BIOCHEMISTRY CHARACTERIZATION OF α - AND β -AMYLASES FROM ZEA MAYS MALT AND STATISTICAL ANALYSIS...

BIOCHEMISTRY CHARACTERIZATION OF a- AND b-AMYLASESFROM ZEA MAYS MALT AND STATISTICAL ANALYSIS

APPROACH OF THE DEGRADATION OF MANIOC STARCH

JOSÉ CARLOS CURVELO-SANTANA1,3, GRAZIELA BATISTA FERREIRA2,JOANA PAULA MENEZES BIAZUS2, ROBERTO RODRIGUES DE SOUZA2 and

ELIAS BASILE TAMBOURGI1

1Department of Engineering of Chemical SystemsSchool of Chemical Engineering

State University of Campinas, University Campus “Zeferino Vaz”Av. Albert Einstein, 500, Post Code 6066

13081-970, Barão Geraldo, Campinas, São Paulo

2Department of Chemical EngineeringFederal University of Sergipe

University City, Prof. José Aloísio de Campos, Av. Marechal Rondon, S/NRosa Elze, São Cristóvão, SE, Brazil

Accepted for Publication July 23, 2007

ABSTRACT

This work examined the degradation of manioc starch by amylases frommaize malt and the biochemistry characterization of enzymes. Enzymecharacterization showed that the maize malt amylases have optimal pHbetween 4.5 and 6.5, with optimal temperature between 50 and 90C;Michaelis-Menten constant (Km) and maximum velocity of starch hydrolysis(Vmax) values for starch hydrolysis by maize malt were 7.69 ¥ 10-2 g/L and7.69 ¥ 102 g/L/min, respectively. Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis analysis of the eluted sample from ion-exchange expanded bedchromatography showed that molecular weights of a- and b-amylases frommaize malt were of 67.4 and 47.5 kDa, respectively. The hydrolysis times hadbeen found between 20 and 30 h, and the higher hydrolysis yield values wereabout 31%; it was higher than 27% (cited by literature) and was obtained at65C and at 9 and 11% (w/v) of any starch concentration studied.

3 Corresponding author. TEL: 55 19 35213952; FAX: 55 19 35213910; EMAIL: [email protected]

Journal of Food Process Engineering 31 (2008) 694–710. All Rights Reserved.© Copyright the AuthorsJournal Compilation © 2008 Wiley Periodicals, Inc.DOI: 10.1111/j.1745-4530.2007.00183.x

694

PRACTICAL APPLICATIONS

The results of this work have practical uses in the hydrolysis process ofstarch by food industries (beers, beverages, bakeries, syrups and others). Thiswork shows optimal conditions of using the amylases from maize malt instarch hydrolysis. Recently, a search for new energetic sources has beenunleashed after the pronounced governing of various countries on a possibleenergetic crisis. At present, Brazil is in search of methods that will increase theethanol offer by production of combustible alcohol. Thus, this study shows theoptimal conditions to produce fermentable syrup from manioc starch and as apotential substitute to sugarcane in alcohol production.

INTRODUCTION

Being a renewable fuel and a lower pollutant compared with petroleumfuels, ethylic alcohol is a combustible car fuel widely used in Brazil, and isdesired by various countries. Recently, a search for new energetic sources hasbeen unleashed after the pronounced governing of various countries on apossible energetic crisis. At present, Brazil is in search of methods that willincrease the ethanol offer by production of the combustible alcohol (Oliveiraand Vasconcelos 2006).

Starch, the main reserve carbohydrate of several crops, is highly abundantin nature and can be easily extracted with high purity and at low cost (Ceredaet al. 2001). The food industry has used starch for a long time as an ingredientbecause of its functional properties. Depending on the source, starches havedifferent applications, improving consistency, stability and other properties(Smith 1982). The most common plant sources for food starches used all overthe world are maize, wheat, potato and manioc. However, there is a constraintbecause of the desire of the industries in bringing new products to the marketand improving others (Takizawa et al. 2004). All starches have structural andcompositional differences, and a great number of studies are being conductedto show each particularity. These differences directly affect the yield of starchhydrolysis (Cereda et al. 2001). An example of these differences is the pres-ence of amylose in potato, sweet potato, maize and manioc starches, which are25.4% (w/w), 19.1% (w/w), 24.5% (w/w) and 18.6% (w/w), respectively(Cereda et al. 2001; Takizawa et al. 2004; Ferreira et al. 2005).

The hydrolysis of starch to products with low molecular weight, cata-lyzed by amylases, is the most important commercial enzyme process. Thehydrolyzed products are widely applied in food, paper and textile industries(Konsula and Liakopoulou-Kyriakides 2004). Because of the increasingdemand for these enzymes in various industries, there is enormous interest in

BIOCHEMISTRY CHARACTERIZATION OF ENZYMES 695

developing enzymes with better properties, such as raw starch-degrading amy-lases suitable for industrial applications and their cost-effective productiontechniques.

a-Amylases (EC 3.2.1.1.) are starch-degrading enzymes that catalyze thehydrolysis of internal a-1,4-D-glycosidic bonds in polysaccharides with theretention of a-anomeric configuration in the products. Most of the a-amylasesare metalloenzymes, which require calcium ions (Ca2+) for their activity,structural integrity and stability. These enzymes account for about 30% of theworld’s enzyme production (Sivaramakrishnan et al. 2006). It has an isoelec-tric point of 5.4, an optimum pH between 4.7 and 6.0, and an optimumtemperature of 75C (Dixon and Webb 1971; Wiseman 1987; Biazus et al.2005, 2006a).

b-Amylase (EC 3.2.1.2) is an extracellular enzyme that hydrolyzesa-D-(a-1,4)-glycosidic linkage of starch components, but it always leaves noreductor boundaries of the molecules. The enzyme has an excellent pHbetween 4.5 and 6.5, an excellent temperature between 55 and 57C, and attemperatures above 60C, this enzyme is inactive speedily. Its isoelectric pointis 4.8, and its molecular weight is below 50 kDa. This enzyme is found inseveral cereals (Dixon and Webb 1971; Wiseman 1987; Reguly 1996; Biazuset al. 2005, 2006a).

Maize (Zea mays) seeds and manioc (Manihot sculenta) root are agricul-tural products very common in Brazil, are commercialized in nature forms,or are minimally processed for low cost. Because of this, obtaining a- andb-amylases from maize malt and syrup of manioc starch will result in aggre-gate price of the maize and manioc cultures (Biazus et al. 2005, 2006a), andwill produce a new technology of alcohol production from this syrup.

This work examined the effect of temperature and starch concentration onthe yield of the process hydrolysis of manioc starch by amylases from maizemalt in an agitated bioreactor and maize malt amylase characterization. Aresponse surface methodology (RSM) analysis was made to obtain the optimalconditions to produce fermentable syrup from manioc starch and as a substi-tute to sugarcane in alcohol production.

MATERIALS AND METHODS

Materials

Reagents. An Amberlite IRA 410 ion-exchange resin from Vetec(São Paulo, Brazil) was used. Maize seeds and manioc roots were provided byEmbrapa (Aracaju, Sergipe, Brazil). Sodium acetate, di-nitric salicylic acid(DNS), acetic and phosphoric acids, and potassium sodium tartrate were

696 J.C. CURVELO-SANTANA ET AL.

provided by Vetec. Potassium hydrogen phosphate, di-sodium hydrogenphosphate, Tris, sodium dodecyl sulfate, Coomassie brilliant blue G-250 andchlorine acid were provided by Merck (Darmstadt, Germany). A Bio-Radelectrophoresis kit was provided by Bio-Rad (Hercules, CA).

Obtaining Manioc Flour. Manioc roots were washed with water at2 ppm of chlorine concentration; their rinds were removed, and roots were cutin pieces of about 2 cm3. They were dried at 100C every 8 h in a drier with aircirculation. Drying roots were crushed in a Willye TE 650 flour mill (Tecnal,São Paulo, Brazil) to obtain manioc flour (Takizawa et al. 2004; Ferreiraet al. 2005). The percent composition of manioc flour was determined bymethodologies showed by Ascar (1985), Bradford (1976), Ferreira et al.(2005), Leonel and Cereda (2000) and Takizawa et al. (2004).

Enzyme Obtained from Maize Malt. Maize seeds were selected,weighed and washed. These seeds absorbed between 40 and 45% of themoisture and germinated in the laboratory at room temperature and pressure(27C and 1 atm, respectively). The maize malt was dried at 54C for 5 h andstored at 5C (Curvelo-Santana 2003; Biazus et al. 2005, 2006a).

Methods

Enzyme Assays. Amylase was assayed using the Milles laboratorymethod (Miller 1959; Aguilar et al. 2000; Nirmala and Muralikrishna 2003;Biazus et al. 2006b; Toledo et al. 2007). Twenty milliliters of 2% solublestarch at pH 4.8 (0.1-M acetate buffer) was hydrolyzed by 500 mL of enzymesample for 10 min at 30 � 2C. Total protein concentration was determinedaccording to the dye binding method of Bradford (1976) with bovine serumalbumin (BSA) as protein standard. One unit of enzyme activity was defined asmicromole of glucose released per minute under the assay conditions. Thespecific activity was calculated as activity per milligram protein.

Amylase Purification. An expanded bed adsorption system in a glasscolumn of 1 ¥ 30 cm at 22C was used for amylase purification. The maize maltwas loaded into a chromatographic column by 0.05-M phosphate buffer atpH 7.0, and enzyme adsorption occurred on 2 g of Amberlite IRA 410 ion-exchange resin in an expanded bed of 4.2–8.0 cm. The elution was profiledwith 0.25-M NaCl at 14 mL/min in descendent flux (Kalil 2000; Biazus et al.2006b; Toledo et al. 2007). The activity and the protein content were measuredat various times during the adsorption, washing and elution periods (Biazuset al. 2006b; Toledo et al. 2007). Molecular mass was determined by sodium


dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmile1970).

Enzyme Characterization. The optimum pH of the enzyme was deter-mined by measuring its activity, after 10 min of reaction, in the pH rangevarying from 3.0 to 10.0 at 30 � 2C, using 5 mL of distilled water and 20 mLof a 2% (w/v) soluble starch solution in the following buffers: 0.1-M acetate(pH 3.0–5.0), 0.1-M phosphate (pH 5.5–7.5) and 0.1-M ammonium (pH 8.0–10). The optimum temperature was obtained by measuring its activity after10 min, in the temperature range varying from 10 to 95C, using some solublestarch solution in 0.1-M acetate buffer at pH 4.8 (Aguilar et al. 2000; Duedahl-Olsen et al. 2000; Ben Ali et al. 2001; Nirmala and Muralikrishna 2003;Mohamed 2004; Wanderley et al. 2004; Toledo et al. 2007). Activation energyof the soluble starch hydrolysis by amylases from maize malt was calculatedby the Arrhenius law (Aguilar et al. 2000; Toledo et al. 2007). Initial rates ofstarch hydrolysis were determined at various substrate concentrations (0.01–20 mg/mL). The kinetic constants Michaelis-Menten constant (Km) andmaximum velocity of starch hydrolysis (Vmax) were estimated by theLineweaver–Burk method (Aguilar et al. 2000; Duedahl-Olsen et al. 2000;Ben Ali et al. 2001; Nirmala and Muralikrishna 2003; Mohamed 2004;Wanderley et al. 2004; Toledo et al. 2007). SDS-PAGE was performed ona mini-PROTEAN II cell (Bio-Rad) with 12% acrylamide gel, using a bufferprotein molecular weight marker (Laemmile 1970).

Experimental Planning of Starch Degradation. A 22 full factorialplanning was used to evaluate the effects of bioreactor temperature, T (x1, C),and starch concentration, CStarch(x2, g/100 mL), on the hydrolysis yield (Y)(Marshal et al. 1999; Barros Neto et al. 2001; Biazus et al. 2005, 2006a).Assays are shown in Table 1 and variables coded are shown as follows:

xT

1

65

5= −

(1)

and x C2 10= −Starch (2)

Model fit was evaluated by analysis of variance and the optimization of starchhydrolysis by RSM in software statistic for Windows 6.0 (Barros Neto et al.2001).

Making of Bioreactors. The experiment of starch degradation wasassayed in a bioreactor with 2 L of total volume, with a mechanical agitator


for mixing, and a jacket for heating at operation temperatures. Bioreactorswere operated in boatload, which contained 1 L of manioc starch solutionand 5 g of maize malt, at constant agitation of 70 rpm and pH 5, as shownin Fig. 1 (Marshal et al. 1999; Konsula and Liakopoulou-Kyriakides 2004;Ferreira et al. 2005). Operation temperature and manioc starch contents werevaried according to Table 1. The glucose concentration (CGlucose, g/L)released into the bioreactor was measured by DNS method (Miller 1959).

TABLE 1.FACTORIAL PLANNING TO STUDY THE STARCH

HYDROLYSIS BY MAIZE MALT AMYLASES

Variables Levels

-1 0 +1

T (C), x1 60 65 70CStarch (%, w/v), x2 9 10 11

T, temperature; CStarch, starch concentration.

FIG. 1. BIOREACTOR PHOTO USED IN HYDROLYSIS ASSAYS OF MANIOC STARCH BYAMYLSES FROM MAIZE MALT


Theoretical glucose concentration (CStarch glucose, g/L) contained in maniocstarch is given in Eq. (3), and the hydrolysis yield (Y, %) is given in Eq. (4),as follows:

C CStarch glucose Starch= ⎛⎝

⎞⎠ ×180

162(3)

YC

C=

⎛⎝⎜

⎞⎠⎟

×glucose

Starch glucose

100 (4)

where 162 and 180 are molecular masses of dextrin monomers and glucose,respectively, and CStarch is the starch concentration into the bioreactor (Sleimanand Venturi Filho 2004; Ferreira et al. 2005).

RESULTS AND DISCUSSION

Biochemistry Characterization

Figure 2 shows that the maize malt amylases kept 80% of the activity ina pH range between 4.5 and 6.5; this large range pH is very important for theuse of this malt in several industrial processes. Literatures have reported thatpH optimum of the greater number of amylases had been found between thepH 4.5 and 6.5; this validates our assays (Dixon and Webb 1971; Wiseman

FIG. 2. pH EFFECT ON THE ACTIVITY OF THE MAIZE MALT AMYLASES


1987; Reguly 1996; Konsula and Liakopoulou-Kyriakides 2004; Mohamed2004; Wanderley et al. 2004; Sivaramakrishnan et al. 2006; Toledo et al.2007); however, there is neutral pH optimum of amylases in Lipomycesherperus and Lygus lineolaris, from Ascaris suum, Hyalomma dromedarii(Mohamed 2004) or basic pH optimum of a-amylases in Spodoptera fru-giperda, Spodoptera littoralis (pH 9.6) and Toxoplasma gondii (pH 8.4)(Duedahl-Olsen et al. 2000).

Figure 3 shows that there are two optimum temperatures: one at 50C, dueto b-amylase activity, and the other at 90C, due to that of a-amylase. Thisshows that maize malt can be used in hydrolysis process at temperature aboutor below 90C (Dixon and Webb 1971; Wiseman 1987; Reguly 1996; Konsulaand Liakopoulou-Kyriakides 2004; Mohamed 2004; Wanderley et al. 2004;Sivaramakrishnan et al. 2006; Toledo et al. 2007). However, we can observethat 80% of the activity of maize malt amylase was kept in temperatures lowerthan 60C for every 70 min. This thermal stability is a good factor to justify theuse of this enzyme in several industrial processes. Literatures have cited that70–75C and 50–55C are the optimum temperatures of the greater number ofa- and b-amylases, respectively (Dixon and Webb 1971; Wiseman, 1987;Reguly 1996; Sivaramakrishnan et al. 2006; Toledo et al. 2007).

Nirmala and Muralikrishna (2003) characterized the amylase from amalted cereal called finger millet, and optimal temperatures found were at 30and 70C, also due to a- and b-amylases, respectively. Aguilar et al. (2000) andWanderley et al. (2004) found amylases with optimal temperature lower thanthose of maize malt amylases. However, optimum temperature varies based onthe a-amylase source. Examples are as follows: a-amylase activity is at 40Cfor a-amylases from Heterorhabditis bacteriophora, A. suum or S. littoralis;50C for a-amylases from Cryptococcus flavus, Schwanniomyces alluvius

FIG. 3. TEMPERATURE EFFECT ON THE ACTIVITY OF THE MAIZE MALT AMYLASES


American Type Culture Collection 26074, Lipomyces kononenkoae orCandida antarctica CBS 667 (Wanderley et al. 2004); 65C for a-amylasesfrom T. gondii (Mohamed 2004); 55C for a-amylases from Lactobacillusmanihotivorans (Aguilar et al. 2000); 70C for a-amylases from Thermomyceslanuginosus (Ben Ali et al. 2001); and 135C for a-amylases from Bacillussubtilis (Konsula and Liakopoulou-Kyriakides 2004).

In Fig. 4, the Arrhenius law was used to obtain the activation energy ofstarch hydrolyses by amylases from maize malt, and it shows a value of about44.5 kJ/mol. Aguilar et al. (2000) found an activation energy of 32.6 kJ/molfor amylases from Lactobacillus manihotivorans LMG 18010T.

Figure 5 shows the kinetic curve of starch degradation by maize maltamylases by the Michaelis–Menten method. Equation (5) shows the derivationof the Km and Vmax constants using the Lineweaver–Burk method; their valueswere 7.69 ¥ 10-2 g/L and 7.69 ¥ 102 g/L/min (or 2.48 ¥ 104 U/mg), respec-tively. The Km value was lower than those for the amylases from finger millet(5.3–14.3 g/L) studied by Nirmala and Muralikrishna (2003), species ofgranivorous coleopterans (0.42 g/L) and lepidopterans (0.38 g/L), from A.suum (0.48–3.33 g/L), from H. dromedarii (10.0 g/L), from H. bacteriophora(6.5 g/L) (Mohamed 2004; Wanderley et al. 2004), from B. subtilis (7.79–13.41 g/L) (Konsula and Liakopoulou-Kyriakides 2004) and from L. maniho-tivorans (3.44 g/L) (Aguilar et al. 2000). The Vmax value was higher than thoseof their cited amylases. This shows that maize malt amylases have high affinityto the soluble starch.

FIG. 4. DETERMINATION OF ACTIVATION ENERGY OF STARCH HYDROLYSES BYAMYLASES FROM MAIZE MALT AT pH 4.8

T, temperature; V, instantaneous velocity of starch hydrolysis by enzymes.


10 0013 0 0001

10 9831

0

2

V SR= +

[ ]=. . . (5)

After the expanded bed chromatography process, the eluted samples wereanalyzed by SDS-PAGE for the determination of enzyme molecular weightsand purified degree (Fig. 6). The analysis of this figure shows that elutedmaterial is pure (two proteins were captured) and their molecular weights are67.4 and 47.5 kDa, respectively, due to a- and b-amylases; according to Dixonand Webb (1971), Reguly (1996) and Wiseman (1987), a-amylase molarweight is between 50 and 120 kDa, and b-amylase molecular weight isbetween 20 and 50 kDa. Some authors also verified these findings for themolecular weight of a-amylase source, such as from a-amylase from A. suum(83 kDa), H. dromedarii (106 kDa) (Mohamed 2004), Bacillus stearothermo-philus (59 kDa) (Ben Ali et al. 2001), L. manihotivoras (135 kDa) (Aguilaret al. 2000) and Aspergillus niger (116 kDa) (Toledo et al. 2007).

Study of Degradation of Manioc Starch

Composition of manioc root and flour is shown in Table 2. It can beobserved that starch concentration in drying manioc root (flour) is about 71%(w/w). According to Cereda et al. (2001), Holloway et al. (1985), Neves et al.(2006) and Smith (1982), the starch composition in manioc flour is higher thanthose in rice (62%, w/w) and wheat flours (60%, w/w); thus, it shows thatmanioc flour is a good starch source.

FIG. 5. SOLUBLE STARCH HYDROLYSIS KINETIC CURVE BY THE MAIZEMALT AMYLASE

V, instantaneous velocity of starch hydrolysis by amylases; [S], substractum concentration.


FIG. 6. MOLECULAR WEIGHT DETERMINATION BY SODIUM DODECYLSULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS

MWS is composed of the following proteins: phosphorylase B (94 kDa), bovine serum albumin(67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and

a-lactoalbumin (14.4 kDa).MWS, molecular weight standard; MM, maize malt proteins; EBC, eluted amylases of the expanded

bed chromatography.

TABLE 2.RESULTS OF DETERMINATION OF THE COMPOSITION OF

MANIOC ROOT AND FLOUR

Variable Average valor Deviation

Initial moisture (%, w/w) 54.17 3.12

Manioc flour in drying base (%, w/w)Ashes 0.81 0.31Starch 71.65 6.34Total sugars 0.55 0.04Total proteins 0.50 0.09


In Table 3, the values of experimental and calculated hydrolysis yields ofmanioc starch by amylases from maize malt are shown for each assay. Thesevalues were calculated by substituting the data of CStarch (Table 3) and glucoseconcentration (CGlucose) into the bioreactor at zero hydrolysis rates, in Eqs. (3)and (4). Figure 7 shows the hydrolysis curves of manioc starch by amylasesfrom maize malt, and it shows that the starch degradation process endsbetween 20 and 30 h for all assays. There is a higher conversion of starch to

TABLE 3.PLANNING MATRIX OF THE DEGRADATION STUDY OFMANIOC STARCH BY AMYLASES FROM MAIZE MALT,WITH THE EXPERIMENTAL (Yexp) AND CALCULATED

(Ycalc) YIELDS

Assays T (C) CStarch (%) Yexp (%) Ycalc (%)

1 60 9 22.6359 20.60682 70 9 13.6412 11.61203 60 11 14.3315 16.36084 70 11 13.8287 15.85805 65 10 29.8427 31.78166 65 10 31.3112 31.78167 65 10 34.1908 31.7816

T, temperature; CStarch, starch concentration.

FIG. 7. HYDROLYSIS CURVES OF MANIOC STARCHES BY AMYLASES FROM MAIZEMALT AT pH 5.0

Y, hydrolysis yield.


glucose, of about 31% of hydrolysis yield, in assay, while the bioreactor wasworking at 65C and 10% of starch concentration. Hydrolysis yield wasreduced for other assays.

Neves et al. (2006) observed that a hydrolysis yield reduces 27.3% (w/w)for 14.8% (w/w), while they worked with amylases from microorganism todegrade the starch of wheat flour at 55 and 75C, respectively. Leonel andCereda (2000) showed the hydrolysis yield for starch of manioc flour similarto that in Neves et al. (2006); it shows that an increase in the temperaturereduces the hydrolysis yield of starch of some source. The hydrolysis yield ofthis work is higher than those in Leonel and Cereda (2000) and Neves et al.(2006).

According to Barros Neto et al. (2001), the first F test (Fcalc/Ftab) andsecond F test (Ftab/Fcalc) must be higher than 1, for the model to be significantand predictive; R2 and explaining variance must be next to 1.0 and 100.0,respectively. Table 4 lists the significant parameters and statistical test resultsof the models. Thus, the F values and explaining variance values of the modelequation show that this model is significant, and the model multiple correlation(R2) indicates a good response between model prediction and experimentaldata (Marshal et al. 1999; Barros Neto et al. 2001; Biazus et al. 2005, 2006a).

The following equation shows the fitting model (Eq. 6) to data of starchdegradation, while the temperature effect on the hydrolysis yield is square andhigher than starch concentration. In Fig. 8, the RSM analysis shows that at 60Cand any starch concentration, the hydrolysis is higher.

Y T T T C= − × − × + × ×31 7816 2 3744 15 6722 2 12302. . . . Starch (6)

Amylases in Sivaramakrishnan et al. (2006) show substrate specificity. Thesubstrate specificity of the a-amylase was evaluated on soluble starch,

TABLE 4.ANALYSIS OF VARIANCE FOR EVALUATION OF FIT OF EMPIRIC MODEL USED TODESCRIBE THE EFFECTS OF TEMPERATURE AND STARCH CONCENTRATION ON

HYDROLYSIS YIELD AT 95% CONFIDENCE LEVEL

Regression Square sum Degree offreedom

Square mean Fcalc Ftab

Residual 461.639 3 153.880Fitting fault 26.256 3 8.752 17.582 9.28Error 16.471 1 16.471Total 9.785 2 4.892 3.367 18.5Regression 487.896 6% Explaining variance 94.618Multiple correlation (R2) 0.9461

Fcalc and Ftab are calculated and tabled test F, respectively.


amylose, amylopectin, glycogen, maltodextrins, and a- and b-cyclodextrins.Amylose was found to be the best substrate for L. manihotivorans LMG18010T a-amylase (Aguilar et al. 2000). The relative activity observed onamylose was 1.6 times higher than that obtained on starch. The hydrolysis ofamylopectin was 90% in relation to starch. Glycogen and dextrins with 10 and20 DP were hydrolyzed at low rates reaching 6.7, 7.3 and 8.9%, respectively,and no activity was found on a- and b-cyclodextrins. Natural starches such asmaize, raw sago, corn and wheat starch increased a-amylase activities (Sivara-makrishnan et al. 2006). Manioc starch is composed of 18.6% (w/w) amyloseand about 80% of amylopectins; this composition is more important to obtainhigher starch degradation by amylases from maize malt (Cereda et al. 2001;Takizawa et al. 2004; Ferreira et al. 2005; Sivaramakrishnan et al. 2006).

CONCLUSIONS

Enzyme characterization showed that the maize malt amylases haveoptimal zone of pH between 4.5 and 6.5, with optimal temperatures between 50and 90C. The Km and Vmax values for starch hydrolysis were 7.69 ¥ 10-2 g/L and7.69 ¥ 102 g/L/min, respectively. SDS-PAGE showed that the molecular weightof a- and b-amylases from maize malt were of 67.4 and 47.5 kDa, respectively.

The hydrolysis time was between 20 and 30 h, and higher values wereabout 31%, which was higher than 27% (cited by literature), and it wasobtained at 65C at any starch concentrations studied (9 and 11%, w/v).

FIG. 8. RESPONSE SURFACE METHODOLOGY EVALUATION OF THE EFFECTS OFSTARCH CONCENTRATION AND TEMPERATURE ON HYDROLYSES YIELD

CStarch, starch concentration; T, temperature.


This work showed optimal conditions to produce fermentable syrup frommanioc starch by amylases from maize malt, as a substitute to sugarcane inalcohol production by industries in this field.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the financial supports received fromPIBIQ-UFS/CNPq (São Cristóvão, SE, Brazil), from CNPq (Brasilia, DF,Brazil) and from FAPESP (São Paulo, Brazil).

REFERENCES

AGUILAR, G., MORLON-GUYOT, M., TREJO-AGUILAR, B. andGUYOT, J.P. 2000. Purification and characterization of an extracellulara-amylase produced by Lactobacillus manihotivorans LMG 18010T, anamylolytic lactic acid bacterium. Enzyme Microb. Technol. 27, 406–413.

ASCAR, J.M. 1985. Alimentos: Aspectos Bromatológicos e Legais. AnalisePercentual, Vol. 1, 1st Ed., Unisinos Editora, São Leopoldo-RS, Brazil.

BARROS NETO, B., SCARMINIO, I.S. and BRUNS, R.E. 2001. Como FazerExperimentos: Pesquisa e Desenvolvimento na Ciência e na Indústria,Vol. 1, 1st Ed., Edunicamp, Campinas, São Paulo, Brazil.

BEN ALI, M., MHIRI, S., MEZGHNI, M. and BEJAR, S. 2001. Purificationand sequence analysis of the atypical maltohexaose-forming a-amylase ofthe B. stearothermophilus US 100. Enzyme Microb. Technol. 28, 537–542.

BIAZUS, J.P.M., SOUZA, A.G., SANTANA, J.C.C., SOUZA, R.R. andTAMBOURGI, E.B. 2005. Optimization of drying process of Zea maysmalt to use as alternative source of amylolytics enzymes. BABT48(special issue), 185–190.

BIAZUS, J.P.M., SOUZA, R.R., CURVELO-SANTANA, J.C. and TAM-BOURGI, E.B. 2006a. Drying optimization of Zea mays malt. CTA26(4), 787–792.

BIAZUS, J.P.M., SEVERO, J.B., JR., SANTANA, J.C.C., SOUZA, R.R. andTAMBOURGI, E.B. 2006b. Study of amylases recovery from maize maltby ion-exchange expanded bed chromatography. Process Biochem. 41,1786–1791.

BRADFORD, M.M. 1976. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein. Utilizing the principle of protein-dyebinding. Anal. Biochem. 72, 248–254.


CEREDA, M.P., FRANCO, C.M.L. and DAIUT, E.R. 2001. PropriedadesGerais Do Amido, Vol. 1, 1st Ed., Fundação Cargill, Campinas, Brazil.

CURVELO-SANTANA, J.C. 2003. Recuperação das enzimas a e b-amilasesem SBA PEG/ CaCl2 para uso como biocatalizador amiláceos. MScThesis, p. 232, School of Chemical Engineering, UNICAMP, Campinas,SP, Brazil.

DIXON, M. and WEBB, E. 1971. Enzymes, 2nd Ed., Longmans and Co. Ltd.,London, U.K.

DUEDAHL-OLSEN, L., KRAGH, M.M. and ZIMMERMANN, A. 2000.Purification and characterization of a malto-oligosaccharide-formingamylase active at high pH from Bacillus clausii BT-21. Carbohydr. Res.329, 97–107.

FERREIRA, G.B., MELO, V.V., ALMEIDA, J.B.O., EVANGELISTA, A.F.and SOUZA, R.R. 2005. Characterizing of obtaining process of a maniocspirit. BJFT 8, special issue of 5th SIPAL, 2–7.

HOLLOWAY, W.D., MONRO, J.A., GURNSEY, J.C., POMARE, E.W. andSTACE, N.H. 1985. Dietary fiber and other constituents of some tonganfoods. J. Food Sci. 50, 1756–1757.

KALIL, S.J. 2000. Inulinase production from Kluyveromyces marxianus andenzyme purification by ion-exchange expanded bed chromatography.PhD Thesis, School of Chemical Engineering, UNICAMP, Campinas,São Paulo, Brazil.

KONSULA, Z. and LIAKOPOULOU-KYRIAKIDES, M. 2004. Hydrolysisof starches by the action of an a-amylase from Bacillus subtilis. ProcessBiochem. 39, 1745–1749.

LAEMMILE, U.K. 1970. Cleavage of structural proteins during the assemblyof head of Bacteriophage T4. Nature 227(5259), 680–685.

LEONEL, M. and CEREDA, M.P. 2000. Avaliação da concentração de pec-tinase no processo de hidrólise-sacarificação do farelo de mandioca paraobtenção de etanol. CTA 20(2), 15–28.

MARSHAL, L.M., JONKERS, J., FRANKE, G.T.H. and TRAMPER, J. 1999.The effect of process conditions on the hydrolysis of amylopectin potatostarch: an experimental design approach. Biotechnol. Bioeng. 62, 348–357.

MILLER, G.L. 1959. Use of dinitrosalicylic acid reagent for determination ofreducing sugar. Anal. Chem. 31(3), 426–428.

MOHAMED, M. 2004. Purification and characterization of a-amylase fromthe infective juveniles of the nematode Heterorbditis bacteriophora.Comp. Biochem. Physiol, B 139, 1–9.

NEVES, M.A., KIMURA, T., SHIMIZU, N. and SHIIBA, K. 2006. Produc-tion of alcohol by simultaneous saccharification and fermentation oflow-grade wheat flour. BABT 49(3), 481–490.


NIRMALA, N. and MURALIKRISHNA, G. 2003. Three a-amylase frommalted finger millet (Ragi, Eleusine coracana, Indaf-15) – purificationand partial characterization. Phytochemistry 62, 21–23.

OLIVEIRA, M. and VASCONCELOS, Y. 2006. Revolução no canavial.Pesquisa FAPESP 122, 62–70.

REGULY, J.C. 1996. Biotecnologia dos Processos Fermentativos, Vol. 1, 1stEd., EDUFPel, Pelotas-RS, Brazil.

SIVARAMAKRISHNAN, S., GANGADHARAN, D., NAMPOOTHIRI,D.K., SOCCOL, C.R. and PANDEY, A. 2006. a-Amylases from micro-bial sources – an overview on recent developments. Food Technol.Biotechnol 44(2), 173–184.

SLEIMAN, M. and VENTURI FILHO, W.G. 2004. Use of malt extracts inbrewing: Physico-chemistry and sensory evaluations. BJFT 7(2), 145–153.

SMITH, P.S. 1982. Starch derivatives and their use in food. In Food Carbo-hydrates (D.R. Lineback and G.E. Inglett) pp. 237–269, AVI PublishingCompany, Westport, CT.

TAKIZAWA, F.F., SILVA, G.O., KONKEL, F.E. and DEMIATE, I.M. 2004.Characterization of tropical starches modified with potassium permanga-nate and lactic acid. BABT 47(6), 921–931.

TOLEDO, A.L., SEVERO, J.B., JR., SOUZA, R.R., CAMPOS, E.S.,SANTANA, J.C.C. and TAMBOURGI, E.B. 2007. Purification byexpanded bed adsorption and characterization of an a-amylasesFORILASE NTL® from A. niger. J. Chromatogr., B 846, 51–59.

WANDERLEY, K.J., TORRES, F.A.G., MORAES, L.P.M. and ULHOA, C.J.2004. Biochemical characterization of a-amylase from the yeast Crypto-coccus flavus. FEMS Microbiol. Lett. 231, 165–169.

WISEMAN, A. 1987. Handbook of Enzyme Biotechnology, 2nd Ed., JohnWiley Sons, New York, NY.


BIOCHEMISTRY CHARACTERIZATION OF α - AND β -AMYLASES FROM ZEA MAYS MALT AND STATISTICAL ANALYSIS...

Documents

Transcript of BIOCHEMISTRY CHARACTERIZATION OF α - AND β -AMYLASES FROM ZEA MAYS MALT AND STATISTICAL ANALYSIS...