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Extracellular Reducing Enzyme Produced during Biobleaching
of Hardwood Kraft Pulp by White-Rot Fungi
片桐, 誠之; 堤, 祐司; 西田, 友昭
木材学会誌. 41(8), p. 780-784
1995-08-25
http://hdl.handle.net/10297/4797
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Copyright © 日本木材学会
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Note
lMokuzai Gakkaishi Vol.41, No.8, p.780-TB4 (1995)l
Extracellular Reducing Enzyme Produced during
Biobleaching of Hardwood Kraft Pulp by
White-Rot Fungi*l
I.{obuyuki KarecIRI*z, Yuji Tsursunal*2 and Tomoaki NIsHne*2
白色腐朽菌 を用 いたバ イオ ブ リー チ ング時 に産生 され る
菌体外還元系酵素 *1
片桐誠 之
*2,堤 祐 司 *2,西 田友昭 *2
Pみ αηθ
″ε力αθtt θ
んOlsθ ψ ″協π 及 び カ ワ ラ タ ケ を用 い て 低 窒 素 ―高 炭 素 (LN― HC)及 び 高 窒 素 ―
高 炭 素 (HN― HC)条 件 下 で 広 葉 樹 未 晒 ク ラ フ トパ ル プ (UKP)の 団体 培 養 を行 い ,バ イ オ ブ リ
ー チ ン グ にお け る還 元 系 酵 素 の 役 割 を検 討 した 。 Pθ ん郷 θψ θガ%物 を用 い た バ イ オ ブ リー チ ン グ
にお い て ,MnPと 還 元 系 酵 素 の産 生 時期 が 異 な っ て お り,両 者 が 相 補 的 に作 用 して リグ ニ ン生 分
解 を行 っ て い る可 能 性 が 考 え られ た 。しか しなが ら,Pθ ″野 θψ ttπ 及 び カ ワ ラ タ ケ を用 い た 処
理 にお い て 白色 度 上 昇 と累積 MnP活 性 との 関 係 は,LN― HC及 び HN― HCの 両 培 養 条 件 下 で 同
一 直 線 を示 し相 関 が 認 め られ た の に対 し,還 元 系 酵 素 の場 合 に は こ の よ うな相 関 は認 め られ ず
,
累積 の 還 元 系 酵 素 活 性 と UKP中 の セ ル ロ ー ス 減 少 との 間 に相 関 が あ っ た。以 上 の 結 果 か ら,還 元
系 酵 素 は UKP中 の残 留 リグ ニ ン の 分 解 に は関与 して お らず ,セ ル ロ ー ス 分 解 に 関 与 して い る と
推 察 され た 。
The role of reducing enzyme in the biobleaching of hardwood unbleached kraft pulp (UKP)by
Pん α
ηι
λαθ
ηθ
tt
θ
んηsθ ψθηZπ Burds.and rγ απι
′
θ
s υ
ι
簿′
θ
θ′
θ/(L.:Fr.)Pilat in the s01id‐ state fermen―
tation system with 10w_nitrogen and high― carbon(LN― HC)and high― nitrogen and high―
carbon(HN―
HC)culture rlledia、 vas investigated. The profiles of lnanganese peroxidase(Ⅳ InP)and reducing
enzyme productions during the biobleaching using 2
ありsθ ψθ7ZZ夕 22 ヽ
Vere very different from each
σ
other,suggesting the complementary function of both enzymes for the lignin degradation. Although
a positive relatiOnship bet、 veen cumulative 1/1nP activity and brightness increase、 vas observed in the
treatment with P θ
んηsθ ψθηπtt and■ υι
6た θゐγunder both LN― HC and HN― HC conditions,no
positive correlatiOn was Observed in the case of the cumulative reducing enzyme activity. On the
other hand,a positive cOrrelation bet、 veen cumulative reducing enzyme activity and the degradation
of cellulose in UKP was observed. These results suggest that reducing enzyme may not be involved
in the degradation Of residual lignin in UKP and is related to the degradation Of cellulose.
κのプ
ι
ク
ds:
θγ
white_rot fungi,biobleaching,reducing enzyme,lignin degradation,ceHulose degrada―
tiOn.
1.INTRODUCT10N
The white― rot fungi, including P/2α ηθγθ αι′
ι
Received ⅣIarch 15, 1995.
静 岡大 学 農 学部 Faculty Of Agriculture,Shizuoka
University,Shizuoka 422
ηsθ ttθ η%π
`乃
Burds.and r彫解θ′θs
`力
υθ恣たθわγ (L.:
Fr.)Pilat, are knOwn as the most effective lignin‐
Vol.41, No. 8,
19951
Extracellular Reducing Enzyme Produced during Biobleaching
degrading microorganisms.'-3)
that laccase,
It
has been suggested
manganese peroxidase (MnP), and
lignin peroxidase (LiP) produced by the white-rot
fungi are involved in the oxidative breakdown of
lignin. Hammel and Moen reported that crude LiP
preparations catalyze at least the partial depolymer-
ization of synthetic syringyl/guaiacyl lignin.n) Wariishi et a/. showed that MnP catalyzes the partial
depolymerization of four different synthetic lignin
preparations.s) However, in uitro,the synthetic lignin
is polymerized by LiP and MnP rather than it
is
depoiymerized.s'6) On the other hand, polymerization
of lignin is not prominent in
uiuo,7-s)
indicating that
15.5) in the solid'state fermentation system with low
nitrogen-high carbon (LN-HC) and high nitrogenhigh carbon (HN-HC) culture media was performed
as described
in a previous
paper.14)
2.2 Enzynte assals
In the assays of MnP and reducing enzyme activities, 0.19 (as bone dry weight) of the fungus-treated
50 ml of the reaction mixtures
containing substrates, the mixtures were homogenizedby a high-speed mixer (HM-5SA, NRK, Japan)
for 30 sec at 10,000 rpm, and the enzyme activities
puip was added to
were determined as follows: MnP activity was determined by the method described previously.rn) Reduc-
the white-rot fungi may have an ability that prevents
ing enzyme activity was measured at 30"C by monitor-
of lignin and phenolic products by
oxidizing enzymes. Kirk and Farrell proposed
ing the reduction of 2,6-dichlorophenol-indophenol
(sodium salt) at 600 nm.15) The reaction mixture
polymerization
these
pM
that phenols are oxidized rapidly past the phenoxy
radical step or that the radicals are reduced back to
the phenols by the enzyme that prevents poly-
contained
merization.l0) Westermark and Eriksson suggested
that phenoxy radicals might be reduced back to
amount of enzyme that changes the absorbance by 0.1
per min, and enzyme activity was expressed in units
by the enzyme cellobiose: quinone oxidoreductase (CBQase) tt) which cataTyzes the oxidation
per gram of treated pulp. Data are means of triplicate
of cellobiose with simultaneous reduction of
2.3 Pwlp properties and deterynination of
phenols
a
quinone.") Ander et al. reported that the polymerization
of kraft lignin by LiP is decreased in the
of CBQa5s.13r However, Kirk and Farrell
50
2,6
- dichlorophenol-indophenol
(sodium salt), 100 pM cellobiose in 20 mM phosphate
buffer (pH 6.0). One unit of activity is defined
as the
analyses.
cellulose
in the UKP
After incubation with fungi, pulp samples
content
were
have reported that CBQase does not prevent polymer-
washed with water, and pulp sheets were prepared
with a Buchner funnel (diameter, 11mm) and then air
ization of phenols by LiP or by
presence
horseradish
dried. Brightness was determined with a colorimeter
peroxidase.lo)
(model CR-300
In a previous paper,'n) we reported the investigation
of the role of oxidizing enzymes, MnP, LiP, and lacc-
determined with the colorimeter were multiplied by a
ase, in biobleaching, and showed
that MnP is the most
important enzyme in brightening and delignification
of hardwood
unbleached
kraft pulp (UKP) by
In this paper,
chrysosporium and T. aersicolor.
P.
to
clarify the role of reducing enzymes, we examine the
relationship between the brightening of UKP and the
cumulative activity of reducing enzyme produced by
P. ckrysosporium and T. uersicolor
in the solid-state
fermentation system with two different culture media.
2. MATERIALS AND METHODS
2.7 Microorganisms and biobleaching of kraft PulP
P. ckrysosporiurn ME-446 and T. (Coriolus) uersicolor IFO-30340 were used in this study. Biobleaching of hardwood UKP (brightness, 29?6; kappa no.,
; Minolta, Tokyo, Japan).
The values
coefficient to adjust them to ISO brightness values.
The kappa number is defined as the amount (in
milliliters) of a 0.1N KMnOn solution consumed by 1
g of moisture-free pulp under standard conditions
(Standard T 236 of the Technical Association of the
Pulp and Paper Industry, Atlanta, Ga.).
Cellulose content was obtained by the following
equation: cellulose:dry weight of UKPx (1-kappa
number x 0.15/100)
.
3. RESULTS AND DISCUSSION
The hardwood UKP was inoculated with
P.
in the solid-state fermentation system with LN-HC and HN-HC culture
media and incubated for six days. After incubation,
ckrysosporium and T. uersicolor
the manganese peroxidase (MnP) activity, the reduc-
NobuvuKi KATAGIRI. Yuii TSUTSUMi and TomoaKi NISHIDA
ing enzyme activity, and the pulp brightness were
determined. In our reducing enzyme assay system,
2,
[Mo力 %`α ブ G″ 力αたん′
activity of reducing enzyme was much more under the
latter. In P. ckrysosporiwm, lignin is degraded only
as the substrates which have been reported for the
assay of cellobiose: quinone oxidoreductase
(CBQase) and/or cellobiose oxidase (CBO)
during secondary metabolism,tu'tt) which is triggered
by a limitation of an essential nutrient such as nitrogen. This was consistent with the result that a
greater brightness increase was obtained with an LN-
activity.ts)
HC condition. On the other hand, the
reducing
Figure 1 shows the changes in MnP activity, reducing enzyme activity, and brightness observed during
the treatment with P. chrysosforium. The profiles of
enzyme was produced extensively under an
HN-HC
6-dichlorophenol-indophenol and cellobiose were used
MnP and reducing enzyme productions during the
biobleaching were very different from each other. As
we showed previously MnP is involved in the brightening of UKP,") the different profiles of both enzymes
suggested that the phenoxy radicals produced by MnP
might be reduced back to the phenols by the reducing
enzyme as proposed by
Kirk
and Farrell,'o) and reduc-
ing enzymes may interact with oxidizing enzyme(s)
such as MnP in the lignin biodegradation.
Figure 2 shows the time courses of the brightness
of UKP and cumulative reducing enzyme
activity during treatment with P. chrysosporium under
LN-HC and HN-HC culture conditions. Although
increase
condition in which sufficient nitrogen was supplied
and showed a smaller brightness increase.
Previously,")
a linear relationship was
observed
between the brightness increase and the cumulative
activity of MnP produced by P. cfuysosporium and T.
in the solid-state fermentation system with
different culture media, and the similar relationship
was obtained in this study (Fig. 3A). However, any
positive correlation was not observed in the case of
cumulative reducing enzyme activity (Fig. 38).
These results indicate that reducing enzyme may not
be involved in the brightening of UKP.
As mentioned above, the reducing enzyme activity
uersicolor
was assayed with 2,6-dichlorophenol-indophenol and
cellobiose; therefore, this activity might be due to
the brightness increase under an LN-HC condition
CBQase and/or CBO. Renganathan et a/. reported
was greater than that under an HN-HC condition, the
that CBQase and CBO bind strongly to microcrystal-
Ｃ一
〇２︶００“Φ﹄
ｏＣ¨
∽∽●Ｃ一
〓０■ｍ
，
４
３
２
ｏ
︵
∽∽ｏＣ一
●︶
〓０■ｍ
ヽ
２ １
１
０コ■０匡
りヽ一
︵
Ｅコ︶、〓ン３０●ＯＥ、Ｎｃｏｏ〓一
͡
ｐ言 ｃコ︶ゝ一¨
＞一
一ＯＣ ●Ｅ 、ＮＥ０ ０Ｅ一
０コ﹁０﹄０＞一
コＥ コ０
］Ｃ一
却υ ３
＝
lncubation time (daYS)
Fig.2.
activity during treatment with
Fig.1. Changes in the MnP activity, reducing
enzyme activity, and brightness of UKP
during treatment with P. ckrysosforium in
the solid-state fermentation system with
LN-HC culture medium.
Legend: O: MnP;I:
brightness.
reducing enzyme;A:
Time courses of the brightness increase of
UKP and cumulative reducing enzyme
ckrysosporiuna
in the solid-state
P.
fermenta-
tion system with LN-HC and HN-HC culLegend
:
ture media.
O : brightness, LN - HC ; I : brightness,
C: reducing enzyme, LN-HC;
HN-HC;
n:
reducing enzyme, HN-HC.
Voi.41, No.8,
Extracellular Reducing Enzyme Produced during Biobleaching
19951
15
reducing enzyme activities during treatment with P.
chrysospori.wm and with ?. aersicolor in the solid-state
fermentation system with LN-HC and HN-HC culture media were examined. A linear relationship was
observed between the cellulose loss and cumulative
activity of reducing enzyme produced by the two
fungi (Fig. 4), indicating that the reducing enzyme
may not be involved in the lignin degradation but in
０ ５
りヽ一
︵
Ｅコ︶
０
B
３
２ １
﹁＞一
にコ︶
︵
ゝだ ＞ 〓 ０ ”
、〓ン〓０” Ｌ Ｅ〓︼０＞〓僣一
コＥ コ０
コＥ コ０ ０Ｅ 、Ｎｃ● ｏＥ一
０コ一●﹄ ０＞〓颯一
radation by cellulase.'*) Therefore, the relationships
between the cellulose degradation and cumulative
A
11O2
the cellulose degradation of UKP. This was coincident with the result that the fungal reducing ability
which was detected by the colorization of tetrazorium
salts is appeared in the primary metabolism and does
n
oon
oo
oI
‐
a
0。
a
oortt
a '
a
'lo 20
not correlate directly with the degradation of lignin in
30
secondary metabolism.
le)
Brightness increase (Point)
Fig.3.
Relationship between
the
cumulative
enzyme activity and brightness increase in
the solid-state fermentation system.
Legend:
LN-HC;O: P.
chrysosporium, HN-HC; l: T. aersicolor,
LN-HC; n : 7. uersicolor, HN-HC. (A)
O:
P.ckrysosporiwrn,
activity.
Cumulative MnP
No. 06660208 from the Ministry of Education, Science,
and Culture of Japan.
REFERENCES
(B) Cumulative
reducing enzyme activity.
1) Ander, P.; Eriksson, K.
-E.
: Physiol. Plant.,
41,
g, 185-210
(1971).
3) Kirk, T.K.; Shimada, M.: "Biosynthesis
４
Biodegradation
of
and
Wood Components", Aca-
demic Press, 1985, p.579.
３
4) Hammel,
K. E. ; Moen, M. A. :
Technol., 13, 15-18 (1991) .
２
１
︵０＞ ¨
コＥ コ０
Ｅヨ︶ゝ居＞〓０” ｏ日 、Ｎｃ● ｏＥ一
０コ●●﹄ ０＞〓”中
I97n.
2) Kirk, T. K.: Annu. Reu. Phytopathol.,
239-248
1l
a./
‐
0
Biofhys.
tr
o
Microb.
Biochem.
Res. Commun., 176, 269-275 (1991).
6) Haemmerli,
10
S. D. ; Leisola, M. S. A. ; Fiechter,
A.: FEMS Microbiol. Lett.,35,33-36 (198G).
7) Chua, M. G. S. ; Choi, S. ; Kirk, T. K. : Holzfors-
20
chumg, 37, 55-61 (1983).
(Y")
Relationship between the cumulative reducing enzyme activity and cellulose loss in the
solid-state fermentation system.
Legend: O: P.chrysosporiu?n, LN*HC;O:
P.
): T. aersicolor,
uersicolor, HN-HC.
8) Faix, O. ; Mozuch, M. D. ; Kirk, T. K.
: ibid.,3g,
203-208 (1985).
9) Reid, I. D. ; Abrams, G. D. ;
Pepper, J. M.
;
Can.
J. Bot., 60, 2357-2364 (1982).
chrysosporium, HN-HC;
LN-HCi n: I
Enz.
5) Wariishi, H.; Valli, K.; Gold, M. H. :
rO
Cellulose loss
Fig.4.
Acknowledgment This research was supported
in part by a Grant-in-Aid for Scientific Research CI
10)
Kirk, T.K.; Farrell, R.L.; Ann. Rea. Microbiol., 41, 465-505 (1987)
line cellulose, and that these enzymes may be involved
in the cellulose degradation process.'u) Bao et
showed
that CBO enhances crystalline cellulose
al.
deg-
11) Westermark, U. ; Eriksson, K. -E.: Acta Chem.
Scand., B.28, 209-ZL4
(197 4)
.
12) Westermark, U.; Eriksson, K. -E.: ibid., B2g,
Nobuyuki KATAGIRI, Yuji TsursUMI and Tomoaki
419-424(1975).
Bliθ
″磁ηθ′
.,13,189198(1990).
14)Katagiri,N.;Tsutsumi,Y.;Nishida,T.:ノ 化妙′
.
E4υ ′
夕η.」7グ θ ι′
.,61,617-622(1995).
θ′
15)Renganathan,V.; Usha,S.N.; Lindenburg,F.:
lθ
lMokuzai Gakkaiski
277285(1978).
13)Ander, P.;Chittra, ⅣI.;Farrell, R.L.;Erik-
sson,KE.:ェ
NISHIDA
,lθ
4Йク
′
.ノサ
イ
Jθ 御ろ
′
.B′ θ
んπθ′
θι
″θ
.,32,609-613(1990).
16)Kirk,T.K.:Schultz,E.;Connors,ヽV.J.;Lorenz,L.F.;Zeikus,J.G.: 4,lθ み. 」
7Jε %θ み
′
., 117,
θι
17)Keyser,P.;Kirk,TK.;Zeikus,J.G.:ェ
Bα ι
′
件
θ
ノ
.,135,790797(1978).
θ′
18)Bao, lV.;Usha, S.N.;Renganathan, V.:
“Biotechnology in Pulp and Paper lndustry",
Kuwahara,Ⅳ
I.and Shilnada, 171。
eds., Uni Pub‐
lishers,1992,p.377-382
19)Hirai, H.:Kondo, R.;Sakai, K.:Proc.39th
Lignin Symp.,Fukuoka,1994,p.17-20.
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