J. Korean Soc. Appl. Biol. Chem. 53(3), 356-363 (2010) Article

Molecular Cloning and Functional Expression of an ExtracellularExo-β-(1,3)-glucanase from Pichia guilliermondii K123-1.

Jai-Hyun So and In-Koo Rhee*

Department of Agricultural Chemistry, Kyungpook National University, Daegu 702-701, Republic of Korea

Received January 19, 2010; Accepted March 16, 2010

The molecular cloning of the exo-β-(1,3)-glucanase gene from Pichia guilliermondii K123-1 was

achieved by polymerase chain reaction amplification using oligonucleotides designed according to

the N-terminal amino acid sequence of purified exo-β-(1,3)-glucanase and the conserved regions in

exo-β-(1,3)-glucanase from different yeast species. This gene predicts an open reading frame that

has no intron and encodes a primary translation product of 408 amino acids. This preproprotein

processes a mature protein of 389 amino acids by signal peptidase and a Kex2-like endoprotease.

The mature protein shares 54% to 68% amino acid identity with other yeast exo-β-(1,3)-glucanases

of the glycosyl hydrolase family 5. The eight invariant amino acid residues of the active site and

signature pattern (IGIEALNEPL) which existed in all Family 5 members were shown in the

mature protein of exo-β-(1,3)-glucanase but the fifth amino acid (LIVMGST) in the Family 5

signature pattern was changed to A. The cloned exo-β-(1,3)-glucanase gene was successfully

overexpressed in Pichia pastoris X-33 and purified by Ni-NTA His-bind resin chromatography. The

molecular mass of the overexpressed enzyme was determined to be approximately 44 kDa. The

optimum pH and temperature for activity was 4.5 and 45oC, respectively. This enzyme showed the

highest activity toward laminarin (apparent Km, 5.24 mg/mL; Vmax, 7.75 U/μg protein) among

the physiological substrates and 4-methylumbelliferyl-β-D-glucoside (apparent Km, 8.67 mM;

Vmax, 8.99 U/μg protein) among the chromogenic substrates.

Keywords: Pichia guilliermondii K123-1, exo-β-(1,3)-glucanase, isoflavone glycoside

In order to increase the aglycone form of isoflavones in

fermented soyfood, it is necessary to select an appropriate

microbial strain that converts the isoflavone glycoside

form to its aglycone form effectively. The Pichia

guilliermondii (P. guilliermondii) K123-1 has good

ability to convert the isoflavone glycoside form to the

isoflavone aglycone form. It was resistant to 15% NaCl

and was isolated from Korean traditional soybean paste

[Kim et al., 2009]. The enzyme that converts the

isoflavone glycoside form to its aglycone form effectively

was also purified from the cell extracts of P. guilliermondii

K123-1 and the N-terminal amino acid sequence of the

enzyme was determined [So, 2007]. Based on the N-

terminal amino acid sequence, it was deduced that this

enzyme belonged to exo-β-(1,3)-glucanase. The most

abundant cell wall hydrolases in yeasts carrying β-glucan

as the major structural component of their cell envelope

are the 1,3-β-glucanases. Their action is known in the

structural alteration of rigid cell walls to accommodate

changes in morphology such as budding, cell separation,

mating and sporulation [del Rey et al., 1980]. These

enzymes can be classified as exoglucanases or

endoglucanases according to their hydrolytic mode of

action. Exo-β-(1,3)-glucanase liberates glucose from the

1,3-β-glucan. This enzyme has been shown to be rather

non-specific because it is able to hydrolyze the synthetic

derivative p-nitrophenyl-β-D-glucoside as a substrate for

β-glucosidases and acts on 1,6-β-linkages with less

efficiency. On the other side, endoglucanase produces a

mixture of laminaridextrins by the hydrolysis of laminarin

[Nombela et al., 1988 Cid et al., 1995]. Many exo-β-

(1,3)-glucanases from yeast have been purified from

extracts or culture fluids of Saccharomyces cerevisiae

[Farkas et al., 1973; del Rey et al., 1980; Sánchez et al.,

1982; Hien and Fleet, 1983; Cenamor et al., 1987],

Kluyveromyces fragilis, Hansenula anomala [Abd-El-Al

and Phaff, 1968], K. lactis [Tingle and Halvorson, 1971],

P. polymorpha [Villa et al., 1975], Cryptococcus albidus

[Notario et al., 1975], Candida utilis [Notario et al.,

*Corresponding authorPhone: +82-53-950-5718; Fax: +82-53-953-7233E-mail: ikrhee@knu.ac.kr

doi:10.3839/jksabc.2010.055

exo-β-(1,3)-glucanase of Pichia guilliermondii K123-1 357

1976], K. aestuarii [Lachance et al., 1977], K.

phaseolosporus [Villa et al., 1978] and Candida albicans

[Ram et al., 1984; Molina et al., 1989; Luna-Arias et al.,

1991]. Also the molecular characterization of functional

homologues of exo-β-(1,3)-glucanases from yeast has

been reported in C. albicans (CaXOG1), P. angusta

(PaEXG1), K. lactis (KlEXG1), Debaryomyces occidentalis

(DoEXG1) [Chambers et al., 1993; Esteban et al., 1999]

and P. pastoris [Zhiwei et al., 2006]. In this paper, the

cloning and overexpression of the exo-β-(1,3)-glucanase

from P. guilliermondii K123-1 and the properties of this

enzyme were reported.

Materials and Methods

Strains and plasmids. P. guilliermondii K123-1 was

isolated from Korean traditional soybean paste [Kim et

al., 2009]. For the genomic library, Escherichia coli

ER1647 was used for λ phage transfection [Woodcock et

al., 1989]. E. coli BM25.8 was used as a strain expressing

cre recombinase [Palazzolo et al., 1990]. Bacteriophage

λBlueSTAR (Novagen, Madison, WI) was used as a

vector for construction of the gene library. For the expression

of recombinant exo-β-(1,3)-glucanase, P. pastoris X-33

which was used as a host strain, plasmid vector pPICZ

and E. coli TOP10F' cells were purchased from

Invitrogen (Carlsbad, CA).

Growth media and culture. E. coli TOP10F' cells

were grown aerobically at 37oC in a low salt Luria-

Bertani medium. P. guilliermondii K123-1 and P. pastoris

X-33 were cultured at 30oC in YPD medium composed of

1% yeast extract, 2% peptone and 2% dextrose. Competent

cells of P. pastoris X-33 were prepared according to the

supplier’s instructions. pPICZ and its derivates were

transformed into these cells using electroporation methods

with a electroporator II (Invitrogen, Carlsbad, CA). The

transformants were selected on YPDS plates (YPD plus 1

M sorbitol) containing zeocin (100 µg/mL) after

incubation for 2 days at 30oC. For the cultivation of the

transformants, MG medium (1.34% yeast nitrogen base,

4×10−5% biotin and 1.0% glycerol) was used. BMM

medium (1.34% yeast nitrogen base, 4× 10−5% biotin, and

0.5% methanol in 100 mM potassium phosphate, pH 6.0)

was used for the overexpression of exo-β-(1,3)-glucanase.

Cloning of the exo-β-(1,3)-glucanase gene by

Polymerase Chain Reaction (PCR). The genomic DNA

was extracted from P. guilliermondii K123-1 grown

overnight in YPD medium at 30oC as described by

Sambrook et al. [1989]. Based on the experimentally

determined exo-β-(1,3)-glucanase N-terminal amino acid

sequence (GLNWDYDN) of P. guilliermondii K123-1

[So, 2007] and the conserved C-terminal sequence

(GEWSAALTDCAR) from exo-β-(1,3)-glucanases of D.

occidentalis (Q12700), Debaryomyces hansenii (XP_458825),

P. stipits (XP_001385760), C. albicans (XP_721216), C.

oleophila (Q8NKF9), Pichia anomala (ABK40520), P.

pastoris (AAY28969) and Saccharomyces kluyveri

(Q875R9), the primers were prepared for the amplification

of exo-β-(1,3)-glucanase gene. PCR products were amplified

from the chromosomal DNA of P. guilliermondii K123-1

as a template at the following temperature profile: 30

cycles of 95oC for 20 s, 50oC for 40 s and 72oC for 1 min

30 s with sense (5'-GGIYTIAAYTGGGAYTAYGAYAA-

3') and antisense (5'-TCTGTCAAAGCAGCAGCACAT

TCACC-3') primers. A final extension was carried out at

72oC for 5 min after the last amplification cycle. The

sequence of first round PCR product was analyzed by

Bioneer Co. (Chungwon, Korea) and used as the probe

for cloning. The chromosomal DNA library of P.

guilliermondii K123-1 was constructed using λBlueSTAR

BamH arms kit (Novagen, Madison, WI) according to the

supplier’s instructions. The probes were radio-labeled

with [α-32P] dCTP with the Rediprime DNA labeling

system (Amersham-Pharmacia Biotech, Chalfont, England).

The chromosomal DNA of P. guilliermondii K123-1 was

partially digested with Sau3AI to yield fragments with an

average size of 15 kb. These fragments were ligated in the

λBlueSTAR phage, which had been completely digested

with BamHI and dephosphorylated with an alkaline

phosphatase. In vitro packaging and infection into E. coli

ER1647 were carried out and the plaque hybridization

was performed as described by Sambrook et al. [1989]

using a Hybond-N nylon membrane (Amersham-Pharmacia

Biotech., Chalfont, England). The positive clones were

transformed into the competent cells of E. coli BM25.8

(Novagen, Madison, WI). The complete sequence of exo-

β-(1,3)-glucanase gene was read by the primer walking

method [Sambrook et al.,2001]. The GenBank/EMBL/

DDBJ accession number of the exo-β-(1,3)-glucanase

sequence in P. guilliermondii K123-1 is FJ648609.

The information of the primer walking results from the

exo-β-(1,3)-glucanase gene was used to design oligo-

nucleotide primers for PCR amplification of the exo-β-

(1,3)-glucanase gene directly from the chromosomal

DNA of P. guilliermondii K123-1. The oligonucleotide

primers were as follows: Sense primer (5'-GGAATTC

ATGCTTCCATACTTCTTTATGATG-3') with a EcoRI

restriction site and antisense primer (5'-GGGGTACCGA

ATTTACATTGGTTGGGATAGG-3') with a KpnI restriction

site. PCR amplification of the exo-β-(1,3)-glucanase gene

for the expression was carried out as described above and

the sequence of 1.2 kb DNA fragment of PCR product

contained exo-β-(1,3)-glucanase gene was analyzed by

Bioneer Co. (Chungwon, Korea). For the expression of

358 Jai-Hyun So and In-Koo Rhee

recombinant enzyme, the 1.2 kb PCR product of the exo-

β-(1,3)-glucanase gene was digested with EcoRI and KpnI

and cloned in the vector pPICZ having a polyhistidine tag

(Invitrogen, Carlsbad, CA) and named as pPICZBG.

Expression and purification of recombinant exo-β-

(1,3)-glucanase of P. guilliermondii K123-1. The

subsequent transformation of pPICZBG into competent

cells of P. pastoris X-33 and transformant selection were

performed as described above. In order to perform

overexpression, a selected transformant was cultured in

25 mL of MG medium for 16 h at 30oC. Following

centrifugation, the pellet was resuspended in 100 mL of

BMM medium. Methanol was added to a final

concentration of 0.5% methanol every 24 h to maintain

the induction of the recombinant the exo-β-(1,3)-

glucanase. After incubation on the shaker at 30oC for 48

h, the cells were removed by centrifugation. Thereafter,

solid ammonium sulfate was slowly added with continuous

stirring to the supernatant up to 70% saturation. After

incubation for 24 h at 5oC, precipitate was collected by

centrifugation and dissolved in 10 mL of 100 mM

sodium phosphate buffer, pH 8.0. Finally the exo-β-(1,3)-

glucanase was purified by Ni-NTA His-bind resin

(Novagen, Madison, WI) according to the supplier’s

instructions. The purified enzyme preparation was

analyzed by 10% sodium dodecyl sulfate-polyacrylamide

gel electrophoresis (SDS-PAGE) to determine the

apparent molecular size of the enzyme with Coomassie

brilliant blue R-250 staining.

Enzyme assays. β-D-Glucosidase activity was determined

by measuring the rate of 4-methylumbelliferone and p-

nitrophenol liberation from 4-methylumbelliferyl-β-D-

glucoside (MUG) and p-nitrophenyl-β-D-glucoside (PNPG),

respectively. One hundred microliter of enzyme solution

and 200 µL of 10 mM MUG or PNPG were added to the

2.7 mL of 100 mM citrate phosphate buffer, pH 4.5 and

then incubated for 30 min at 37oC. After incubation, the

reactions were terminated by adding 1 mL of 1 M sodium

carbonate and the released 4-methylumbelliferone or p-

nitrophenol was measured spectrophotometically with a

spectrofluorophotometer (4-methylumbelliferone, excitation

at 363 nm, emission at 440 nm; p-ntrophenol, 420 nm) in

conjunction with a standard curve. One unit of activity

was represented as the amount of enzyme required to

produce 1 µmol of 4-methylumbelliferone or p-nitrophenol

for one min per µg of enzyme protein under the above

conditions. To determine enzyme activity toward

nonchromogenic substrates, 100 µL of enzyme solution

and 100 µL of 10 mM substrate (in the case of laminarin,

8 mg/mL) were added to the 800 µL of 100 mM citrate

phosphate buffer, pH 4.5. After incubation for 30 min at

37oC, the reactions were terminated by thermal inactivation

for 2 min at 100oC. Liberated glucose was determined by

a glucose hexokinase assay kit (Sigma-Aldrich, St. Louis,

MO) in conjunction with a standard curve. One unit of

activity was defined as the amount of enzyme required to

produce 1 µmol of glucose from the substrate for one min

per µg of enzyme protein.

Effect of temperature and pH on exo-β-(1,3)-

glucanase. The optimum pH for the exo-β-(1,3)-glucanase

was determined with 10 mM MUG as a substrate in

citrate phosphate buffers (pH 3.0 to 7.0), sodium phosphate

buffers (pH 6.5 to 8.0) and Tris-HCl buffers (pH 8.0 to

9.5) at 100 mM final concentration. The optimal temperature

of exo-β-(1,3)-glucanase was investigated at 30 to 60oC in

100 mM citrate phosphate buffer, pH 4.5 with 10 mM

MUG as a substrate.

Results and Discussion

Molecular cloning of exo-β-(1,3)-glucanase gene of

P. guilliermondii K123-1. To prepare the probe for the

genomic library, a sense primer based on the empirical N-

terminal sequence (GLNWDYDN) confirmed from the

purified enzyme [So, 2007] was designed. An antisense

primer based on the conserved C-terminal motif

GEWSAALTDCAR exhibited by the other yeast exo-β-

(1,3)-glucanases retrieved from NCBI (http://www.ncbi.

nlm.nih.gov/) was also designed. About 900 bp of PCR

product was obtained. Its sequence was analyzed to verify

whether the PCR product corresponded to the majority of

the exo-β-(1,3)-glucanase or not. To obtain its 5'- and 3'-

ends, a genomic library was constructed using 900 bp of

PCR product as a probe and primer walking was undertaken.

As shown in Fig. 1, the exo-β-(1,3)-glucanase gene

consists of 1,227-nucleotide open reading frame (ORF)

terminated by a TAG stop codon without intron and the

ORF encodes a deduced polypeptide of 408 amino acids.

A BLAST search using this ORF as query sequence

indicated that this enzyme had high sequence identity in

the range between 54 and 68% and similarity in the range

between 68 and 79% to the exo-β-(1,3)-glucanases (EC

3.2.1.58) from other yeasts such as Debaryomyces

occidentalis (identity, 68%; similarity, 79%; accession no,

Q12700), Candida albicans (identity, 64%; similarity,

76%; accession no, XP_721216), Kluyveromyces lactis

(identity, 55%; similarity, 71%; accession no, XP_452436)

and Candida glabrata (identity, 54%; similarity, 68%;

accession no, XP_447274). This enzyme belonged to

Glycosyl Hydrolase Family 5, which included the endo-

1,4-β-glucanases(cellulases), β-xylanases, and β-mannanases

and was also known as Cellulase A (http://afmb.cnrs-

mrs.fr/~cazy/CAZY/index.html).

Despite the considerable divergence, this family has

exo-β-(1,3)-glucanase of Pichia guilliermondii K123-1 359

two structural features shared by all the members of this

group. The first is the presence of the consensus motif

[LIV]-[LIVMFYWGA](2)-[DNEQG]-[LIVMGST]-X-

N-E-[PV]-[RMTNSTLIVFY] as a common primary

signature pattern in the part of their sequence involved in

substrate hydrolysis [Zhiwei et al., 2006]. The second is

the strict conservation at eight amino acid positions which

were composed of the active sites in the Glycosyl

Hydrolase Familiy 5. Among these amino acids, two of

the eight amino acid positions are glutamic acid residues

which are involved in glycosidic bond cleavage, one

acting as the proton donor [Baird et al., 1990; Py et al.,

Fig. 1. Nucleotide sequence and deduced amino acid sequence of exo-(1,3)-β-glucanase gene in P. guilliermondiiK123-1. In the coding region, the first and second arrowhead indicates the putative processing sites for the signal peptidaseand the Kex2-like endoprotease, respectively. The signal peptide (MLPYFFMMAATFAAA) and consensus sequence(signature pattern) of Glycosyl Hydrolases Family 5 (IGIEALNEPL) lie within the gray box. The eight invariant aminoacid residues exhibited by members of Glycosyl Hydrolases Family 5 are circled. The GenBank/EMBL/DDBJ accessionnumber of the exo-β-(1,3)-glucanase sequence in P. guilliermondii K123-1 is FJ648609.

360 Jai-Hyun So and In-Koo Rhee

1991; Navas and Béguin, 1992] and the other as the

nucleophile [Macarrón et al., 1993; Wang et al., 1993;

Mackenzie et al., 1997].

In the exo-β-(1,3)-glucanase of P. guilliermondii K123-

1, the signature pattern of Glycosyl Hydrolase Family 5

can be found between residues 195 and 204 (IGIEALNEPL),

but the fifth amino acid residue (LIVMGST) was

changed to [A]. Eight invariant amino acid positions

(circled amino acid residues in Fig. 1) corresponded to

the acid residues of Glu202 and Glu301 and the polar

planar groups of Arg102, His145, Asn201, His 262,

Tyr264, and Phe371 (Fig. 1) which might be composed of

the active site in the enzyme of P. guilliermondii K123-1.

The deduced ORF contained an additional 19 amino

acids compared to purified exo-β-(1,3)-glucanase, but the

likely cleavage site of an N-terminal signal peptide for

exo-β-(1,3)-glucanase was anticipated to be between

positions 15 and 16 (i.e., AAA-IT) by both Neural

Networks and Hidden Markov Models of SignalP 3.0

(http://www.cbs.dtu.dk/services/SignalP/). This implies

that the exo-β-(1,3)-glucanase of P. guilliermondii K123-

1 was synthesized as a preproprotein that was initially

processed by a signal peptidase when it entered into the

secretory pathway and then the proprotein was cleaved

immediately after the RR dipeptide by a Kex2-like

endoprotease, which resulted in a mature protein with the

empirically determined N-terminal sequence. These

kinds of processing were also observed in yeast exo-β-

(1,3)-glucanase and C. albicans exo-β-(1,3)-glucanase

[Vázquez et al., 1991; Navas and Béguin, 1992] where

exo-β-(1,3)-glucanase maturation involved the removal

of the N-terminal hydrophobic portion by means of the

signal peptidase followed by an additional proteolytic

cleavage after a Lys-Arg dipeptide by a Kex2-endoprotease.

The molecular mass and pI of mature protein was

predicted to be 44,417.06 Da and 4.64, respectively,

following computations performed with the pI/Mw

program (http://www.expasy.ch/cgi-bin/pi_tool).

Overexpression and purification of active recombinant

exo-β-(1,3)-glucanase. The complete exo-β-(1,3)-glucanase

gene ORF was cloned into the pPICZ vector and named

as pPICZBG. After that, plasmid pPICZBG was transformed

into P. pastoris X-33 for the overexpression. After

methanol induction for 48 h, the exo-β-(1,3)-glucanase

activity produced in the culture medium of pPICZBG/P.

pastoris X33 was 42-fold higher than that observed in

control (empty vector) cultures (data not shown). To

verify the protein expression, SDS-PAGE was carried out

with culture filtrate every 24 h. As time went on, the

protein expression increased till 96 h (data not shown) but

it was obvious that the activity decreased after 72 h.

Therefore the pH of culture medium was checked. The

pH of culture medium after 72 h had decreased to 3.1

(data not shown). As a result, it was presumed that the

decrease of exo-β-(1,3)-glucanase activity after 72 h was

caused by the instability of enzyme in the low pH of the

culture medium (Fig. 4b).

Subsequent purification of exo-β-(1,3)-glucanase to

apparent homogeneity was accomplished in a single step

by Ni-NTA His-bind resin chromatography after

Fig. 3. Optimum temperature and thermal stability ofthe over-expressed exo-(1,3)-β-glucanase from pPICZBG/P. pastoris X-33. The enzyme activity for the optimaltemperature was measured in the standard reaction mixtureat the indicated temperature for 30 min at pH 4.5. For thethermal stability, the enzyme was incubated in 100 mMcitrate phosphate buffer, pH 4.5, for 30 min at eachtemperature and then remaining activity was measured inthe standard reaction mixture as described in Materials and

Methods. �-�, thermal stability; �-�, optimum temperature

Fig. 2. SDS-PAGE analysis of the purified exo-(1,3)-β-glucanase from pPICZBG/P. pastoris X-33. Protein wasstained with Coomassie brilliant blue R-250. Lane 1,molecular mass marker; lane 2, exo-(1,3)-β-glucanasepurified by Ni-NTA His-bind resin; 66 kDa, bovine serumalbumin; 45 kDa, ovalbumin; 36 kDa, glyceraldehyde-3-phosphate dehydrogenase; 29 kDa, carbonic anhydrae; 24kDa, trypsinogen.

exo-β-(1,3)-glucanase of Pichia guilliermondii K123-1 361

precipitation with ammonium sulfate. This step yielded a

homogeneous protein, as judged by a single band of

approximately 44 kDa molecular mass upon SDS-PAGE

with Coomassie brilliant blue R-250 staining (Fig. 2).

The molecular mass of recombinant exo-β-(1,3)-

glucanase on the SDS-PAGE was similar to the molecular

mass (44,417.06 Da) calculated with the pI/Mw program

(http://www.expasy.ch/cgi-bin/pi_tool). The molecular

mass of exo-β-(1,3)-glucanase of K. lactis, Hansenula

polymorpha and Schwanniomyces occidentalis was

49,815, 49,268, and 49,132 Da, respectively [Esteban et

al., 1999]. The molecular mass of exo-β-(1,3)-glucanase

of P. guilliermondii K123-1 was somewhat low compared

to other yeasts as known before.

Characterization of the recombinant exo-β-(1,3)-

glucanase. The recombinant exo-β-(1,3)-glucanase was

stable at temperatures between 30 and 45oC but unstable

above 50oC. The optimum temperature for the enzyme

was 45oC at pH 4.5 (Fig. 3). The recombinant exo-β-

(1,3)-glucanase was stable over a broad pH range from

4.0 to 9.0 but its stability was rapidly decreased under pH

3.5 (Fig. 4b). The maximal activity was observed at pH

4.5 in a 100 mM sodium citrate buffer (Fig. 4a). The

optimal pH of recombinant exo-β-(1,3)-glucanase was

slightly low compared with other exoglucanases purified

from P. anomala (pH 5.5) [Jijakli and Lepoivre, 1988], K.

aestuarii (pH 5.2) [Lachance et al., 1977], C. albicans

(pH 5.5-6.0) [Molina et al., 1989] and P. pastoris (pH 6.0)

[Zhiwei et al., 2006].

Substrate specificity of the recombinant exo-β-(1,3)-

glucanase. To investigate the substrate specificity of the

recombinant exo-β-(1,3)-glucanase for glycosides, its activity

toward several p-nitrophenyl and 4-methylumbelliferyl

glycosides were tested. As shown in Table 1, the enzyme

was active toward MUG (100%), 4-methylumbelliferyl-

β-D-xyloside (15%), and 4-methylumbelliferyl-β-D-

cellobioside (5%), but failed to hydrolyze 4-methyl-

umbelliferyl-β-D-galactosides. Also, the enzyme was less

active toward PNPG (19%), p-nitrophenyl-β-D-xyloside

(3.0%), and p-nitrophenyl-β-D-cellobioside (1.5%), but

failed to hydrolyze p-nitrophenyl-β-D-galactoside. The

Km and Vmax values of the recombinant exo-β-(1,3)-

glucanase for MUG was determined to be 8.67 mM and

8.99 U/µg protein, respectively. As a result of Table 1, it

was deduced that the purified exo-β-(1,3)-glucanase was

more active when the sugar moiety of glycoside was

glucose and the size of aglycone was more bulky than

that of phenyl groups.

A relative assessment was performed to determine the

specificity of the purified enzyme for nonchromogenic

Fig. 4. Optimum pH (A) and pH stability (B) of theover-expressed exo-(1,3)-β-glucanase from pPICZBG/P.pastoris X-33. For the optimal pH, the enzyme activitywas measured at the each indicated pH in the standardreaction mixture. For the pH stability, the enzyme wasincubated in each pH buffer at 4 for 24 h and thenenzyme activity was measured in the standard reaction

mixture as described in Materials and Methods. �-�,sodium citrate buffer; �-�, sodium phosphate buffer; �-�, tris-HCl buffer.

Table 1. Relative activity of the purified exo-(1,3)-β-glucanase toward the selected chromogenic substrates

Substrate Specific activity (unit/μg protein) Relative activity (%)

4-Methylumbelliferyl- β-D-glucoside 4.90 100

4-Methylumbelliferyl-β-D-xyloside 0.72 15

4-Methylumbelliferyl-β-D-cellobioside 0.25 5

4-Methylumbelliferyl-β-D-galactoside 0.02 0

p-Nitrophenyl-β-D-glucoside 0.92 19

p-Nitrophenyl-β-D-xyloside 0.15 3

p-Nitrophenyl-β-D-cellobioside 0.08 1

p-Nitrophenyl-β-D-galactoside 0.04 0

362 Jai-Hyun So and In-Koo Rhee

natural substrates (Table 2). Among the nonchromogenic

substrates, laminarin (100%) which is a linear

polysaccharide made up of β(1→3)-glucan with β(1→6)

linkages was most rapidly hydrolyzed. The cyanogenic

diglucoside amygdalin (84%) which has β(1→6)

linkages and the coumarin glucoside esculin (24%) were

also hydrolyzed, but little activity was shown toward

salicin (1.0%) and cellobiose (8.0%). The purified

enzyme was able to favorably hydrolyze the β-1,3-

glucosidic bond, compared with the β-1,4 and the β-1,6-

glucosidic bonds. With laminarin as a substrate, the

apparent Km was 5.24 mg/mL, and the Vmax was 7.75

U/µg protein.

Little is known about the biological roles of this

enzyme or the chemical composition of Pichia cell walls

yet [Bartnicki-Garcia, 1968; Villa et al., 1980]. But, from

the reports for other well-studied yeast exo-β-(1,3)-

glucanases [del Rey et al., 1982; Nombela et al., 1988;

Cid et al., 1995], it can be deduced that this exo-β-(1,3)-

glucanase may play a role in cell wall modification

during growth, development, and division because of its

extracellular location and activity toward laminarin. In

the S. cerevisiae, six different β-glucanases were purified

[Nebreda et al., 1988] and three different β-glucanases

were purified from the culture medium of P. polymorpha

[Villa et al., 1975].

Another heat-labile β-glucosidase was found in the

culture filtrate of P. guilliermondii K123-1 during the

purification of the enzyme [So, 2007]. Therefore, P.

guilliermondii K123-1 may have additional extracellular

exo-β-(1,3)-glucanase. The exo-β-(1,3)-glucanase existed

in the periplasm of P. guilliermondii K123-1 [So, 2007]

but the overexpressed exo-β-(1,3)-glucanase in P. pastoris

X-33 was secreted mostly into the culture medium and

also existed in the periplasm less than 20% (data not

shown). It was thought to be due to differences in cell

wall composition between P. guilliermondii K123-1 and

P. pastoris X-33 or excessive expression of exo-β-(1,3)-

glucanase.

Acknowledgments. This research was supported (in

part) by Kyungpook National University Research Fund,

2009 (Code 200910470000).

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