J. Biol. Macromol., 4 (2), 57-66 (2004)

ArticlePurification and characterization of an endo-1,3-β-glucanasefrom Arthrobacter sp.
Zhongcun Pang,a,* Kodo Otaka,b Yasunori Suzuki,a Kyoji Goto,c Masatake Ohnishi aaLaboratory of Cellular Macromolecules, Graduate School of Agricultural Science, The Prefecture University of
Kyoto, Sakyo-ward, Kyoto, 606-8522,
Japanb
Department of Research, Sun Biorex Co., Ltd., Sakyo-ward, Kyoto,606-0805, Japanc
Daiwa Kasei Co., Tennoji-ward, Osaka, 520-0225, Japan

Received January 13, 2004; Accepted March 18, 2004
An endo-1,3-β-glucanase from Arthrobacter sp. was purified by ammonium sulfate precipitation,
anion-exchange and gel-filtration chromatographies. SDS/PAGE and gel-filtration chromatographysuggested a monomer enzyme with molecular mass of 32,500. N-terminal sequence for 10 residues was
APGDLLWSDE. Stable and optimum pHs were 5 to 8 and 6.5, respectively. Optimum temperature
was 55℃, however, above 50℃ its activity was lost rapidly. The Michaelis constant Km forlaminaritetraose, laminaripentaose, laminarihexaose, laminariheptaose, and laminarin were 0.12, 0.11,
0.067, 0.066mM, and 0.16mg/ml, respectively. Curdlan and lichenan were also hydrolyzed. The
purified enzyme did not give product by digestion with endoglycosidase H (Streptomyces griseus),
indicating that the carbohydrate moiety was little consisted in the enzyme protein, if any, with N-linked
chain or other type linkage. It was suggested that the enzyme belong to the family 16 of glucosylhydrolase from the properties as above mentioned.
keywords: Endo-1,3-β-glucanase; Arthrobacter sp.; Purification; Molecular/Enzymatic properties
described by Gueguen et al.1 In these studies,

Introduction
β-1,3-glucanases are widely distributed among
bacteria,

physiological roles, and practical uses. For example,

physiological roles, such as involvement in cellthe enzymes could be convenient for the degradationdifferentiation and defense against fungal pathogensof cell wall in fungi, yeasts, and higher plants.

been

and

suggestedhigherinplants,plants.1-3andβ-1,3-glucanases have interesting and important

their

have

fungi

fungi,

The application of 1,3-β-glucanases is also well

β-1,3-glucanases seem to have different functions inestablished in preparation of protoplasts, 4,5 in analysis

morphogenetic processes, in β-glucan mobilizationof fungal cell wall structures, in clarifying of slimy

and in fungal pathogen-plant interactions. Recently,

must, in degradation of barley β-glucan thatthe first metazoan β-1,3-glucanase, which may be

accumulates during the brewing process, and in

involved in the early embryogenesis, has beentreatment of fungal diseases. Furthermore, theseIn

glucanases are important tools for use in yeast*
Corresponding
author:Tel.&+81-75-7035763
E-mail address: a2752003@kpu.ac.jp

Fax:

biotechnological processes such as cell fusion,transformation, and extraction of protein products.

58

endo-1,3-β-glucanase from Arthrobacter sp.

Some but not all of this class of enzymes are also

as Arthrobacter sp. strain YCWD3(55k),16 Oerskovia

found to be capable of inducing lyses of viable yeast

xanthineolytica

LL

G109

(Cellulomonasdegradation of fungal mycelia (lytic activity),

cellulans)( βglⅡ , 27k and βglⅡ A, about 30k),4,17 andutilization as biomass resources of yeast cell walls

Oerskovia xanthineolytica TK-1(40k)18 have also been

wasted by brewing industries, or control of pathogenic

obtained from the genus of Arthrobacter (Oerskovia).

fungi infecting plants and rotting foods during

The molecular mass range of these purified glucanases

cells,

4,6

7

storage.

from Arthrobacter (Oerskovia) was found to be 12k to

On the other hand, β-1,3(1,6)-glucans are major

57k. 6,16,19-22

components in cell wall of yeasts and fungi. In mostBased on the action patterns of glucanase-catalyzed

yeast, especially Saccharomyces, the cell wallhydrolysis reactions, β-1,3-glucanases are classified

polysaccharide

into

glucanisconsistedwithexo- β-1,3-glucanases

the(β-1,3-glucan

predominantly β -1,3-linked backbone having someglucanohydrolase

branch via β -1,6-linkages.

Crude preparations ofendo-β-1,3-glucanases (β-1,3-glucan glucanohydrol-

yeast β-glucans have been known for 40 years toase (EC3.2.1.6 and EC3.2.1.39)).1,22 By using an

stimulate animal defense mechanisms. It is now

enzyme preparation Tunicase® , which is a productgenerally believed that the active component in thesefrom

preparations is β-1,3-glucans. β-glucan is known to

endo-1,3-β-glucanase and found that molecularpossess the antimicrobial and antitumorial activities

properties of the purified enzyme were different fromby enhancing the host immune function, andthose of the enzyme samples reported about

4,6,8,910

11

activating macrophages, neutrophils and NK cells.

In recent years, the function of glucan found in

Arthrobacter(EC3.2.1.58))

sp.,

we

and

have

the

purifiedanArthrobacter (Oerskovia).4,6,16-18,23-30
In

this

paper,

theexperimental

resultsofbaker’s yeast cell wall has also been widely noticed

purification and molecular properties of the enzyme

for its action of immune stimulation; that is, by

preparation obtained from Arthrobacter sp. have beenutilizing β- glucan of the yeast cell wall to stimulate

described.

host immune function for antimicrobe, antitumor andsoon.

Meanwhile,followingthesestudies,Materials

1,3-β-glucanases are also thought to play more and

Crude preparation Tunicase® was purchased from

more important roles not only in food production, but

Daiwa Kasei Co., Japan, for purification of thealso in more effective utilization of resource what not

enzyme endo-1,3-β-glucanase. All of the otherused and wasted. Thus, it is supposed to be interestingmaterials, saccharides, enzymes, and chemicals wereto make use of β-glucanases and β-glucans to produce

purchased

functional oligomers for bioactive and immune

available and guaranteed products and used without

12

studies.

For this purpose, it is necessary to makeasfollows

that

were

commerciallyfurther purification: Soluble laminarin from Eisenia

clear of the molecular and reactionary properties ofbicyclis

endo-1,3-β-glucanases, as a first step, here we arelaminari-oligosaccharides

concerned with purification and characterization of an(Seikagaku Kogyo Co., Tokyo, Japan), lichenan fromendo-1,3- β-glucanases from Arthrobacter sp. In fact,

Cetraria islandica (Sigma Chemical Co., USA),several enzymes closely related to food production

curdlan from Alcaligenes faecalis, pustlan fromhave been isolated from Arthrobacter sp., such as

Umbilicaria

maltooligosyl

maltooligosyl(CMC), and chitin from crab shells (Wako Puretrehalose trehalohydrolase, glucoseisomerase, andChemical Industries, Ltd., Osaka, Japan). The

13-15

lipase.

trehalose

synthase,

Some kinds of endo-1,3-β-glucanases, such

(NacalaiTesque,papullosa,Inc.,Kyoto,

from

Poria

carboxymethyl

Japan),
cocos

cellulose

following materials were also the commercial

59
Z. Pang et al.

products: DEAE Toyopearl 650M (Tosoh Co., Tokyo,(exclusion limit 20,000, φ 62mm). All operations

Japan), Sephacryl S-200 HR (Amersham Pharmacia

were carried out at 4℃. The concentrated sample was

Biotech Co., USA), the kit of the low-range molecularloaded to the gel filtration chromatography, Sephacryl

standard proteins and enzymes for molecular mass

S-200 HR column (φ 2.5×120cm long) and eluted

estimation (Bio-Rad Lab, USA), endoglycosidase H

with a 300mM NaCl/phosphate buffer solution at flow

(abbreviatedtoendo-H,

endo-β-N-acetylgluco-

rate about 0.28ml/min. Fractions having laminarinase

saminidase

H,

EC3.2.1.

96,

activity were pooled and concentrated by the samemannosyl

glyco-

protein-1,4- N- acetamidodeoxy-β-Dglycohydrolase)from Streptomyces griseus (Seikagaku Kogyo Co.,

procedures as described above.
Protein concentration was determined by themethod of Bradford 32 for all protein samples in theTokyo, Japan).

purified steps. When the kinetic examination was
Enzyme activity assays

carried out with the purified sample, concentration of

The enzyme-catalyzed reactions were measured

the enzyme was determined by methods of Lowry 33

with 2.5mg/ml soluble laminarin as a substrate in aand of Bradford simultaneously, for measurement of

40mM potassium phosphate buffer, pH 7.0, at 30℃,

exact quantity. In the both assays, bovine serumfor 15 min to obtain the reaction-time course. The

albumin was used as the standard.

reducing power caused by the enzymatic reaction
from

the

substratewas
31

Somogyi-Nelson methods,

determined

For the SDS-Polyacrylamide gel-electrophoresis

by(SDS/PAGE), protein samples were mixed with equalof which absorbance atvolume of a 125mM Tris-HCl buffer pH 6.8,
4%

(w/v)

SDS,

10%

β

600nm was measured by a Shimadzu UV-1600

containing(v/v)

Spectrophotometer. One unit of enzyme was defined-mercaptoethanol, 20% (v/v) glycerol, and 0.1%

as the amount of enzyme, which liberates 1μ mol ofbromophenol blue (BPB). After the mixture was

reducing sugar equivalent to glucose per 1min underincubated in a Dry Thermo Unit DTU-1C (TAITEC)

the standard assay conditions.

at 90℃ for 3min, the samples were run on the gel(composing of 10% or 12.5% separating gel pH8.8
and 4% stacking gel) at 20mA as described by

Enzyme purification®

The crude preparation Tunicase was dissolved in a

Laemmli.34

Protein

bands

were

stainedwith

10mM phosphate buffer pH7.0 (abbreviated to

Coomassie brilliant blue (CBB). A kit of thephosphate buffer, below) at 5% (w/v), mixed with

low-range molecular standard-proteins/enzymes was(NH4)2SO4 up to 65% saturation with stirring, andadopted for determination of molecular weight.

stayed for overnight at 4℃. After centrifugation at
20,000×g for 30 min at 4℃, the precipitation was
dialyzed against the same buffer. The sample dialyzed
DEAE Toyopearl 650M column (φ 2.5×35cm long)and eluted by the stepwise gradient with 0, 50, 100,
200, 400mM NaCl/phosphate buffer solutions at flow

protein (280nm)

was loaded on the anion-exchange chromatography,

0.7

1.4

0.6

1.2

0.5
0.4
0.3

1
280nm
600nm

0.8
0.6

0.2

0.4

rate about 1.67ml/min. Fractions having laminarinase

0.1

0.2

activity were pooled and concentrated at 2.5-3.00

kgf/cm2 with the Amicon TAITEC ultra-filter andTomoegawaPaper-Advantec

UP-20

membrane0
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86

fraction number
Fig.1. Anion-exchange chromatography on DEAE Toyopearl
650M;each fraction was 20mL, eluted by stepwise gradient of NaCl
concentration (0, 50, 100, 200, 400mM).

activity (600nm)

recovered, dissolved with phosphate buffer, and60

endo-1,3-β-glucanase from Arthrobacter sp.

Table 1
Summary of purification of the endo-1,3-β-glucanase from Arthrobacter sp.
Purification step

Total activity(U)

Total protein(mg)

Specific activity(U/mg)

Purification factor(fold)

5% Tunicase® solution

188

50.0

3.76

1.00

ammonium sulfate
precipitation

135

28.2

4.79

1.27

DEAE Toyopearl 650M
anion-exchange

111

11.5

9.65

2.57

Sephacryl S-200 HR
gel-filtration

26.0

2.06

12.6

3.35

deviation were obtained from the plots [S]0/v-[S]0

Sequencing of N-terminal amino acids
N-terminal amino-acid sequence of the purified

using an NEC PC-9801 personal computer andenzyme was analyzed for 10 residues after the

Bio-graph software (Kyoto Software Co., Japan) for

SDS/PAGE without staining and with blotting to

non-linear least-squares method.

PVDF membrane for 1h at 100mA. The blotted
protein sample was stained by CBB and analyzed by
the Shimadzu Model PPSQ-219 amino acid sequencer

Analysis of hydrolyzed products
Products from the enzymatic degradation werecomposed of automated Edman degradation system;

analyzedbythin-layer

chromatography(TLC)

Shimadzu LC-10AS Liquid Chromatography andtechniques using Silicagel 70 F254 Plate (Wako Co.,

Shimadzu UV-VIS detector Model SPD-10A.

Japan) and ethyl acetate/acetic acid/water (2:2:1, byvol.) as solvent at about 30-40℃. Oligosaccharides on

Enzyme kinetic analysis
Initialvelocitiesv

the plate were visualized with the orcinol ferricoftheenzyme-catalyzedhydrolysis reaction were analyzed under the different
concentrations

[S]0

of

chloride spray reagent (Sigma Co., USA) at 100℃ for
15 min.

laminari-oligosaccharides(G4-G7, concentration range in 0.04-1.0mM) andsoluble laminarin (range in 0.3125-5.0mg/ml) as

Hydrolysis reaction of saccharides
Laminari-oligosaccharides

G2-G7

at

a

substrates in a 40mM phosphate buffer pH 7.0 at 25℃.

concentration 0.5mM, and chitin, laminarin, lichenan,

The kinetic parameters, Michaelis-Menten constants

curdlan, pustlan and CMC at a concentration 1 mg/ml

Km and molar activity k0, with the value of standard

were examined for the enzyme-catalyzed hydrolysis

0.25

1.8
1.6

0.2

1.4
1.2
1

0.15

280nm
600nm

0.05

0.8
0.6
0.4

0

0.2
0

0.1

activity (600nm)

protein (280nm)

M.W.

1

2

(k)
97.4
66.2
45.0
31.0
21.5

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96
fraction number
Fig.2. Gel-filtration chromatography on Sephacryl S-200 HR; eachfraction was 7.0mL, eluted by 0.3M NaCl/phosphate buffer.

Fig.3. SDS/PAGE of the purified enzyme; lane1:
molecular mass marker; lane2: the purified enzyme, MW
was 32.5k.

61
Z. Pang et al.

Table 2Kinetic parameters of the endo-1,3-β-glucanase from Arthrobacter sp.a
Substrate

Km

k0

k0 /Km(mM or mg / ml )(1/sec)(1/sec·mM or ml /sec·mg)

laminarin
laminariheptaose
laminarihexaose
laminaripentaose
laminaritetaroselaminaritriose
laminaribiose

0.157mg/ml
0.066
0.067
0.107
0.120
n.d.
n.d.

3.5
19
30
43
47
n.d.
n.d.

22.3 ml /sec·mg
288
442
402
392
n.d.
n.d.

a

Reaction conditions: enzyme concentration was 1.5×10-8M (by the method of Lowry ), at pH 7.0,
with 40mM potassium phosphate buffer for 10 min, 25℃. n.d. =not determined.
reaction as a substrate under the experimental

10 min. Thermo-stability was evaluated by theconditions described above. Prior to this test for the

residual activity after incubation of the enzyme at 10,

substrate specificity, lichenan, curdlan, pustlan and20, 30, 40, 45, 50, 55, 60, 70℃ and 80℃ for 2h.

chitin were treated with boiling water at about 95℃
for 15min to make a gel state, then cooled to room
temperature. On the other hand, for the product

Endoglycosidase H treatment
De-N-glycosylation

was

for

the

purifiedcarriedout

withanalysis the enzyme-catalyzed hydrolysis reactions for

enzymelaminarin, laminari-oligosaccharides, curdlan, andglycochain-scission enzyme endo-H: 5μ l of 1U/ml

lichenan were carried out in a 5-8mM phosphateendo-H commercial preparation was dissolved into

buffer pH 7.0 at 25℃ with the enzyme concentration

250μ l of a buffer solution, in which 10μ l of 100mM-7

molecule

reactionaof 1.5×10 M. The reaction was stopped in the boilingphenylmethylsulfonyl fluoride (PMSF) and 3.3μ l ofwater at 15, 30, 60, 90, and 120min after the

3mg/ml Pepstatin A were mixed in 500ml of 100mM

beginning of the reaction.

citrate/phosphate buffer pH5.0. Endo-H solution 50ml
was added to equal volume of the purified enzyme

Effects

of

pH

and

temperature

on

the

enzyme-catalyzed reaction and enzyme stability
The optimal pH of the purified enzyme wasdetermined by running the standard enzymatic assay

solution and was incubated at 37℃ for 22h. Then, thefinalreaction

mixturewasanalyzedbythe

SDS/PAGE to detect the molecular mass after endo-H
treatment.

at 30℃ for 10min in a 30mM sodium pyrophosphatephosphoric acid buffer, of which pH ranges are at

Results and discussion

3.0-10.0. Effect of pH on stability of the enzyme wasanalyzed at pH 4.0 to 10.0 with the pyrophosphate

Purification and N-terminal amino acid sequence

buffer. After incubation at 30℃ for 2h, the residual

analysis

enzymatic activity was measured by the standard
kinetic assay procedures at 30℃.
For analysis of the optimal temperature, the

5%Tunicase®, was precipitated with 65%-saturated
ammoniumsulfatesolution,

followedbycentrifugation, dialysis, and finally anion-exchange

enzyme-catalyzed hydrolysis reactions were carried

andout by running the standard kinetic assay in a 40mM

purification steps, the enzyme activities and protein

potassium phosphate buffer (pH 7.0) at 10 to 90℃ for

quantities of all samples are summarized in Table 1.

gel-filtration

chromatographies.

In

every

62

endo-1,3-β-glucanase from Arthrobacter sp.

Fractions eluted from the anion-exchange column

G109 (Cellulomonas cellulans), a 27k enzyme protein

are shown in Fig.1, indicating that the fractions fromβglⅡ reported by Parrado et al.,4 and 2) an Oerskovia

No.21 to 31 were in the high level of the hydrolytic

xanthineolytica TK-1 glucanase, a 40k enzyme protein

enzyme activity. Protein samples in these fractions

reported by Saeki et al.18 This amino acid sequence,

were pooled and concentrated to about 5ml using the

however, was different from those of 1) an

ultra-filtration apparatus. Subsequently, the sample

Arthrobacter

concentrated was loaded on a column with Sephacryl

submitted (Dec, 1993) to the GenBank databases

S-200 HR for the gel-filtration and its chromatogram(SWISS-PROT,

was obtained as illustrated in Fig.2, clearly indicating

Watanabe et al. and 2) an Oerskovia xanthineolyticathat a single peak of protein is consistent with that of

glucanase, reported by Shen et al.7 Moreover, thehydrolytic enzyme activity. The protein samples in the

N-terminal amino acid sequence of a glucanase fromfractions from No.61 to 67 were collected andArthrobacter sp. strain YCWD3, which is coincided

concentrated by using the ultra-filtration apparatus.

with Arthrobacter citreus, is almost all the same as

Thus obtained enzyme preparation was analyzed bythat

the SDS/PAGE as shown in Fig.3, clearly indicating axanthineolytica, MW of these enzymes are 5,5000 andsingle band of protein, which was, a pure preparation

5,7000, respectively.

of the enzyme obtained by the purification procedures
employed. N-terminal sequence of amino acids was

of

sp.

(strainaccessionanother

YCWD3)
numberglucanaseglucanase,Q59146)frombyOerskovia

Anyway, the purified enzyme obtained here from
Tunicase® is called ENG in this study.

analyzed for the initial 10 residues of the pure enzyme
preparation by using a sequence analyzer and found toProteochemical and molecular properties of thebeenzyme ENG

APGDLLWSDE,

namelyAla-Pro-Gly-Asp-

Leu-Leu-Trp-Ser-Asp-Glu-, of which sequence is theMolecular mass 32,500 was evaluated for the ENG

same as those of 1) Oerskovia xanthineolytica LL

obtained here using the SDS/PAGE procedures asshown in Fig.3. Moreover, another experimental trial120

was carried out as follows: The ENG obtained was

activity (%)

100

examined using the gel-filtration chromatography with

80

Sepahcryl S-200 HR. Based on the results, its

60
pH activity
40

molecular mass of the native state for the ENG was

pH stability

20

roughly evaluated to be 21,700, suggesting that the

0
3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

8

8.5

9

9.5

10 10.5

ENG is a monomer protein. In this study, it waspH
Fig.4. Effect of pH on activity and stablity of the purified enzyme (30℃).

Table 3
Substrate specificity of the endo-1,3-β-glucanasefrom Arthrobacter sp.a120
100
te m p e r a tu r e a c tiv ity
te m p e r a tu r e sta b ility

activity (%)

80
60
40
20
0
10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

T e m p e r a tu r e ( ℃ )Fig .5 . E f f e c t o f te m p e r a tu r e o n a c tiv ity a n d sta b ility o f th e p u r if ie d e n z y m e( p H 7 .0 ) .

Substrate

Linkage type

Relative rate (%)

laminarin
curdlanlichenan
CMC
chitinpustlan

β-1,3
β-1,3
β-1,3-1,4
β-1,4
β-1,4
β-1,6

100
53
64
0
0
0

a
Reaction conditions: all substrates concentration was 1mg/ml,
curdlan,lichenan, pustlan, chitin were boiled (about 95℃) for 15
min before the assay, enzyme concentration was 1.5×10-8M (bythe method of Lowry ), at pH 7.0, 25℃, with 40mM potassium
phosphate buffer for 10 min.

63

Z. Pang et al.

concluded that the ENG is composed of a monomer

laminarin under various pHs as the results are shown

having the molecular mass of 32,500.

in Fig.4, where the hydrolytic reaction rates are

The ENG was treated with endo-H, at 37℃ in thepresented by a relative value (%). The ENG-catalyzed

phosphate buffer pH4.5 for 22h and the reaction

reactions were observed to maximum at pH 6.5, thus

mixture was analyzed by the SDS/PAGE procedures.

6.5 is concluded to the optimum pH of the enzyme

The protein band of the reaction mixture on

ENG.

SDS/PAGE

enzyme-catalyzed reaction was observed at pH 5.0-8.0

chromatogram was compared with that of theat 30℃ for 2h as shown in Fig.4. Thus, stable pH ofreference protein, which was the ENG not treated with

the ENG-catalyzed reaction is concluded to be in theendo-H. No difference in these protein bands was

range of 5.0 to 8.0. Effects of temperature on thefound between the sample and the reference,

enzyme-catalyzed reaction and on the stability of thesuggesting that the ENG could be little digested (theenzyme were observed for the substrate laminarin bydata not shown) by the endo-H-catalyzed reaction.

the steady-state kinetics under various temperatures in

The endo-H enzyme employed has a characteristic

the phosphate buffer pH 7.0, as the results are shown

property on the substrate specificity, of which

in Fig.5. At 55 ℃ , the ENG-catalyzed reaction

catalyzed-reaction

theexhibited in maximum, and the ENG molecule was

3-continuous mannose sequence in the substrate

stable at less than 40℃ and was very unstable at

glycochain molecule. In this stage of our investigation,higher than 50℃.

rate

is

very

slow

for

More

than

80%

activityofthe

we cannot deny this possibility for the glycochain
attached to the ENG obtained. The saccharide attached

Kinetic properties of the ENG-catalyzed reactions

to ENG was supposed to be not exist, because, in the

The steady-state kinetics of the ENG-catalyzed

present studies, the presence of saccharide linkage wasreaction was carried out for laminarin, lichenan,

endo-1,3- β

curdlan, and laminari-oligosaccharides Gn (degree of

not

known

in

-glucanasefrompolymerization n=2-7) as substrates at the enzyme

procaryote(bacteria).
Effects of pH on the ENG-catalyzed reaction and on

concentration 1.5 × 10-8M. Under the experimental

the molecular stability of ENG were examined by theconditions employed, it was found that all thesteady-state kinetic measurement for the substratesacchari- des examined were hydrolyzed by the

(A)
M 30

(B)
60

120minM 15 30 60 90 120min

ENG-catalyzed reaction. The reactions with laminarin
and laminarioligosaccharides were confirmed to beobeyed the Michaelis-Menten kinetics over theconcentration

range

laminari-oligosaccharides

0.04-1.0mM

for(G4-G7),

andG1

0.31-5.0mg/ml for soluble laminarin. The steady-stateG2kinetic parameters, the Michaelis constant Km andG3
G4

molar activity k0 values were evaluated for the

G5

substrate laminari-oligosaccharides and laminarin as

G6summarized in Table 2, where the k0/Km values wereobtained with Km and k0. Laminarihexaose G6 has theFig.6. TLC of reaction products obtained from laminarin
and lichenan hydrolyzed by ENG.
(A)
laminarin(5mg/ml),(B)lichenan(2mg/ml).
Reaction conditions:ENG1.5×10-7M, phosphate buffer

largest k0/Km, thus it should be concluded that G6 is

5-8mM, pH7.0, 25℃. M=maker.

for a substrate and were found not to be hydrolyzed by

the

best

oligosaccahride

substrate

for

the

ENG-catalysed reaction. G3 and G2 were examined
ENG at any degree of hydrolysis, even though at64

endo-1,3-β-glucanase from Arthrobacter sp.

higher concentration of enzyme (examined by 1.5×
-7

performed．The major products for every substrates

10 M) for 2h under the experimental conditions

were detected as G3 and G2. The TLC pattern, in case

employed.

of G7, was shown in Fig.7 as an example.
Meanwhile, when G6 and G7 were employed as

Substrate specificity of ENG

substrates for the ENG-catalyzed reaction, theThe substrate specificity was analyzed by thelaminari-oligosaccharides products that degree of

steadystate kinetic procedures for the polysaccharides

polymerization larger than 6 and 7 were alsowith β-1,3-, -1,4-, and -1,6-glucans as the results are

confirmed in the TLC chromatograms. This finding

summarized in Table 3, where the relative values aresuggests that transglucosylation reaction would be

represented instead of the kinetic parameters. Theseoccurred in the ENG-catalyzed reaction.

data show that laminarin is the best in the substrate

These findings on the steady-state kinetics and thepolysaccharide examined, and lichenan and curdlan

product analysis give the action pattern of theare good substrates for ENG. The ENG-catalyzed

ENG-catalyzed reaction, which is endo-type.

reaction was examined for pustlan, carboxymethyl
cellulose (CMC), and chitin as substrates, however,
the enzymatic reactions were little observed for these

Identification of the enzyme ENG obtainedA

yeast-lytic

actinomycete

Oerskovia

polysaccharides (Table 3). According to the TLC

xanthineolytica is now considered a subjective

analysis, major saccharides, which are initiallysynonym of Cellulomonas cellulans and previously

produced by the ENG-catalyzed reaction for the

known as Arthrobacter luteus.17 According to thesubstrate laminarin, lichenan, were found to be G3

previous descriptions, the N-terminal amino acidand G6, and G3, respectively, as shown in Fig.6. On

sequence of the initial 10 residues of the ENG is justthe other hand, G2 was found to produce mainly fromthe same as that of Oerskovia xanthineolytica LL

curdlan (data was not shown). These findings suggest

G109 glucanase (βglⅡ ), 4 not only the amino acidthat β-1,3- and also β -1,3-1,4-glucosidic bonds aresequence but also the other properties are very similarsusceptible to hydrolysis with the enzyme ENG. The

in these two enzymes. For example, lichenan is a kind

TLC

from

of suitable substrate for β -1,3-glucanase as well aslaminali-oligosaccharides (G2 to G7) was also

β-(1,3-1,4)-glucanase-cata- lyzed reactions. However,analysisofhydrolyzed

products

molecular mass, optimal temperature, and temperature
G1

stability of the Oerskovia enzyme is very different

G2

from those of ENG. The molecular mass of βgl Ⅱ is

G3

about 27,000 as determined by SDS/PAGE, theG4

optimal temperature is 65℃, and the enzyme is stable

G5
G6
G7

at 55-75℃ at least 80% of its optimal activity butvery unstable above 75℃. Thus, the ENG is different
from the βgl Ⅱ , that obtained from Oerskovia
xanthineolytica LL G109. N-terminal sequence for the
initial 10 amino acid residues of the ENG obtained

M 15 30 60 90 120minof products obtained fromlaminariheptaose hydrolyzed by ENG.
Reaction conditions: laminariheptaose 10mM,
the others were same as described Fig.6.
M=maker.

Fig.7

TLC

here is also the same as that of Oerskovia
xanthineolytica TK-1 glucanase, but the later MW is40,000. Thus, those glucanases reported by several
researchers have just different proteochemical andenzmymatic properties, MW, optimum and stable pH,

65

Z. Pang et al.

optimum and stable temperature, were different from

2. Hrmova, M.; Fincher, G.B. Biochem. J. 1993,
289, 453-461.

the ENG obtained here.
The results described above suggest that an intact

3. Boller, T. in Plant-Microbe Interaction: Molecularenzyme molecule could be built as a part with theand Genetic Perspectives, Kosuge, T.; Nester,

enzymes from Oerskovia xanthineolytica LL G109
(βgl Ⅱ MW 27,000), and from Arthrobacter sp.
(MW32,500), which is studied here. Glucanase from
Oerskovia xanthineolytica TK-1 could be regarded as

E.W., Eds.; Macmillan, New York, 1987; Vol.2,
pp385-413.
4. Parrado, J.; Escuredo, P.R.; Conejero-Lara, F.;

the intact enzyme, thus the other glucanases are

Kotik, M.; Ponting, C.P.; Asenjo, J.A.; Dobson,derivedC.M. Biochimica et Biophysica Acta, 1996,

fromOerskovia

xanthineolytica

TK-1,

possibly. In this stage we do not understand how the
enzymes are derived from the intact, maybe, byde-amination or proteolysis.
endo- β-1,3-glucanase

5. Kitamoto, Y.; Kono, R.; Shimotori, A.; Mori, N.;
Ichikawa, Y. Agric. Biol. Chem. 1987, 51,

In bacteria, a metabolic function has been reported
for

1296,145-151.

andendo- β-1,3-1,4-glucanase, which are polysaccharide endohydrolases

3385-3386.
6. Aono, R.; Hammura, M.; Yamamoto, M.; Asano, T.

with closely related specificities. β-1,3-glucanases

Appl. Envtron. Microbiol. 1995, 61, 122-129.

hydrolyze 1,3- β -glucosyl linkages, but they usually

7. Shen, S.H.; Chrétien, P.; Bastien, L.; Slilaty, S.N.

requirea

1,3- β-linked

regionofglucoseβ-1,3-1,4-glucanases

unsubstituted,
residues.

catalyzethecontiguousIn

contrast,hydrolysis

of

1,4- β-glucosyl linkages only when the glucose
residue itself is linked at the O-3 position.17 Genes
encoding β-1,3-and β-1,3-1,4-glucanases have beencloned and sequenced from different bacterial species,
including Oerskovia xanthineolytica LL G109 (βglⅡ ,NCBI accession number S72212 and AF052745,

8. Ballou, C.E. in The Molecular Biology of the Yeast
Saccharomyces, Strathern, J.N.; Jones, E.W.;
Broach, J.R., Eds.; Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY, 1982; pp 335-360.
9. Manners, D.L.; Masson, A.J.; Patterson, J.C.
Biochem. J. 1973, 135, 19-31
10. Zimmerman, J.W.; Lindermuth, J.; Fish, P.A.;
Palace, G.P.; Stevenson, T.T.; DeMong, D.E.

Japan).
Many

J. Biol., Chem., 1991, 266, 1058-1063.

bacterialendo- β-1,3-glucanases(laminarinases) have been known to date sharesequence similarity with endo- β-1,3-1,4-glucanases(lichenases) and classified in the same family 16 of
glycosyl hydrolases.1,35 It is still unclear for the geneand full amino acid sequence of the ENG now,

J.Biol. Chem. 1998, 273, 22014-22020.
11. Lee, D.Y.; Ji, I.H.; Chang, H-I.; Kim, C-W.
Biosci.

Biotechnol. Biochem. 2002, 66, 233-238.

12. Otaka, K.; Hidaka, K.; Nakajima, A.; Maoka, T.
J. Biol. Macromol., 2002, 2, 64-74.

however, based on the properties of the ENG

13. Ohnishi, M. Glucoenzymes, Japan Scientificdescribed previously, suggesting that ENG belong toSocieties Press, KARGER, Japan, 2000,

the family 16 of glycosyl hydrolase.

pp217-221.
14. Sangyouchousakai Jitenshuppansenta. Foods

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Communicated by Yoshihiko Amano