JOURNAL OF BACTERIOLOGY, Dec. 1995, p. 6937–6945 Vol. 177, No. 23
0021-9193/95/$04.0010
Copy igh q1995, Ame ican Socie y o Mic obiology
A No el Endo-b-1,3-Glucanase, BGN13.1, In ol ed in he
Mycopa asi ism o T ichode ma ha zianum
JESU
´SDE LA CRUZ,
1
JOSE
´A. PINTOR-TORO,
2
TAHI
´A BENI
´TEZ,
3
ANTONIO LLOBELL,
1
*
AND LUIS C. ROMERO
2
Ins i u o de Bioquı´mica Vege al y Fo osı´n esis, Consejo Supe io de In es igaciones Cien i icas y Uni e sidad de
Se illa,
1
Ins i u o de Recu sos Na u ales y Ag obiologı´a, Consejo Supe io de In es igaciones Cien i icas,
2
and
Depa amen o Gene´ ica, Uni e sidad de Se illa,
3
Se ille, Spain
Recei ed 20 June 1995/Accep ed 25 Sep embe 1995
The mycopa asi ic ungus T ichode ma ha zianum CECT 2413 p oduces a leas h ee ex acellula b-1,3-
glucanases. The mos basic o hese ex acellula enzymes, named BGN13.1, was exp essed when ei he ungal
cell wall polyme s o au ocla ed mycelia om di e en ungi we e used as he ca bon sou ce. BGN13.1 was
pu i ied o elec opho e ic homogenei y and was biochemically cha ac e ized. The enzyme was speci ic o b-1,3
linkages and has an endoly ic mode o ac ion. A syn he ic oligonucleo ide p ime based on he sequence o an
in e nal pep ide was designed o clone he cDNA co esponding o BGN13.1. The deduced amino acid sequence
p edic ed a molecula mass o 78 kDa o he ma u e p o ein. Analysis o he amino acid sequence indica es
ha he enzyme con ains h ee egions, one N- e minal leade sequence; ano he , nonde ined sequence; and
one cys eine- ich C- e minal sequence. Sequence compa ison shows ha his b-1,3-glucanase, i s desc ibed
o ilamen ous ungi, belongs o a amily di e en om ha o i s p e iously desc ibed bac e ial, yeas , and
plan coun e pa s. Enzyma ic-ac i i y, p o ein, and mRNA da a indica ed ha bgn13.1 is ep essed by glucose
and induced by ei he ungal cell wall polyme s o au ocla ed yeas cells and mycelia. Finally, expe imen al
e idence showed ha he enzyme hyd olyzes yeas and ungal cell walls.
b-1,3-Glucanases a e widely dis ibu ed among bac e ia,
ungi, and highe plan s (49), and many o hem ha e been
pu i ied and cha ac e ized (10, 26, 40, 63, 73). They a e clas-
si ied as exo-b-1,3-glucanases (b-1,3-glucan glucanohyd olase
[EC 3.2.1.58]) and endo-b-1,3-glucanases (b-1,3-glucan gluca-
nohyd olase [EC 3.2.1.6 o EC 3.2.1.39]). The co esponding
b-1,3-glucanase genes om he a o emen ioned o ganisms
ha e also been isola ed and cha ac e ized, wi h he excep ion
o genes om ilamen ous ungi (29, 56, 58, 61, 70, 73). The
physiological unc ions o b-1,3-glucanases depend on hei
sou ce. In plan s, hese enzymes a e hough o be a ype o
de ense sys em agains ungal pa hogens (25, 37), al hough
oles in cell di e en ia ion ha e also been sugges ed (6, 20, 42).
In bac e ia, which lack b-1,3-glucan, a nu i ional ole has been
assigned (73). In ungi, b-1,3-glucanases seem o ha e di e en
unc ions. Fi s , a physiological ole in mo phogene ic-mo -
pholy ic p ocesses du ing ungal de elopmen and di e en ia-
ion has been indica ed (47). Second, b-1,3-glucanases ha e
been ela ed o he mobiliza ion o b-glucans unde condi ions
o ca bon and ene gy sou ce exhaus ion, unc ioning as au o-
ly ic enzymes (48, 62). b-1,3-Glucanases a e also in ol ed in
ungal pa hogen-plan in e ac ions, deg ading callose (b-D-1,3-
glucan) in he hos ’s ascula issues du ing pa hogen a ack
(56). Finally, a nu i ional ole in sap ophy es and mycopa a-
si es has been sugges ed (9, 35, 59).
Soilbo ne ungi o he genus T ichode ma ha e been espe-
cially s udied o bo h hei celluloly ic ac i i y (38) and hei
an agonis ic p ope ies agains ungal plan pa hogens (46).
The deg ada ion and u he assimila ion o phy opa hogenic
ungi, namely, mycopa asi ism, has been p oposed as he majo
mechanism accoun ing o hei an agonism agains plan
pa hogens (19). A numbe o T ichode ma isola es p oduce
hyd oly ic enzymes such as chi inases, b-1,3- and b-1,6-glu-
canases, and p o eases when g own on lamina in (b-1,3-glu-
can), chi in, o ungal cell walls as he ca bon sou ce (14, 19).
This obse a ion, oge he wi h he ac ha chi in and b-1,3-
glucan a e he main s uc u al componen s o ungal cell walls
(47), sugges s ha chi inases and b-1,3-glucanases p oduced by
some T ichode ma spp. a e he key enzymes in he lysis o cell
walls du ing hei mycopa asi ic ac ion agains phy opa ho-
genic ungi (12, 13, 19, 58).
In his s udy, we epo he pu i ica ion, molecula cloning,
and cha ac e iza ion o an endo-b-1,3-glucanase, namely,
BGN13.1, om he mycopa asi ic ungus T ichode ma ha zia-
num, which has been desc ibed as an agen o biological con-
ol o phy opa hogenic ungi (11). Exp ession o he gene and
enzyme sec e ion occu when T. ha zianum g ows in ungal cell
wall componen s, and hese p ocesses a e speci ically induced
by g owing he ungus in au ocla ed mycelia om di e en
ungi. These condi ions ha e been desc ibed as simula ing
mycopa asi ism (24, 34). Also, on he basis o sequence com-
pa ison, BGN13.1 may be classi ied as a new class o b-1,3-
glucanase enzyme. A possible ole o his enzyme in mycopa a-
si ism is discussed.
MATERIALS AND METHODS
Fungal ma e ial and g ow h condi ions. T. ha zianum CECT 2413 (Spanish
Type Cul u e Collec ion, Valencia, Spain) was used in his s udy. Po a o dex ose
aga was used o he main enance o he cul u e. P oduc ion o he enzyme was
ca ied ou unde he wo-s ep cul u e condi ions p e iously desc ibed (14).
Au ocla ed cells om Saccha omyces ce e isiae (bake ’s yeas ; La Cin a Roja,
Se ille, Spain) o mycelia om Bo y is cine ea CECT 2100, Gibe ella ujiku oi
IMI 58289 (Impe ial Mycological Ins i u e, Kew, Su ey, Uni ed Kingdom),
Phy oph ho a sy ingae CECT 2351, Rhizoc onia solani CECT 2815, o T. ha zia-
num CECT 2413 we e used as ca bon sou ces. Mycelia om he di e en ila-
men ous ungi we e ob ained as desc ibed elsewhe e (33). Mycelia we e lyoph-
ilized and added o T. ha zianum second-s ep cul u es.
b-1,3-Glucanase assay and p o ein de e mina ion. b-1,3-Glucanase ac i i y
was de e mined by measu ing he amoun o educing suga eleased om
* Co esponding au ho . Mailing add ess: Ins i u o de Bioquı´mica
Vege al y Fo osin esis, CSIC y Uni e sidad de Se illa, Apa ado 1113,
E-41080 Se ille, Spain. Phone: (34) (5) 4557080. Fax: (34) (5) 4620154.
6937
on July 25, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om
lamina in (Sigma Chemical Co., S . Louis, Mo.). The s anda d assay mix u e (1
ml) con ained enzyme solu ion p ope ly dilu ed, 4 mg o lamina in, and 50 mM
po assium ace a e bu e , pH 5.5. The eac ions we e un o 30 min a 378C and
hen s opped by boiling o 5 min, and he educing-suga con en was de e -
mined by he p ocedu e o Somogyi (60) and Nelson (43). Enzyme and subs a e
blanks we e also included. A uni o b-1,3-glucanase ac i i y is de ined as he
amoun o enzyme ca alyzing he elease o 1 mmol o glucose equi alen pe
min.
P o ein concen a ion was measu ed by he me hod o B ad o d, wi h o albu-
min as he s anda d (5). Fo mycelium g ow h de e mina ion, o al p o ein was
measu ed as desc ibed elsewhe e (14, 36).
Enzyme pu i ica ion. (i) S ep 1. Unless o he wise indica ed, all enzyme pu i-
ica ion s eps we e ca ied ou a 48C. Supe na an luids (200 ml) om 48-h T.
ha zianum chi in cul u es we e ob ained by il e ing he media h ough Wha man
no. 1 pape and cen i uging a 10,000 3g o 10 min. The supe na an was hen
p ecipi a ed wi h (NH
4
)
2
SO
4
(80% sa u a ion) a 48C o 2 h. The p ecipi a e was
eco e ed by cen i uga ion (12,000 3g o 20 min a 48C), dissol ed in he
minimal amoun o dis illed wa e , and dialyzed ex ensi ely agains 2 li e s o 50
mM po assium ace a e bu e , pH 5.5.
(ii) S ep 2. Dialyzed aliquo s we e hen adso bed o pus ulan (b-1,6-glucan;
Calbiochem, La Jolla, Cali .) as desc ibed elsewhe e (13). Adso bed ac ions
we e washed h ee imes wi h 1 M NaCl–70 mM phospha e bu e , pH 6.0;
esuspended in 50 mM po assium ace a e bu e , pH 5.5, con aining 1 mM
sodium azide and 1 mM phenylme hylsul onyl luo ide; and incuba ed a 378C
o e nigh . Cla i ied solu ions, a e pus ulan diges ion, we e dialyzed ex ensi ely
agains 2 li e s o 25 mM imidazole-HCl bu e , pH 7.4.
(iii) S ep 3. The dialyzed solu ion (named pus ulan diges ion) was subjec ed o
ch oma o ocusing (pH 7.4 o 4) on a Polybu e exchange column, PBE 94 (1 by
20 cm; Pha macia, Uppsala, Sweden), equilib a ed in 25 mM imidazole-HCl
bu e , pH 7.4. P o ein elu ion and g adien condi ions we e exac ly as desc ibed
elsewhe e (13). Mos ac i e ac ions we e pooled and concen a ed o app ox-
ima ely 0.5 ml on Cen icon 10 concen a o s (Amicon, Be e ley, Mass.).
(i ) S ep 4. The concen a ed pool was il e ed h ough a Sephac yl S200 HR
column (1.6 by 40 cm; Pha macia) equilib a ed wi h 1 M NaCl–100 mM po as-
sium ace a e bu e , pH 5.5. Elu ion was pe o med in he same bu e a a low
a e o 7 ml/h, collec ing ac ions o 0.65 ml. Mos ac i e ac ions we e pooled,
washed, and concen a ed in 50 mM po assium ace a e bu e , pH 5.5, on Cen-
icon 10 concen a o s and inally s o ed a 2208C.
Basic ch oma o ocusing. Basic ch oma o ocusing was pe o med by using a
column (1 by 20 cm) o PBE 94 esin equilib a ed in 25 mM e hanolamine-
NaOH, pH 9.4. The column was elu ed a a low a e o 9 ml/h wi h Polybu e
96, which had been dilu ed eigh old wi h wa e and adjus ed o pH 9.4 wi h
NaOH. The ch oma o ocusing was moni o ed by measu ing he pH o he elu ed
ac ions.
Gel elec opho esis. Elec opho esis unde dena u ing condi ions (sodium
dodecyl sul a e-polyac ylamide gel elec opho esis [SDS-PAGE]) was pe o med
by he me hod o Laemmli (31) wi h 4% ac ylamide in he s acking gel and 12%
ac ylamide in he sepa a ing gel. Gels we e s ained wi h Coomassie R-250 b il-
lian blue. Low-molecula -mass s anda d p o eins (Bio-Rad, He cules, Cali .)
we e used o molecula mass de e mina ion.
Glycop o ein s ain assays we e done in he gels by using ei he he pe iodic
acid-Schi eagen p ocedu e (Sigma) o he sil e ni a e p ocedu e (18). Ex-
acellula yeas in e ase (Sigma) was used as a glycop o ein posi i e con ol.
Also, o es ima ing possible mobili y changes, deglycosyla ion eac ions p io o
SDS-PAGE we e pe o med wi h endo-b-N-ace ylglucosaminidase) H (endo-H)
(Boeh inge , Mannheim, Ge many) (70) o wi h a-mannosidase (74). The p es-
ence o ca bohyd a es in he p o ein was also es ima ed by he binding o pu i ied
b-1,3-glucanase o a ious amoun s o concana alin A-Sepha ose 4B (Pha ma-
cia) (54).
Isoelec o ocusing was pe o med a a pI ange o 3.5 o 10 as desc ibed
elsewhe e (51). P o eins we e isualized by Coomassie s aining. b-1,3-Glucanase
ac i i y was de ec ed a e isoelec o ocusing by using Ampholine PAG pla es,
pH 3.5 o 9.5 (Pha macia), in a Mul ipho sys em (Pha macia) acco ding o he
manu ac u e ’s ins uc ions. The ac i i y was assayed by using he lamina in–
2,3,5- iphenyl e azolium chlo ide me hod p e iously desc ibed (45). The e-
agen s we e pu chased om Sigma.
Wes e n blo s (immunoblo s) we e incuba ed wi h an ise um agains T. ha -
zianum b-1,6-glucanase II (13) o an ise um o obacco b-1,3-glucanase an i-
PR2 o obacco basic b-1,3-glucanase (26). Immunological de ec ion o he
an igen-an ibody complexes was ca ied ou as desc ibed p e iously (13). An i-
bodies agains obacco b-1,3-glucanases we e a gene ous gi om B. F i ig,
Ins i u de Biologie Mole´culai e des Plan es, S asbou g, F ance.
N- e minal and in e nal amino acid pep ide sequencing. N- e minal and in-
e nal amino acid pep ide sequencing was pe o med by using an Applied Bio-
sys em 470 A Gas Phase P o ein Sequence , equipped wi h a 120 A on-line
phenyl hiohydan oin analyze , as desc ibed elsewhe e (4).
Kine ic pa ame e s, empe a u e op imum, and he mal s abili y. Michaelis-
Men en cons an s we e de e mined om Linewea e -Bu k ep esen a ions o
da a ob ained by measu ing he ini ial a e o lamina in hyd olysis unde he
assay condi ions desc ibed abo e and using a ange o 20 o 0.5 mg o lamina in
pe ml. The e ec o d-gluconolac one (Sigma) on he ac i i y o he pu i ied
enzyme was measu ed as desc ibed p e iously (44).
The empe a u e op imum and he mal s abili y we e de e mined as desc ibed
elsewhe e (13), by adding lamina in as he subs a e. The inac i a ion empe a-
u e was de ined as he one a which he speci ic ac i i y was educed by 50%
unde he condi ions desc ibed abo e.
Subs a e speci ici y and analysis o b-1,3-glucanase eac ion p oduc s. The
spec um o subs a es hyd olyzed by he enzyme was es ed wi h o he a- and
b-glucans a a inal concen a ion o 5 mg/ml. Reac ion p oduc s we e de e -
mined by he s anda d assay desc ibed abo e.
b-1,3-Glucanase eac ion p oduc s we e also de e mined by high-pe o mance
liquid ch oma og aphy (HPLC) on an HPX 42-A column (Bio-Rad) a 608Cas
desc ibed elsewhe e (13).
Hyd olysis o ungal cell walls. Ly ic ac i i y by he b-1,3-glucanase on ungal
cell walls was moni o ed as desc ibed p e iously (13, 65).
Ex ac ion and blo ing analysis o DNA and RNA. To al DNA om T.
ha zianum was p epa ed as desc ibed elsewhe e (41) and analyzed by Sou he n
blo ing unde condi ions desc ibed p e iously (32).
To al RNA was isola ed acco ding o he me hod o Wadswo h e al. (71).
RNA was sepa a ed in 1% aga ose– o maldehyde gels by s anda d p o ocols (53,
71). Du ing No he n (RNA) analysis, he amoun o RNA loaded on o he gel
was moni o ed by me hylene blue s aining o he memb anes. Equal amoun s o
RNA we e loaded in o he wells.
PCR condi ions and sc eening o he cDNA lib a y. A cDNA lib a y on
lg 11-S i-No was cons uc ed om poly(A)
1
RNA isola ed om T. ha zianum
g own o 33 h on a chi in medium as a second-s ep cul u e (32). To al DNA was
isola ed om his lib a y and used as a empla e o PCR using as a sense p ime
a degene a ed oligonucleo ide [59-AATACICA(A/G)AATGGITATACIG
GIGG-39] co esponding o an in e nal sequenced pep ide o he p o ein (p12)
and as an an isense p ime a comme cial lg 11 e e se p ime . The ampli ica ion
p o ocol used was as ollows: 958C, 1 min; 578C, 1.5 min; 728C, 2.0 min. This
s anda d cycle was epea ed 35 imes and was ollowed by incuba ion a 728C o
5 min. The single ampli ied band was pu i ied om 0.8% aga ose gels, subcloned
in o pGEM-T ec o (P omega), and analyzed by sequencing.
App oxima ely 4 310
4
PFU om he lg 11-S i-No cDNA lib a y was
sc eened by plaque hyb idiza ion, wi h he
32
P-labeled PCR-ampli ied cDNA
agmen desc ibed abo e used as a p obe. Hyb idiza ions we e ca ied ou by a
s anda d p o ocol p e iously desc ibed (32).
DNA sequencing. Nucleo ide sequencing was pe o med by he dideoxynucle-
o ide chain e mina ion me hod (55) wi h a Pha macia ALF au oma ic sequenc-
ing sys em. Compu e analyses o sequencing da a we e pe o med by using he
Uni e si y o Wisconsin Gene ics Compu e G oup p og ams (15).
Nucleo ide sequence accession numbe . The nucleo ide sequence epo ed in
his a icle has been submi ed o he GenBank/EMBL Da a Bank unde acces-
sion numbe X84085.
RESULTS
Enzyme p oduc ion. Enzyme p oduc ion was a emp ed un-
de wo-s ep cul u e condi ions. In he i s s ep, minimal me-
dium con aining 10% glucose ( ep ession condi ions) was used
o g ow he mycelia; in he second s ep, minimal medium
con aining he selec ed ca bon sou ce (induc ion condi ions)
was used and he le els o ex acellula b-1,3-glucanase ac i i y
we e measu ed. Table 1 shows he b-1,3-glucanase-speci ic ac-
i i y ound a e 48 h o induc ion. The b-1,3-glucanase ac i -
i y was de ec ed in abundance in chi in, pus ulan, and au o-
cla ed yeas cells and in ilamen ous ungal mycelia used as
ca bon sou ces. Ac i i y, hough less abundan , was also de-
ec ed when 0.1 and 2% glucose, 1% lamina in, and chi in
supplemen ed wi h 2% glucose we e used; inally, a a concen-
a ion o 10% glucose he e was no de ec ion o he ac i i y.
Resul s, he e o e, sugges ep ession a a high glucose con-
cen a ion, basal ac i i y a a lowe glucose concen a ion, and
induc ion in he p esence o ungal cell wall polyme sou ces,
yeas cells, o ungal mycelia.
In o de o es whe he he induc ion de ec ed in hese
expe imen s is due o he p esence o di e en b-1,3-glucanase
isoenzymes, enzyma ic de ec ion on isoelec o ocusing gels was
ca ied ou wi h ammonium sul a e-concen a ed supe na an s
o he p e ious cul u es a e 48 h o induc ion. As Fig. 1
shows, no b-1,3-glucanase iso o ms we e de ec ed in he p es-
ence o 2 and 10% glucose. Unde s a a ion condi ions (0.1%
glucose), a sligh basic band was occasionally de ec ed; in he
p esence o di e en ungal cell wall polysaccha ides, he basic
isoenzyme and a leas h ee addi ional acidic b-1,3-glucanases
6938 DE LA CRUZ ET AL. J. BACTERIOL.
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we e ound. Finally, in he p esence o ei he au ocla ed yeas
cells o di e en au ocla ed ilamen ous ungi he basic iso-
o m was p e e en ially induced. Thus, he measu ed b-1,3-
glucanase ac i i y is due o a leas ou p o eins, he mos basic
enzyme being speci ically induced unde condi ions ha could
simula e mycopa asi ism.
Comme cially a ailable chi in was a good induce o b-1,3-
glucanase ac i i y (Table 1). Figu e 2 shows mycelial g ow h
and b-1,3-glucanase p oduc ion du ing he 96 h o induc ion in
chi in as he ca bon sou ce. The appea ance o ex acellula
b-1,3-glucanase ac i i y was ime dependen bu was no e-
la ed o mycelial g ow h. In addi ion, h ee peaks o b-1,3-
glucanase ac i i y (pI o .7.4, pI o 6.2, and pI o 5.3, espec-
i ely) we e esol ed when concen a ed ex acellula samples
we e ac iona ed by acidic ch oma o ocusing (Fig. 3). Resul s
indica ed ha in his case he measu ed b-1,3-glucanase ac i -
i y is due o a leas h ee p o eins, he mos basic being also
he mos ac i e one. Fo hese easons, u he s udies we e
ocused on he basic b-1,3-glucanase iso o m, namely,
BGN13.1 (b-glucanase-1,3 i s iso o m).
Pu i ica ion o BGN13.1 and size de e mina ion. Pu i ica-
ion o BGN13.1 was based on he a ini y o b-glucanases on
pa icula ed b-glucans. The supe na an s o chi in-supple-
men ed cul u es (200 ml) we e concen a ed by 80% sa u a ion
ammonium sul a e, and concen a ed p o eins we e hen sub-
jec ed o pus ulan adso p ion and u he diges ion. Mos o
he eco e ed enzyma ic ac i i y in he pus ulan diges ion was
b-1,6-glucanase, as expec ed wi h an a ini y adso p ion, bu
7% o he o al b-1,3-glucanase ac i i y was also p esen .
Acidic ch oma o ocusing sepa a ed he pus ulan diges ion in o
wo ac ions, a nonadso bed ac ion which e ained 10 o
15% b-1,6-glucanase ac i i y and he o al b-1,3-glucanase ac-
i i y and a second ac ion as a peak a pH 5.8, which con ains
he es o he b-1,6-glucanase ac i i y (13). Ac i e ac ions o
he nonadso bed olume we e pooled, concen a ed by using
Cen icon 10 concen a o s, and inally pu i ied by gel il a ion
on a Sephac yl S-200 HR ch oma og aphy column. Figu e 4
shows he elu ion pa e n o b-1,3-glucanase and b-1,6-glu-
canase ound by S-200 HR ch oma og aphy, and Table 2 sum-
ma izes he pu i ica ion p ocedu e. The inal pu i ied p epa-
FIG. 1. b-1,3-Glucanase isoenzymes on isoelec o ocusing gels. Ca bon
sou ces used we e as ollows: lane 1, glucose, 0.1%; lane 2, glucose, 2%; lane 3,
glucose, 10%; lane 4, chi in, 1%; lane 5, lamina in, 1%; lane 6, pus ulan, 1%; lane
7, au ocla ed yeas cells, 1%; lane 8, au ocla ed mycelia om B. cine ea, 1%; lane
9, au ocla ed mycelia om G. ujiku oi, 1%; lane 10, au ocla ed mycelia om T.
ha zianum, 1%. Ac i i y was de eloped as indica ed in Ma e ials and Me hods.
FIG. 2. Analysis o ex acellula p oduc ion o b-1,3-glucanase ac i i y om
T. ha zianum g owing in chi in. Shown is a ime cou se cu e o b-1,3-glucanase
ac i i y p oduced by T. ha zianum du ing g ow h in 1.5% chi in as he sole ca bon
sou ce. F,b-1,3-glucanase ac i i y; E, ex acellula p o ein; Ç, g ow h exp essed
as o al p o ein.
FIG. 3. Ch oma o ocusing analysis o ex acellula b-1,3-glucanase isoen-
zymes om T. ha zianum g owing wi h chi in. An 80% (NH
4
)
2
SO
4
ac ion om
a 48-h chi in T. ha zianum cul u e luid was loaded on o he column. F,b-1,3-
glucanase ac i i y; Ç, p o ein measu ed as A
280
;E, pH.
TABLE 1. Ex acellula b-1,3-glucanase ac i i y p oduc ion by
T. ha zianum p eg own in 10% glucose and incuba ed o 48 h wi h
di e en ca bon sou ces
Ca bon sou ce
a
(% [w / ol])
b-1,3-Glucanase
ac i i y (mU/mg o
p o ein)
Glc (0.1).................................................................................. 10
Glc (2)..................................................................................... 75
Glc (10)................................................................................... BD
b
Chi (1) ................................................................................... 640
Chi (1) 1Glc (2)................................................................. 50
Lam (1)................................................................................... 78
Pus (1) ................................................................................... 340
Yeas (1)................................................................................. 1,000
Bo (1) .................................................................................... 800
Gib (1).................................................................................... 790
Rhizo (1) ................................................................................ 785
Phy o (1)................................................................................. 700
T icho (1) ............................................................................... 670
a
Glc, glucose; Chi , chi in; Lam, lamina in; Pus , pus ulan; Yeas , au ocla ed
cells om S. ce e isiae; Bo , au ocla ed mycelia om B. cine ea; Gib, au ocla ed
mycelia om G. ujiku oi; Rhizo, au ocla ed mycelia om R. solani; Phy o,
au ocla ed mycelia om Phy oph ho a ci oph ho a; T icho, au ocla ed mycelia
om T. ha zianum.
b
BD, below limi o de ec ion (,0.5 mU/mg o p o ein).
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a ion mig a ed as a single band on SDS-PAGE, indica ing a
homogeneous p o ein wi h an appa en molecula mass o 66
kDa (Fig. 5). When he molecula mass was calcula ed by gel
il a ion, a e calib a ion o he Sephac yl S-200 HR column,
he alue ob ained was 5 o 10 kDa.
Biochemical analysis o he pu i ied BGN13.1. The isoelec-
ic poin s o he pu e enzyme, de e mined by bo h elec o o-
cusing and basic ch oma o ocusing (pH ange, 9.4 o 6), we e
es ima ed o be pH 7.7 and pH 8.0, espec i ely. Da a we e
con i med a e enzyma ic de e mina ion in isoelec o ocusing
gels, since he pI o he pu i ied enzyme was a basic one and
a ound pI 8.0 (da a no shown).
Pu i ied BGN13.1, subjec ed o SDS-PAGE, was no s ained
by he sil e ni a e ca bohyd a e p ocedu e (18) o by he
Schi ’s eagen ( esul s no shown). endo-H o a-mannosidase
ea men also ailed o al e he appa en size on SDS gels
( esul s no shown). Also, he enzyme was no e ained by
concana alin A-Sepha ose a concen a ions om 0 o 1.4
mg/ml ( esul s no shown). Posi i e con ols wi h highly glyco-
syla ed ex acellula yeas in e ase we e included o he ex-
pe imen s desc ibed in his sec ion.
The op imum empe a u e o he enzyma ic ac i i y was
408C. A e a 30-min p eincuba ion a a ious empe a u es,
he inac i a ion empe a u e was 558C. The K
m
alue wi h
lamina in as he subs a e was 3.3 mg/ml. A he same ime, a
V
max
o 75 mmol o p oduc pe min zmg o p o ein was cal-
cula ed.
The pu i ied b-1,3-glucanase was no de ec ed by immuno-
blo ing wi h ei he an an ise um agains plan acidic ex acel-
lula b-1,3-glucanase o basic acuola b-1,3-glucanase (da a
no shown). Fu he mo e, he e was no c oss- eac ion when
an i-T. ha zianum b-1,6-glucanase II an ise um was used.
N- e minal and in e nal amino acid sequences. The NH
2
-
e minal sequence, de e mined wi h a con idence le el up o 11
esidues, is shown in Table 3. Fou in e nal pep ide sequences
a e also shown.
Subs a e speci ici y and hyd oly ic ac ion. As shown in
Table 4, he enzyme speci ically hyd olyzed molecules con ain-
ing he b-1,3-glucoside bond, including lamina in and pe io-
da e-oxidized lamina in, pachyman, and yeas glucan. A sligh
ac i i y was also ound wi h pus ulan (a b-1,6-glucan), p obably
because o b-1,3 bonds in he pus ulan b anch poin s. Sligh
hyd olysis was obse ed wi h he assayed ilamen ous ungus o
yeas pu i ied cell walls. In addi ion, no clea ing ac i i y o he
b-1,3-glucanase p o ein alone, measu ed as desc ibed in Ma-
e ials and Me hods, was obse ed in aga pla es con aining
ungal cell walls (da a no shown). Howe e , when he enzyme
was combined wi h T. ha zianum b-1,6-glucanases I and II,
ac i i y was de ec ed in aga pla es con aining yeas cell walls
(13).
The mode o ac ion o BGN13.1 owa d lamina in was ex-
amined by measu ing he a e o glucose p oduc ion e sus
ha o educing-suga p oduc ion a di e en imes and also
analyzing he co esponding hyd olysis p oduc s by HPLC
(Fig. 6). Incuba ion o lamina in wi h he enzyme esul ed in
he p oduc ion o la ge amoun s o educing suga s han o
glucose (da a no shown). A e HPLC analysis, a se ies o
oligosaccha ides was de ec ed, lamina i e aose, lamina ibiose,
and glucose being he inal p oduc s o hyd olysis. When he
enzyme was incuba ed wi h lamina ibiose, no u he hyd oly-
sis was ob ained ( esul s no shown). In addi ion, no inhibi ion
was obse ed wi h d-gluconolac one (1 o 20 mM), which has
been desc ibed as a po en inhibi o o exo-b-glucanases a low
concen a ions (44). Thus, all hese esul s indica e an endo-
ype mode o ac ion o he pu i ied enzyme.
Molecula cloning o he bgn13.1 gene. The molecula clon-
ing o he b-1,3-glucanase bgn13.1 gene was a emp ed by PCR
echniques, on he basis o he amino acid sequence o an
FIG. 4. Gel il a ion ch oma og aphy h ough a Sephac yl S-200 HR col-
umn. F,b-1,3-glucanase ac i i y; E,b-1,6-glucanase ac i i y; Ç, p o ein mea-
su ed as A
280
.
FIG. 5. SDS-PAGE o he pu i ied endo-b-1,3-glucanase. s d., molecula
mass s anda ds (10 mg); 1, pu i ied b-1,3-glucanase (10 mg). P o eins we e
s ained wi h Coomassie blue. Numbe s on he le e e o molecula masses o
p o ein s anda ds.
TABLE 2. Pu i ica ion o a b-1,3-glucanase om T. ha zianum
S ep Vol (ml) To al p o ein
(mg) To al ac i i y
(U) Sp ac
(U/mg) Yield
(%) Pu i ica ion
( old)
80% (NH
4
)
2
SO
4
18.0 121 428 3.5 100 1
Pus ulan diges ion 9.0 4.5 29 6.4 7 2
Ch oma o ocusing elua e 0.4 0.6 10 16.6 2.3 5
Sephac yl S-200 HR elua e 0.4 0.05 2 40.0 0.5 12
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in e nal pep ide o he p o ein. Fo PCRs, DNA om a uni-
di ec ional cDNA lib a y in lg 11-S i-No ec o was used as
he empla e and a degene a e oligonucleo ide, co esponding
o he in e nal sequence NTQNGYTGG (p12 [Table 3]), and
he lg 11 e e se p ime s we e used. Unde he condi ions
desc ibed in Ma e ials and Me hods, a single 250-bp agmen
was epe i i ely ampli ied and was u he subcloned and se-
quenced. The ampli ied DNA was he C- e minal egion o he
b-1,3-glucanase p o ein, co esponding mos ly o he 39un-
ansla ed egion o he gene. This PCR-ampli ied band was
used as a p obe o sc eening he a o emen ioned cDNA li-
b a y. Upon sc eening a ound 40,000 plaques, se e al posi i e
clones we e selec ed and isola ed and he one con aining he
longes inse was chosen o u he cha ac e iza ion. The
cDNA inse o his phage (named bgn13.1) was cloned in he
EcoRI-No I si es o he pBluesc ip SKII (1) ec o (P o-
mega) and hen was sequenced.
Sequence analysis and homologies wi h o he b-glucanases.
Nucleo ide and amino acid sequences a e shown in Fig. 7. The
cloned cDNA has a size o 2,484 bp, o which 32 bp co e-
sponds o he 59un ansla ed egion, 164 bp co esponds o he
39un ansla ed egion, and 2,288 bp co esponds o he unique
open eading ame p esen in he cDNA. T ansla ion o he
open eading ame indica es ha he bgn13.1 gene p oduc
(BGN13.1) has 762 amino acids wi h a p edic ed molecula
mass o 81,246 Da. Compa ison o he deduced sequence wi h
he N- e minal amino acid sequence om he isola ed b-1,3-
glucanase p o ein (Table 3) indica es ha he ma u e p o ein
s a s a esidue 34, immedia ely p eceded by he basic esidues
Lys and A g, which ha e been desc ibed as he ecogni ion
sequence o KEX2-like endop o einases (22). The e o e, he
ma u e b-1,3-glucanase con ains 728 amino acid esidues and
has a p edic ed molecula mass o 77,972 Da and an es ima ed
isoelec ic poin o 7.3. The p edic ed p o ein con ains he i e
pep ides sequenced om he pu i ied p o ein, demons a ing
ha he bgn13.1 gene codes o he b-1,3-glucanase desc ibed
abo e (Fig. 7 and Table 3).
Compa ison o he nucleo ide and amino acid sequences o
BGN13.1 wi h sequences p esen in he da abases does no
show any signi ican homology o any a-o b-glucanases o
o he ela ed p o ein amilies. This esul clea ly indica es ha
BGN13.1 ep esen s a new class o b-1,3-glucanase. In ac ,
alignmen o BGN13.1 wi h known b-1,3-glucanase sequences
om bac e ia, yeas s ains, and plan s shows ha BGN13.1
has none o he conse ed domains p esen in he o he p o-
eins. A phylogene ic ee c ea ed wi h he algo i hms DIS-
TANCE and GROWTREE by compa ison o 63 glucanase
sequences indica es ha BGN13.1 is axonomically a om
sequences o bac e ial, yeas , o plan b-1,3-glucanase amilies;
ha i is no ela ed o ei he he b-glucosidase (BGL1 [3]) o
he b-1,6-glucanase II (BGN16.2 [33]) genes om T ichode ma
spp., and ha i may mo e likely be ela ed o T ichode ma
cellulases (da a no shown).
Analysis o he p o ein clea ly dis inguishes h ee di e en
egions. The i s 33 amino acids ma k a hyd ophobic sequence
and co espond o he leade pep ide esponsible o sec e ion
and possible KEX2-media ed ac i a ion o he p o ein. I had
been demons a ed ha se e al sec e ed enzymes a e syn he-
sized in an inac i e o m and a e ac i a ed p io o sec e ion by
a KEX2 endop o einase (22). Al hough he p esence o
KEX2-like p o einase in T. ha zianum has no been demon-
s a ed, o he sec e ed enzymes, like CHIT42 chi inase (23),
BGN16.2 b-1,6-glucanase (33), and PRB1 p o ease (24), as
well as XYN2 xylanase om T. eesei (67), con ain a simila
leade pep ide ending in he pai Lys-A g. The egion be ween
amino acids 33 and 520 has no appa en s uc u al ele ance,
and so i could con ain he ca aly ic si e. E en hough he
p o ein con ains se e al Glu esidues, he lanked conse ed
FIG. 6. HPLC analysis o b-1,3-glucanase ac ion on lamina in. Lamina in (5
mg/ml) was incuba ed as desc ibed in Ma e ials and Me hods wi h 2 mgo he
pu i ied enzyme, o he indica ed imes. G1, G2, and G4 indica e glucose
oligome s; numbe s indica e he deg ee o polyme iza ion.
TABLE 3. NH
2
- e minal and pep ide sequences om BGN3.1
Pep ide Sequence
a
N e minal ........................ATSFYYFNMDHV
p5.......................................NPTYGDVTSSNTRPSALAPG
p12.....................................LGXGTFINTQNGYTGGXTPGGGDVXP
p13.....................................FNTDTILMGDPTN
p14.....................................XVGEA(M)ATITGNGNFFK
a
The mos p obable amino acids a e in pa en heses. X, unknown amino acid.
TABLE 4. Subs a e speci ici y o he pu i ied b-1,3-glucanase
om T. ha zianum
Subs a e
a
Main linkage ype
(monome ) b-1,3-Glucanase
ac i i y
b
(%)
Lamina in b-1,3 (Glc) 100
IO
4
-oxidized lamina in b-1,3 (Glc) 100
Glucan (S. ce e isiae)b-1,3 (Glc) 97
Pachyman b-1,3 (Glc) 46
Pus ulan b-1,3:b-1,6 (Glc) 5
Ca boxyme hyl cellulose b-1,4 (Glc) 0
Colloidal chi in b-1,4 (GlcNAc) 0
Glycol chi osan b-1,4 (GlcN) 0
Nige an a1,3:a-1,4 (Glc) 0
Soluble s a ch a-1,4:a-1,6 (Glc) 0
Dex an a-1,6 (Glc) 0
B. cine ea cell walls Glucan:chi in 1
G. ujiku oi cell walls Glucan:chi in 1
P. sy ingae cell walls Glucan:cellulose 4
S. ce e isiae cell walls Glucan 2
a
All subs a es we e used a a concen a ion o 5 mg/ml.
b
100% ac i i y co esponds o 28 U/mg o p o ein.
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esidues a ound he Glu ca aly ic si e desc ibed o he b-glu-
canase s udied (8) a e absen in BGN13.1. The hi d egion is
localized a he C- e minal end and ex ends om esidue 520
o he end o he p o ein. This egion con ains eigh o he nine
cys eine esidues p esen in he p o ein.
Finally, acco ding o DNA-Sou he n blo hyb idiza ions ca -
ied ou a high and low s ingencies he e appea s o be only
one copy o he bgn13.1 gene in he T. ha zianum genome, and
no o he ela ed sequences we e de ec ed (Fig. 8).
Exp ession pa e n o bgn13.1. No he n analysis was pe -
o med o es ablish whe he he induc ion o bo h ac i i y and
p o ein could also be ound a he RNA le el. As Fig. 9 shows,
a e 48 h o induc ion he mRNA le els we e below he de-
ec ion limi when glucose and pus ulan we e used as ca bon
sou ces and he mRNA was de ec ed when lamina in and
chi in we e used. Also, mRNA accumula ion was obse ed
when au ocla ed yeas cells o mycelia we e used. Thus, hese
da a a e in ag eemen wi h hose ob ained when enzyma ic
ac i i y and p o ein we e de e mined (Table 1 and Fig. 1) and
clea ly indica e ha bgn13.1 is con olled a he ansc ip ional
le el. The e o e, bgn13.1 is subjec ed o bo h ep ession by
glucose and induc ion by ungal cell wall polyme s, yeas cells,
o ungal mycelia. Howe e , when pus ulan and lamina in we e
used as ca bon sou ces, a lack o co ela ion be ween he
ac i i y and he mRNA le els was obse ed, sugges ing ha
egula ion a he ansla ional le el may also occu . This phe-
nomenon has also been obse ed wi h a b-1,6-glucanase
(BGN16.2) om he same mic oo ganism (33) and could be
FIG. 7. Nucleo ide and p edic ed amino acid sequences o he bgn13.1 cDNA
clone. A ailable pep ide sequences om he pu i ied p o ein a e unde lined.
Cys eine esidues a e ma ked by ci cles. Ma u e BGN13.1 s a s a he sequence
ATS a e he boxed KEX2 ecogni ion sequence.
FIG. 8. Sou he n blo analysis o he bgn13.1 gene. The expe imen was
pe o med by using 15 mg o ch omosomal DNA diges ed wi h EcoRI (ERI),
BamHI (BHI), HindIII (HIII), o XbaI (XI). High- and low-s ingency condi-
ions show he same band pa e n.
FIG. 9. No he n blo analysis. Ca bon sou ces used we e as ollows: glucose
(Gluc), 0.1 o 2%; lamina in (Lam), 1%; pus ulan (Pus ), 1%; chi in (Chi ), 1%;
yeas au ocla ed cells (Yeas ), 1%; au ocla ed mycelia om P. sy ingae (Phy ),
1%; au ocla ed mycelia om B. cine ea (Bo ), 1%; au ocla ed mycelia om R.
solani (Rhiz), 1%; au ocla ed mycelia om T. ha zianum (T ic), 1%. Equal
amoun s o RNA we e loaded in o he wells and es ed by me hylene blue
s aining.
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explained by he high le el o s abili y o hese enzymes in he
cul u e media. The enzyma ic ac i i y a 48 h in pus ulan would
be due o an ea lie inc ease o mRNA le els, u he e-
p essed by glucose p oduced by ac ion o he enzyme o e i s
subs a e.
DISCUSSION
T. ha zianum CECT 2413 is a mycopa asi ic ungus ha
sec e es a leas h ee enzymes wi h b-1,3-glucanase ac i i y in
a chi in-con aining medium. The unc ion o hese ungal en-
zymes is unknown, bu hey migh ha e a nu i ional ole wi h
b-glucans as subs a es (62), a di ec ole in mycopa asi ism
(9), o mo phogene ic unc ions in g ow h and de elopmen
(47). We epo he ein he ollowing: i s , he exp ession pa -
e ns o hese enzymes in di e en ca bon sou ces (Table 1 and
Fig. 1), and second, he pu i ica ion, molecula cloning, and
cha ac e iza ion o he b-1,3-glucanase p o ein, named
BGN13.1, co esponding o he majo ex acellula ac i i y
om supe na an s o T. ha zianum g owing in chi in as he sole
ca bon sou ce.
By he enzyma ic de ec ion o b-1,3-glucanase on isoelec o-
ocusing gels (45), we ha e shown he di e en condi ions o
induc ion o b-1,3-glucanase isoenzymes om T. ha zianum
CECT 2413. Acidic and basic b-1,3-glucanases we e induced in
he p esence o a ious polysaccha ides, bu he basic enzyme
(BGN13.1) was speci ically induced by au ocla ed yeas cell
walls o ilamen ous ungi (Fig. 1). P elimina y esul s ob ained
wi h he mycopa asi ic ungus S achybo ys elegans ha e also
shown di e en induc ion condi ions o some ex acellula
chi inase and b-1,3-glucanase p o eins (68). Al hough an-
sc ip ion o he bgn13.1 gene co ela ed wi h he da a ob ained
om enzyma ic assays (Table 1, Fig. 1, and Fig. 8), he egu-
la ion appea s o be a ei he he ansc ip ional o he ans-
la ional le el. In addi ion, b-1,3-glucanases om T. ha zianum
we e p oduced coo dina ely wi h b-1,6-glucanases, p o eases,
and chi inases, e en hough he medium wi h chi in does no
con ain b-glucan o p o ein as a ca bon sou ce (14, 24). In
T ichode ma longib achia um, he b-1,3-glucanase ac i i y is
cons i u i ely p oduced and depends on he ungal g ow h
(66), which sugges s a mo phogene ic ole. In o he ungi, such
as B. cine ea (62), Penicillium i alicum (55), Penicillium oxali-
cum (10), and Scle o ium glucanicum (48), a de ep ession o
he b-1,3-glucanase ac i i y in s a a ion is associa ed wi h he
mobiliza ion o b-glucans and in ol ed in he au olysis o cell
walls. Howe e , his is no he case in T. ha zianum (Table 1,
Fig. 1, and Fig. 8). A coo dina ed induc ion o chi inases,
b-1,3- and b-1,6-glucanases, and p o eases may occu in T.
ha zianum when mycopa asi ic ac ion is es ablished, as i oc-
cu s in plan s o chi inases and b-1,3-glucanases du ing de-
ense esponses agains pa hogens (72). As p e iously dis-
cussed (13, 14, 16), a coo dina ed induc ion o he enzymes by
an isola ed polysaccha ide would be o biological ele ance,
since he subs a es o hese enzymes a e p esen simul a-
neously in ungal cell walls (47).
The pu i ica ion me hod included speci ic adso p ion o pus-
ulan (b-1,6-glucan) and diges ion, ollowed by ch oma o ocus-
ing and gel il a ion. This p ocedu e is no el, quick, and easy,
and e en i he yields seem o be poo , i is good enough o
pu i y p o ein o an ex ensi e biochemical cha ac e iza ion. In
addi ion, he p ocess allows pu i ica ion o wo o he p o eins
wi h b-1,6-glucanase ac i i y. Da a om SDS-PAGE and S-200
HR gel il a ion indica ed appa en molecula masses o 66
and 5 o 10 kDa, espec i ely. This esul indica ed an a ini y
o Sephac yl suppo ha has p e iously been obse ed o a
numbe o ex acellula enzymes, including o he ungal cell
wall hyd olases (12, 13, 50, 66). In addi ion, he molecula mass
p edic ed om he amino acid sequence is 77.9 kDa. Dispa i y
be ween calcula ed and deduced molecula weigh s has also
been obse ed o he endo-b-1,3-glucanase om S. ce e isiae
(BGL2), which shows an appa en size o 29 kDa by SDS-
PAGE and a p edic ed one o 33.5 kDa om he amino acid
sequence. In his las case, i has been demons a ed ha he
di e ence in he molecula size is due o anomalous mig a ion
o he p o ein in he gel a he han o pos ansla ional p o-
cessing o he polypep ide chain (40).
The compa ison o he amino acid sequence o BGN13.1
wi h hose o BGL2 and o he b-glucanases did no show any
signi ican homology. Compa ison be ween sequences o yeas
and plan b-glucanases showed ha ele an egions o he
deduced p ima y s uc u e a e highly conse ed, especially
a ound he glu amine esidues in ol ed in he ca alysis. The
boxes VXEXGWP and EXXXGXF loca ed a he nucleophilic
and acidic ca aly ic si es, espec i ely, a e no p esen in
BGN13.1 (8, 27). Likewise, he box EXDXE p esen in he
bac e ial coun e pa is also absen in BGN13.1. The e o e, he
p ima y s uc u e o BGN13.1 showed wo signi ican di e -
ences wi h ega d o o he b-glucanases: (i) BGN13.1 lacks he
conse ed egions p esen in he s udied b-glucanases, and (ii)
BGN13.1 con ains a cys eine- ich mo i absen in o he known
b-glucanases, excep o he b-1,3-glucanase om he bac e-
ium Oe sko ia xan hineoly ica. Dele ion o he cys eine- ich
ca boxyl- e minal egion in he bac e ial p o ein does no a -
ec he enzyma ic ac i i y bu abolishes he capabili y o in e -
ac wi h he yeas cell wall and he e o e exe i s ly ic ac i i y
(58). A simila Cys- ich mo i has also been ound in o he
p o ein amilies which a e able o bind speci ic memb ane
componen s, like he saccha ide-binding plan lec ins (e.g.,
whea ge m agglu inin); he lipid ans e p o eins o animals,
ungi, and plan s; and class I chi inases (7, 17). The Cys- ich
domain o BGN13.1 may unc ion by in e ac ing wi h o he
componen s o ungal cell walls. In ac , BGN13.1 is he only
b-1,3-glucanase om T. ha zianum able o in e ac wi h poly-
saccha ides which a e no subs a es o he enzyme (like pus-
ulan). Since he enzyma ic ac i i y o BGN13.1 is speci ic only
o b-1,3 linkages, his pu a i e cell wall binding domain may
ha e unc ional ele ance in he mycopa asi ism o T. ha zia-
num by ecognizing a cell wall componen o he a ge ungi.
This hypo hesis is cu en ly being s udied in ou labo a o ies.
The unique s uc u e o he basic b-glucanase om T. ha -
zianum was con i med by he ac ha se a aised agains b-1,3-
and b-1,6-glucanases om plan s (26) and ungi (13), espec-
i ely, did no c oss- eac wi h any o he b-1,3-glucanase en-
zymes om T. ha zianum. Fu he mo e, ex acellula hyd o-
lases o ungi a e no mally glycosyla ed (76), as a e cellulases
om T ichode ma eesei (30, 52). Ou esul s, including hose
ob ained wi h speci ic ca bohyd a e s ains, endo-H o a-man-
nosidase ea men s, and concana alin A-binding assays, indi-
ca ed ha ei he he pu i ied BGN13.1 is no glycosyla ed o
he glycosyla ion le el is so low as o be no de ec ed unde he
condi ions used. These esul s, howe e , a e simila o hose
ound wi h o he ex acellula b-glucanases and chi inases
om T. ha zianum (12, 13).
The measu ed K
m
o lamina in (3.3 mg/ml) o he pu i ied
enzyme was subs an ially highe han hose epo ed o o he
b-1,3-glucanases (21, 54, 66, 69), wi h he excep ion o one
b-1,3-glucanase o he ungus S. glucanicum (48) and ha o
Rhizopus chinensis (75). The enzyme pu i ied in his wo k was
speci ic o b-1,3 linkages in polysaccha ides (Table 4), hyd o-
lyzing lamina in and yeas glucan wi h he same e iciency and
pachyman less e icien ly. The sligh ac i i y ound wi h pus u-
lan (linea b-1,6-glucan) could be ela ed o he binding o his
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on July 25, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om
enzyme o his subs a e and he p esence o some b-1,3 link-
ages in i . Unlike he exo-b-1,3-glucanases p e iously desc ibed
o T. ha zianum (28) and T ichode ma eesei (2), he enzyme
pu i ied in his wo k beha es as an endo-spli ing hyd olase as
judged om he libe a ion o mo e educing suga s han o
glucose om lamina in, he p esence o ac i i y on pe ioda e-
oxidized lamina in (unable o be hyd olyzed o exo-b-1,3-
glucanases), he noninhibi o y e ec o d-gluconolac one on
he enzyma ic ac i i y, he inabili y o hyd olyze subs a es such
as lamina ibiose, and he pa e n o eac ion p oduc s de ec ed
by HPLC. In con as wi h some ungal endo-b-1,3-glucanases
ha deg ade b-1,3-glucans o gene a e oligosaccha ides o
high molecula mass and ha a e classi ied as la ge -oligosac-
cha ide-p oducing ypes o enzyme (1), he endo-b-1,3-glu-
canase cha ac e ized in his wo k hyd olyzes b-1,3-glucans in
an endo manne wi h glucose, lamina ibiose, and lamina i e-
aose as end p oduc s and he e o e, as a e o he bac e ial and
ungal endo-b-1,3-glucanases, is classi ied as a smalle -oligo-
saccha ide-p oducing ype (39, 64).
Finally, he enzyme alone eleases educing suga s bu is
unable o o m hyd oly ic haloes when incuba ed wi h pu i ied
cell walls o se e al ilamen ous ungi, including phy opa ho-
genic ones. Fu he mo e, when i was combined wi h o he
ungal cell wall hyd olases om T. ha zianum, such as b-1,6-
glucanases and chi inases (13), no only did he enzyme gi e
clea ing ac i i y on ungal cell walls bu also an inhibi ion o
he g ow h o phy opa hogenic ungi was obse ed. Simila
esul s ha e been ound wi h T. ha zianum P1 and also wi h
o he o ganisms. A syne gis ic, inhibi o y e ec o chi inases,
N-ace yl-b-glucosaminidases, and b-glucanoly ic enzymes om
s ain P1 o T. ha zianum has been desc ibed (34, 35). In
plan s, a combina ion o b-1,3-glucanases wi h chi inases is
essen ial o obse a ion o inhibi o y e ec s agains some phy-
opa hogenic ungi (37). Also, in he ly icase om he bac e-
ium O. xan hineoly ica ab-1,3-glucanase and a p o ease ac i -
i y ac in combina ion in yeas cell lysis (57).
In conclusion, because o he speci ic induc ion o his en-
zyme unde simula ed mycopa asi ic condi ions, as well as i s
an i ungal ac ion when combined wi h o he ungal cell wall-
deg ading enzymes om T. ha zianum, a con ibu ion o his
b-1,3-glucanase du ing he i s s eps o mycopa asi ism could
be expec ed, al hough he esul s did no disca d he possibili y
o he implica ion o his b-1,3-glucanase in o he , unknown
sap ophy ic oles. A p esen , ou wo k is ocused on he o e -
exp ession o his enzyme in ansgenic T. ha zianum, which
could esul in he imp o emen o his ungus as a biocon ol
agen .
ACKNOWLEDGMENTS
We hank F. Dominguez o his help ul assis ance du ing all bio-
chemical expe imen al wo k. We also hank E. Ma inez-Fo ce o his
help wi h HPLC expe imen s and R. Rhe o p oo eading he manu-
sc ip . We hank R. Ge emia and G. Bauw a he Labo a o ium oo
Gene ika (Gen , Belgium) o he N- e minal and pep ide amino acid
de e mina ions.
J.C. is a ecipien o a ellowship om he Minis e io de Educacio´n
y Ciencia. This wo k was suppo ed by g an BIO91-1078 om CICYT
(Spain) and g an TS3-CT92-0140 om he Eu opean Communi y.
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