Purification, partial characterization, and covalent ...

Purification, partial characterization, and covalent immobilization–stabilization of an extracellular α-amylase from Aspergillus niveus Tony Marcio Silva, André Ricardo de Lima Damásio, Alexandre Maller, Michele Michelin, Fabio M. Squina, João Atílio Jorge, et al. Folia Microbiologica Official Journal of the Institute of Microbiology, Academy of Sciences of the Czech Republic and Czechoslavak Society for Microbiology ISSN 0015-5632 Volume 58 Number 6 Folia Microbiol (2013) 58:495-502 DOI 10.1007/s12223-013-0230-1

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Author's personal copy Folia Microbiol (2013) 58:495–502 DOI 10.1007/s12223-013-0230-1

Purification, partial characterization, and covalent immobilization–stabilization of an extracellular α-amylase from Aspergillus niveus Tony Marcio Silva & André Ricardo de Lima Damásio & Alexandre Maller & Michele Michelin & Fabio M. Squina & João Atílio Jorge & Maria de Lourdes Teixeira de Moraes Polizeli

Received: 22 December 2011 / Accepted: 10 February 2013 / Published online: 6 March 2013 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2013

Abstract An extracellular amylase secreted by Aspergillus niveus was purified using DEAE fractogel ion exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5 % polyacrylamide gel electrophoresis (PAGE) and 10 % sodium dodecyl sulfate (SDS-PAGE). The enzyme exhibited 4.5 % carbohydrate content, 6.6 isoelectric point, and 60 and 52 kDa molar mass estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The amylase efficiently hydrolyzed glycogen, amylose, and amylopectin. The end-products formed after 24 h of starch hydrolysis, analyzed by thin layer chromatography, were maltose, maltotriose, maltotetraose, and maltopentaose, which classified the studied amylase as an α-amylase. Thermal stability of the α-amylase was improved by covalent immobilization on glyoxyl agarose (halflife of 169 min, at 70 °C). On the other hand, the free α-amylase showed a half-life of 20 min at the same temperature. The optima of pH and temperature were 6.0 and 65 °C for both free and immobilized forms. T. M. Silva : M. Michelin : J. A. Jorge : M. d. L. T. d. M. Polizeli (*) Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, Monte Alegre, 14.040-901, Ribeirão Preto, SP, Brazil e-mail: [email protected] A. R. d. L. Damásio : A. Maller Departamento de Bioquímica e Imunologia (FMRP), Universidade de São Paulo, Ribeirão Preto, Brazil F. M. Squina Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, Brazil

Introduction The enzymatic hydrolysis of starch is one of the most important biocatalytic reactions, which are carried out in industrial scale. α-Amylase (1,4-α-D-glucan glucanohydrolase; EC 3.2.1.1) is the key enzyme in the metabolism of a wide variety of living organisms that use starch as carbon and energy sources. It belongs to the family GH13, the major glycoside hydrolase family acting on substrates containing α-glucoside linkages (Janecek 1997; MacGregor et al. 2001; van der Maarel et al. 2002). In this family, there are not only hydrolases (EC 3) but also transferases and isomerases (EC 2 and 5, respectively) (Horvathova et al. 2000). α-Amylases are endo-acting enzymes which randomly hydrolyze α-1,4-glycosidic bonds between adjacent glucose units in a starch polymer leading to the formation of linear and branched oligosaccharides (Regulapati et al. 2007). These enzymes are the most important ones because of their potential application in industrial processes such as in fermentation, textiles, pharmaceuticals, detergent, brewing, baking, paper, and food industries (Gupta et al. 2003). α-Amylase is essential during starch processing and it plays an important role in the liquefaction of this polymer and in the subsequent saccharification where larger carbohydrate chains are hydrolyzed and converted into smaller carbohydrates (Chaplin and Bucke 1990; Baks et al. 2006). α-Amylases are originated from different sources such as plants, animals, and microorganisms, but the microbial amylase is the most useful industrial enzyme due to their high productivity (Tanyildizi et al. 2005). A number of reports are available on α-amylase produced by different bacteria and fungi, but the most thoroughly studied and industrially useful sources are Bacillus and Aspergillus sp. (Jensen and Olsen 1992; Nigam and Singh 1995; Crabb and Mitchinson 1997). Extremophiles

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α-amylases were recently reported by Sharma et al. (2012). It is necessary to find more stable enzymes that work under adverse conditions, such as the presence of organic solvents, salts, and pH variations. Although enzymes are excellent catalysts, having high specificity and selectivity, most of them operate in a narrow range of pH and temperature, which hinders their use in processes where conditions are extreme. Many of these adversities can be overcome by the immobilization of the catalysts that are not stable. Immobilized enzymes are used in biotechnology, food industries, analytical chemistry, and biomedicine. Immobilized enzymes have various advantages over free enzymes including easy separation of the reactants, products, and reaction media, easy recovery of the enzyme, and repeated or continuous reuse besides an improvement in thermal stability (Vandamme 1983; Varavinit et al. 2002). In the present study, we purified and immobilized a thermostable α-amylase, which produces glucose and malto-oligosaccharides from glycogen, soluble starch, and related polysaccharides. Biochemical characterization of the free enzyme and also from an immobilized form on a glyoxyl agarose support was carried out. The current work is the first study of the properties of α-amylase from Aspergillus niveus, an excellent producer of other enzymes such as xylanase and α-glucosidase (Peixoto-Nogueira et al. 2008; Betini et al. 2009; Silva et al. 2009).

Materials and methods Organism and growth conditions A. niveus was isolated in our laboratory from Mangifera indica. The microorganism was identified and deposited in the culture collection at Pernambuco Federal University (PE, Brazil). It was maintained on slants of potato dextrose agar medium covered with mineral oil and stored at 4 °C. Approximately 107 conidia per milliliter from 3-day-old cultures were inoculated into 125 mL Erlenmeyer flask containing 25 mL of Khanna medium modified (Khanna et al. 1995) as follows: 0.1 % yeast extract, 5 % Khanna salt solution (2 % NH4NO3, 1.3 % KH2PO4, 0.36 % MgSO4.7H2O, 0.1 % KCl, 0.07 % ZnSO 4 .H 2 O, 0.014 % MnSO 4 .H 2 O, 0.007 % Fe2(SO4)3.6H2O, and 0.006 % CuSO4.5H2O), and 1 % starch, and initial pH6.5. After inoculation, the cultures were incubated under agitation, or static condition, at 40 °C, for up to 72 h. Culture filtrates were obtained by filtration through filter paper no. 1 in a Büchner funnel. The filtrate was used as a source of amylase crude extracellular. Purification of α-amylase All steps were carried out at 4 °C. The culture filtrate was dialyzed overnight against 10 mmol/L Tris–HCl buffer, pH

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7.5, and a volume of 100 mL was applied to a DEAE Fractogel TSK 650 M column (20×70 mm) equilibrated with the same buffer, and eluted at a flow rate of 100 mL/h with 200 mL of a linear gradient (0–1 mol/L) of sodium chloride in the same buffer. The fractions showing amylolytic activity were pooled, dialyzed against distilled water, lyophilized, and suspended in 2 mL of 100 mmol/L sodium acetate buffer, pH5.0. This sample was applied to a Sephacryl S-200 gel filtration column (20×850 mm) equilibrated and eluted in 100 mmol/L sodium acetate buffer, pH5.0. Fractions of 1 mL were collected at a flow rate of 18 mL/h and the active fractions were pooled and used for enzyme characterization. Elution of protein was monitored by measuring the absorbance at 280 nm. Enzymatic assays, determination of protein, sugar content, and kinetic constant α-Amylase activity was determined by measuring the production of reducing sugar using 3,5-dinitrosalicylic acid as described by Miller (1959). The assay was carried out at 65 °C, for 5 min, using a 1.0 % starch solution in 0.1 mol/L sodium acetate buffer, pH5.0. One unit of α-amylase activity was defined as the amount of enzyme that releases 1 μmol of maltose per minute. When p-nitrophenyl-α-Dglucopyranoside was used as substrate, the activity was measured in a mixture containing 0.20 mL of 0.1 mol/L sodium acetate, pH5.0, 0.05 mL of a 2 mol/L substrate solution, and 0.1 mL of enzyme. After 1 min of incubation at 65 °C, the reaction was stopped with 1 mL 2 mol/L NaCO3, and the phenolate released was quantified by spectrophotometer at 410 nm. For the specific determination of α-amylase, p-nitrophenyl-β-D-maltoheptaoside was used as substrate and the activity was measured in a mixture containing 0.20 mL of 0.1 mol/L sodium acetate, pH5.0, 0.05 mL of a 2 mmol/L substrate solution, and 0.1 mL of the enzyme in the presence of 16 U of αglucosidase (Silva et al. 2009). After 5 min of incubation at 65 °C, the reaction was stopped with 1 mL 2 mol/L NaCO3, and the phenolate released was quantified by spectrophotometer at 410 nm. Protein concentration was estimated as described by Lowry et al. (1951) using bovine serum albumin as a standard. Total neutral carbohydrate was quantified by the phenol–sulfuric acid method (Dubois et al. 1956) using D-mannose as a standard. In order to determine which substrate was more efficiently hydrolyzed, the apparent kinetic parameters KM and kcat of the free and immobilized enzyme were determined using starch and glycogen (1–60 mg). The reactions were carried out at 65 °C (optimum temperature), and the apparent KM values were calculated by Hanes (1932) plots.

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Polyacrylamide gel electrophoresis analysis Polyacrylamide gel electrophoresis performed under nondenaturing conditions (5 % polyacrylamide gel electrophoresis (PAGE)) was carried out by Davis (1964) method and under denaturing conditions (10 % sodium dodecyl sulfate (SDS)-PAGE) according to Laemmli (1970). The proteins were stained with 0.25 % Coomassie Brilliant Blue R-250 and destained with methanol–acetic acid–water (3:1:6). α-Amylase activity on polyacrylamide gels was detected using 1 % soluble starch as substrate. The gel containing starch was placed in a bath at 60 °C for 30 min and soon after that immersed in an iodine solution. Isoelectric focusing was carried out using Pharmalyte Ampholyte (pH3.0–10.0; O’Farrel et al. 1977). Mass spectrometry Protein band was excised, reduced, alkylated, and submitted to in-gel chymotrypsin digestion. An aliquot (4.5 μL) of the resulting peptide mixture was separated by C18 (75 μm× 100 mm) RP-nanoUPLC (nanoAcquity, Waters) coupled with a Q-Tof Ultimamass spectrometer (Waters) with nano-electrospray source at a flow rate of 0.6 μl/min. The gradient was 2–90 % (v/v) acetonitrile in 0.1 % (v/v) formic acid over 45 min. The instrument was operated in the “top three” mode, in which one MS spectrum is acquired followed by MS/MS of the top three most-intense peaks detected. The spectra were acquired using software MassLynx v.4.1 (Waters Corporation, MA, USA) and the raw data files were converted to a peak list format (mgf) by the software Mascot Distiller v.2.3.2.0, 2009 (Matrix Science Ltd.). The MS/MS profile was searched against predicted protein sequence of A. niveus α-amylase using engine Mascot v.2.3 (Matrix Science Ltd.) with carbamidomethylation as fixed modification, oxidation of methionine as variable modification, one chymotrypsin missed cleavage, and a tolerance of 0.1 Da for both precursor and fragment ions. Enzymatic characterization The optimum pH was determined at 65 °C using citrate phosphate buffer (pH range, 3.0–7.0). The pH stability was determined at 25 °C, for 24 h, after the pre-incubation of the diluted enzyme in citrate phosphate buffer at different pH (range, 3.0–9.0). The optimum temperature was determined with 0.1 mol/L sodium acetate buffer, pH5.5. The thermostability was determined by measuring the residual activity after incubating the free and the immobilized enzymes in the absence of substrate at 70 °C in 0.1 mol/L sodium acetate buffer, pH5.0, for 6 h. It was prepared a support suspension (100 mg/mL) in acetate buffer and under controlled-agitation it was collected 50 μL of the suspension

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for enzyme activity measurement. In order to avoid the support retention, we cut the tips. The free or the immobilized enzymes were incubated at 70 °C for different periods of time, cooled in ice bath, and after that all enzymatic assays were carried out as described. For the determination of pH and temperature stabilities, the enzymatic assays were carried out using 1 % soluble starch as substrate. For the determination of the effect of metallic ions on α-amylase activity, assays were performed in 1 and 10 mmol/L final concentration of salts, where the activity in the absence of salts was defined as the control. Chromatography of hydrolysis products Chromatographic analysis of the reaction end products of αamylase activity on soluble starch was carried out using thin layer chromatography. A volume of 10 μL of the reaction mixture was applied on silica gel plates (DC-Alufolien Kieselgel 60, Merck), and subjected to two sequential ascending chromatography runs using butanol/ethanol/water (5:3:2) as the solvent system. After air drying the plate, the spots were developed by spraying with a solution of H2SO4 and methanol (1:9) containing 0.2 % orcinol, and heating at 100 °C (Fontana et al. 1988). Immobilization on DEAE Cellulose The procedure was carried out by mixing 1 g of DEAE cellulose in 10 mL of α-amylase solution (crude enzyme at 0.9 mg/mL) and incubated in Tris-buffer pH7.0 with constant stirring at room temperature for 30 min. Then, the DEAE cellulose was filtered and washed with 100 mL of distilled water. After that, the immobilized enzyme was used for activity measurements. The washing was found to be free of enzyme activity. Immobilization on CNBr-activated agarose In order to perform this experiment, the instructions from GE Healthcare Life Science were followed. About 1 g of washed CNBr activated agarose 4BCL was prepared and it was incubated with 10 mL of 0.1 mol/L sodium phosphate buffer at pH 7.0 and 10 mL of alpha-amylase solution, for 15 min at 4 °C under gentle stirring. After that, the immobilized preparation was filtered and the remaining reactive groups were blocked with 1 mol/L ethanolamine at room temperature for 2 h with gentle stirring. Finally, the derivative was washed with 0.1 mol/L sodium phosphate buffer, pH7.0. Immobilization on glyoxyl agarose Immobilizations were performed at pH10.0–10.5 (0.1 mol/L sodium bicarbonate buffer) and the pH was controlled


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Results and discussion

150

Purification of extracellular α-amylase and properties

130

100

70

55

40

35

a

b

c

d

Fig. 1 Electrophoresis analysis of purified α-amylase: a molecular weight ladder, b purified α-amylase in 10 % SDS-PAGE, c developed for α-amylase activity using iodine solution, d 5 % PAGE of the purified α-amylase revealed with Coomassie blue

through the addition of 2 mol/L sodium bicarbonate pH 10.5, when necessary the pH was lower up to 10. The procedure was carried out as follows: about 1 g of glyoxyl support (Guisán et al. 1993) was added to 10 mL of αamylase solution. The mixture was maintained at 25 °C for 12 h. A reference suspension under identical conditions, using reduced glyoxyl agarose, was used as a control. Finally, the immobilized preparation was reduced for 30 min at 25 °C with sodium borohydride (1 mg/mL). After that, the preparation was washed with an excess of distilled water and assayed. All the derivatives were assayed as described above using 50 μL of a solution at 100 mg/mL of derivative. Fig. 2 Amino acid sequence of the α-amylase from A. fumigatus. The protein α-amylase from A. niveus was identified by mass spectrometry using the amino acid sequences of tryptic peptides and they are indicated in bold and underlined

A volume of 100 mL of culture filtrate was applied to a DEAE fractogel column and the α-amylase activity was eluted at approximately 0.3 mol/L NaCl (data not shown). After pooling the fractions containing amylolytic activity, the concentrated sample was applied to a Sephacryl S-200 column equilibrated and eluted as described in “Materials and methods” section. This α-amylase pool was used for biochemical studies. The purified protein migrated as a single polypeptide under 10 % SDS-PAGE (b in Fig. 1) and 5 % PAGE (c and d in Fig. 1). The molecular mass estimated by SDS-PAGE and gel filtration was 60 and 52 kDa, respectively, which is similar to the molecular mass of 52.5 kDa by the α-amylase from Aspergillus flavus (Khoo et al. 1994) and 52 kDa by the α-amylase from Aspergillus terreus ATCC76080 (Chang et al. 1995). Other fungal α-amylases described (Wanderley et al. 2004; Balkan and Ertan 2010) presented an apparent molecular mass of 75 and 32.5 kDa, respectively. Electrofocusing of the purified α-amylase of A. niveus revealed a pI of 6.6, in contrast, the α-amylase from A. flavus presented a pI of 3.5 (Khoo et al. 1994). The A. niveus α-amylase is a glycoprotein and contains 4.5 % carbohydrate. Another α-amylase from Aspergillus tamarii presented a carbohydrate content of 32 % (Moreira et al. 2003). The triptic peptide sequence analyzed by mass spectrometry showed identity to A. fumigatus α-amylase, as can be observed in Fig. 2. Hydrolysis of different substrates The α-amylase activity from A. niveus against various substrates is shown in Table 1. The enzyme hydrolyzed some polysaccharides, such as soluble starch, amylose, amylopectin, and glycogen. Among the malto-oligosaccharides, the enzyme preferentially hydrolyzed maltopentaose, maltotriose, maltotetraose, and malto-oligosaccharide (G10). Sucrose, trehalose, α-cyclodextrin, β-cyclodextrin, and p-nitrophenyl α-D-glucopyranoside were not hydrolyzed. The classification of the A. niveus enzyme as an α-amylase was based on the


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Table 1 Hydrolysis of different composts by α-amylase from A. niveus

Table 2 Effect of metal ions, EDTA, and β-mercaptoethanol on α-amylase activity

Substrate

Relative activity (%)

Ions

Soluble starch Amylose Amylopectin Glycogen Maltooligosacharide (G10) Maltopentaose Maltotetraose Maltotriose p-Nitrophenyl-β-D-maltoheptaoside α-PNPG α-Cyclodextrin β-Cyclodextrin Trehalose

100 82 (±0.3) 84 (±0.2) 110 (±0.4) 23 (±0.3) 108 (±0.4) 80 (±0.2) 106 (±0.3) + 0 0 0 0

Sucrose

0

+ presence of enzymatic activity Results are mean values of three replicates

starch hydrolysis products and also on the hydrolysis of pnitrophenyl-β-D-maltoheptaoside (Table 1). After 30 min of reaction with 1 % soluble starch, maltose, maltotriose, maltotetraose, and maltopentaose were detected as the endproducts (Fig. 3). According to Henrissat (1991), the α-amylases are classified into family13 glycosyl hydrolases. This group of enzymes share a number of common characteristics such as a

Relative activity (%) 1 mmol/L

10 mmol/L

Control CaCl2 BaCl2 NH4F NaBr KH2PO4 MnCl2.4H2O MgCl2.6H2O HgCl2 Pb(C2H3O2)2.3H2O

100 (±0.3) 109 (±0.4) 100 (±0.6) 117 (±0.3) 114 (±0.7) 100 (±0.4) 180 (±0.7) 108 (±0.3) 100 (±0.2) 100 (±0.4)

100 109 95 123 112 120 144 114 10 90

(±0.2) (±0.3) (±0.4) (±0.4) (±0.5) (±0.6) (±0.5) (±0.4) (±0.3) (±0.3)

NH4Cl CuCl2 KCl NaCl EDTA NaH2PO4.H2O β-Mercaptoethanol CoCl2.6H2O AgNO3 Fe2(SO4) ZnCl2

107 (±0.5) 100 (±0.3) 106 (±0.4) 100 (±0.4) 100 (±0.4) 128 (±0.6) 161 (±0.7) 100 (±0.3) 70 (±0.5) 90 (±0.4) 139 (±0.6)

116 92 110 112 100 111 100 116 46 55 100

(±0.4) (±0.2) (±0.3) (±0.5) (±0.6) (±0.4) (±0.4) (±0.5) (±0.4) (±0.6) (±0.3)

Results are mean values of three replicates. Control (490 U/mg protein) was considered as 100 %

(β/α)8 barrel structure, the hydrolysis or formation of glycosidic bonds in the conformation, and a number of conserved amino acid residues in the active site (van der Maarel et al. 2002). In a more recent division, the fungal α-amylases

G

Table 3 Stability factor of different α-amylase derivatives

G2

Support

Incubation time (min)

Remained activitya

Stability factorb

DEAE-cellulosec CNBr-activatedd Glyoxyle

15 15 240

100 100 90

1.2 2.7 8.2

G3 G4

G5

a

Residual α-amylase activity after incubation with different supports

b

Stability factor is the ratio between half-life times of different derivatives (inactivated at 70 °C and pH7.0) and the half-life time of soluble enzyme c

S

T0

a

b

c

Fig. 3 Thin layer chromatography analysis of the end products of hydrolysis of purified α-amylase under soluble starch: S standard (G glucose, G2 maltose, G3 maltotriose, G4 maltotetraose, and G5 maltopentaose); T0 zero time of assay; a 10 min, b 30 min, and c 60 min of hydrolysis

Enzyme immobilization on DEAE-cellulose by ionic interaction

d

Enzyme immobilization on CNBr-activated agarose (15 min, pH7.0, and 4 °C). Very likely, the enzyme is immobilized by a one-point attachment involving the amino terminal residue e Enzyme immobilization on glyoxyl agarose. The enzyme is multipoint covalently immobilized, at pH10, through the region having the highest amount of Lys groups


a

b 120

120

Relative activity (%)

100 Relative activity (%)

Fig. 4 Effect of pH and temperature on activity and stability of the soluble and immobilized α-amylase. a pH activity, b temperature activity, c pH stability, and d termal stability. The thermal inactivation was determined at 70 °C. Filled square soluble enzyme, empty square glyoxyl agarose derivative, empty circle DEAE cellulose derivative, and filled triangle CNBr-activated agarose derivative

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80 60 40

100

80

60

20 40

0 3

4

5

6

7

40

8

50

c

70

80

90

d 110

100

100

90

90

80

Residual activity (%)

Residual activity (%)

60

Temperature (0C)

pH

80 70 60 50 40 30

70 60 50 40 30

20

20

10

10 0

0 3

4

5

6

7

8

9

10

0

50

100

pH

of the GH13 family are classified into subfamilies GH13_1 and GH13_5. This latter subfamily, GH13_5, comprises an intracellular α-amylase with low similarity to fungal α-amylases of family GH13_1 (van der Kaaij et al. 2007). Then, our results suggested that A. niveus amylase is an enzyme with endoamylolytic character, classified as α-amylase (EC 3.2.1.1 1,4-α-glucan glucanohydrolase) of the GH13_1 subfamily, which include the extracellular fungal amylolytic enzyme. Influence of salts, β-mercaptoethanol and EDTA Many fungal amylases described in the literature are activated by metal ions (Ali and Moneim 1989; Aquino et al. 2003). The α-amylase from A. niveus showed a slight increase in its activity in the presence of many salts. This enzyme was activated 17, 14, 80, 28, 39, and 61 % in presence of 1 mmol/L of NH 4F, NaBr, MnCl 2 .4H 2O, NaH 2PO 4H 2O, ZnCl 2 and

150

200

250

300

350

400

Time (min)

β-mercaptoethanol, respectively (Table 2). In the concentration of 10 mmol/L, the α-amylase activity was increased in 23, 20, 16, 12, and 16 %, in the presence of NH4F, KH2PO4, NH4Cl, NaCl, and CoCl2.6.H2O, respectively. HgCl2, AgNO3, and Fe2 (SO4) drastically inhibited the enzyme activity. Immobilization of α-amylase We tried to improve the α-amylase stability at 70 °C carrying out immobilization assays. The used supports were DEAEcellulose, CNBr-activated agarose, and glyoxyl agarose. The remaining activity after the incubation with these supports was 100 % for DEAE cellulose and CNBr activated agarose, and 90 % for glyoxyl agarose with stabilized factor of 1.2, 2.7, and 8.2 for each derivative, respectively (Table 3). The free and the immobilized α-amylase presented identical pH (pH 6.0, Fig. 4a) and optimum temperature of 65 °C (Fig. 4b). The

Table 4 Kinetic parameters of the α-amylase from A. niveus Enzymatic state

Substrate

KM (mgmL−1)

Vmax (U mg−1 protein)

kcat (s−1)

kcat/ KM (s−1 mg−1 mL−1)

Free enzyme

Starch Glycogen Starch Glycogen

4.2±0.3 3.2±0.2 5.7±0.5 4.0±0.3

168±7 260±9 138±5 193±8

250 387 205.4 287.2

59.5 120.9 36 71.8

Glyoxyl–agarose derivative


immobilized α-amylase on glyoxyl agarose was more pH stable (range, pH4–7) compared to the free enzyme, DEAE cellulose derivative, and CNBr-activated agarose derivative (pH4.5–6.5, Fig. 4c). The thermal inactivation profiles of α-amylase showed that except with DEAE cellulose, the derivatives (enzyme + support) presented improved stability profiles at 70 °C, when compared to the soluble enzyme (Fig. 4D). The derivatives formed through uni-point (CNBr activated agarose) and multipoint covalent bounds (glyoxyl agarose) showed higher stability when compared to the soluble enzyme and the derivative glyoxyl agarose was the most stable, due to its multipoint covalent bounds. The CNBr derivative was about three times more stable than the free enzyme at 70 °C; moreover, the glyoxyl derivative was 8.2 times more stable than the free enzyme. DEAE cellulose derivative showed a similar stability with soluble enzyme, probably due to the weak ionic interaction enzyme support. The improved stabilization of the derivative α-amylase glyoxyl agarose increases the perspectives for using this immobilized catalyst in biotechnological process. The substrate (starch and glycogen) concentration required for the maximum activity or for the saturation of the immobilized α-amylase was higher than that of the free enzyme. The KM obtained with the immobilized enzyme, calculated by Hanes plot (Hanes 1932) was increased as compared to the free enzyme, indicating the decreased affinity of the enzyme towards the substrate (starch and glycogen) after the immobilization. The kcat/KM values for the immobilized α-amylase were lower than those of the free enzyme for both substrates used (starch and glycogen), which indicates a smaller efficiency catalytic after immobilization (Table 4).

Conclusion Here, we have described the purification, biochemical characterization, and immobilization by ionic and covalent attachment of an α-amylase from A. niveus. The catalytic properties suggest that the enzyme can be used as a tool for industrial applications, where its properties (thermostability and pH range) are useful. This enzyme efficiently hydrolyzed starch, glycogen, amylose, amylopectin, and malto-oligosaccharides. The products of hydrolysis of 1 % soluble starch, as maltose, maltotriose, maltotetraose, and maltopentaose suggested that this enzyme is an α-amylase (1,4-α-glucan glucanohydrolase, EC 3.2.1.1). The use of enzymes with high stability in the presence of different metal ions, salts, and the enzyme immobilization by covalent attachment reinforce their importance for industrial applications where these characteristics are demanded.

501 Acknowledgments This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho de Desenvolvimento Científico e Tecnológico (CNPq). João Atílio Jorge; Maria de Lourdes Teixeira de Moraes Polizeli are Research Fellows of CNPq. Tony Márcio da Silva was a recipient of a FAPESP Fellowship and this work was part of his Doctoral Thesis. This project is part of National Institute of Science and Technology of the Bioethanol and National System for Research on Biodiversity (Sisbiota-Brazil, CNPq 563260/2010-6/FAPESP no 2010/52322-3). We thank Ricardo Alarcon, Mauricio de Oliveira for technical assistance and Mariana Cereia for technical assistance and English review. We also appreciate the availability of using of the Mass Spectrometer from National Laboratory Biosciences by coordination of Dr. Adriana Franco Paes Leme.

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