Covalent immobilization of Aspergillus niger amyloglucosidase (ANAG) with ethylenediamine-functionalized and glutaraldehyde-activated active carbon (EFGAAC) obtained from sesame seed shell

Journal Pre-proofs Covalent immobilization of Aspergillus niger amyloglucosidase (ANAG) with ethylenediamine-functionalized and glutaraldehyde-activated active carbon (EFGAAC) obtained from sesame seed shell Yakup Aslan, Yousif Mohammed Sharif, Ömer Şahin PII: DOI: Reference:

S0141-8130(19)32289-5 https://doi.org/10.1016/j.ijbiomac.2019.09.226 BIOMAC 13522

To appear in:

International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

29 March 2019 22 September 2019 24 September 2019

Please cite this article as: Y. Aslan, Y.M. Sharif, O. Şahin, Covalent immobilization of Aspergillus niger amyloglucosidase (ANAG) with ethylenediamine-functionalized and glutaraldehyde-activated active carbon (EFGAAC) obtained from sesame seed shell, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.09.226

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Covalent immobilization of Aspergillus niger amyloglucosidase (ANAG) with ethylenediamine-functionalized and glutaraldehyde-activated active carbon (EFGAAC ) obtained from sesame seed shell Yakup Aslan1*, Yousif Mohammed Sharif2, Ömer Şahin3 1

Department of Food Engineering, Faculty of Engineering, Siirt University, Kezer Campus, 56100, Siirt, Turkey 2 Department of Petroleum Engineering, College of Engineering, University of Zakho, 42000, Duhok, Kurdistan Region-Iraq 3 Department of Chemical Engineering, Faculty of Engineering, Siirt University, Kezer Campus, 56100, Siirt, Turkey

*E-mail: [email protected], Tel: +90 484 212 11 11 / 3020, Fax: +90 484 223 19 98 Abstract This study was aimed the covalently immobilization of Aspergillus niger amyloglucosidase (ANAG) onto activated carbon (AC) obtained from sesame seed shell. AC was firstly functionalized with ethylenediamine, and after then activated with glutaraldehyde. 99.80% immobilization yield and 99.83% activity yield were obtained as the result of optimization of immobilization conditions (pH and molarity of immobilization buffer, AC amount, and reaction time). The optimum pH (5.5) and the optimum temperature range (55-60 oC) for ANAG were not affected by immobilization. After immobilization, Vmax value decreased from 1464.1 μmol D-glucose / L.min to 1342.3 μmol D-glucose / L.min, while Km value decreased from 116.3 g maltodextrin / L to 109.9 g maltodextrin / L. The immobilized enzyme retained 99.30% and 98.30% of its initial activity, respectively after twenty repeated uses and after twenty days of storage in 5 mL sodium phosphate buffer (0.1 M, pH 5.5) at +4 o

C in a refrigerator. Finally, glucose syrup was produced from maltodextrin solution having

1% (w/v) concentration by using the immobilized ANAG. Maltodextrin was completely converted to glucose after four hours. Consequently, it can be said that the immobilized ANAG obtained in this study can be used in the industrial production of glucose syrup. Keywords: Activated carbon; amyloglucosidase; covalent immobilization

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1. Introduction Amyloglucosidases (AG) (EC 3.2.1.3) are important enzymes mainly used in the hydrolysis of starch and in other many applications, on a large scale [1]. Initially the term amyloglucosidase was used originally to designate enzymes capable of hydrolyzing α-1,4glucosidic bonds from amylose, amylopectin, glycogen and their degradation products [2]. They break down the bonds between adjacent glucose units in these polysaccharides, yielding products characteristic of the particular enzyme involved. Enzymes that hydrolyze the α-1,4 and α-1,6 linkages, e.g. amyloglucosidase (glucoamylase) and the exiting amylase [3]. ANAG is an exo-amylase that cleavages α-1,4-linkages at the non-reducing ends of starch and dextrins and is also able to cleavage α-1,6-linkages [3]. When an exo-amylase reached an α1,6 bond, cuts also this bond but at a lower rate than the α-1,4 bond [4]. After enzymatic hydrolysis, the substrates are mainly converted to D-glucose as end product [5]. AG have wide application areas such as, liquefaction of insoluble starch granules, Manufacture of maltose, high fructose containing syrups, dextrins and maltodextrins, maltotetraose syrup, removal of starch sizer from textile (desizing), Direct fermentation of starch to ethanol, Treatment of starch processing waste water (SPW) [3]. Therefore, immobilization of AG stil very important research area. The enzyme produced from active cells is a highly efficient catalyst. Compared to the chemical catalyst, it has many advantages, such as high specificity, high catalytic efficiency, and an adjustable activity that greatly encourages its use in the pharmaceutical, chemical and food industry. However, its low stability, low reusability and high cost of single use limit its use in industrial production [6]. Immobilization of enzymes is often required to solve the problem of enzyme solubility and the difficulties of enzyme reuse in industrial applications [7-10]. However, a suitable immobilization protocol may also improve some enzyme properties (in many cases, physiological ones) which can be a disadvantage in the industrial reactor, such as moderate stability, inhibition by substrates or products, and moderate selectivity or specificity to industrially related substrates [11]. In recent years, enzyme immobilization technology has not only improved catalytic properties and reusability, but has also provided an effective method to overcome these problems to facilitate multiple reuse, separation and continuous automatic operation of enzymes in industrial production [12]. There are several reasons for using an enzyme in immobilized form. In addition to more 2

convenient use of the enzyme, it provides easy separation from the product, thereby minimizing or eliminating protein contamination of the product. Immobilization also facilitates efficient recovery and reuse of expensive enzymes, a condition that is not economically feasible in many applications, and enables them to be used in continuous, fixed bed operations. A further advantage is often improved stability against denaturation, both under storage and operational conditions, for example by heat or organic solvents or autolysis. Improved enzyme performance through improved stability and repeated reuse is then reflected in higher catalyst yields (kg product / kg enzyme) which determine enzyme costs per kg product. As a general rule, enzyme costs should not be more than a few percent of total production costs [13]. Therefore, the priority targets of an enzyme immobilization study are obtaining higher immobilization yield and activity yields, higher reusability and longer storage stability. Up to day, various supports and methods have been used for enzyme immobilization in order to

improve

the

properties

of

free

enzyme

[14].

Adsorption,

cross-linking,

entrapment/encapsulation and covalent attachment are four main principal methods for enzyme immobilization [13]. Each one of these methods have some advantages and some disadvantages. Adsorption which includes reversible surface interaction between carrier and enzyme [15] can be accomplished by mixing anzyme and support in adequate buffer solution at optimum conditions such as pH and ionic strength [16]. This method is easy, cheap and fast, but weak linkages between enzyme molecules and support can be resulted in the losing the catalytic activity of enzyme due to desorption of enzyme molecules from support under the catalytic reaction conditions. Entrapment of enzyme in a polymeric gel during the formation of gel matrice can be performed by dropping the mixture of enzyme and polyanyonic polymers into the solution of polyvalent metal ions under optimum conditions such as ratio of enzyme / polymer, pH and ionic strangth [17]. This method is also easy, cheap and fast but mass transfer limitations for the large substrates and product molecules can lead to lower catalytic activity of enzymes [16]. Cross-linking method is linking of enzyme molecules to each other with covalent bonds by using cross-linking reagent such as glutaraldehyde, carbodiimide and diisosyanate to prepare carrierless macroparticles. This method is not required using any support material. The clear advantages of this method are highly concentrated catalytic enzyme activity, high stability and low production costs due to 3

the exclusion of an additional (expensive) carrier [13]. Covalent immobilization of enzyme may have some advantages over physical adsorption, such as inhibition of enzyme desorption during processing and increased stability from multiple points covalent binding [18, 19]. However, it also has a problem: both enzyme and support should be discarded after enzyme inactivation [18]. On the other hand, covalent immobilization is generally resulted in higher reusability, due to low leakage level of enzyme molecules from support [13]. But some supports haven't functional groups on their surface for covalent attachment of enzymes. Therefore, these supports are functionalized and activated befor covalent attachment. For the functionalization of support there are a lots of reagents carrying functional groups such as amine, epoxy, hydroxy, carboxy sulfhydryl etc. After functionalization of support, these functional groups are pre-activated by using bifunctional reagents such as glutaraldehyde, carbodiimide, diisosyanate [20]. Glutaraldehyde is mostly used one, among these reagents. Glutaraldehyde is a five-carbon linear dialdehyde pungent oily liquid exhibiting excellent solubility in all proportions in water, alcohol and organic solvents [21]. The methodology is quite simple [22] and effective, and in some cases even allows to increase enzyme stability by multi-point or multi-channel immobilization [23, 24]. Furthermore, since glutaraldehyde activation remarkably reducing the leaving of enzyme molecules from the supports thorough by promoting strong support-protein bonding, enzymes immobilized via glutaraldehyde show higher operational and storage stabilities [25]. The exact structure of glutaraldehyde on the support was not exactly understood [21], but when considering the high stability of the aminoglutaraldehyde bond, the formation of some kind of cyclic structures appears likely [21, 26, 27]. Glutaraldehyde promoted crosslinking in 2 ways – intermolecularly and intramolecularly [28]. Although glutaraldehyde reacts mainly with other protein groups (thiols, phenols, and imidazoles), it can react with different enzyme moieties that essentially contain primary amino groups of proteins [21, 29-31]. The chemical covalent bonding process using glutaraldehyde is particularly attractive because it provides a carefully regulated link with specific groups present in proteins under mild pH conditions [27]. Furthermore, the chemical reactivity indicated by glutaraldehyde means that reduction with sodium borohydride is not absolutely necessary [11]. Although glutaraldehyde can react mainly with other groups (thiols, phenols and imidazoles), it can react with different portions of the enzyme, which essentially contain the primary amino 4

groups of proteins [11, 27]. It is disclosed that supplements functionalized with glutaraldehyde groups are readily formed from different supplements containing primary amino groups (i.e., ethylenediamine-activated supplements). However, the structures of protein crosslinking or enzyme immobilization as well as glutaraldehyde chemistry in aqueous solution have not yet been fully understood [21]. Some constructs have been proposed for aqueous glutaraldehyde solutions which report that the glutaraldehyde reagent may be present as monomer at pH 3.0 to 8.0 and under dilute conditions, however, oligomeric hemiacetals are formed under (25)% or acidic conditions at concentrations above 25. The balance between linear and cyclic monomeric forms is directed linearly as the temperature increases. Under basic conditions, oligomeric aldehydes can be formed by intermolecular aldol condensation [11, 21, 27]. Depending on the conditions applied during the immobilization process with glutaraldehydeactivated supports, proteins can be immobilized by three different mechanisms. When very high ionic strength is used, the protein can be immobilized by hydrophobic adsorption before the formation of covalent bonds first, whereas when the low ionic force is used, primary immobilization occurs through anion exchange. It has been described that protein immobilization mainly results from covalent bonds when the applied ionic strength is moderate [11, 26, 27, 32, 33]. As shown in Table 1, immobilization of ANAG has been widely investigated by using various natural and synthetic supports and traditional immobilization methods: cross-linking [1, 34], adsorption [35-39], entrapment [40] and covalent bonding [5, 35, 41-50]. The highest activity yield (100%) achieved by Sanjay and Sugunan [49] in the covalent immobilization of ANAG onto Montmorillonite K-10. But the immobilized activity obtained in this study have been preserved only 100 hours. On the other hand, the longest operational stability (100% of retained activity after 30 cycle) for immobilized ANAG have been obtained by Tanrıseven and Zehra [45] in the covalent immobilization of ANAG on polyglutaraldehyde-activated gelatin particles in the presence of polyethylene glycol and soluble gelatin. But the activity yield was 85%. Sustainable high activity yield is the most important goal of the enzyme immobilization. Therefore, the studies aimed to obtain higher activity and stability using different supports and methods in the immobilization of ANAG are also on going today.

5

Selection of the suitable supporting matrix is the first consideration towards enzyme immobilization. Activated carbon is predominantly an amorphous solid with a large internal surface area and pore volume [51]. The chemical reactivity of these is due to the existence of active sites at the edges of the aromatic planes. The ratio of these active sites in relation increases as the surface area increases [52]. Mesoporous activated carbon (MAC) is well suited as a support due to its large pore size, strong adsorption capacity, good mechanical properties, inertness and non-toxicity [53]. Enzyme loading and activity expression is largely dependent on the size of the pores in the carrier bead and the accessible surface [54]. A variety of agricultural waste was used to prepare activated carbon. Sesame seed shells are a by-product of sesame seed production and are therefore a ready and abundant natural material that can be regarded as a raw material for producing an effective adsorbent. According to statistics, sesame seeds worldwide production of about 6.111.548 tons in 2016, while Turkey is 19.521 tonnes of production [55]. MAC produced from sesame seed shells that used in this study posses hydroxyl and carbonyl groups on its surface and cavities [55]. These groups may be used to functionalize the MAC before covalent immobilization of enzymes. MAC have been used for enzyme immobilization with adsorption [51, 56] and covalent [53, 57] methods. As a result of our literature investigation, there are no any study related to the covalent immobilization of ANAG with EFGAAC. Therefore, this study was aimed the following objectives: (1) to investigate the possibility of the obtaining the higher activity yield, higher resuability and longer storage stability than previous studies available in the related literature by optimizing the covalent immobilization conditions of ANAG with EFGAAC, and (2) to investigate the usability of immobilized ANAG for the production of glucose syrup from maltodextrin. 2. Materials and Methods 2.1. Materials Active carbon (AC) beads having 1254 m2/g surface area was produced from sesame seed shell according to the method of Sharif et al [55]. ANAG which have 143.7 IU/g activity, is a commerical powder enzyme preparation, was provided as a gift by Bio-Cat (Troy, USA). UVVIS Spectrometer (UV-6300PC) was purchased from VWR (Radnor, USA). pH meter (Hanna HI 2020 edge), was purchased from Hanna Instruments Ltd. (Bedfordshire, UK). Magnetic 6

stirrer (Heidolph MR Hei-Standard) was purchased from Heidolph UK-Radleys (Shire Hill, UK). Pure water appliance (Mini Pure 1, MDM-0170) was purchased from MDM Co. Ltd. (Suwon-si, South Korea). Precision scale (Shimadzu-ATX224) was purchased from Shimadzu Corporation (Kyoto, Japan). Orbital shaking heated incubator (Mipro-MCI) was purchased from Protek Lab Group, professional laboratory solutions company (Ankara, Turkey). Vacuum pump (Biobase, GM-0.50A) was purchased from Biobase Biodustry Co., Ltd. (Shandong, China). Bovine Serum Albumin (BSA), sodium hydroxide, sodium dihydrogen phosphate,

hydrochloric

acid,

lactose,

sodium

sulphite,

phenol,

glutaradehyde,

ethylenediamine, aceton and D-glucose were purchased from Sigma-Aldrich (Taufkirchen, Germany). 3,5-dinitrosalicilic acid (DNS) was purchased from Alfa Aesar (Kandel, Germany). Sodium potasium tartrate (Roechelle salt) was purchased from VWR Prolabo Chemicals (Leuven Belgium). Sodium azide was purchased from Merck Millipore (Darmstadt, Germany). 2.2. Methods 2.2.1. Functionalization of active carbon The amine solution (2.5%, v / v) was prepared by mixing 2.5 ml of ethylenediamine (EDA) with 97.5 ml of acetone. Then, amino functionalizing of AC was carried out by adding 30 g of dried (no moisture) AC to the amine solution and by stirring the mixture for 10 minutes according to method described by Ramani et al [57]. The glutaraldehyde solution was also prepared by mixing 100 ml of acetone with 100 ml of glutaraldehyde (25%, v/v) and then the mixture was added to the AFAC and stirred gently for 30 minutes using a magnetic stirrer. To facilitate strong immobilization of the enzyme, the amino-functionalized active carbon was activated with the aldehyde group by the addition of glutaraldehyde solution. The above material was dried under vacuum until the solvent had completely evaporated and heated at 150 °C for 24 hours. The prepared material was washed with distilled water to remove unbonded chemicals. The eluate was dried at 110 oC for 6 hours to obtain the final product, which was labeled as EFGAAC for further work [57].

7

2.2.2. Immobilization procedure Immobilization of ANAG was performed by reacting 100 mg of AFAC beads with 200 μL (0.21 IU) of ANAG in 5 mL of sodium phosphate buffer (0.5 M, pH 5.5) at 25 ◦C for 3 h in an incubator with gentle shaked at 150 rpm according to procedure of Ramani et al [57]. After immobilization, the beads were filtered and washed with 15 mL of sodium phosphate buffer (0.1 M, pH 5.5) and 15 mL distilled water as three aliquots respectively, on a sintered glass filter by suction under vacuum. After then, immobilized enzymes have been stored in 5 mL of sodium phosphate buffer (0.1 M, pH 5.5) in a refrigerator at +4 oC until next use. 2.2.3. Optimization of immobilization procedure Optimum conditions for immobilization were determined by changing individually the conditions, (pH range was from 4.0 to 7.0; buffer concentration range was from 0. 5 to 2.0 M; amounts of activated carbon were from 100 to 400 mg; and duration of immobilization were from 0 h to 24 h). 2.2.4. Verification of covalent immobilization 0.312 g immobilized ANAGs containing 0.21 IU activity were incubated at growing ionic strength (2.5, 5.0, 7.5, and 10%) by using NaCl according to [58]. After immobilized enzymes were washed with plenty of distilled water, protein in the filtrate and immobilized ANAG activity was determined by using standard activity assay method. 2.2.5. Protein assay The amounts of proteins present in the immobilization buffer before and after immobilization were determined by using Bradford Protein Assay Method [59]. The protein (enzyme) concentrations in the immobilization solutions and in the filtrates were calculated Using Equation 3 obtained from BSA standard plot. Y = 0.29958X

(1)

8

2.2.6. Determination of ANAG activity Maltodextrin solutions (1 % w/v) prepared 5 mL 25 mM sodium phosphate buffer (pH 5.5) was reacted with 200 μL of free or 0.312 g immobilized ANAG containing 0.21 IU activity at 55 ◦C for 60 min in an incubator with gently shaking. 200 μL of aliquots from reaction mixture was added to 1800 μL of distilled water and boiled for 10 min to inactivate the enzyme. The amounts of D-glucose in the diluted samples were determined by measuring its absorbance using a UV spectrophotometer at 575 nm, according to slightly modified method of Miller [60]. One IU ANAG activity was defined as the amount of enzyme forming 1 µmol D-Glucose from maltodextrin per minute, under optimum activity assay conditions. ANAG activity was calculated by using following equation. IU / mg Enzyme 

Released D - Glucose (μmol) Enzyme used (mg) x Duration of reaction (min)

(2)

2.2.7. Determination of immobilization yields Immobilization was monitored by measuring the enzyme activity in the suspension and in the supernatant. Immobilization yields were determined in terms of bound protein (IYP) and expressed activity (IYA) according to the method of Urrutia et al. [61]: IYP =

IYA =

PI x100 PC

AI x100 AC

(3)

(4)

where PI is the immobilized protein per gram of support (difference between contacted protein and unbound protein in the supernatant) and PC is the contacted protein per gram of support, AI is the activity expressed by the immobilized biocatalyst (measured by suspending the immobilized and washed biocatalyst in the activity buffer), AC is the enzyme activity contacted per gram of support.

9

2.2.8. Characterization of free and immobilized enzyme 2.2.8.1. Optimum pH The effect of pH on enzyme activity was investigated by performing the activity assay for the free and immobilized enzymes with 1 % (w/v) buffered maltodextrin solutions, at different pHs, at 55 oC according to method of Aslan and Tanrıseven [62]. 2.2.8.2. Optimum temperature The effect of temperature on enzyme activity was found by conducting the activity assay with 1 % (w/v) buffered maltodextrin solutions (pH 5.5) at different temperatures according to method of Aslan and Tanrıseven [62]. 2.2.8.3. pH stability 200 μL of free or 0.312 g immobilized ANAG containing 0.21 IU activity were incubated in sodium dihydrogen phosphate buffer solutions at various pH ranges (3.0–8.0) at room temperature for 1 h and the retained activities were determined under standart assay conditions according to method of Aslan and Tanrıseven [62]. 2.2.8.4. Thermal stability 200 μL of free or 0.312 g immobilized ANAG containing 0.21 IU activity were incubated in sodium dihydrogen phosphate buffer solutions (25 mM, pH 5.5) at temperatures from 30 to 80 ◦C for 1 h and then the retained activities were determined using the standart assay method according to method of Aslan and Tanrıseven [62]. 2.2.8.5. Kinetic constants Initial velocities for kinetic parameters were determined by performing the reactions between 200 μL of free or 0.312 g immobilized ANAG containing 0.21 IU activity and maltodextrin solutions at different concentrations (5 to 80 g/L) for 5 min. Km and Vmax values were calculated from Lineweaver–Burk plot.

10

2.2.8.6. Operational and storage stabilities of the immobilized ANAG Operational and storage stabilities of the immobilized enzyme were determined by measuring the retained activities using standard activity assay method, after each use in repeated 20 batch experiments, and two days intervals for thirty days, respectively according to method of Aslan and Tanrıseven [62]. 2.2.9. Production of glucose syrup from maltodextrin by using immobilized ANAG 0.312 g immobilized ANAG containing 0.21 IU activity was reacted with the buffered 5 % (w/v) maltodextrin solution (pH 5.5) at 55 ◦C for 6 h and D-glucose content was determined with 60 minutes intervals by using UV spectrophotometer. 2.2.10. Statistical Analysis Each value represents the mean for three independent experiments performed in triplicates. Data were analyzed by using Microsoft Windows Excell. 3. Results and Discussions 3.1. Protein assay The enzyme concentration in a 5.2 mL immobilization solution containing 200 μL of free ANAG was calculated to be 6.842 mg / mL. The enzyme concentration in the powdered ANAG preparation was also calculated as 684.2 mg / g by using dilution factor (100). This result is consistent with the declaration (AG content is between

62 – 82%) of the

manufacturer company [63]. 3.2. Determination of ANAG activity Free ANAG activity was calculated as 0.21 IU / mg. Furtheremore, since 1 g of powder ANAG preparation contains 684.2 mg of free ANAG, the activity of powdered ANAG was also calculated to be 143.7 IU/g for maltodextrin as substrate. This result was agreed with the declaration (100-100 IU/g) of the producer company [64]. The amount of free ANAG having 1 IU activity was also calculated as 32.30 mg.

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3.3. Optimization of immobilization procedure Immobilization conditions such as, pH and molarity of immobilization buffer, enzyme/support ratio and immobilization time, should favor the enzyme–support reaction [65, 66]. On the other hand, a good immobilization protocol should keep high catalytic activity after enzyme immobilization. However, in some cases, the enzyme may have two forms with very different activity, and if we were able to fix the enzyme form with higher activity, the final immobilized preparation may be more active than the native one [67]. As a result, after optimization of the immobilization conditions, 99.80% of immobilization yield and 99.83% of activity yield were achieved. Among the previous studies, the best activity yield (100%) achieved by Sanjay and Sugunan [35] in the covalent immobilization of ANAG onto Montmorillonite K-10 is nearly equal with ours. 3.3.1. Effect of immobilization buffer pH on immobilization efficiency Table 1 shows the influence of pH on immobilization. Since glutaraldehyde is not good stable at alkaline pHs [27], immobilization was performed at pH values are around of optimum pH of ANAG. The maximum immobilization yield (83.46%) and the maximum activity yield (76.56%) were obtained at optimum pH (5.5) of ANAG. As seen in the Table, both of the immobilization yield and the activity yield were increased by increasing pH until 5.5 but the activity yield was decreased at higher pHs. The highest immobilization yield at pH 7 may be due to more reactivity of terminal amino groups whose pK is between 7 and 8 that are than the reactivities of all Lys amino groups at neutral pH values [68]. The decreasing activity at pH 6-7, may be result of te establishement of some new covalent enzyme-support bonds between the different groups available at high concentration on the support and some nucleophiles of protein that are in the area exposed to the support [69]. Furthermore, pH can encourage the denaturation of enzymes taht resulted in the decreased activity [18, 21, 70]. On the other hand, the catalytic activity of enzyme depends on conformational structure of the protein, even minor alterations in the tertiary structure of the protein resulted in loss of its catalytic activity [71]. Similar results can be seen in the related literature. For example, Ramani et al. [56] obtained the maximum activity yield at the optimum pH (5.0) in the immobilization of Pseudomonas gessardii acidic lipase with MAC. In another study, Chen et al. [72] also obtained the maximum immobilization and activity yields at he optimum pH of in the immobilization of β-galactosidase on glutaraldehyde activated chitosan beads. 12

3.3.2. Effect of immobilization buffer molarity on immobilization efficiency Table 2 shows that the maximum immobilization yield (83.46%) and the maximum activity yield (76.56%) were obtained with immobilization buffer of 0.5 M. Moreover, it is seen that both of immobilization yield and activity yield were decreased when the molarity of immobilization buffer increased. Ionic strength can promote the denaturation of enzymes [18, 21, 70]. At low ionic strength, the enzyme will first be immobilized on the support by ionic exchange, thus nucleophiles of protein exposed to the support can are reacted with glutaraldehyde moieties on the support [27, 35, 73]. But, the higher salt concentrations can lead to decrease in the activity by distording the three dimensional structures of enzyme molecules that responsible for the enzyme activity [74]. Moreower, very high ionic strength prevents ionic exchange between support and enzyme molecules and immobilzation occured by hydrophobic interaction which is faster than covalent attachment [26]. Therefore, the decreased activity versus increased ionic strength probably resulted from leacking of enzymes immobilized by hydrophobic interaction during washing the beads with buffer and pure water. From another perspective, high ionic strength may force a stronger interaction between the immobilized enzyme and the support perhaps closing the gap between enzyme active center and support surface and thus, increasing the steric hindrances to the entry of the substrate to the active center of the enzyme [75]. 3.3.3. Effect of AFAC amount on immobilization efficiency According to Table 3, it can be see that the immobilization yield of 83.46 % and the activity yield of 76.56 % were achieved for 100 mg of EFGAAC. Furthermore, according to the table by increasing amounts of EFGAAC the activity yield was decreased while immobilization yield was incresed. This result can be expected. Because, it is well known that enzyme immobilization yield and activity yield is largely depands on the amount of support used. Similar results can be seen in literature. For example, Ramani et al. [57] reported that the amount of lipase immobilized was increased with the increased amount of functionalized mesoporous activated carbon (FMAC) from 1 to 25 g. The decreases of activity yield despite increases of immobilization yield by increasing amount of EFGAAC is possibly the result of the deterioration the active structure of enzyme molecules due to multipoint attachment of enzyme molecules to the support via new interaction between enzyme and support [72, 76]. 13

On the other hand, blocking of the active site of the enzyme molecules by support can also lead to decreasing the activity, due to restricted substrate transfer to the active center of the enzyme molecules [18]. 3.3.4. Effect of immobilization time on immobilization efficiency The effects of the immobilization time on the immobilization efficiency are shown in Table 4. The maximum immobilization yield (99.80%) and activity yield (99.83%) were achieved for 12 hours and then the maximum activity yield gradually decreased to 90.87% at the end of 24 hours. Although immobilization may be very rapid, multipoint interaction between the noncomplementary enzyme and support surfaces is a slow and time-dependent process [67]. The gradually decreasing of obtained maximum activity is probably due to unfavourable changing the three dimentional structure of enzyme molecules due to multipoint attachement of enzyme to the support via excess glutaraldehyde groups available on the activated support [72, 76]. 3.3.5. Verification of covalent immobilization Based on literatures, presence of this salt can lead to breakage non-covalent bond of enzyme and support, by way of ion exchange or washing out the hydrogen bond [58]. The results in Table 5 showed that bounded ANAG onto un-activated AC lost 34.73, 57.64, 66.27, and 100% of its activity, respectively, after incubating at 2.5, 5.0, 7.5, and 10% sodium chloride for 24 hours. But bounded ANAG onto activated AC preserved nearly all of its activity at all ionic strength tested after incubating 24 hours. Furthermore, as seen in the Table 5, enzyme amount in the filtrate was gradually increased by increasing ionic strength. This mean is that only non-covalently bounded enzyme molecules leaks from support. Therefore, it can be said that almost of ANAG was covalently immobilized onto EFGAAC. Moreover, the support can also immobilize enzymes, even if they are very weakly activated, because the enzyme is covalently fixed to the support by only one point since the glutaraldehyde-protein bonds are stable [27].

14

3.3.6. Characterization of immobilized enzyme 3.3.6.1. Optimum pH According to Fig. 1, the optimum pH (5.5) of ANAG was not affected by immobilization. Unchanged optimum pH may indicate that no major conformational change occurred in the immobilized ANAG after immobilization [44]. There are only one study in literature in which maltodextrin used as substrate for determining the ANAG activity. Tanrıseven end Olcer [45], reported that optimum pH of ANAG 4.0. This differency is probably caused from natüre and size of buffer ions used [77]. On the other hand, as seen in Fig. 1, the immobilized ANAG is more active than free counterpart at all pH range tested. The decreased activity at more acidic and alkaline pHs is resulted from denaturation of enzymes with the effects of acids and bases [9, 78]. The higher activity of immobilized ANAG was resulted from increased stability bu multi-point attachment during immobilization [79-82]. 3.3.6.2. Optimum temperature Fig. 2 shows that the optimum temperature range (55-60 °C) was not affected by immobilization. The unchanged optimum temperature of ANAG after immobilization (Fig. 2) agree with two of previous studies [7,17]. Furtermore, according to Fig. 2, it is also clear that the immobilized ANAG exhibits higher activity than the free ANAG at the entire temperature range tested. It is well known that immobilized enzymes exhibit higher activity than free enzymes at high temperatures due to the limited conformational mobility of molecules following immobilization [81, 83] as a result of interaction between enzyme and the support [84]. 3.3.6.3. pH stability and thermal stability According to Fig. 3, it is seen that the immobilized ANAG is more stable than the free enzyme at all tested pH values. It is seen in Fig. 4, the immobilized ANAG more stable than the free enzyme at the higher temperatures than 55 oC. It is generally known that immobilization improves the pH and thermal stabilities of enzymes [85]. Furtermore, the higher stability of ANAG at alkaline pHs, may be result of multipoint attachment of enzyme molecules to the support [50, 81, 86] due to increased number of deprotonated amine groups 15

of protein. Multi-point attachment of the enzyme to the support material, which may increase the resistance of the enzyme from denaturation conditions [50]. 3.3.6.4. Kinetic constants The kinetic constants of free and immobilized ANAG were calculated using the LineweaverBurk plot (Fig. 5). After immobilization, Vmax value has been decreased from 151.5 to 147.1 μmol D- Glucose / L.min, while Km value decreased from 116.3 to 109.9 g maltodextrin / L. while Km value decreased from 116.3 to 109.9 g maltodextrin / L. Km value indicates the affinity of an enzyme to a substrate. It is well known that when the Km value increases, the affinity of the enzyme to substrat decreases [85]. 3.3.6.5. Operational and storage stabilities of the ımmobilized ANAG The activity of immobilized ANAG decreased to 99.3 % of initial value after the repeated twenty uses under optimum conditions (Fig. 6). The activity of immobilized ANAG decreased to 98.3 % of initial value after thirty days under optimum storage conditions (Fig. 7). The results in Fig. 6 and Fig. 7 show that the immobilized ANAG show high operational and storage stabilities. Therefore, the activity / stability properties of the enzymes immobilized on glutaraldehyde-activated supports depend on the exact immobilization protocol used. For example, different groups may provide some differences in stability, the short intermediate arm (monomer) may provide a higher stiffness, and conversely the longer intermediate arm (dimer) may allow more groups to react and give the best results [79]. The fixation of enzyme molecules to a surface often leads to the highest stabilizing effect on enzyme activities, since the active form of the immobilized enzyme is stabilized by the formation of a multipoint bond between the substrate and the enzyme molecules [44, 87]. Among the previous studies, the longest operational stability (100% of retained activity after 30 cycle) for immobilized ANAG have been obtained by Tanrıseven and Zehra [45] in the covalent immobilization of ANAG on polyglutaraldehyde-activated gelatin particles in the presence of polyethylene glycol and soluble gelatin. But their activity yield (85%) is lower than ours (99.83%). Therefore, it can be said that the operational stability achieved in this study is the best among the previous studies.

16

3.3.7. Production of glucose syrup from maltodextrin by using immobilized ANAG. The changes in the maltodextrin and D-glucose concentration during the production of glucose syrup were evaluated by investigating Fig. 8. As seen in Fig. 8, all of the maltodextrin has converted to D-glucose in four hours by using immobilized ANAG. 4. Conclusions In the present study, firstly 99.80% of immobilization yield and 99.83% activity yield were achieved by optimizing the immobilization conditions. This result is among the best results in the immobilization studies of ANAG in the literature. Secondly, the immobilized ANAG obtained in the present study showed high operational and storage stabilities. Thirdly, immobilization increased the affinity of ANAG to its substrate maltodextrin. Lastly, maltodextrin has completely converted to glucose by using immobilized ANAG. Consequently, it can be said that immobilized ANAG obtained in the present study can be used in the industrial production of glucose syrup and in the other industrial applications. Acknowledgement The authors acknowledge to Bio-Cat Company for their kindly gift amyloglucosidase. The authors also acknowledge to Scientific Research Projects Coordinatorship of Siirt University for their financial support. 5. References [1] K. Gupta, A.K. Jana, S. Kumar, M.M. Jana, Solid state fermentation with recovery of Amyloglucosidase from extract by direct immobilization in cross linked enzyme aggregate for starch hydrolysis, Biocatalysis and Agricultural Biotechnology 4(4) (2015) 486-492. [2] P. Bernfeld, Amylase, α and β, Methods in Enzymology 1 (1955) 149-158. [3] P. V. Aiyer, Amylases and their applications, African Journal of Biotechnology 4(13) (2005) 1525-1529. [4] J. Emnéus, G. Nilsson B.sc, L. Gorton, A Flow Injection System for the Determination of Starch in Starch from Different Origins with Immobilized α‐Amylase and Amyloglucosidase Reactors, 45(8) (1993) 264-270.

17

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24

Table 1 Comparrison of the results obtained in this study with the results that obtained in previous studies related to immobilization of ANAG

Matrix

Method

Substrate

Activity Yield (%)

Operational Stability (Retained Activity %)

Stotage Stability (Retained Activity %)

90 after 25 cycle 98 after 60 days

Reference

Without support

Cross-Linking

Starch

65

Without support

Cross-Linking

Soluble starch

41.2

unstudied

unstudied

[34]

Montmorillonite K-10

Adsorption / Covalent

Starch

95 / 100

95 after 100 h / 100 after 100 h

unstudied

[35]

Charcoal

Adsorption

Dekstrin

90

unstudied

100 after 14 days

[36]

Nonporous polystyrene/poly(sodium styrene sulfonate) (PS/PNaSS) microspheres

Adsorption

Dekstrin

89.7

unstudied

unstudied

[37]

Bulk catalytic filamentous carbon (bulk CFC)

Adsorption

Dextrin

45.4

50 after 350 h

unstudied

[38]

Magnetic Cu2+-chelated particles

Adsorption

Soluble starch

84

100 after 10 cycle

95 after 6 weeks

[39]

Alginate Beads

Entrapment

Starch

92

unstudied

unstudied

[40]

Chitin

Covalent

Dekstrin

98

unstudied

100 after 25 days

[41]

Spacer-arm attached magnetic poly(methylmethacrylate) microspheres

Covalent

Dekstrin

73

Macroporous copolymers of glycidyl methacrylate and ethylene glycol dimethacrylate (poly(GMA-co-EGDMA))

Covalent

Macroporous copolymers of glycidyl methacrylate and ethylene glycol dimethacrylate (poly(GMA-co-EGDMA))

Covalent

92 after 60 hours 88 after 21 days

[1]

[42]

9

unstudied

unstudied

[43]

Soluble starch

98.2

unstudied

100 after 4 weeks

[44]

Covalent

Maltodextrin

85

Highly activated Glyoxyl-agarose supports

Covalent

Starch

80

89 after 50 h

unstudied

[46]

Poly(GMA-co-EGDMA) microspheres

Covalent

Soluble starch

64.25

unstudied

unstudied

[47]

Magnetic nanoparticle (MNP)

Covalent

Starch

92.8

95 after 22 cycle

unstudied

[48]

Mesoporous silica (SBA-15)

Covalent

Starch

87.71

unstudied

unstudied

[49]

2-Hydroxyethyl methacrylate/ Ethylene glycol dimethacrylate (pHEMA/EGDMA)

Covalent

Dekstrin

71

96 after 50 h

unstudied

[5]

Poly(methyl methacrylateglycidyl methacrylate) [Poly(MMA-GMA)] cryogels

Covalent

Starch

24.5

Soluble starch

100 after 30 cycle 75 after 90 days

93 after 20 cycle 68 after 30 days

[45]

[50]

25

Table 2 Effect of immobilization buffer pH on immobilization efficiency Immobilization Buffer pH 4.0 5.0 5.5 6.0 7.0

Immobilization Yield (%) 74.58 ± 0.04 79.87 ± 0.02 83.46 ± 0.03 84.30 ± 0.05 87.17 ± 0.02

Activity Yield (%) 70.17 ± 0.02 72.44 ± 0.03 76.56 ± 0.05 69.28 ± 0.04 66.15 ± 0.02

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Table 3 Effect of immobilization buffer concentration on immobilization efficiency Buffer Concentration (M) 0.5 1.0 1.5 2.0

Immobilization Yield (%) 83.46 ± 0.03 79.57 ± 0.03 74.22 ± 0.04 58.90 ± 0.05

Activity Yield (%) 76.56 ± 0.05 73.91 ± 0.03 72.50 ± 0.05 71.13 ± 0.02

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Table 4 Effect of EFGAAC amounts on immobilization efficiency EFGAAC Amounts (mg) 100 200 300 400 500

Immobilization Yield (%) 83.46 ± 0.03 87.62 ± 0.04 92.31 ± 0.05 97.21 ± 0.02 99.42 ± 0.02

Activity Yield (%) 76.56 ± 0.05 71.93 ± 0.05 67.68 ± 0.02 64.01 ± 0.03 63.21 ± 0.03

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Table 5 Effect of immobilization time on immobilization efficiency Immobilization Time (hours) 3 6 9 12 15 18 21 24

Immobilization Yield (%) 83.46 ± 0.03 91.63 ± 0.03 97.38 ± 0.04 99.80 ± 0.05 99.80± 0.02 99.80± 0.03 99.80± 0.05 99.80± 0.02

Activity Yield (%) 76.56 ± 0.05 89.94 ± 0.02 94.35 ± 0.03 99.83 ± 0.05 97.32 ± 0.03 96.97 ± 0.04 94.35 ± 0.02 90.87 ± 0.02

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Table 6 Desorption of ANAG molecules that bounded onto non-activated AC and onto activated AC, after incubating at growing ionic strength for 24 hours. GA Activation

No

Yes

Ionic Strength (%, w/v) Protein in the filtrate (mg)*

Retained Activity (%)**

0.0

0.053

98.27

2.5

1.976

75.27

5.0

3.951

52.36

7.5

4.534

23.73

10.0

6.842

0.00

0.0

0.001

99.99

2.5

0.004

99.94

5.0

0.007

99.89

7.5

0.011

99.84

10.0 0.012 99.83 *After incubating 0.312 g immobilized ANAG containing 0.21 IU activity at growing ionic strength, protein in the filtrate was determined by using Bradford protein assay method. **After incubating 0.312 g immobilized ANAG containing 0.21 IU activity at growing ionic strength, ANAG activity was determined by using standard activity assay method described in the section 2.2.4.

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Fig. 1. Optimum pH of free and immobilized ANGA.

31

Fig. 2. Optimum temperature of free and immobilized ANGA.

32

Fig. 3. pH stability of free and immobilized ANGA.

33

Fig. 4. Thermal stability of free and immobilized ANGA

34

Fig. 5. Lineweaver–Burk plots of free and immobilized ANGA.

35

Fig. 6. Operational stability of immobilized ANGA.

36

Fig. 7. Storage stability of immobilized ANGA.

37

Fig. 8. Production of glucose syrup from maltodextrin by using immobilized ANGA.

38

HIGHLIGHTS 1. ANAG was covalently immobilized on AFAC with 100% efficiency. 2. Immobilized ANAG's initial activity was retained after 20 use and 20 days storage. 3. Maltodextrin was completely converted to glucose by using immobilized ANAG. 4. Immobilized ANAG obtained can be used in industrial production of glucose syrup.

39