isolation and characterization of a novel thermostable α-amylase from korean pine seeds

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New Biotechnology �Volume 26, Numbers 3/4 �October 2009 RESEARCH PAPER

Isolation and characterization of a novelthermostable a-amylase from Koreanpine seedsMd. Abul Kalam Azad1, Jae-Han Bae1, Jong-Sang Kim1, Jin-Kyu Lim1,Kyung-Sik Song2, Beom-Soo Shin3 and Hak-Ryul Kim1

1Department of Animal Science and Biotechnology, Kyungpook National University, Daegu 702-701, Republic of Korea2 School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea3Department of Biological Systems Engineering, Kangwon National University, Chuncheon 200-701, Republic of Korea

Amylases have significant importance in broad industrial application including bio-ethanol production.

Although amylases are widely distributed in microbes, plants and animals, it has been sought for new

amylases from various sources with special industrial potential. In this study we firstly isolated and

characterized a novel thermostable a-amylase from Korean pine seed. Enzyme was purified to

homogeneity level with purification fold of 1286.1 using several techniques such as self-precipitation,

(NH4)2SO4 fractionation, DEAE anion exchange and starch affinity chromatography. The purified a-

amylase showed two bands in SDS-PAGE with molecular weight of 44 and 45 kDa. The apparent

molecular weight of native enzyme was calculated to be 46.7 kDa. Internal peptide sequencing

confirmed that the purified a-amylase was a novel enzyme. The optimum pH and temperature for

enzyme activity were pH 4.5 and 65 8C, respectively. This enzyme was fully stable for 48 h at 50 8C and

retained 80% activity up to 96 h. The Km and Vmax were 0.84 mg/ml and 3.71 mmol/min, respectively. On

the basis of high thermal stability and a broad range of pH stability, the pine seed a-amylase showed a

good prospect of industrial application.

IntroductionAmylases (EC 3.2.1.0) widely distributed in microbes, plants and

animals [1,2] are classified into four major groups such as a-amylase

(endoamylase), b-amylase, glucoamylase (exoamylase) and pullu-

lanase. Among these enzymes, a-amylase (EC 3.2.1.1) plays an

important role in the digestion of starch in animals. It plays also

an important role in seed germination of plant. The germinating

embryo obtains the necessary energy from the enzymatic hydrolysis

of storage carbohydrate of plant seed. Alpha-amylase in the endo-

sperm is responsible for mobilization of the stored carbohydrate

reserves by initiating the degradation process [3,4]. Although other

amylolytic enzymes participate in the process of starch breakdown,

however, the breaking of a-1,4 linkage of polyglucan by a-amylase is

a prerequisite for the initiation of this process [5,6].

Corresponding author: Kim, H.-R. ([email protected])

1871-6784/$ - see front matter � 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2009.09.006

Amylases have a significant commercial importance and they

occupied about 25–33% of the world enzyme market [7]. In many

industries, the hydrolysis of starch by a-amylase is used as the basic

step to produce glucose, maltose or a mixture of malto-oligosac-

charides from starch [8]. These products have significant industrial

uses in nutritional, cosmetic and pharmaceutical application [9].

However, the most common uses of a-amylase are in the prepara-

tion of glucose syrup, bread making and brewing [10]. Recently,

the hydrolysis of starch by a-amylase has been increasingly used as

a key step to produce carbohydrate substrate for bio-ethanol

production. At now about 60% of the ethanol is produced by

fermentation process [11]. The major advantages of the enzymatic

route are the selectivity with its associated high yield and exclu-

sivity toward the desired product. However, a least expensive,

economical and environmentally favored process is always desired

for large-scale production of bio-ethanol [12,13].

www.elsevier.com/locate/nbt 143

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http://dx.doi.org/10.1016/j.nbt.2009.09.006

RESEARCH PAPER New Biotechnology � Volume 26, Numbers 3/4 �October 2009

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Accordingly, it has been sought for new amylases from various

sources with special industrial potential. So far, amylases for

industrial purposes are mainly produced from microbial system.

Plant sources had not been considered with enough significance as

the source of these enzymes yet. As an attempt to seek novel

industrially valuable a-amylase, we have chosen the pine seeds

as the source of a-amylase since there is no study done yet on the

isolation of a-amylase from pine seeds. In this study, we firstly

isolated and characterized a novel highly thermostable a-amylase

from Korean pine seed.

Materials and methodsMaterialsFresh seeds of Korean pine tree (Pinus koraiensis Sieb. et Zucc., KPS)

were purchased from a local market. Protein markers for size

exclusion chromatography and DEAE–Sepharose anion exchange

resins were purchased from Sigma (St. Louis, MO, USA). Purifica-

tion column was purchased from Amersham Biosciences (Uppsala,

Sweden). Other chemicals were purchased from Sigma, unless

mentioned otherwise.

Preparation of crude protein extractCrude protein extract was prepared from Korean pine seeds as

followed. The outer seed coat of healthy pine seeds were removed

carefully and nude pine seeds were stored in airtight poly-bag at

4 8C. About 350 g of pine seeds was homogenized in a blender with

1400 ml of buffer A (sodium acetate buffer 20 mM, pH 5.5, 10 mM

CaCl2) for 3 min at 4 8C. After homogenization, the homogenate

was centrifuged at 13,000 rpm (19,650 � g) for 30 min at 4 8C.

Finally, the supernatant was filtered through the filter paper

(150 mm, 5B, Advantec Toyo, Japan) to get the clear crude extract

of pine seeds.

Enzyme purificationA portion of crude extract was taken in Falcon tube with tight

sealing and kept at 25 8C for 36 h in BOD incubator. Suspended

dissolved substances and unstable proteins of crude extract were

precipitated during this heat treatment step. The precipitate was

removed from the solution by centrifugation (7,155 � g for 30 min

at 4 8C) and the supernatant was concentrated under 65% satura-

tion of (NH4)2SO4 overnight at 4 8C. The precipitated protein was

collected by centrifugation (19,650 � g, 30 min at 4 8C) and dis-

solved in buffer A. After dialysis overnight in the same buffer with

five buffer changes, the collected enzyme was loaded on DEAE–

Sepharose CL-6B column and the elution was carried out by

50 mM Tris–HCl buffer (pH 7.6) with a stepwise gradient of NaCl

(0–500 mM). The highest active fractions were pooled and dia-

lyzed for 5 h in buffer A and loaded on starch affinity column

(1.4 cm I.D. � 12.0 cm). The starch affinity column was prepared

with 40.0 g of insoluble corn starch following a method described

by Mendu et al. [14] with some modifications. In brief, the starch

was washed several times with distilled water and finally with

buffer A before packing. The packed column was kept overnight at

4 8C to settle down the starch and the column was equilibrated

with 200 ml of buffer A. The starch column was then transferred to

room temperature and kept for 3 h before use. After loading

protein sample, the unbound proteins were removed by extensive

washing of the column with the equilibrium buffer till no protein

144 www.elsevier.com/locate/nbt

was detected in the effluent. The bound proteins were then eluted

with the same sodium acetate buffer using a gradient concentra-

tion of NaCl (100–400 mM) at a flow rate of 5.6 ml/h. The purified

amylase from this step was used for the characterization study. The

purity of protein from each step was confirmed by SDS-PAGE

under the denaturing condition. Protein concentration was mea-

sured by Bradford method [15] using the Bio-Rad protein assay

reagent (Richmond, VA). Bovine serum albumin was used as the

standard protein.

SDS-PAGE and native-PAGEThe purity and molecular mass of amylase was determined by SDS-

PAGE with 12% gel in mini gel electrophoresis (Bio-Rad, Rich-

mond, CA, USA). The gel was stained with Coomassie Brilliant Blue

R-250 and destained with a solution composed of methanol, acetic

acid and water (50:10:40, v/v/v). Discontinuous native-PAGE was

done in 8% acrylamide gel with Bio-Rad mini gel electrophoresis.

All the buffer system used for native-PAGE was prepared without

SDS.

Enzyme assayThe amylase activity was determined using dinitrosalicylic acid

(DNSA) method by measuring the reducing sugars released from

the soluble potato starch [16]. The reaction mixture contained

50 ml of enzyme solution, 450 ml of buffer B (sodium acetate buffer

20 mM, pH 5.5) and 500 ml of 1% (w/v) soluble potato starch

dissolved in buffer B. Enzymatic reaction started by addition of

enzyme solution to the assay mixture at 60 8C and continued for

5 min. After the reaction was stopped by the addition of a few

drops of 0.5 M HCl, 1.0 ml of dinitrosalicylic acid reagent was

added and the reaction mixture was incubated at 90 8C in water

bath for 5 min. The released reducing sugar changed the original

color of dinitrosalicylic acid and the intensity of color change was

spectrophotometrically measured at 540 nm. The amount of sugar

released from starch was calculated from the standard curve of

glucose. One unit of enzyme activity was defined as the amount of

enzyme required to release 1 mmol of glucose per min at 60 8C.

Data presented in this study were average of duplicate and error

range was within 10%, unless mentioned otherwise.

Analysis of hydrolytic end productsThe end product of starch degradation by amylase was identified

by following the method of Kimura and Horikoshi [17] with slight

modification. About 1.0 ml enzyme solution and 1.0 ml soluble

potato starch solution (5%, w/v) were taken in a test tube and the

reaction mixture was incubated at 60 8C for 36 h in a water-bath.

An aliquot of reaction mixture (10 ml) was spotted on silica gel 60

F254 TLC plate (Merck, Germany) and TLC was run with a solvent

system of 1-butanol:ethanol:water (5:3:2, v/v/v). The plate was

first air-dried and the spots were visualized by spraying 50%

sulfuric acid followed by heating at 110 8C for 45 min. Finally,

the end products were identified by comparing the spots with

standard carbohydrates.

Characterization of the enzymeThe optimum pH for enzyme activity was determined by changing

the pH of assay mixture using the following buffers: glycine–HCl

(20 mM, pH 2.0–3.0), sodium acetate buffer (20 mM, pH 4.0–5.5),


TABLE 1

Purification of a-amylase from Korean pine seeds

Purification stepa Total protein (mg) Total activity (unit) Specific activity (unit/mg) Purification (n-fold) Recovery (%)

Crude extract 3230.3 2200.9 0.7 1.0 100.0

Self-precipitation 2067.0 1906.4 0.9 1.4 86.6

Ammonium sulfateprecipitation

228.0 1082.7 4.8 6.9 49.2

DEAE–Sepharose 21.3 311.0 14.6 21.4 14.1

Starch affinity 0.2 128.6 874.6 1286.1 5.8

a See ‘Materials and Methods’ for detailed description of purification steps.

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sodium phosphate buffer (20 mM, pH 6.0–7.0), Tris–HCl (20 mM,

pH 8.0–9.0), and glycine–NaOH (20 mM, pH 10.0–11.0). For pH

stability of the purified enzyme, enzyme sample was diluted 4

times with proper buffers and incubated at 4 8C. An aliquot

amount of enzyme was withdrawn at a given time and the activity

was measured using standard way. Optimum temperature for

enzyme activity was measured by determination of enzyme activ-

ity at different temperatures ranged from 50 to 80 8C in buffer B.

The thermal stability of the purified enzyme was determined by

incubating the purified enzyme in 20 mM sodium acetate buffer

(pH 5.5) at different temperatures (4–80 8C) in a water-bath. After a

given incubation period, a small portion of enzyme was with-

drawn and the enzyme activity was measured by the standard way.

This procedure measured the irreversible inactivation of enzyme

by heat treatment [18]. The effect of metal ions and enzyme

effectors on enzyme activity was determined using the enzyme

purified with metal ion-free buffer system. Metal ions and other

compounds (1 mM) were added to assay mixture independently

before enzyme assay started.

A variety of carbohydrate substrates were tested to find out the

substrate specificity of the purified enzyme. A 1% solution of each

carbohydrate was prepared in 20 mM sodium acetate buffer (pH

5.5) and used as substrate and the enzyme activity was measured

by the standard way. For kinetic analysis of the purified enzyme,

the initial rates of pure enzyme on soluble potato starch were

determined at various substrate concentrations (1–10 mg/ml) by

the standard way. The rate constant Km and the maximum reac-

tion rate Vmax were calculated from Lineweaver–Burk plot.

Determination of internal peptide sequenceFor partial internal amino acid sequencing of the purified enzyme,

the protein band run on a 12% SDS-PAGE was cut off from the gel

and digested with trypsin. The peptides obtained from digestion

were analyzed using ESI-QUAD-TOF-MS/MS (QTOF II, Micromass,

UK) at Korea Basic Science Institute (KBSI, Daejon, Korea). The

spectra obtained were used to determine amino acid sequence

using MassLynx software (ver 3.5). The amino acid sequences

determined were quarried to NCBI databases to find out homo-

logous protein.

ResultsEnzyme purificationThe target protein was purified to homogeneity level by four steps;

self-precipitation, ammonium sulfate fractionation, DEAE–Sephar-

ose anion exchange chromatography and starch affinity chromato-

graphy. The results from each purification step are summarized in

Table 1. In DEAE–Sepharose anion exchange chromatography, the

most active fractions were eluted with 100 mM NaCl in 50 mM Tris–

HCl buffer, pH 7.6 (data not shown). The starch affinity chromato-

graphy among four different purification techniques used in this

study was most effective to increasepurification fold. From this step,

a single active peak was obtained and the purification fold increased

60 times over the previous step (data not shown). Consequently, the

purified protein yielded a specific activity of 874.6 U/mg, represent-

ing purification fold of 1286.1 and 5.8% recovery. This protein

presented two bands closely located at 44 and 45 kDa in SDS-PAGE

(Figure 1A). When this protein sample was run on native PAGE, two

bands were appeared with different enzyme activities (Figure 1B).

The gels corresponding to each band were cut off followed by

protein extraction for activity determination. The lower band repre-

sented 82.3% activity whereas upper band contained 17.7% activity

of the total enzyme activity. The protein isolated from lower band

was co-chromatographed in SDS-PAGE with the purified protein

from the last step (Figure 1A, lane 7). The apparent molecular mass

of the native protein from the last step was calculated to be 46.7 kDa

using size exclusion chromatography (data not shown). From these

results, it was concluded that the purified protein was amylase and

existed in two isozymes in the active form. As the purified enzyme

was identified to be amylase, enzymatic action of the purified

enzyme was examined. After incubation of the protein sample from

each purification step with soluble starch as a carbohydrate sub-

strate, reaction products were analyzed by TLC. As shown in

Figure 2, three major products such as glucose, maltose and mal-

totriose were identified as end products from the samples of all

purification steps. On the basis of this result, the purified amylase

was confirmed to be a-amylase.

Effect of pHThe optimum pH for enzyme activity was determined with pH

range from pH 2.0 to pH 9.0. Although maximum activity was

detected with pH 4.5 (Figure 3), relative activities with between pH

4.0 and pH 7.0 were retained over 60% compared to maximum

value. However, when the purified enzyme was incubated in the

buffer with different pHs for six days, activities at between pH 4.1

and pH 8.8 were retained over 95% compared to the starting point

suggesting that the purified enzyme was highly stable at these pH

ranges (Figure 4). At acidic pH 2.2 and 3.2, enzyme lost its total

activity within 12 and 48 h, respectively. Similarly at alkaline pH

9.7 and 10.7, the enzyme activity was lost completely within 24

and 3 h, respectively.



FIGURE 1

Analysis of the protein samples in SDS-PAGE (A) and in native PAGE (B). Lane

1, standard proteins; lane 2, crude enzyme extract; lane 3, self-precipitation;

lane 4, 65% (NH4)2SO4 precipitation; lane 5, DEAE–Sepharose CL-6Bchromatography; lane 6, starch affinity chromatography; lane 7, protein

collected from the native PAGE.

FIGURE 3

OptimumpH for activity of the purifieda-amylase. Activity was determined at60 8C using different pH buffers such as 20 mM glycine–HCl (pH 2.0–3.0),

20 mM Na–acetate (pH 4.0–5.5), 20 mM Na–phosphate (pH 6.0–7.0), and

20 mM Tris–HCl (pH 8.0–9.0).

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Effect of temperatureTo determine the effect of temperature on enzyme activity,

enzyme assay was performed under varied temperatures ranging

from 50 to 80 8C. In general, temperature effect on the purified a-

amylase was mild in that enzyme activity was recorded over 60%

compared to the maximum value for all the temperatures tested in

this study although optimum temperature was 65 8C (Figure 5).

Even at 80 8C, relative activity was about 80% of the maximum

value. On the basis of these results, heat stability was determined

with broad temperature range (Figure 6). When incubated at 40 8Cand 50 8C, activity increased by 50% and 32% during the first 24 h,

respectively, and decreased thereafter. However, after 72 h incuba-

tion, activity was retained to be 103% and 92%, respectively. At

60 8C, 61% of the original activity was remained after five-day

FIGURE 2

TLC analysis of the hydrolysis products of soluble potato starch by the protein

samples. Protein samples from each purification step in Table 1 were used for

hydrolysis of soluble potato starch. Lane 1, standard sugars; lane 2, crudeextract; lane 3, self-precipitation; lane 4, (NH4)2SO4 precipitation; lane 5,

DEAE–Sepharose chromatography; lane 6, starch affinity chromatography.

Reaction conditions are described in ‘Materials and Methods’ section.


incubation. Half-life of the enzyme activity at 70 8C and 80 8C was

36 and 10 h, respectively. From these results, the purified a-amy-

lase was confirmed to be thermally stable.

Effect of metal ionsOut of nine metal ions tested, none of them was specifically

required for catalytic activity of a-amylase (Table 2). The calcium

ion, generally known to be required for amylase activity, had no

positive effect on a-amylase activity (99.81%). The enzyme activity

was partially inhibited by Na+ (85.31%), K+ (80.28%), Mg2+

(72.14%), Mn2+ (64.24%), Zn2+ (58.73%) and Co2+ (50.82%).

Fe3+ and Cu2+ inhibited completely the enzyme activity.

FIGURE 4

pH stability of the purified a-amylase. All the enzyme samples prepared in

adjusted pH were stored at 4 8C. At each given time period, protein sample

was withdrawn and enzyme activity was measured by standard way

described in ‘Materials and Methods’ section.


FIGURE 5

Optimum temperature for activity of the purified a-amylase at pH 5.5 (20 mM

sodium acetate buffer).

FIGURE 6

Thermal stability of the purified a-amylase. After a fixed time of interval, a

small portion of enzyme was withdrawn and left at room temperature for 1 hfollowed by activity determination. The enzyme activity was measured at

60 8C.

TABLE 2

Effects of metal ions on a-amylase activity

Metal ions (1 mM) Relative activity (%)

Controla 100.0

Na+ 85.3

K+ 80.3

Ca2+ 99.8

Mg2+ 72.1

Fe3+ n/db

Mn2+ 64.2

Cu2+ n/d

Zn2+ 58.7

Co2+ 50.8

a Control represents enzyme activity without metal ion.b Not detected.

TABLE 3

Effects of enzyme effectors on a-amylase activity

Effector (1 mM) Relative activity (%)

Controla 100.0

EDTA 74.9

DTT 111.7

b-Mercaptoethanol 124.8

Urea 93.9

SDS n/db

PMSF 90.2

a Control represents enzyme activity without effectors.b Not detected.

TABLE 4

Substrate specificity of a-amylase

Substrate (1%) Relative activity (%)

Controla 100.0

Corn starch 41.3

Amylose n/db

Amylopectin 48.3

Glycogen (Oyster) n/d

Pullulan n/d

Dextran n/d

Cellulose n/d

a Soluble potato starch was used as control.b Not detected.

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Effect of enzyme effectorsAmong the different enzyme effectors tested, DTT and b-mercap-

toethanol increased the enzyme activity by 11.7% and 24.8%,

respectively (Table 3). However, EDTA (74.9%), urea (93.9%) and

PMSF (90.2%) slightly reduced the activity and SDS caused total

loss of the enzyme activity.

Substrate specificitySpecificity of the purified a-amylase was examined for several

carbohydrate substrates such as potato starch (soluble), corn

starch, glycogen (oyster), amylose, amylopectin, pullulan, dextran

and cellulose (Table 4). This enzyme was highly specific to soluble

potato starch for its enzymatic hydrolysis. Corn starch and amy-

lopectin showed relative activities of 41.26% and 48.33% com-

pared with soluble potato starch, respectively, while other

carbohydrates did not show any activities. These results might

come from the different solubilities of substrates tested.

Enzyme kineticsThe purified a-amylase showed a typical Michaelis–Menten reac-

tion rate curve when soluble potato starch was hydrolyzed at pH

5.5 and 60 8C (data not shown). Kinetic values of Km and Vmax were

calculated from Lineweaver–Burk plot as to be 0.84 mg/ml and

3.71 mmol/min, respectively (Figure 7).



FIGURE 7

Lineweaver–Burk plot of enzyme activity of the purified a-amylase over

varied concentration of soluble potato starch.

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Partial internal sequencingFor internal amino acid sequencing, sequences of two peptides

were obtained (Table 5). Sequences of peptide-I and II determined

from Mascot search were Arg-Leu-Lys-Asp-Ser-Asn-Gly-Lys-Pro-

Ser-Gly-Leu-Ile-Gly-Val-Leu-Pro-Gln-Lys-Ala and Arg-Ile-Ile-Thr-

Ala-Glu-Gly-Asp-Leu-Tyr-Met-Ala-Ala-Ile-Asp-Glu-Lys-Ile, respec-

tively. NCBI Blast searches (http://blast.ncbi.nlm.nih.gov/Blas-

t.cgi) for those sequences were performed over 60% homology

identity to find out any homologous protein from plant sources.

The sequence of peptide-I showed varied homology identities with

six a-amylases of plant sources such as Citrus reticulata (80%),

Phaseolus vulgaris (70%), Ipomoea nil (70%), Arabidopsis thaliana

(70%), Zea mays (65%) and Oryza sativa (65%) while the sequence

of peptide-II showed homology identities over 60% with four plant

a-amylases such as Phaseolus vulgaris (70%), Vigna angularis (64%),

TABLE 5

Sequence alignment of internal peptides of a-amylase from Korean

aIdentical residues at each position are highlighted.


Vigna mungo (64%) and Malus domestica (61%). The sequences of

both peptide-I and II peptides showed 70% homology identity

with a-amylase of Phaseolus vulgaris. These results confirmed that

the purified a-amylase of Korean pine seed was a novel enzyme.

DiscussionThermostable a-amylase is very important for food, paper, textile,

distillery and brewing industries [19–21]. Microbial sources are

solely used for the commercial production of this enzyme for

simple preparation process. However, new trials need to be focused

on finding new sources of this enzyme with distinct properties for

diverse industrial application. In this view point, we isolated and

characterized a novel thermostable a-amylase from Korean pine

seeds. Initially, we faced severe problems to get clear crude enzyme

solution from the pine seeds owing to high content of fat and

dissolved carbohydrate materials in the seed. This problem was

solved by the addition of 10 mM CaCl2 in extracting buffer and

heat-treated precipitation of crude extract by incubation at 25 8Cfor 36 h. After this step, clear enzyme solution was obtained. Most

of the amylases are known to be metal ion dependent, especially

calcium ion [21]. However, pine seed a-amylase activity was not

influenced by the presence of calcium ion although EDTA, the

chelating agent, reduced the activity by 25% explaining partially

the importance of basal or intrinsic metal ions for the activity.

The purified a-amylase of Korean pine seed represented appar-

ent molecular weight of 46.7 kDa and exhibited two close bands at

44 and 45 kDa in SDS-PAGE. These molecular sizes were similar to

other a-amylases from plant sources such as pea seedlings and

shoots 43.5 kDa [22], barley 43 and 44 kDa [23], rice seeds 44 kDa

[24], poplar leaves 44 kDa [25] and maize 44.5 kDa [3]. Two close

bands from SDS-PAGE and two bands from native PAGE with

relative distinct enzyme activities suggested that the a-amylase

from Korean pine seed existed in nature as two possible isozymes.

This was supported by the fact that treatment of the enzyme with

the reducing agents such as DTT and b-mercaptoethanol did not

give any negative effects on enzyme activity but even increased

enzyme activity. In plants and cereal seeds, most of the a-amylases

pine seed with plant a-amylases

http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://blast.ncbi.nlm.nih.gov/Blast.cgi


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are found to consist of several isozymes [26]. Four isozymes from

maize seeds (44.5–47.5 kDa), two isozymes from barley (43–

44 kDa) [3,23,24], and three isozymes from finger millet

(45 � 2 kDa) have been reported [27].

The optimum temperature for enzyme activity and thermal

stability of a-amylase of Korean pine seed were higher than any

other plant a-amylases reported so far. The optimum temperature

of a-amylases from finger millet and wheat were reported to be 45–

50 8C and 40–50 8C, respectively [27,28]. As like as thermal stabi-

lity, the pine seed a-amylase had a good stability in wide range of

pH (4.0–7.0). On the basis of wide range of pH stability and high

thermal stability, a-amylase of Korean pine seed showed a good

prospect in industrial application. Enzymatic hydrolysis of starch

generally involves liquefaction and saccharification of starch

molecules at high temperature to precook and dissolve the parti-

cles [29,30]. In the case of barley a-amylase, the most suitable

condition for enzyme reaction to liquefy the starch is pH 4.5 at

45 8C [28]. Comparing to this enzyme, a-amylase of Korean pine

seed showed similar optimal pH but higher thermal stability

suggesting that it contained better useful properties for starch

liquefaction for industrial scale.

In conclusion, this study identified a novel thermostable a-

amylase from Korean pine seeds. The a-amylase was purified to

homogeneity level through the two cost-effective simple steps,

DEAE anion exchange and starch affinity chromatography. The

purified a-amylase of Korean pine seed showed a higher thermal

stability than any other a-amylases isolated from plants. Consid-

ering a wide distribution of pine trees in the temperate region of

the world, the pine seeds would have a good prospect to be used as

a source of a-amylase in future.

AcknowledgementThis work was supported by the Research Foundation Grant

funded by the Korean Government (KRF-2005-211-C00091).

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isolation and characterization of a novel thermostable α-amylase from korean pine seeds

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