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IndianJournal of Biochemistry & BiophysicsVol.42, October 2005, pp. 287-294

Purification and characterization of a-amylase from Bacillus amyloliquefaciensNCIM 2829

Mithu De', Kali P Das2 and P K Chakrabartty"'Department of Microbiology, Bose Institute, PlI12 C.LT. Scheme VII-M, Kolkata-700 054 and 2Department of Chemistry,

Bose Institute, 93/1 A. P. C. Road, Kolkata 700 009

Received 3 December 2004; revised 20 July 2005

a-Amylase (EC 3.2.1.1) was purified to homogeneity (specific activity 58,000 umole minot mg protein") from theculture filtrate of Bacillus amyloliquefaciens NCIM 2829. Its molecular mass was found to be 67.5 kDa. The activity of theenzyme increased by almost 50% in the presence of Co+2 ion. Hg2+ and Cu2+ acted as strong inhibitors of the enzyme. Thetryptophan moities of the enzyme were fairly protected from the aqueous environment. However, the globular interior of theprotein was somewhat loosely packed. The protein had nearly an equal amount of a-helical and l3-sheet structure in dilutesolution. In concentrated solution, its secondary structure had a higher proportion of l3-sheet at the expense of some randomcoil structure. The protein showed a molten globule state at a low concentration of chaotropic agent. The denaturationprofile of the protein showed no cooperativity. C02+ enhanced the structural stability of the enzyme.

Keywords: a-Amylase, Bacillus amyloliquefaciens NCIM 2829, circular dichroism, fluorescence measurement,Ff-IR spectroscopy, secondary structure, guanidine hydrochloride, metal ions, denaturation.

IPC Code: C12N 9/28

a-Amylase (EC 3.2.1.1) catalyzes the hydrolysis ofa-D-(l,4) glycosidic linkages in starch or relatedcarbohydrates releasing oligosaccharides and glucose.It has extensive commercial applications in starchliquefaction, brewing, sizing in textile industries, andpaper and detergent manufacturing processes' .Various aspects of a-amylases such as its production,purification, properties, protein structure and functionand applications have been investigatedi". Severalspecies of Bacillus including B. subtilis andB. amyloliquefaciens are versatile producers of theenzyme? a-Amylases from B. amyloliquefaciensstrains F, P, N, T, SB were shown to bephysiologically as well as biochemically differentfrom that of B. subtilis strains ATCC 6051, ATCC4529, ATCC 7067, ATCC 9466, W 23 and 168 r.Theformer strains are characterized by production ofliquefying ce-amylase''.

a-Amylase from B amyloliquefaciens was reportedtohave temperature and pH optima at 65°C arid pH5.9, respectively? However, a-amylases from

B. amyloliquefaciens strains F, P, N, T, SB werefound to differ with respect to activity on varioussubstrates, pH, temperature stability, Km and energyof activation, reflecting conformational differences ofthe enzymes". The molecular mass of the a-amylaseof B. amyloliquefaciens N was found to be 50 kDa9•

The a-amylase gene of B amyloliquefaciens E18 wascloned and sequenced and from the nucleotidesequence molecular mass of the protein was deducedto be 54.778 kDalO

• Similarities in the primarystructure among a-amylases and existence of fourhighly conserved regions in their primary sequenceswere observed" and similarities in their secondarystructures were proposed'<.

B. amyloliquefaciens strain NCIM 2829 has aunique property of using pectin as the sole carbonsource, in contrast to the type strain of the speciesATCC 2335013

• Despite extensive investigations ona-amylase from different sources, reports onsecondary structure and structural stability amonga-amylases are very lirnited!2. In order to understandthe secondary structure of the enzyme and to use it asa model system in elucidation of secondary structure,in this communication, we report the characterizationof a-amylase :purified from the culture filtrate ofB. amyloliquefaciens NCIM 2829.

*Authorfor correspondenceEmail: krishna@boseinst.erneLinFax: 91-33-2334 3886Tel:(033) 2337 9416

288 INDIAN 1. BIOCHEM. BIOPHYS., VOL 42, OCTOBER 2005

Materials and MethodsOrganism and growth conditions

Bacillus amyloliquefaciens NCIM 2829 wasprocured from the National Collection of IndustrialMicroorganisms, National Chemical Laboratory,Pune, India. It was grown in dextrin-peptone mediumwith 2% dextrin, 2% peptone, 1% corn steep liquor,0.01 % KCI, 0.05% MgS04. 7H20, pH 7.0, at 37°C.

Assay of a-amylaseAssay of ex-amylase was carried out by incubating

an appropriately diluted enzyme with 5 ml ofgelatinised 1% potato starch in 50 mM phosphatebuffer, pH 7.2, at 70°C. Aliquots were taken out ato and 10 min of incubation. Saccharifying activitywas estimated by measuring the increase in reducingpower of the reaction mixture as determined bydinitrosalicylic acid method 14using glucose as thestandard. One unit of saccharifying activity wasdefined as the amount of enzyme that liberated1 umole of reducing sugar per min under the assayconditions.

Purification of enzymeA 48 hr grown culture was centrifuged at

10,000 x g for 15 min at 4°C and the supernatant wasused as the source of the enzyme. The supernatantwas brought to 30-60% (N~hS04 saturation. Theprecipitate obtained was dissolved in a small volumeof 5 mM phosphate buffer (PB), pH 7.2. The solutionwas dialyzed at 4° C twice against 5 mM PB over aperiod of 4 hr. The dialyzate was then applied to aDEAE-Sepharose column (2.5 x 10 em) equilibratedwith 5 mM PB. The column was initially washed with20 ml of 0.2 M NaCl in 5 mM PB, pH 7.2. Theenzyme was eluted with 80 ml of 0.2-1.5 M lineargradient of NaCI in the same buffer at a flow rate of0.5 ml min-I and 2 ml fractions were collected.Amylase activity and protein contents of the fractionswere determined. The active fractions were pooledand precipitated with 80% saturation of (NH4hS04,dissolved in 50 mM PB, pH 7.2, and applied to acolumn of Sephadex G-75 (150 x 1.5 em). Theenzyme was eluted with the same buffer at a flow rateof 5 ml min-I. The active fractions were pooled andused for further experiments.

Molecular mass and isoelectric point determinationSDS-PAGE was carried out as described by

Laemmli'", with 8% polyacrylamide slab gel (1 mmthick). Protein bands were visualized by silver nitrate

staining". Marker proteins (29-205 kDa) were run ina parallel lane. The molecular mass of purifiedamylase was estimated from its position relative to thestandard proteins and was further confirmed by SDS-capillary electrophoresis (Beckman Instrument Inc.).

Analytical isoelectric focusing was performed inthe pI range of 3-10 using a P/ACE system 5010capillary electrophoresis (Beckman). Markers havingpI 9.45 (RNase A), 5.9 (carbonic anhydrase), 5.1(f3-lactoglobulin) and 2.75 (CCK peptide) were usedas standards.

Effect of temperatureThe amylase activity was determined at various

temperatures ranging from 35 to 100°C in thepresence or absence of 1 mM CoCh. For studying thetime course of thermal inactivation, the enzyme in50 mM PB (PH 7.2) was pre-incubated at varioustemperatures ranging from 40 to 90°C and the residualactivity was determined at an interval of 10 min.

Effect of metal ions and chemical reagentsEnzyme assays were performed in the presence of

various metal ions such as Na+, K+, Cs+, Ca2+, Mg2+,Mn2+, C02+, Pb2+, Sr2+, Hg2+, Ba2+ as chloride salts,Ag+, C02+, Bi2+ as nitrate salts, C02+, Ni2+, Cu2+,A13+

as sulphate salts, or of chemical reagents such asEDT A and EGT A at 1 mM concentration. Therelative activity of the enzyme was calculated bycomparison of its activities in the presence and in theabsence of reagent.

Fluorescence measurementsFluorescence quenching experiments were done to

obtain information about structural integrity of theprotein. The measurements were carried out in aHitachi 4500 spectrofluorimeter. Tryptophanfluorescence emission spectrum of the purifiedenzyme was taken using an excitation wavelength of295 nm and scanning between 310-400 nm using5 nm slit widths for both excitation and emissionchannels. Appropriate solvent blank was digitallysubtracted from the sample spectrum. For thetryptophan fluorescence quenching experiments,2.0 ml protein solution (0.1 mg/mI, in 50 mM PB, pH7.0) was taken in a 3 ml quartz cuvette. The solutionwas titrated with small aliquots of either acrylamideor potassium iodide from a 5 M stock solution.Decrease in the fluorescence intensity at 337 nmusing an excitation wavelength of 295 nm was notedfor each addition 17. The intensity values were

appropblank,

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De et al.: CHARACTERIZATION OF (X-AMYLASE FROM BACILLUS AMYLOLIQUEFACIENS 289

Table I-Purification of rx-amylase from culture filtrate of Bacillus amyloliquefaciens 2829

Enzyme fraction Total protein (mg) Total activity Specific activity Yield (%) Fold purification(saccharifying unit) (unit/mg protein)

Culture filtrate 62 41,000 661 100 1.0

30-60% NH4S04 precipitate 15.5 34,QOO 2266 83 3.4

DEAE-Sepharose 1.6 13,505 8440 33 12.8

Sephadex G-75 0.048 2,784 58056 6.8 88

appropriately corrected for the contribution of theblank,dilution effect and inner filter effect.

Circulardichroism (CD) spectroscopyCD measurements 18,19 were done to obtain

information at the level of secondary structure of theprotein. The measurements were carried out using aJASCO J-600 model CD spectrometer using a 1 mmpath length cylindrical quartz cuvette. Spectrum ofa-amylase was taken in the range 200-250 nm in5 mM PB, pH 7.0. Usually 5 scans were averagedusing a time constant of 1 sec. All spectra werecorrected by subtracting control buffer solution. TheCD data were smoothed by application of asmoothening function. Curve fitting by K2Dprograrrr" was applied to both smoothed andunsmoothed data.

FT·IRspectroscopyFf-IR measurements were done to complement CD

data on secondary structure of a-amylase. Theexperiments were done in a Nicolet Impact 400 modelinfrared spectrometer. Protein solution (l0 mg/ml)was prepared in 50 mM phosphate buffered D20solutionand 10 III was placed between two CaF2 disksseparated by a 50 11m teflon spacer. The samplechamber was flushed with dry nitrogen during theexperiments. Spectrum was recorded in theabsorbance mode averaging 64 scans in the range of1500-1800 em" at 2.0 cm' resolution after recordingthe background. IR spectrum of the protein wascorrected for contributions for buffer and watervapour by subtracting suitable control spectra. Allspectral subtractions and other computationsincluding Fourier self-deconvolution and curvefittings were done to resolve the contributing bandsdue to various secondary structural elements" usingtheOmnic software supplied with the instrument. Therelative percentage of secondary structure elementswasobtained by curve fitting of the original amide Iband, using the peak positions from the deconvolvedspectrum.

Stability of u-amylase against chemical denaturantThe structural stability of the a-amylase against a

chemical denaturant guanidine hydrochloride(Gu-HCI), was studied by fluorescence and CDspectroscopy'+". Stability of the purified enzymeagainst Gu-HCl was studied by monitoring eithertryptophan fluorescence emission intensity at 337 nmusing 295 nm as excitation radiation or ellipticity at222 nm in CD spectroscopy, as a function of theincreasing concentrations of the denaturant. Typically,2.0 ml of the a-amylase solution was taken in a celland either the fluorescence intensity or the ellipticityreading was taken. Concentration of a-amylase was0.1 mg/ml and 0.2 mg/ml for the fluorescence and CDexperiments, respectively. A fixed volume (20 Ill) ofthe solution was then withdrawn from the cell andwas replaced with the same volume of proteinsolution of same concentration in 6 M Gu-HCI. Thisway the concentration of a-amylase was kept constantand only the denaturant concentration was allowed tovary. The solution was allowed to equilibrate bygently stirring magnetically for 3 min, before takingreadings.

ResultsPurification of o-amylase

The a-amylase from culture filtrate ofB. amyloliquefaciens NCIM 2829 was purified tohomogeneity with a yield of 6.8%. The culturesupernatant contained 660 units of the enzymeactivity mg protein", Following (NH4)2S04precipitation and chromatography on DEAE-Sepharose, the protein was subjected to gel filtrationand the enzyme was eluted as a broad peak. Thepooled protein had a specific activity of 58, 000 unitmg' with 88-.fold purification (Table 1). The isolatedprotein was homogenous with a molecular mass of67.5 kDa as judged by SDS-PAGE (Fig. 1), whichwas further confirmed by SDS-capillaryelectrophoretic analysis. The purified proteinexhibited a single peak upon isoelectric focusing (data

290 INDIAN J. BIOCHEM. BIOPHYS., VOL 42, OCTOBER 2005

xua

1 2 3 4 5

Fig. 1-S0S-PAGE at different purification stages of o-amylasefrom the culture of B. amyloliquefaciens NCIM 2829 [Proteinswere visualized by silver nitrate staining. Lane 1, molecular massmarkers (in KDa); lane 2, crude protein from the culturesupernatant; lane 3, protein after (NH4hS04 precipitation; lane 4,pooled protein after OEAE-Sepharose chromatography; and lane5, pooled protein after Sephadex G-75 chromatography]

not shown) and the pI of the protein was estimated tobe 5.1. A single band upon SOS-PAGE and a singleprotein peak upon capillary electrophoresisdemonstrated the purity of the enzyme.

Effect of temperatureThe enzyme activity was assayed in 50 mM PB,

pH 7, at various temperatures in presence or absenceof 1 mM CoCh (data not shown). The activity wasfound to be almost 50% higher in the presence ofCoCl»; the highest activity was observed at 70°C,irrespective of the presence or absence of CoCh. Theenzyme remained stable at 70°C for 20 min.

Effect of metal ions and other chemicalsNa+, K+, Cs+, Mg2+, Ni2+ and Ae+ did not have any

effect on the a-amylase activity of the strain NCIM2819. The enzyme activity was reduced to 80-90%upon addition of Mn2+ and Ag+ and to 60-70% uponaddition of Pb2+, Sr2+, Ba2+ and Bi2+. However, Hg2+and Cu2+ were highly inhibitory to the enzyme andreduced the enzyme activity to less than 10% of thecontrol. C02+ (as chloride, sulphate or nitrate salts)increased the enzyme activity by 50%. But,

298.9 A.~'";::Q):sQ)u;::e•.Q)...o

"G: 0.001- •.••.•••...•.•...J... __ .••....••••..•••.••...••-'--'-.L.... •.......••....•...J.......L.- ...•••••.......L...300.0 320.0 340.0 360.0 380.0 400.0

where [(F, andabsenceVolmerFigs. 2constantthe data

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Concentration of Acrylamide, 1M)

Fig. 2-Fluorescence studies on characterization of the foldedstate of o-amylase [(A): Tryptophan fluorescence emissionspectrum of n-amylase. Excitation wavelength was 295 nm, c-amylase concentration was 0.1 mg/ml in 50 mM PB, pH 7.0; (B):Stern- Volmer plot of quenching of tryptophan fluorescenceemission of u-amylase by potassium iodide; and (C): SternVolmer plot of quenching of tryptophan fluorescence emission byacrylarnide. In both (B) and (C) emission intensity at 337 nm weremeasured using excitation wavelength of 295 nm and u-amylaseconcentration of 0.1 mg/ml in 50 mM PB, pH 7.0]

surprisingly Ca2+ had no such effect, indicating thatpossibly the enzyme was purified in its Ca2+ ladenstate. The enzyme activity was reduced to less than25% of the control by treatment with EDTA as wellas EGT A; however, DTT had no effect on theenzyme.

Characterization of folded state by fluorescence spectroscopyThe fluorescence emission spectrum of the

purified a-amylase is shown in Fig. 2A. Theemission maximum (Amax) as determined byderivative analysis of the spectrum was found to be337 nm. Using either acrylamide or potassiumiodide as quencher the environment of tryptophanswas also probed by fluorescence quenching. Thequenching efficiency expressed as quenchingconstant (KQ) is given by the Stern Volmer equation(1) shown belowl7.

~400.0..--

~ 05

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d (C): Sterne emission by:337 nm werend a-amylase

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to less than>TAas wellfeet on the

spectroscopyurn of the~. 2A. Theermined by: found to beIf potassium. tryptophansmching. The; quenchingmer equation

05

De et al.: CHARACTERIZATION OF a-AMYLASE FROM BACILLUS AMYLOLIQUEFACIENS

FolF = 1 + KQ [Q] ... (1)

. where [Q] is the molar concentration of the quencher,Fo and F are fluorescence intensity either in theabsence or presence of the quencher. The SternVolmer plots for iodide and acrylamide are shown inFigs. 2B and 2C, respectively. The quenchingconstant for the acrylamide determined by linear fit ofthe data according to Eq. (1) is 3.3 MI.

Secondary structure of a-amylaseThe far-UV CD spectrum of the protein is shown in

Fig. 3A. The spectrum shows a minimum around222 nm. Fitting of the spectrum with K2D programyields 26% ex-helix, 25% ~-sheet and 49% randomcoil. In such a fit, the contributions from turns andbends are included in the estimate of the random coil.

The amide I band of IR spectrum of ex-amylasebetween 1700 and 1600 ern", as determined by FT-IRspectroscopy is shown in Fig. 3B. It shows a peakaround 1630 ern". The Fourier self-deconvolved

9

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Fig. 3-Determination of secondary structure of a-amylase [(A):Circular dichroic spectrum of a-amylase (0.2 mg/ml) in 5 rnMPB, pH 7.0; and (B): Ff-IR spectrum of the amide I region of a-amylase. Concentration of a-amylase was 10 mg/ml in 50 mM PBin D20. Dashed curve in panel B refers to the Fourier selfdeconvolution of the spectrum. Bandwidths of 20 cm' andresolution enhancement factor 2.0 were used as deconvolutionparameters]

291

spectrum (dashed curve, Fig. 3B) shows distinctbands at 1670, 1662, 1653, 1642, 1635, 1628 and1606 em". Bands at 1670 and 1662 cm' are assignedto turns and bends, 1653 em" to ex-helix, 1642 cm-I torandom coil, 1635 and 1628 cm' to ~-sheet and 1606em" to carboxylic acids24

•25

• The relative percentageof secondary structure elements obtained was: ex-helix20%, ~-sheet 44%, turns and bends 6% and random30%.

Stability of a-amylase against chemical denaturantsSince C02+ enhanced the enzymatic activity, we

checked the stability of ex-amylase in the presence ofCo2+. Tryptophan fluorescence of the purifiedex-amylase as a function of increasing concentrationsof Gu-HCI in the absence or presence of C02+ isshown in Fig. 4A. The denaturation profiles (Fig. 4A)lacked sign of cooperativity, indicating gradual loss ofstructure, resulting in the exposure of tryptophanresidues. There was a decrease in the tryptophanfluorescence intensity at 0.5 M Gu-HCl. In theabsence of C02

+, fluorescence intensity was highest at1M Gu-HCI and thereafter, it progressively declined

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292 INDIAN J. BlOCHEM. BIOPHYS., VOL 42, OCTOBER 2005

as the concentration of Gu-HCI increased. In thepresence of C02+, the increase of intensity above 1 MGu-HCI was uniform. At 6 M Gu-HCI, tryptophanfluorescence intensity reached 70 arbitrary units, bothin the presence and absence of C02+. However, theintensity was always lower in the presence of C02+throughout the range of Gu-HCI.

For a-amylase, the ellipticity at 222 nm withincreasing Gu-HCI concentration in the absence orpresence of C02+ is shown in Fig. 4B. The variationof ellipticity with Gu-HCI concentration followedsimilar trends as in Fig. 4A, except that nodistinctive change at 0.5 M Gu-HCI was observed.In fact, 0.5 M Gu-HCI induced very little change ofsecondary structure either in the presence or inabsence of C02+.

DiscussionPurification and characterization of a-amylase

from different species of Bacillus includingB. amyloliquefacien were reported in theliterature26-28.The enzyme from the strain NCIM 2829was purified by a combination of ion-exchange andgel-filtration yielded about 6.8% of the total secretedprotein. The specific activity of the purified a-amylase (58,000 unit mg protein") of the strain NCIM2829 appears to be very high, as compared toa-amylase from various other microbialsources2,7,26,29.The enzyme has higher molecular mass(67.5 kDa) than from the other strains of B.amyloliquefacienst+"; The molecular mass of a-amylase from B. amyloliquefaciens E18 was predictedto be 54.778 kDaIO from cloned gene sequence. Themolecular mass is, however, similar to that of B.licheniformis (62.5 kDa)30 and B. acidocaldarius(68 kDa)3I. The molecular mass of a-amy lase from B.subtilis strain YY 88 and the strain NRRL B 3411was 67 kDa32 and 48 kDa33, respectively, and fromBacillus spp. SAM 1606 was 68.886 kDa34. It appearsthat molecular mass of the a-amylase varies evenamong closely-related organisms.

The isoelectric point of the purified amylase of thestrain NCIM was within the reported pI of 5.09 to5.18 of the a-amy lases from other strains ofB. amyloliquefaciens'", It has a temperature optimumof 70°C, similar to that from B. stearothermophilus'i ,B. coagulans" and B. caldoiyticus": Although, theenzyme is less heat stable than that ofB. stearothermophilus"; it appears to be more heatstable than the a-amylase from other

B. amyloliquefaciens strains, which were reported tobe rapidly inactivated above 45°C26_

Activity of the a-amylase of the strain NCIM 2829was increased significantly in presence of Co2

+.

H . . - d b C 2+ M 2+ dowever, It IS not activate y a or g, aninhibited in the presence of Cu2+ or Ag+_ Earlier,interaction of C02+ with the a-amylase fromB. amyloliquefaciens procured from a commercialsource (Sigma Chemical, USA) was reported tosignificantly increase enzyme activity'". Also,activation of maltotetraose-forming alkalinea-amylase from an alkalophilic Bacillus strainGM8901 by Ca2+, Mg2+, Cu2+, C02+or Ag+ wasreported". Inhibition of the enzyme activity by EDTAas well as EGT A indicated the enzyme to be ametallo-protein containing Ca2+ ion, Saccharifyingamylases from Bacillus spp. A-40-240 andB. alcalothermophilus A3_84I were reported to bestable in response to EDT A treatment, whereas theliquefying enzymes from Bacillus strains requiredCa2+ion for the enzyme activity.

A folded protein is characterized by the masking ofits hydrophobic residues from the contact with aqueousmedium, and tryptophan residue is often used as a

k ;: hi 1742 Fl ..mar er lor t s purpose ' _ uorescence errussionspectrum of the purified a-amylase fromB. amyloliquefaciens NCIM 2829 was studied to probethe environment of tryptophans in it An excitationwavelength of 295 om was used for the purpose, so thatonly tryptophan and no other aromatic residues gotexcited and gave emission'". Globular proteins withburied tryptophans show Amax in the range 330-340 run.Proteins with exposed tryptophans have Amax above 345nm24,42.Thus, the Amax of the purified a-amylase at 337nm (Fig. 2A) indicated hydrophobic environment oftryptophans in it

Florescence quenching studies were carried outusing acrylamide or iodide to probe the environmentof tryptophans in the rx-amylase. Acrylarnide, aneutral quencher, is able to penetrate the interior ofglobular protein, while iodide, a bulky ionic quenchercan only quench tryptophans close to the proteinsurface22,24. Also, buried tryptophans are quenchedwith low efficiency, while exposed tryptophans arequenched efficiently 17_In these experiments, the valueof KQ for acrylamide (3_3 M-1

) was somewhat higherthan, when tryptophans would be tightly packedinside globular interior of proteins, but was much

17 hsmaller than that of fully exposed tryptophans w enKQ would exceed 8 MI. Thus, the data indicated that

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De et al.: CHARACTERIZATION OF a-AMYLASE FROM BACILLUS AMYLOLIQUEFACIENS 293

a-amylase interior was somewhat loosely packed.The relatively low KQ (104 M-') for iodide indicatedthat most tryptophans were away from the proteinsurface. Thus, the data presented in Fig. 2 wereindicative of a globular nature of a-amylase with asomewhat loose interior.

A minimum around 222 nm in the far-UV CDspectrum (Fig. 3A) is typical of a protein containingsubstantial number of residues in a-helicalconfiguration". However, probing of secondarystructure of the purified amy lase by FT -IRspectroscopy using a concentrated solution gave anestimate of 20% a-helix, 44% p-sheet, 6% turns andbends and 30% random coil. These estimates werequite different from those obtained from CDspectroscopy, indicating structural differences ofa-amylase in dilute and concentrated solutions.

Measurement of tryptophan fluorescence intensityof a-amylase as a function of concentration of Gu-HCI in the presence or absence of C02

+ furnisheddenaturation profile, which lacked cooperativity(Fig.4A). The data indicated gradual loss of structure,resulting in the exposure of tryptophan residues.However, lower fluorescence intensity in the presenceof C02

+ than in its absence throughout the range ofGu-HCl indicated that C02+ had a stabilizing effect onthe structure of the a-amylase. Negative ellipticity at222 nm gives a crude measure of the global secondarystructure of a proteinI8

•19

• No distinctive changeobserved in the fluorescence measurement at 0.5 MGu-HCI concentration reflected a 'molten globulestate' of a-amylase, characterized by native-likesecondary structure with compromised tertiarystructure43

•44

. More negative ellipticity values ofa-amylase at all other Gu-HCI concentrations in thepresence of C02+ than those in its absence alsoindicated a stabilizing effect of Co2

•

Fluorescence spectral results (Fig. 2) indicated afolded protein interior of the purified a-amylase withburied hydrophobic residues without a highlycompact core. The loose interior has implications inthegeneral lack of stability. This was indeed observedby studies of structural stability against chemicaldenaturants (Fig. 4). Destabilization started at lowGu-HCl concentration «0.5 M). The lack of co-operativity in the chemical denaturation profileindicated that the molecule had very little resistanceagainst structural destabilization by chaotropic agents.The 'molten globule state' reported here at 0.5 mM

G HCI" . . 4344 Iu- IS quite common in many protems . . tindicated that transitions to denatured state proceededvia this structural intermediate. Despite its poorstability against chemical denaturants, its thermalstability, though lower than a-amylases of somethermophilic organisms, in general, was higher thanmany enzymes (Tm = 70°C). This indicated thathydrophobic interactions played an importantstructure-stabilizing role in amylases with increasingtemperature up to 70°C.

The secondary structure determined by CD andFT-IR showing considerable differences mayoriginate from difference in solution concentrations ofa-amylase used in the two experiments (0.2 mg/mlfor CD and 10 mg/ml D20 for FT-IR). It seems thereis a packing induced conformational change ina-amylase. The difference may partly arise due to useof D20 in FT-IR instead of H2021

,45. It is also seenthat much of the additional p-sheet structure estimatedby FT-IR comes at the expense of the randomstructure, observed under dilute solution conditions byCD.

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