Nano co-immobilization of α-amylase and maltogenic amylase by nanomagnetic combi-cross-linked enzyme aggregates method for maltose production from corn starch

Journal Pre-proof Nano co-immobilization of α-amylase and maltogenic amylase by nanomagnetic combi-cross-linked enzyme aggregates method for maltose production from corn starch Homa Torabizadeh PII:

S0008-6215(19)30594-4

DOI:

https://doi.org/10.1016/j.carres.2019.107904

Reference:

CAR 107904

To appear in:

Carbohydrate Research

Received Date: 9 October 2019 Revised Date:

1 December 2019

Accepted Date: 21 December 2019

Please cite this article as: H. Torabizadeh, Nano co-immobilization of α-amylase and maltogenic amylase by nanomagnetic combi-cross-linked enzyme aggregates method for maltose production from corn starch, Carbohydrate Research (2020), doi: https://doi.org/10.1016/j.carres.2019.107904. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Nano co-immobilization of α-amylase and maltogenic amylase by nanomagnetic combi-cross-linked enzyme aggregates method for maltose production from corn starch Homa Torabizadeh1* 1

Department of Chemical Technologies, Food Science and Technology group, Iranian Research Organization for Science and Technology (IROST), Mojtama Asre Enghelab Building, Shahid Ehsanirad Street, 33535111, Tehran, P.O. Box 37575-111, Iran *

[email protected]

ABSTRACT Starch hydrolysis to maltose by nano-magnetic combined cross-linked enzyme aggregates of α-amylase and maltogenic amylase (NM-Combi-CLEAs) is an important step to open new perspectives for special food and pharmaceutic production. Improvement of mass transfer, thermostability, functional specificity, and reusability of combined enzymes was performed. The obtained results exhibited that, 1:9 ratio of α-amylase/maltogenic amylase, use of tertbutanol as precipitant, 2 mM glutardialdehyde, 1:0.75 ratios of combined enzymes to lysine, 20 h crosslinking at 3–4ºC are well-suited conditions. The dynamic light scattering (DLS) results implied that the nanomagnetites diameter was about 81.9–88.9 nm, with polydispersity index (PDI) of 0.242 and a Ȥ-potential of -21 mV. Moreover, the particle size, PDI, and Ȥ-potential of NM-Combi-CLEAs were around 99.6 nm, 0.088, and -32 mV respectively. The NM-Combi-CLEAs kept 80.4% of its original activity after 10 cycles, its Km value exhibited about 1.5 folds reduction with about 1.5 times enhance in thermostability at 95ºC than free one. Immobilization activity yield revealed about 84% of activity retaining by NM-Combi-CLEAs strategy. Accordingly, this efficacious nanobiocatalyst with high thermostability and reusability recommended for starch conversion to maltose. Keywords : Nanoco-immobilization; α-amylase; Maltogenic amylase; NM-Combi-CLEAs; Maltose syrup. 1. Introduction α-amylases (α-1, 4-glucan-glucanohydrolase, EC 3.2.1.1) are endo-hydrolase enzymes that acts on starch (polysaccharides) and degrades α-1,4-glucosidic linkages in an endo fashion and produce oligosaccharides. Maltogenic amylase (EC 3.2.1.133) is an exo-acting amylase belongs to glycoside hydrolase family 13 (GH13) that provides exohydrolysis of 1,4-αglucosidic linkages in amylose, amylopectin and related glucose polymers. This enzyme works from the non-reducing end of oligosaccharides and catalyzes the hydrolysis of the second α-1,4 glycosidic bond, cleaving off two glucose units (maltose) at a time [1]. Maltose, is a disaccharide contains two D-glucose units that binded by an α-1,4-glycosidic bond together. It applies in food, soft drinks, and pharmaceuitical industries. Industrial maltose production from corn starch is a two-step process included , liquefaction and saccharification. [2, 3]. Maltose is an important component in brewing beer and distilling alcohol, and provides a distinct flavor to malted beverages. It has a variety of technological properties such as bulking agent, body, mouthfeel, binder, chewiness, crispiness, crunchiness, light sweetness, helps to balance the sweetness in food formulations, replaces sucrose, texture

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stability, anti-crystallizing agent, processability, cooking tolerance, fermentation a substrate, and fat reduction. Due to its special properties, maltose is widely used in the food and pharmaceutical industries. At the industrial scale, enzymes should have several features such as easy handling, operational stability, and reusability to be beneficial for use. For this reason, enzyme immobilization has introduced as an effective technique for fabrication of new biocatalysts because it provides several advantages such as the possibility of enzyme reuse, improvement in enzyme stability against harsh conditions (high temperatures, pH variations, high substrate concentration, polar solvents, and mechanical shear) volumetric activity and selectivity increment. In general, there are two types of enzyme immobilization methods: physical (adsorption, entrapment, and encapsulation) and chemical (covalent attachment to a support, and cross-linking of native, crystallized, or aggregated enzyme molecules) procedures [4]. It should be noted that one method is not suitable for immobilization of all enzymes [5] and selection of the best technique for enzyme immobilization depends on the nature of enzyme molecules and expected use of it. In simple and efficacious cross-linked enzyme aggregates (CLEA) technique, enzyme molecules are subjected to aggregation by altering their dielectric constant using organic solvents or by using salts or non-ionic polymers [6, 7]. Cross-linking is occurred via formation of aldol condensations or Schiff’s base binding in the structure of aggregated enzyme through bonding between accessible lysine amino acid and aldehyde groups of crosslinkers [8-11]. Elimination of the expensive carriers, ease in enzyme extraction from crude fermented liquid, operational and storage stability improvement against heat, organic solvents, and autoproteolysis denaturation owing to the tertiary structure rigidification of the enzyme [6, 7, 12, 13], high productivity and yield also combining of two or more enzymes in a single CLEAs structure besides, enhancement of structural stability and enzyme selectivity versus free enzymes are the main advantages of this technique [5, 6]. Accordingly, CLEAs technology has been presented as an uncomplicated, economical, and easy to optimize alternative immobilization procedure with several advantages in industrial applications in various medium and different reaction conditions. These features turn CLEAs into favorable insoluble biocatalyst for industrial applications [14]. Many types of research have been performed on the improvement of amylases immobilization [15]. Peroxidase and α-amylase were immobilized through CLEAs technique by Jiang and coworkers [16]. A combined CLEAs composed of α-amylase, pullulanase, and amyloglucosidase was fabricated by Talekar and coworkers via aggregation in saturated ammonium sulphate and cross-linking for starch hydrolysis [17]. Easa et al. immobilized amylase from super mealworm by CLEAs techniques and its kinetic performance were evaluated [18]. Calcium and sodium ions assisted CLEAs of thermostable α-amylase from Bacillus licheniformis was studied by Torabizadeh [19]. Sercan Sahutoglu et al. prepared α-amylase and glucoamylase CombiCLEAs by the application if glutardialdehyde and dextran polyaldehyde [20]. The particle diameter of CLEAs has an substantial efficacy on its mass transfer limitations and filterability at industrial scale. In general CLEAs particle diameter is differed from 0.1 to 200 micrometer. Use of filtration and centrifugation during separation processes causes an increase in particle diameter , mass transfer limitation, and reduction in catalytic efficiency [4, 6, 21]. The most important factors that are affecting the formation of particle size are an enzyme and cross-linker concentration (e.g., glutaraldehyde). These factors can influence on the final features of the fabricated immobilized enzyme [4, 22]. Large non-uniform particles might have diffusional problems and downward catalytic performance while fine particles may not be retrieved. Thus, particle size control is necessary for achieving uniformity in CLEAs particles to prevail in these disadvantages [8]. For improvement of CLEAs structure and function several strategies have been utilized such as use of feeder proteins (e.g., bovine serum albumin and soy protein isolate) to reduce diffusion problems [23, 24] likewise poly-

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lysine and polyamine compounds [8, 25, 26]. The use of these methods has had a favorable effect on the functional stability of formed CLEAs [21]. Commonly, magnetic materials are composed of Fe3O4, CoFe2O4, Pt, Ni, and Co. Among them, amino functionalized Fe3O4 is widely used for magnetic immobilization of enzymes due to the positive effect on the operational stability, specific activity, reusability, and easiness in separation by magnet [27, 28]. Xie also Guo and coworkers have immobilized Lipase and α-amylase on magnetic carriers and were reported that the thermal stability, pH stability, and storage stability of enzymes were significantly enhanced [29, 30]. Uygun et al. used magnetic poly (2hydroxyethyl methacrylate-N-methacryloyl-(l)-phenylalanine) to immobilize α-amylase. They mentioned that substrate affinity of the enzyme enhanced after immobilization in such a way that, after ten cycles, 85% of the enzyme specific activity was remained. Hence, in this present work, a new nanobiocatalyst is fabricated by using of co-immobilization of αamylase and maltogenic amylase in nano scale. This method consists of the simultaneous nano aggregation of the free amylases in an appropriate solvent and cross-linking of aggregates containing functionalized Fe3O4 by glutaraldehyde via ε-NH2 groups of accessible surface lysine residues [21]. The produced efficient nanobiocatalyst displayed improvement in thermal stability and reusability [25]. Mass transfer limitation, missed fine CLEAs during recovery processes are eliminated [23]. 2. Results and discussion 2.1. Nanomagnetites and Nano-Combi-CLEAs FE-SEM Results The size and morphology obtained nanomagnetites were defined by using of FE-SEM (Fig. 1). As represented in Fig. 1a, nanomanetites exhibited similar distribution with spherical and semi-spherical morphology with about 50–60 nm diameter. While, after enzyme immobilization nanomagnetic combined CLEAs of amylases have a nearly uniform distribution with semi-spherical configurations and less than 100 nm in diameter (Fig. 1b and 1c). Fig. 1 The existence of the nanomagnetites and combined CLEAS of amylases together with Fe3O4 was affirmed by applying EDX, that characterized the composition of elements in the particles (Table 1). Respect to Table 1, nanomagnetites have higher oxygen and iron elements compared to combined CLEAS of amylases whereas, NM-Combi-CLEAs has carbon and nitrogen elements content contrast to Fe3O4 nanoparticles. Therefore, being of the Fe3O4 nanoparticles in the combined CLEAS of amylases was confirmed. Table. 1 2.2. Particle Size, PDI, and Ȥ-Potential of Nanomagnetites and Combined CLEAs For describing the size of particles, PDI, and Ȥ-potential of nanomagnetites and nanomagnetic Combi-CLEAs, dynamic light scattering technique was used. The data outcomes exhibited that, the size of Fe3O4 nanoparticles mainly was around 81.9–88.9 nm (Fig. 2a) while, it was subsequently enhanced to around 99.6 nm after enzyme immobilization (Fig. 2b).

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Fig. 2 DLS results for nanomagnetites and nanomagnetic combined CLEAs acknowledged the SEM image results, due to the shrinkages created along drying the sample also SEM analysis under vacuum environment, the particle size was lightly less than DLS outcomes. Accordingly, the PDI of Fe3O4 was equal to 0.242 with a Ȥ-potential of -21 mV and the nanomagnetic combined CLEAs relevant polydispersity index was 0.088 with Ȥ-potential of -32 mV, respectively. It seems that the PDI index of NM-Combi-CLEAs is reduced compared to functionalized magnetic nanoparticles because of protein binding with nanomagnetites and enhancement of hydrophilicity that leads to improvement of nanoparticles stability. Furthermore, the Ȥ-potential of -32 mV affirms this assertion too.

2.3. Accessible Surface Area Assessment of Enzymes The GETAREA results (Table 2) implied that 6 out of 20 (in α-amylase) and 7 out of 30 (in maltogenic amylase) lysine residues were accessible. On that basis, it seemed that, lysine addition during the aggregation process would increase the strength of fabricated NM-Combi-CLEAs via Schiff base formation by glutaraldehyde; therefore, more stable nanoparticles will be produced. Table. 2

2.4. Optimal Fungamyl/Maltogenase Ratio, Enzyme Activity, and Protein Content At first, free Fungamyl and Maltogenase activity were specified. The results indicated that the hydrolytic activity of Maltogenase is about three times greater than Fungamyl for starch conversion to reducing sugars (Fig. 3a). Optimum Fungamyl/Maltogenase ratio for maltose production was assessed, and the results revealed that the best Fungamyl/Maltogenase ratio is 1:9 which under these conditions, the highest enzyme activity is obtained significantly to convert starch into maltose at 65° C (Fig. 3b). Besides, the activity of free Fungamyl and Maltogenase was separately assessed compared with various combined enzyme ratios. The results revealed that, enzyme activity is enhanced after combination of Fungamyl and maltogenase in 1:9 ratio compared to single enzymes about 4 times for Fungamyl and 1.5 times for Maltogenase. This activity enhancement after enzymes combination is a straight evidence to prove the advantage of the combined-enzyme strategy. Accordingly, the estimated activity of free Fungamyl and maltogenase was 25.76 U/mg and 1069 U/mg, respectively. This value for combined amylases (1:9 Fungamyl/Maltogenase ratio) was 600.18 U/mg that was reduced after immobilization to 503.36 U/mg (around 16%). As it is revealed in Fig. 3c, although by the formation of CLEAs, enzyme activity slightly decreased, the permanence of activity is increased that means, NM-Combined CLEAs of amylases is more active in an extended time relative to free combined enzymes (Fig. 3d).

Fig. 3 The protein content of the free Fungamyl, Maltogenase, free combined amylases (1:9 Fungamyl/Maltogenase ratio) and NM-Combi-CLEAs was 107.17 mg.ml-1, 6.94 mg.ml-1, 114.11 mg.ml-1 and 113.76 mg.ml-1 respectively.

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2.5. Effect of Precipitants on Enzyme Activity When protein structures are subjected to organic solvents, high ionic strength (salts), or nonionic polymers media, they can be physically aggregated. The stability and activity of prepared NM-CLEAs are markedly affected by the precipitation step. Therefore, a designation of the proper precipitating agent in an aggregation of the enzyme molecules is essential, and it pertains to the enzyme molecule character, the manner, holding time, pH and temperature of aggregation process [22]. For this aim, various solvents and precipitants were used afterward; aggregates activity was assigned before the cross-linking process. The outcomes are indicated in Fig. 4a. As it is revealed in Fig. 4a, aggregation by tert-butanol exhibited maximal enzyme activity significantly compared to other solvents used. While the combined amylases aggregates that were formed in saturated ammonium sulphate has the lowest activity value, namely about 7 folds less activity than tert-butanol. In fact, the dielectric constant of water molecules (ɛ=78.3) that are around the enzyme molecule surfaces gradually reduces by replacing tert-butanol (a miscible polar organic solvent with ɛ=12.5). As a result, the hydration layer around the enzyme molecules become lower and aggregation is occurred by dipole forces and electrostatic attractions [31]. Thus, tert-butanol was selected as a proper precipitant in NM-Combi-CLEAs fabrication in the next experiences. 2.6. Lysine and/or BSA Addition Efficacy Regarding that, the protein content in combined free amylases was relatively low (114.11 mg.ml-1), also, the specified accessible surface area of α-amylase and maltogenic amylase revealed that, only 13 lysine residues are accessible from total 50 lysine residues that are present in amylases molecules for bonding with glutaraldehyde. Hence, for the enrichment of combined amylases molecules addition of lysine amino acid and BSA seemed necessary in the cross-linking process. Thus, the effect of various lysine and BSA concentrations with combined enzymes to lysine/BSA ratios of 1:0.25, 1:0.5, 1:0.75, 1:1, 1:2, 1:3, 1:5 on the CLEAs activity after cross-linking was examined. Comparative study for specifying the optimal ratios of combined amylases/lysine also combined amylases/BSA revealed that, the best ratio for achieving higher enzyme activity of prepared CLEAs results in 1:0.75 enzyme/Lysine ratio. This value is two times higher than amylases /BSA activity at an equal ratio (Fig. 4b). Instable NM-Combi-CLEAs that was established at less than optimum enzyme/lysine ratios (<1:0.75) was lost during the washing process. Fig. 4 Contrary, ratios higher than optimum value inhibited the required cross-linking of combined enzymes by the rivalry of ɛ-NH2 of lysine and that is present at the surface of amylases molecules [32]. The optimal selected ratio of enzymes/lysine was utilized in subsequent analysis. 2.7. Cross-linker Amounts and Holding Time Effects The influence of the glutaraldehyde quantity on the outcome immobilized enzyme activity was obtained by specifying the effect of 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mM glutaraldehyde on the CLEAs activity. The results are revealed in Fig. 5a. In this case, when 2 mM glutardialdehyde was utilized, the favorable enzyme activity was attained. The CLEAs formation is affected by glutaraldehyde concentration, and it can influence the catalytic efficiency and performance of immobilized enzymes. When the low concentration of glutaraldehyde (less than 2 mM) was used the fabricated NM-Combi-CLEAs of amylases has a weak and unstable structure and owing to inadequate cross-linking reactions, its activity

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was unfavorable. On the contrary, excessive cross-linking (higher glutaraldehyde values, >2 mM) reduces structure flexibility of the enzyme and raises limitation in a mass transfer that leads to a decrement in enzyme activity. Accordingly, with a gradual increase in glutaraldehyde concentration from 2 to 50 mM, the activity of NM-Combi-CLEAs of amylases is diminished by 26.6%. The holding time (1, 2, 5, 10, 15, 20, 24, 30, and 35 h) results revealed that most activity of the enzyme attained after 20 h CLEAs holding at 2–4° C (Fig. 5b). The activity of the enzyme notably enhanced (about 20%) by rising the holding time up to 20 h, while crosslinking for lengthy periods (more than 20 h) displayed 36.3% reduction in enzyme activity. Fig. 5 2.8. pH and Temperature Efficacy on The Starch Hydrolytic Activity The pH affects on free amylases, and NM-Combi-CLEAs activities were assessed at pH 5, 5.5, 6, 6.5, 7, 7.5, 8, and 9 for 90 minutes at 60º C respectively. The optimal pH of native combined amylases and NM-Combi-CLEAs for starch conversion was recognized equal to 6.0. Accordingly, no significant alteration did not appear optimal pH of the enzymes after immobilization (Fig. 6a). Also, various temperatures ranging from (55, 65, 75, 85, and 95º C) at a pH of 5.5 for 90 minutes were applied for identifying of the effect of temperature on the activity of free and immobilized amylases. The results revealed that, although the optimum temperature of the immobilized enzymes (65º C) did not change significantly, its thermostability at 95º C increased about 1.5 times than that of free one (Fig. 6b). The higher activity of NM-Combi-CLEAs compared to free amylases at 95º C can be attributed to the establishment of new covalent bonds during the cross-linking process and restriction in free enzyme molecules motion and finally, enzyme active site conservation. Fig. 6 2.9. FTIR Results FTIR spectroscopy analysis of the functionalized MNPs and Combi-CLEAs of amylases ranging between wave numbers 4000 and 400 cm−1 were recorded (Fig. 7a). This method provides an understanding of the transmission bands attributable to the different chemical and physical properties of the compounds also explains the interaction of functionalized nanomagnetites with nano Combined CLEAs of amylases. According to the spectra of the nanomagnetites obtained (Fig. 7a), a well-resolved band at 600 cm−1 assigned to Fe-O stretching vibrations and band at 1621 cm-1 related to H2O absorbed on the sample, and a well-resolved band at 3400 cm−1 devoted to OH− stretching vibrations of structural hydroxyl (Fe-OH) groups. The nanomagnetic combined CLEAs exhibited Fe-O stretching vibration band at 602 cm-1. Another two bands at 1067 and 1141 cm-1 ascribed to stretching vibration mode of C-O and C-O-C groups. Other four peaks that are appeared at 1352 cm-1, 1586 cm-1, 2936 cm-1 and 3373 cm-1 assigned to C=C, NH2, OH and or CH, and N-H and alcohol OH stretching vibration bands respectively. A considerable band at 1637 cm-1 can be attributed to the vibrations of the backbone C=O, referred to the vibration of amide groups. 2.10. Reusability of NM-Combi-CLEAs of Amylases Enzyme reusability is an important factor for its affordable in industrial use. The main purpose for the fabrication of nanomagnetic combined α-amylase and maltogenic amylase CLEAs was long-time usage of these industrial-value-added enzymes beside conserving of their biological activity along with easiness in separation from the reaction medium. For this purpose, NM-Combi-CLEAs efficiency of reusing was evaluated up to 14 cycles (Fig. 7b).

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The results revealed that CLEAs of amylases was maintained 80.4% and over 50% of its initial activity after 10 and 13 cycles respectively, which indicates potent operational stability. Fig. 7 2.11. Kinetic features of Native and CLEAs amylases Michaelis constant (Km) and maximum velocity (Vmax) of native (11.40 mg/100 µl) and NMCombi-CLEAs of amylases (11.38 mg/100 µl) were specified by the double reciprocal plot method (Lineweaver-Burk plots) by various concentrations of corn starch as substrate (Table. 3). The assessed Km value for native combined enzymes was 4.86 × 10−4 (M) that after combined CLEAs formation this value decreased to 3.33 × 10−4 (M), namely about 1.5 folds reduction represented that, lesser substrate value is required for the enzyme saturation means that, affinity for substrate is enhanced. Table 3. It represented that, interaction between enzyme and substrate may be consolidate via appropriate direction of the active site of the enzyme than substrate and the surrounding molecular structure lead to minor steric limitations as a consequence, the substrate interact with immobilized enzyme freely. Beside, structural flexibility of the the immobilized amylases is increased after aggregation in tert-butanol and cross-linking in glutaraldehyde. Also can be said that, the substrate has not encountered diffusional limitation due to enzyme insolubilization [33]. Moreover, mass transfer limitation decrement owing to NM-CombiCLEAs fabrication at the nanoscale as well as, presence of nanomagnetic particles enhances CLEAs hydrophobicity and recovers the substrate binding efficiency [34]. In many instances due to molecular structure changes immobilization of the enzyme is accompanied with reduction in enzyme activity. [35]. The provided NM-Combi-CLEAs was also displayed slightly reduction in Vmax compared to the combined native enzymes. In this case, activity of combined native enzymes was reduced from 10.95 µmol min−1 to 9.98 µmol min−1 after CLEAs preparation. The desirable effects of CLEAs fabrication on Michaelis constant improved the enzyme catalytic efficiency (Vmax/Km) up to 1.33 folds, compared to the free combined enzymes as well as kcat/Km that was increased from 0.41×106 M-1s-1 (free combined enzymes) to 0.54×106 M-1s-1 (NM-Combi-CLEAs) about 1.32 folds. 2.12. Assessment of Immobilization Yield Immobilization activity yield (%) was estimated based on equation (2). The results implied that, the NM-Combi-CLEAs of amylases maintained a high amount of activity (83.86%) in comparison to the native combined enzymes after immobilization by nanomagnetic coimmobilization via CLEAs technique. 3. Conclusion In conclusion, a carrier-free nanobiocatalyst is fabricated by co-immobilization of α-amylase and maltogenic amylase onto the lysine-functionalized magnetic Fe3O4 nanoparticles and used to conversion of corn starch to maltose via nanomagnetic combined cross-linked enzyme aggregates method with a high immobilization activity yield. The fabricated nanobiocatalyst possess a stable nanoscale particle size, high thermostability at 95° C, considerable high reusability, lower Km value and higher enzyme affinity to substrate compared to free enzyme. Thus, two drawbacks that are encountered during CLEAs usage in industrial scale namely, mass transfer limitations and enzyme handling will be resolved. Moreover, two stage process integration for starch hydrolysis to maltose become possible. 7

4. Materials and methods 4.1. Materials Fungamyl 800 L (α-amylase, EC 3.2.1.1) from Aspergillus oryzae and Maltogenase L (Maltogenic amylase, EC 3.2.1.133) from Bacillus subtilis were supplied by Novozymes (Bagsvaerd, Denmark). Corn starch was prepared from Arian Glucose company (Tehran, Tehran, Iran). Dinitrosalicylic acid (DNS), sodium potassium tartrate, glutardialdehyde (25% aqueous solution), iron(II) and iron(III) chlorides, D(+) glucose, and L-lysine monohydrochloride (>99%) were purchased from Merck (Darmstadt, Germany). Bovine serum albumin fraction V (BSA) was acquired from Fluka company (Fluka, Buchs, Switzerland). Coomassie brilliant blue (G-250) was procured from GE Healthcare (Uppsala, Sweden). Other used chemicals were from Merck (Darmstadt, Germany). Morphology evaluation was achieved by Tescan Mira II FE-SEM (Kohoutovice, Czech Republic) at 20.0 kV voltage and sample coating with thin gold layer by magnetron sputtering. Perkin Elmer Lambda 25 UV/VIS spectrophotometer (Waltham, MA, USA) was applied for assessment of absorbance intensity (cells with 1 cm path length) versus the blank. The infrared spectra of samples were acquired by employing Fourier transform infrared spectroscopy (FTIR-8300, Shimadzu, Kyoto, Japan). The dynamic light scattering (DLS) technique was used for sample sizing in the liquid phase, applying a BI-200 SM Goniometer Version 2 (Brookhaven Instrument Corp., Holtsville, NY, USA). The light scattered by the nanoparticles was detected at 173º in dynamic laser scattering. Moreover, evaluation of nanoparticle stability versus aggregation was specified by zeta potential using the DLS method. Whole amounts were indicated as average (± standard deviation) of triplicates experiments and were analyzed via general factorial design with regression coefficient (R2) of 0.999 by employing Design Expert, version 10 software (Stat-Ease, Inc., Minneapolis, MN USA). The variables were statistically significant at p<0.05. 4.2. Synthesis and Lysine Functionalization of Magnetic Nanoparticles Chemical coprecipitation was applied for nanomagnetite fabrication [24, 26, 28]. Then 0.1 g of lysine monohydrate was mixed with 50 ml of 0.1 M sodium acetate buffers (pH 5.0), thereafter, the equal amounts (0.1 g) of water-based Fe3O4 magnetic nanoparticles (MNPs) were added to the lysine solution and the mixture was then subjected to ultrasonic waves (300 Hz) for 10 min at room temperature. Then, the mixture was stirred for 12 h at room temperature for directly binding of lysine onto Fe3O4 nanoparticles. Afterward, the acquired functionalized nanomagnetites were isolated at 11357×g by centrifuge, washed with deionized water and dried by vacuum drier. 4.3. Enzyme Activity and Protein Assay Free α-amylase, maltogenic amylase, and a combination of two enzymes in free and immobilized form were assessed for activity assay by quantifying of maltose concentration. By using of a standard calibration curve of maltose via DNS procedure and values were specified in triplicates manner at 575 nm. The activity assay cocktail was included of 100 µL of enzyme (10.72 mg of free Fungamyl, 0.69 mg of free Maltogenase, 11.41 mg of free combined Fungamyl and Maltogenase in 1:9 ratio, and 11.38 mg of NM-Combi-CLEAs / 100 µl) with 900 µL substrate (liquified starch 1% w/v) in 9000 µL of 0.05 M sodium acetate buffer with pH 5.5, at 65° C for 60 min.

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A unit of combined amylases activity corresponds to the released of 1 µmol reducing sugar in one minute under standard assessment conditions [36]. Moreover, by using of Bradford method, the protein quantity was defined by using of Bio-Rad protein strategy, at 595 nm and application of a bovine serum albumin (BSA) calibration curve [37]. 4.4. Accessible Surface Area Assessment of α-amylase and Maltogenic amylase α-amylase (EC 3.2.1.1) crystal characterization from Aspergillus oryzae is determined at a resolution of 1.5 Å and is placed at the Protein Data Bank (PDB Code: 3VX0) website [38]. The crystal structure of maltogenic amylase (EC 3.2.1.133) from Bacillus subtilis is not specified while this amylase from Thermus sp. IM6501is resolved at a resolution of 2.8 Å and is located at the PDB website (PDB Code: 1SMA), [39]. Using of LALIGN program that is located at the Expasy server (The SIB Swiss Institute of Bioinformatics, Lausanne , Switzerland) for detecting multiple matching in two sequences revealed that, maltogenic amylase from Thermus sp. IM6501 has an 83.3% similarity (54.9% identity) with maltogenic amylase from Bacillus subtilis (Fig. 8). For determining of accessible surface lysine residues of α-amylase and maltogenic amylase, GETAREA bioinformatics program (The University of Texas, Medical Branch, Galveston, TX, USA), (with a radius of the water probe 1.4 Å) of each enzyme (the PDB format files) was applied. Fig. 8 4.5. Optimum pH and Temperature of Free and Immobilized Enzyme It was carried out by incubating the reaction mixture containing free and immobilized combined α-amylase and maltogenic amylase at various pH consist of 5, 5.5, 6, 6.5, 7, 7.5, 8, and 9 (pH 5–6.5, 100 mM sodium acetate buffer; pH 7–9, 50 mM phosphate buffer) at 65° C [40]. Under these conditions, enzyme activity was assessed and defined as µmol.min-1 for each of the pH values. For evaluating the performance of free and immobilized combined amylases the combined enzymes in the buffer solution (pH 5.5) were incubated at various temperatures (55, 65, 75, 85, and 95º C) without substrate for 90 minutes. After that, enzyme activity measurement was carried out by dinitrosalicylic acid procedure and displayed in micromole per min at any specified temperature. 4.6. Selection of the best Fungamyl to Maltogenase ratio In this step, at first fungamyl (10.72 mg/100 µl) and maltogenase (0.69 mg/100 µl) activity were quantified using by DNS method. Afterward, 1:1 to 1:50 ratios of Fungamyl:Maltogenase were prepared, and enzymatic hydrolysis was performed by thoroughly mixing 100 µL of each combined enzyme ratio with 900 µL substrate (liquified starch 1% w/v) in 9000 µL of 0.05 M sodium acetate buffer with pH 5.5, at 65° C for 60 min. Then, the maltose concentration was estimated based on maltose standard curve and absorption at 575 nm spectrophotometrically. 4.7. Fabrication of Nano-Magnetic Combi-CLEAs of α-amylase and Maltogenic amylase The concurrently aggregation and cross-linking process was performed by admix 100 µL of desirable enzyme mixture and different combined enzyme to lysine and/or BSA weight ratio (0.25-5 mg/ 1mg of combined enzyme protein) along with functionalized nanomagnetites at 1.7 times of protein content of combined enzymes to 900 µL of the precipitating agents (isopropanol, acetone, tert-butanol , acetonitrile, ethanol, and saturated ammonium sulphate) accompanied by adding of 1–100 mMol glutardialdehyde (25% v/v) for achieving crosslinking process. Afterward, via straightly binding of Combi-CLEAs onto MNPs sonication at 9

200 Hz was employed for 10 minutes at room temperature. Thereafter it was held at 2–4º C for 3–35 hours. Then, magnetic nanobiocatalyst was isolated from the liquid phase by using the centrifuge (11357×g) and washed with 50 mM phosphate buffer pH 7 three times. Finally, the activity of the immobilized enzymes was specified by DNS procedure. 4.8. Optimum Glutaraldehyde Concentration Glutaraldehyde is the most common reagent in biocatalysts fabrication. Accessible lysine amino groups that are present on the surface of the enzyme structure involved in the crosslinking process [41, 42]. The enzyme activity and catalytic performance of fabricated CLEAs are affected by the concentration of glutaraldehyde [43]. For this purpose, various glutaraldehyde concentrations (1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mM) were utilized together with tert-butanol (enzyme /solvent ratio was 1:9) as a precipitant for CLEAs formation under 1–35 h holding time at 2–4°C for producing an efficient, stable, and reusable CLEAs designation. 4.9. Optimal Time Keeping in CLEAs Formation To access the best holding time for formation of CLEAs with a stable and convenient structure, activity assessment was performed for fabricated NM-Combi-CLEAs of amylases after keeping the cross-linking cocktail for various time intervals (1, 2, 5, 10, 15, 20, 24, 30, and 35 h) at 2–4°C respectively. 4.10. Kinetic parameters of Free and NM-Combi-CLEAs amylases The Michaelis constant (Km) and maximum velocity of the enzyme reaction (Vmax) of free combined enzymes and NM-Combi-CLEAs of amylases (11.38 mg/100 µl) were assessed by the Lineweaver-Burk plot procedure for the description of the hydrolysis of corn starch using various concentrations (0.25, 0.5, 1, 3, 5 mg mL-1) of the substrate at 65° C (pH 5.5). For various mentioned substrate concentrations, the activity was determined in triplicates by DNS procedure via spectrophotometer. The acquired values were plotted by the inverse of the reaction rate (1/V) against the inverted value of the substrate concentration (1/[S]). [S]/V0 = 1/Vmax.[S]+ Km/Vmax

(1)

Which [S] is the concentration of the substrate (starch), V0 is the primary enzyme velocity, Vmax is the maximum enzyme velocity, and Km is the Michaelis constant. The assay mixture contained 11.41 mg.ml−1 of free combined Fungamyl 800 L (α-amylase) and Maltogenase L (Maltogenic amylase) with a ratio of 1 to 9 (1 Fungamyl: 9 Maltogenase) and NM-CombiCLEAs and numerous corn starch concentrations (0.25–5 mg mL−1) in sodium acetate buffer (50 mM, pH 5.5) at 65° C for 60 min [44, 45]. 4.11. Reusability of NM-Combi-CLEAs of amylases The reusability assessment of the fabricated nanobiocatalyst was carried out at the ending of each starch conversion cycle by entirely mixing 100 µL of NM-Combi-CLEAs (1:9 Fungamyl/Maltogenase ratio, and 11.38 mg protein/ 100 µl) with 900 µL substrate (liquified starch 1% w/v) in 9000 µL of 0.05 M sodium acetate buffer with pH 5.5, at 65° C for 60 min. Then, CLEAs was isolated from reaction media, and washed with 0.1 M phosphate buffer pH 7.0 and repeatedly was added to the mentioned reaction medium. The enzyme activity was defined after each cycle by assessment of starch conversion to maltose via DNS method at

10

575 nm by employing of maltose standard curve. The remaining activity was evaluated based on the enzyme activity at the first cycle 100 percent. 4.12. Immobilization Yield Activity yield was calculated as the difference between the total units of the native solution and those of the supernatant solution, multiplied by 100 and divided by the total units of the native solution [46].

(2)

4.13. Statistical Methods The values were represented as mean of triplicates and were analyzed via general factorial design with regression coefficient (R2) of 0.999 by employing Design Expert, version 10 software. The parameters were statistically significant at p<0.05.

Abbreviations V0: initial enzyme velocity; [S]: substrate concentration; Km: Michaelis constant; Vmax: maximum enzyme velocity; CLEAs: Cross-linked enzyme aggregates; CLE: Cross-linked enzyme; CLEC: crystallized enzymes; m-CLEAs: magnetic-CLEAs; m-combi-CLEAs: magnetic-combined-CLEAs; NM-Combi-CLEAs: nanomagnetic combined CLEAs; GH family: glycoside hydrolase family; BSA: bovine serum albumin; SPI: soy protein isolate; DNS: dinitrosalicylic acid; DLS: dynamic light scattering; PDI: polydispersity index; Ȥpotential: zeta-potential; FTIR: Fourier transform infrared spectroscopy; PDB: protein data bank; FE-SEM: field emission scanning electron microscope; MNPs: magnetic nanoparticles; EDX: Energy-dispersive X-ray spectroscopy.

Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Conflicts of Interest The author declare that there is no conflict of interest regarding the publication of this paper. Funding This work was supported by the Iranian Research Organization for Science and Technology of Iran, Tehran, Iran [grant 2016/1010291011]. Acknowledgments The financial support provided by the Iranian Research Organization for Science and Technology (IROST) of Iran (grant 2016/1010291011) is appreciated. References 11

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[39] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248-54. [40] M. Sugahara, Crystal Structure of α-amylase from Aspergillus oryzae, PDB data bank. https://doi.org/10.2210/pdb3VX0/pdb. [41] J.S. Kim, S.S. Cha, H.J. Kim, T.J. Kim, N.C. Ha, S.T. Oh, H.S. Cho, M.J. Cho, M.J. Kim, H.S. Lee, J.W. Kim, K.Y. Choi, K.H. Park, B.H. Oh, Crystal structure of a maltogenic amylase provides insights into a catalytic versatility, J. Biol. Chem. 274 (1999) 26279-26286. [42] T. Yewale, R.S. Singhal, A.A. Vaidya, Immobilization of inulinase from Aspergillus niger NCIM 945 on chitosan and its application in continuous inulin hydrolysis, Biocatal. Agric. Biotechnol. 2 (2013) 96-101. [43] O. Barbosa, C. Ortiz, A. Berenguer-Murcia, R. Torres, R.C. Rodrigues, R. FernandezLafuente, Strategies for the one-step immobilization-purification of enzymes as industrial biocatalysts, Biotechnol. Adv. 33 (2015) 435-456. https://doi.org/10.1016/j.biotechadv.2015.03.006. [44] E.T. Hwang, M.B. Gu, Enzyme stabilization by nano/microsized hybrid materials, Eng. Life Sci. 13 (2012) 49-61. https://doi.org/10.1002/elsc.201100225. [45] R. Rodrigues, C. Ortiz, A. Berenguer-Murcia, R. Torres, R. Fernández-Lafuente, Modifying enzyme activity and selectivity by immobilization, Chem. Soc. Rev. 42 (2013) 6290-6307. https://doi.org/10.1039/c2cs35231a. [46] I. Antal, M. Koneracka, M. Kubovcikova, V. Zavisova, I. Khmara, D. Lucanska, L. Jelenska, I. Vidlickova, M. Zatovicova, S. Pastorekova, N. Bugarova, M. Micusik, M. Omastova, P. Kopcansky, D,L-lysine functionalized Fe3O4 nanoparticles for detection of cancer cells, Colloids Surf. B: Biointerfaces. 163 (2018) 236–245. http://doi.org/10.1016/j.colsurfb.2017.12.022.

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Figure Captions: Fig. 1. Scanning electron microscopy images of Fe3O4 and immobilized amylases. (a) magnetic nanoparticles, (b&c) NM-Combi-CLEAs of amylases with different magnifications. Fig. 2. Particle size distribution of magnetic nanoparticles and NM-Combi-CLEAs by DLS methods. (a) magnetic nanoparticles, (b) NM-Combi-CLEAs. Fig. 3. Fungamyl and Maltogenase activity characterization. (a) comparison of free Fungamyl and Maltogenase activity, (b) optimum free Fungamyl to Maltogenase ratio, (c) comparative starch hydrolytic activity of free and immobilized combined amylases, (d) reducing sugars production trend of combined amylases before and after immobilization by NM-Combi-CLEAs method. Each data point represents the mean of triplicate experimental measurements for enzyme activity; error bars represent ±1 standard deviation. Fig. 4. Effect of various precipitants and enzyme to lysine-BSA ratio on the free and immobilized combined amylases activity. (a) precipitating agents, (b) enzyme to lysine ratio, and/or enzyme to BSA. Each data point represents the mean of triplicate experimental measurements for enzyme activity; error bars represent ±1 standard deviation. Fig. 5. Effect of different glutaraldehyde concentrations and holding time on free and immobilized amylases activity. (a) glutaraldehyde concentrations, (b) holding time. Each data point represents the mean of triplicate experimental measurements for enzyme activity; error bars represent ±1 standard deviation. Fig. 6. Effect of various pH and temperatures on free and immobilized amylases activity. (a) different pH, (b) various temperatures. Each data point represents the mean of triplicate experimental measurements for enzyme activity; error bars represent ±1 standard deviation. Fig. 7. FTIR spectra and reusability results for magnetic nanoparticles and NM-CombiCLEAs of amylases. (a) FTIR spectra at 4000-400 cm-1 of magnetic nanoparticles compared to NM-Combi-CLEAs. (b) effect of the enzyme reuses on the activity of NM-Combi-CLEAs of amylases during starch hydrolysis at pH 5.5 and 65ºC for 60 min. Each data point represents the mean of triplicate experimental measurements for enzyme activity; error bars represent ±1 standard deviation. Fig. 8. Similarity results between maltogenic amylase from Thermus sp. IM6501 (tr) and from Bacillus subtilis (sp) amino acid sequences by LALIGN program. (*) represents similar amino acids in two different sequences.

16

17

Table 1 EDX for elemental composition of Fe3O4 and NM-Combi-CLEAs. Elements

Fe3O4

NM-Combi-CLEAs

(W%)

(W%)

C

15.98

32.56

N

10.34

26.06

O

70.84

40.75

Fe

2.84

0.63

Total

100

100

Table 2 Accessible surface lysine residues in α-amylase and Maltogenic amylase molecules. Enzyme

PDB Code

Total amino acids

Mass (Da)

Total Lysine

Accessible Surface Lysine

α-amylase from Aspergillus oryzae

3VX0

499

54,810

20

6

Maltogenic amylase from Bacillus subtilis (Thermus sp.IM6501)

1SMA

588

68,722

30

7

Total

-

1087

123,532

50

13

Accessible Surface Area of α-amylase and Maltogenic amylase were estimated by GETAREA bioinformatics

program for calculation of Solvent Accessible Surface Areas, with radius of the water probe 1.4 Å.

Table 3 Kinetic parameters of free combined amylases and NM-Combi-CLEAs

Free combined enzymes NM-Combi-CLEAs

Vmax (µmol.min-1)

Km (M)

Vmax/ Km

kcat/Km

10.95±0.042

4.86 × 10−4±0.000021

2.25×104±0.042 0.41×106±0.0014

9.98±0.057

3.33 × 10−4±0.000017

3.0×104±0.128

(M-1s-1)

0.54×106±0.0015

All values are averaged with standard deviations collected from at least 3 independently assembled experiments.

Highlights •

A nanomagnetic combi-CLEAs method which converts starch into maltose.

•

Efficient starch conversion to maltose obtaining by nano co-immobilized amylases.

•

Reusable NM-combi-CLEAs of amylases with strong operational stability.

•

Higher affinity for substrate acquiring by NM-combi-CLEAs of amylases.

•

Higher thermostability representing by NM-combi-CLEAs of amylases.

Conflicts of Interest The author declare that there is no conflict of interest regarding the publication of this paper.