyloglucosidase to 698 kU mg−1 (standard error: 29.2 kU mg−1) for agar-agar entrapped enzyme. 4
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Fig. 3. Stability studies of soluble and entrapped amyloglucosidase at various temperatures: 40 °C (A); 50 °C (B); 60 °C (C); 70 °C (D); 80 °C (E).
Fig. 4. Storage stability of soluble and immobilized amyloglucosidase at 37 °C (A) and 4 °C (B).
When comparing performance of immobilized biocatalysts intended for industrial use against soluble enzymes, the portrayal of their storage stabilities is also important. If storage stability is high then the practical application cost of the majority of bioprocesses could be reduced. Storage stability was examined for up to 90 days. As observed from Fig. 4A and B, agar-agar entrapped amyloglucosidase retained around 33% and 57% of its initial activity after 60 days of storage at 37 °C and 4 °C, respectively. Immobilized amyloglucosidase is more stable as compared to its soluble counterpart at these temperatures. These findings revealed that agar-agar provided a shielded effect to amyloglucosidase. Thus, it is suggested that the confined microenvironment of the agar-agar beads would able to prevent the biocatalyst from harsh industrial conditions. Similarly, 50% activity of agar-agar immobilized urease was observed at 4 °C even after 53 days [32].
Fig. 5. Recycling efficiency of immobilized amyloglucosidase.
3.1.7. Recycling studies The main objective of the current study was to design an immobilization method that can proficiently recover amyloglucosidase from the reaction mixture for reuse because for continuous utilization of enzymes, operational stability is among the most important factor. Therefore, reusability potential of entrapped amyloglucosidase was examined at 60 °C up to 10 cycles. Results of current findings showed that agar-agar entrapped amyloglucosidase possess considerable operational stability with a retention of 68% and 46% activity after the 4th and 6th recycles, respectively (Fig. 5). For incessant bioprocessing at industrial scale, such operational stabilities always provide benefit by
protein structure after exposure to higher temperatures which is above its enzyme kinetic maxima is one of the main phenomenon that is involved in deactivation of enzyme's catalytic performance. It is assumed that after immobilization, some conformational changes may have occurred that improves the stability of an enzyme by reducing the unfolding rate [34]. On a commercial scale, the thermostable amyloglucosidase is mostly required [35] and hence, it is suggested that this thermostable amyloglucosidase entrapped in agar-agar could be chosen for starch saccharification at higher temperatures. 5
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Fig. 6. Scanning electron micrograph of agar-agar beads without (A) and with (B) amyloglucosidase entrapment.
increasing the yield of the product through continuous recycling and in the case of starch saccharification, immobilized amyloglucosidase can continuously produce glucose molecules. After multiple cycles the enzyme efficiency was decreased which might be due to the outflow of the enzyme molecules from the beads during several washing steps. Sharma et al. [34] reported that α-amylase entrapped in agar beads lost 80% of its catalytic activity after the 6th cycle but when α-amylase was entrapped in calcium agar beads then it retained 80% of its activity.
Declaration of competing interest Authors declare no conflict of interest regarding publication of this manuscript. Acknowledgment Authors gratefully acknowledge the indigenous financial support from The Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi, Karachi, Pakistan.
3.1.8. Scanning electron microscopy Surface topology of agar-agar matrix beads in the presence and absence of amyloglucosidase was observed under scanning electron microscopy at different magnifications. Results of obtained micrographs exhibited that relatively plane surface was visualized (Fig. 6A) in the case of control beads (without enzyme) whereas, after entrapment of enzyme, heterogeneous spherical aggregates were observed on the surface of agar-agar beads (Fig. 6B). Sattar et al. [30] also observed globular particles when protease was entrapped in agar-agar beads. Previously, amyloglucosidase from Aspergillus fumigatus KIBGE-IB33 has been immobilized using derivative of agar i.e. agarose [13]. However, results indicated that immobilization of amyloglucosidase using agar has more catalytic efficiency as it showed higher activity at wide range of pH and temperatures as compared to agarose. Furthermore, the stability studies in terms of thermal and storage stability was also higher in case of agar entrapped amyloglucosidase as at higher temperatures i.e. at 70 °C and 80 °C agarose entrapped amyloglucosidase retained 25% and 14% activity after 120 min while agar entrapped amyloglucosidase retained 40% and 27% activity after same time at same temperatures, respectively. The stability study profile of agar entrapped amyloglucosidase is also higher as at 4 °C and 37 °C agarose entrapped amyloglucosidase retained 23% and 3% activity after 90 days while agar entrapped amyloglucosidase retained 37% and 13% activity after same time at same temperatures, respectively [13]. Therefore, it is suggested that agar is suitable support for immobilization of amyloglucosidase as compared to agarose.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.carres.2019.107860. References [1] Y.H. Hui, Food Biochemistry and Food Processing, first ed., Wiley-Blackwell, London, 2006. [2] B. Jensen, J. Olsen, Amylases and their industrial potential, in: B.N. Johri, T. Satyanarayana, J. Olsen (Eds.), Thermophilic Molds in Biotechnology, Netherland, Kluwer Academic Publishers, 1999, pp. 115–137. [3] A.G. Glazer, H. Nikaido, Microbial enzymes, in: A.G. Glazer, H. Nikaido (Eds.), Microbial Biotechnology: Fundamental of Applied Microbiology, W.H. Freeman and Company, New York, 1995, pp. 241–263. [4] S. Pervez, A. Aman, S. Iqbal, N.N. Siddiqui, S.A. Qader, Saccharification and liquefaction of cassava starch: an alternative source for the production of bioethanol using amylolytic enzymes by double fermentation process, BMC Biotechnol. 14 (2014) 49. [5] R. Gupta, P. Gigras, H. Mohapatra, V.K. Goswami, B. Chauhan, Microbial α-amylases: a biotechnological prospective, Process Biochem. 38 (2003) 1599–1616. [6] R. DiCosimo, J. McAuliffe, A.J. Poulose, G. Bohlmann, Industrial use of immobilized enzymes, Chem. Soc. Rev. 42 (2013) 6437–6474. [7] A. Liese, L. Hilterhaus, Evaluation of immobilized enzymes for industrial applications, Chem. Soc. Rev. 42 (2013) 6236–6249. [8] Y. Arıca, G. Bayramoğlu, Reversible immobilization of tyrosinase onto polyethyleneimine-grafted and Cu (II) chelated poly (HEMAco-GMA) reactive membranes, J. Mol. Catal. B Enzym. 27 (2004) 255–265. [9] K.F. O'Driscoll, Techniques of enzyme entrapment in gels, in: K. Mosbach (Ed.), Methods in Enzymology, Academic Press, New York, 1976, pp. 169–183. [10] L.E. Rodgers, R.B. Knott, P.J. Holden, K.J. Pike, J.V. Hanna, L.J.R. Foster, J.R. Bartlett, Structural evolution and stability of sol–gel biocatalysts, Phys. B Condens. Matter 385–386 (2006) 508–510. [11] L. Cao, Immobilized enzymes: science or art? Curr. Opin. Chem. Biol. 9 (2005) 217–226. [12] A.H. Clark, S.B. Ross-Murphy, Structural and Mechanical Properties of Biopolymer Gels. Biopolymers, Springer, Berlin, 1987. [13] S. Pervez, M.A. Nawaz, A. Aman, S. Qayyum, F. Nawaz, S.A. Qader, Agarose hydrogel beads: an effective approach to improve the catalytic activity, stability and reusability of fungal amyloglucosidase of GH15 family, Catal. Lett. 148 (2018) 2643–2653. [14] S. Pervez, A. Aman, S.A. Qader, Role of two polysaccharide matrices on activity, stability and recycling efficiency of immobilized fungal amyloglucosidase of GH15 family, Int. J. Biol. Macromol. 96 (2017) 70–77. [15] S. Pervez, N.N. Siddiqui, A. Ansari, A. Aman, S.A. Qader, Phenotypic and molecular characterization of Aspergillus species for the production of starch-saccharifying
4. Conclusions Results of the current findings suggested that immobilization of amyloglucosidase using agar-agar as an entrapment matrix improves the kinetic properties of the biocatalyst for that catalyzes hydrolysis of soluble potato starch into glucose molecules. The thermal and storage stability of amyloglucosidase was also considerably enhanced after entrapment. Thus, it is proposed that amyloglucosidase entrapped in agar-agar has a potential to be applied for saccharification of starch in starch processing industry. 6
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