hydrolysis of water-soluble and water-insoluble cellulosic substrates by endo-,b

297

1 Appl Glyeosei 51297-301 (2004) copy 2004 The Japanese Society of Applied Glycoscience

Hydrolysis of Water-Soluble and Water-Insoluble Cellulosic Substrates by

Endo-B -14-Glucanase from Acetobacter xylinum

(Received April 3D 2004 Accepted June 9 2004)

Fuyu Ito Yoshihiko Amano1 Kouichi Nozaki Inder M Saxena2 Malcolm R Brown Jr 2 and Takahisa Kanda

Department of Chemistry and Material Engineering Faeulry of Engineering Shinshu University (4--17-1 Wakasato Nagano 380-8553 Japan)

School of Biological Science The University of Texas at Austin (Austin Texas 78712 USA)

Abstract Morphology changes in bacterial cellulose produced by Acetobacter xylinum ATCC23769 were obshyserved in the presence of 3 -glucodisaccharides such as gentiobiose and cellobiose Endo-3 -14-glucanase acshytivity in culture broth was higher than that in the absence of those sugars So we have investigated the propshyerties of endo-3 -14-glucanase (AEG) produced by this bacterium This enzyme could hydrolyze water-soluble cellulose such as CMC hydroxyethyl cellulose and cellodextrin and decreased the viscosity of the substrate solution On the other hand AEG could not produce any soluble sugars from water-insoluble cellulose such as Avicel and bacterial cellulose These properties were completely different from endo-glucanase from fungi AEG hydrolyzed cellohexaose and produced cellobiose cellotriose and cellotetraose but in the presence of bacterial cellulose the soluble sugars produced from cellohexaose disappeared in the reaction mixture It is suggested that AEG might have transglycosyl activity though it belongs to glycosidase family 8 It is proposed that this activity is closely related to cellulose synthesis

Key words Acetobacter xylinum endo-3 -14-glucanase Irpex lactells

A xyinum is a very simple and convenient system for bated at 25degC for 15 days under the static condition studying the biochemical genetic aspects of cellulose bioshy Enzymes An extracellular enzyme produced by A synthesis Why does A xylinum synthesize cellulose xyiinum (designated AEG) was obtained by the following One possibility is that this bacterium would use cellulose Be-free cells and the culture supernatant were recovered as a form of glucose storage and its polymerizations cause from the broth by filtration through a nylon membrane a reduction in osmotic strength If this is the case A (l3-XXlOO SEFAR Swiss) The filtrate was centrifuged xyhnum must also encode a cellulase gene Various reshy at 30000 X 9 for 15 min at 4C The supernatant was ports have described the production of cellulolytic enshy treated with ammonium sulfate at 80 saturation the preshyzymes by Aeetobacler strains -) One of their genes enshy cipitates formed were dissolved in 50 TIL sodium acetate codes the endo-5 -1 4-glucanase (CMCax) that can decomshy buffer (pH 50)_ The solution was dialyzed for 24 h at pose cellulose Because cellulose biosynthesis in Acetoshy4 C against distilled water and was used as the crude enshybaeler )ylinum was inhibited by the addition of the antishy zyme solution The fungal endo-type cellulase (designated CMCax serum to its growth medium this enzyme is En- I) used in the present work was obtained from Driseshythought to be essential for cellulose biosynthesis Howshy lase a commercial product of Irpex iaetetts manufactured ever the relation between cellulose biosynthesis and celshy by Kyowa Hakko (Tokyo Japan) according to procedures lulolytic enzyme has not yet been established previously reported_

From this point of view it has been expected to make Substrates Avicel a microcrystalline cellulose powshythe properties of endo-glucanase clear but there are few der (Alt 2331) was purchased from Merck (Darmstadt reports about the mode of action of Aeetobaeler-cellulase Germany) Carboxymethyl cellulose (CMC) and ostazin on various cellulosic materials We have investigated the brilliant red-hydroxyethyl cellulose (OBR-HEC) were purshyaction pattern of this enzyme on water-soluble and chased from Sigma Chemical (USA) Cotton was prepared -insoluble substrates as described by Hoshino et ai) Phosphoric acid-swollen

cotton (HC) and Avicel (HA) were prepared as described MATERIALS AND METHODS by Wood Tamarind xyloglucan was donated by T

Hayashi Kyoto University Cellohexaose (G6) and pshyCulture A xylinum ATCC 23769 was used in this nitrophenyl jJ-D-glucopyranoside (PNPG) were purchased

study by picking up rough colonies in 5-days-old agar from Seikagaku Kogyo (Tokyo Japan) plate For seed culture Hestrin and Schramm (SH) meshy Protein measurements Enzyme protein was detershydium was used The liquid medium (1000 mL) was stershy mined by the Lowry method9

) using bovine serum albushyilized at 121degC for 15 min and inoculated and then incu- min as the standard protein

Saccharification activity on water-soluble cellulose

gt Corresponding author (Tel amp Fax --81--26--269-5394 E-mail The reaction mixture contained O I TIL of I _Owt CMC yoamanogipwcshinshu-uacjp) solution O I mL of enzyme solution and 02 TIL of 005

298 1 Appl Glycosci Vol 51 NO4 (2004)

M sodium acetate buffer (pH 50) in a total volume of 04 mL After incubation at 30T for 24 h reducing sugars produced per mL of the reaction mixture were determined by the method of SomogyiOI-Nelson) One unit was deshyfined as the amount of enzyme producing 1 p mol of glushycose per min

Saccharification activity on water-insoluble cellulose The reaction mixtures contained 01 mL of a substrate

(l wt Avicel 01 wt HA HC or 025wt BC) 01 mL of enzyme solution and 02 mL of 005 1 sodium acetate buffer pH 50 Incubation was conducted at 30

v C with

mechanical shaking (l00 strokesmin) for 24 h and the mixtures were filtered through a glass filter Reducing sugars produced were measured in the same way as the above One unit was defined as the amount of enzyme producing 1 umol of glucose per min

p -Nitrophenyl f3 -glucoside hydrolyzing activity A reaction mixture contained 01 mL of 5 ffiJV1 of pNPG 01 mL of enzyme solution and 02 mL of 005 M sodium acetate buffer pH 50 After incubation at 30degC for 24 h 10 mL of 10wt NaC03 and 20 mL of distilled water were added to the mixture The amount of p-nitrophenol liberated was measured at 420 nm One unit was defined as the amount of enzyme producing I umol of pshy

nitrophenol per min Hydrolyzing activity on aBR -HEe The reaction

mixture contained 035 mL of 10wt OBR-HEC solushytion 035 mL of enzyme solution and 07 mL of 005 M

sodium acetate buffer pH 50 After incubation at 30degC for 24 h 04 mL of the reaction mixture was mixed with 12 mL of acetone The precipitated substrate was reshymoved by centrifugation and the absorbance of the supershynatant was measured at 550 nm One unit was defined as the amount of enzyme changing the absorbance (005h)

Viscometry of degradation of CMC and XG The acshytivity was assayed viscometrically in a viscometer at 30degC with 1 TIL of enzyme solution and 4 mL of 005 M soshydium acetate buffer (pH 50) and 10 mL of 10wt CMC or 05wt XG One unit of activity was defined as the amount of enzyme required to cause a 10 decrease in relative viscosity for 24 h under these conditions

Molecular weight (Mw) distribution of CMC and XG The molecular weight distributions of CMC and XG

were determined using HPLC (801 JASCO Tokyo Jashypan) They were detected using a RI monitor (410 Washyters USA) equipped with a G 3000 PWXL column (78 X 300 mm Tosoh Tokyo Japan) The mobile phase is disshytilled water at the flow rate of 08 mLmin MOlecular weight was also determined using Shodex STANDARD P-82 (Hayashibara Biochemical Laboratories Japan) as the standard carbohydrate

Thin -layer chromatography (TLC) The reaction mixture consisted of 01 mL of enzyme solution 02 mL of 5 mgmL cellohexaose or 5 mgmL cellohexaose to which was added 1 mgmL bacterial cellulose and 01 mL of 005 M sodium acetate buffer at pH 50 After inshycubation for appropriate periods 30uL aJiljuots of reacshytion mixture and authentic sugars (ccllo-oligosaccharides) solution were spotted individually Analytical TLC was performed with silica-gel 60 (05 mm thickness Merck) in the solvent system of chloroform-methanol-water (90

65 15) The resolved sugars were detected by heating the plate at 120degC for 10 min after spraying with 30 sulfushyric acid

RESULTS

Enzyme activity in crude enzyme preparation The protein content of the crude enzyme preparation

(AEG) was determined to be 143 mgmL Table 1 sumshymarizes the hydrolyzing activity of AEG against watershysoluble and insoluble substrates AEG hydrolyzed watershysoluble cellulose but did not hydrolyze water-insoluble cellulose AEG also containing j1-glucosidase as p NPG was degraded On the other hand En-1 hydrolyzed watershysoluble and insoluble cellulose as reported previously2 I

Hydrolysis of CMC by AEG and En-I The time courses of hydrolysis of CMC were compared

with typical endo-glucanase En-I from I laeteus (Fig I) En-1 hydrolyzed CMC rapidly at the initial stage and deshycreased the viscosity of CMC following the typical curve of endo cellulase On the other hand AEG decreased the viscosity very slowly The molecular weight distribution of CMC was determined using HPLC (Fig 2 Table 2) It was estimated at about 180000 initially and decreased to 300 after treatment with En-l for 24 h This suggests that the main products produced by En-l were celJoshyoligosaccharides On the other hand the elution profile of CMC treated with AEG revealed that the original peak of CMC shifted to three clear peaks showing low molecular weights Their molecular weights were estimated as 180000 50000 and 5000 respectively It is suggested this enzyme cleaved specific glycosidic bonds of CMC and then stopped From these results the mode of action of AEG was completely different from En-I

Table 1 Specific activities (SP) of AEG against various subshystrates

Substrate (water soluble)

SP (X 10- Umg)

Substrate (water insoluble)

SP (X 10- Umg)

CMC 066 AviceJ 0

XG 04 HPO-Avicel 0

OBR-HEC

pNPG

068

106

HPO-cotton

BC

0

0

08 ~

E ~

06 ~

AEGC v 04 0

ii 02

En-lf 0

0 [ 2 Time (h)

Fig 1 Viscosity changes of CMC during incubation with AEG and En-I

The activity was assayed viscometrically at 30v

C with an enzyme solution and 10wt CMC One unit of activity was defined as the amount of enzyme required to cause a 100lt decrease in relative visshycosity for 24 h under these conditions

299 Hydrolysis of Cellulosic Substrates by Endo-S -I 4-Glucanase from Acetobacter xylinum

AEG En-I

Oh

~~~)~ ~I reg

JC ~=J[ o 75 150 o 75 150

Retention time (min)

Fig 2 HPLC pattern of hydrolysis products from CMC by AEG and En-I

The molecular weight distributions of CMC were deterrnined usshying HPLC Numbers and arrows indicate molecular weight markers CD 180000 0) 50000 Q) 5000 300

Table 2 Molecular weight (Mw) changes of CMC during inshycubation with AEG and En-I

Mw AEG

o h 48 h Oh

En-l

48 h

(1)180000

0)50000

0)5000

300

60

40

55

25

20

60

40 18

82

Hydrolysis of XG by AEG and En-I The time courses for the hydrolysis of the XG by endoshy

glucanases were examined viscometrically (Fig 3) Both AEG and En-l decreased the viscosity of the XG solution very slowly but AEG decreased to a larger extent than En-I and this result was the reverse of CMC degradation The molecular weight of XG was estimated to be about 9000000 initially using HPLC (Fig 4 Table 3) The moshylecular weight of XG after treatment with AEG or En-l shifted from 9000000 to 380000 120000 and 60000 after 48 h From these results the modes of action of both enzymes were almost similar to each other

Degradation of cellohexaose (G6) with or without bacshyterial cellulose The hydrolysis products from Go by AEG were anashy

lyzed by TLC (Fig 5 (A)) The enzyme produced celshylotriose in the early stage but cellobiose and cellotetraose were also detected after 6 h incubation This enzyme could not attack cellodextrin from cellobiose to cellopenshytaose and accumulated in the reaction mixture though celshylopentaose was degraded after prolonged incubation This suggests that AEG required more than a hexaose unit of glucose for catalysis The degradation pattern of Go in the presence of BC is shown in Fig 5 (B) Although the hyshydrolysis products were similar to those from Go only the

04

~02 o () U)

gt AEG

o o 24 48 72 96

Time (h)

Fig 3 Viscosity changes of XG during incubation with AEG and En-I

Experimental dctails are the same as described in Fig 1

o 75 150 o 75 150


Fig 4 HPLC pattern of hydrolysis products from XG by AEG and En-I

Experimental details are the same as described in Fig I Numshybers and anows indicate molecular weight markers (1) 9000000 0) 380000 Q) 120000 60000

Table 3 Molecular weight (Mw) changes of XG during incushybation with ALG and En-I

Mw AEG

o h 48 h

En-I

oh 48 h

(1)9000000 70 70

0)380000

Q) I20000

30 67

30

30 15vo

70

60000 3 15

amount of the hydrolysis products in the presence of bacshyterial cellulose obviously decreased compared with prodshyucts from Go only Interestingly cello-oligosaccharides produced at the initial stage disappeared after prolonged incubation with AEG It is suggested that AEG has a transglycosyl activity especially in the presence of insolshyuble cellulose

DISCUSSION

Cellulase activity was detected in the culture broth of A xylinum as some researchers have reportedv We inshyvestigated the properties of cellulase especially the mode of action on various cellulosic substrates as the role of

300 J Appl Gycosci Vol 5 l No4 (2004)

(A) (8) _--_------

Gl

G2

G3

G4

G5

G6 S 0 05 3 6 12 24 S S 0 3 6 12 24 48 S

Time (h) Time (h)

Fig 5 Thin-layer chromatograms of hydrolysis products from cellohexaose (A) and cellohexaose in the presshyence of bacterial cellulose (B) by ALG

The reaction mixture consisted of 01 mL of enzyme solution 02 mL of 5 mgmL cellohexaose or 5 mgml cellohexaose with the addition of J mgmL bacterial cellulose and 01 mL of buffer After incubation for approshypriate periods 30 pL aliquots of the reaction mixture and authentic sugar (cello-oligosaccharides) solution were spotted individually Symbols S standard sugars G glucose G cellobiose G cellotriose G cellotetraose

bull

G cellopentaose G6 cellohexaose

cellulase for cellulose synthesis of A xylinum was not esshy

tablished Cellulase is defined as the enzyme that degrades

cellulose and produces cello-oligosaccharides However

Acetobacter cellulase could not degrade ceJloshy

oligosacharides less than DP 5 which is the smallest subshy

strate as a completely soluble sugar Furthermore it could

not produce soluble sugars from insoluble celluloses such

as Avice] bacterial cellulose and phosphoric acid-swollen

cellulose We have the questions of which substrates this

cellulase reacts with and how it degrades cellulose One

possibility was derived from our results of cellohexaose

degradation Three products 0gt02=0 were formed

from 0 6 that was partially solubilized in water but it had

a weak crystalline structure From these results Acelobacshyfer cellulase is an endo-glucanase but is diffcrent from

fungal endo-glucanases such as En-l from lrpex lacleus It is interesting that A xylinum produced enzymes that

have transglycosyl activity because cello-oligosaccharides

were reused and disappeared in the reaction mixture of

cellohexaose and bacterial cellulose This phenomenon

was observed only in the presence of bacterial cellulose

that had the same conditions as the A xylinul11 culture IL

is reported that the endo-glucanase from A xylinum beshy

longs to glycosidase family 8 which is an inverting enshy

zyme (httpafmbcnrs-mrsfrCAZYIOH_8html) As

inverting enzymes merely catalyze the transglycosyl reacshy

tion this reaction rrlight be catalyzed by other enzymes

because we did not purify this enzyme completely Anshy

other possibility of the cellulase action in the culture is acetan degradation as it degrades xyloglucan which is a

sirrlilar structure to acetan Both substrates have the 3shy14-g1ucan backbone attached side chain composed of

rrlixed oligosaccharides Indeed we detected some sugars

composed of side chains such as rhamnose mannose and

gentiobiose (data not shown)

The cellulase from A xylinum did not attack purified

bacterial cellulose produced by itself when we detected

reducing sugars produced However it is repoJ1ed that

cellulose produced under the condition that it produced

more cellulose than usual has a low degree of polymerizashytion We also measured the DP of bacterial cellulose

when it was cultivated in the presence of 3shyglucodisaceharides such as gentiobiose and cellobiose

The DP of bacterial cellulose produced in the presence of

3-glucodisaccharides was lower than that in the absence

of those sugars Furthermore cellulase activity in the culshy

ture broth was also higher than usual (data not shown)

From these results we propose that the cellulase is very

important for cellulose production and it might attack

bacterial cellulose though it does not release soluble prodshy

ucts Matthysse el al 16

17

) reported that an endoglucanase

gene (designated ceIC) homologous to A xylinum ATeC

23769 is contained in an operon of cellulose synthase in

Agrobacferium lumefaciens and that there is a transposon

insertion in celC blocks cellulose synthesis indicating

that the production of CMC-hydrolyzing activity may be

specifically associated with cellulose production In higher

plants the activities of endo-J-l4-glucanase are closely

related to various physiological aspects of plant growth

Moreover Tonouchi et al reported that cellulose proshy

duction by strain BPR2001 was enhanced by the addition

of a small amount of an endo- 3-1 4-glucanase from Bashycillus sublilis Cellulase especially endo-)-I4-glucanase

is somehow related to the biosynthesis of cellulose The

decrease in the DP of a polymer material is generally

thought to influence its quality We expect that AEG can

contribute to the unique physical properties of Be

This work was supposed by Grants-in-Aid for 21st Century COE Program by the Ministry of Education Culture Sports Science and Technology We wish to thank Dr T Hayashi of the Wood

301 Hydrolysis of Cellulosic Substrates by Endo-9-14-Glucanase from AcelObacter xylinum

Research Institute Kyoto University for supplying the xyloglucan cellulose synthesis in Agrobacterium tumefaciens 1 Bacteshyof tamarind riol 177 1069-1075 (1995)

17) AG Matthysse DL Thomas and AR White Mechanism of REFERENCES cellulose synthesis in Agrobacterium tumefaeiens 1 Bacteshy

I) WE Husemann and R Werner Cellulosesynthese durch Aceshytobacter xylinum I Ober Molekulargewicht und Molekulargeshywichtsvel1eillung von Bakteriencellulose in Abhangigkeit von der Synthesedauer Makromo Chem 59 43-60 ([ 963)

2) R Standal TG Iversen DH Coucheron E Fjaervik JM Blantny and S Valla A new gene required for ceJlulose proshyduction and a gene encoding cellulolytic activity in Acerobacshyter xylinum are colocalized with the bcs operon 1 Bacteriol 176 665-672 (1994)

3) T Okamoto S Yamamoto H Ikeaga and K Nakamura Cloning of the Acetobacter xylinum cellulase gene and its exshypression in Escherichia coli and Zymomonas mobilis App Microbio Biotechnol 42563-568 (1994)

4) HM Koo SH Song YR Pyun and YS Kim Evidence that a 9-14-endoglucanase secreted by Acetobacter xylinum plays an essential role for the fonnation of cellulose fiber Biosei Biotechnol Biochem 62 2275-2259 (1998)

5) S Hestrin and M Schramm Synthesis of cellulose by Acetoshybacter xylinum 1 Micromethod for the determination of cellushylose Biochem J 56 163-[66 (1954)

6) T Kanda S Nakakubo K Wakabayashi and K Nisizawa Purification and properties of a lower-molecular-weight endoshycellulase from Irpex lacteus (Polyporus tulipijerae) 1 Bioshychem84 1217-1226 (1978)

7) E Hoshino T Kanda Y Sasaki and K Nisizawa Adsorption mode of exo- and endo-cellulase from Irpex lacteus (Polyshyporus tulipiferae) on cellulose with different crystallinities 1 Biochem 111 600-605 (1992)

8) TM Wood Preparation of crystalline and dyed cellulose subshystrates Methods in Enzymology WA Wood and ST Kelshylogg eds Academic Press New York pp 19-25 (1988)

9) OH Lowry NJ Rosebrough AL Fan and RJ Randall Protein measurement with the Folin phenol reagent 1 Bioi Chem 193 265-275 (1951)

10) M Somogyi Notes on sugar detelmination 1 Bioi ClleIn 195 19-23 (1952)

II) N Nelson A photometric adaptation of Somogyi method for the detennination of glucose 1 Bio Chem 153 375-380 (1944)

12) T Kanda K Wakabayashi and K --Jisizawa Purification and properties of a lower-molecular-weight endo-cellulase from 11shypex lacteus (Polyporus tulipijerae) 1 Biochem 87 1625-shy1634 (1980)

13) T Kanda Y Amano M Shiroishi and E Hoshino Mode of action of exo- and endo-type cellulase from fungi in the hyshydrolysis of various substrates 1 Appl Glycosei 41 273--282 (1994)

14) M Shiroishi Y Amano E Hoshino K Nisizawa and T Kanda Hydrolysis of various cellulose (I ~3) (1-gt4)-9-0shyglucans and xl log lucan by three endo-type cellulases Mokushyzai Gakkaishi 43178-187 (1997)

IS) N Tahara M Tabuchi K Watanabe H Yano Y MOIinaga and F Yoshinaga Degree of polymerization of cellulose from Acetobacter xyLinum BPR2001 decreased by cellulase proshyduced by the strain Biosei Bioteehnol Biochem 61 1862shy1865 (1997)

16) AG Matthysse S White and R Lightfoot Genes required for

riol 1771076-1081 (1995) 18) N Tonouchi N Tahara T Tsuchida F Yoshinaga T Beppu

and S Horinouchi Addition of small amount of an endoglucashynase enhances cellulose production by Acetobacter xylinum Biosci Biotechno Biochem 59 805--808 (1995)

-tz )[ D - 7 ~pound 00 Acetobacter xylinum (J) ~pound 9 Q

I-- ~-fJ -14-1)[177- iZmiddot(J)~t1stV

=1~t1yen~M9Qfl=ffl 1f1ipJ$T Riff Bt~ f)IJjijJj]- Inder M Saxena

Malcolm R Brown Jr 2 t$83r~

181+1IgMm14tIH (380-8553 ampf)mS~ 4-17-])

School of Biological Science The University of

Texas at Austin

(Austin Texas 78712 USA)

1 v 0 - A IL)if Iii -C 06 Ut ~m (I lil1f5H=-L --shy(3 -14- 7 )v j T - --c ~ 5j-ipound -9 6 i t If T t ~ j- - A

-1 0 ~j- -A 7J CO)tJ1l~jJplusmniJJ9=J 11JOz 6 C7f~L~1J~1t

L 2 G1gt(-0) C sect O)m1f5i-~L V-P-14-7v j

T - --cititJ b ~ lt7J 6 - C-11) 1J -J t -f - -c - O)L

r-p-IA-7)v j T - --cO)I1~ ~ ilfflj-Zt ~5ItJ1lO)poundgjf

-c~~0-J~c-~ CMc~~VO~~LT~1~

0- A 7J C0) PJiI1O)tJ1l I 1J07Jlt5t~ 2 h tiJ Be ~ 7

~1 v7J c0)fi110)J1l-cl1J07k5t~ijo1J~- G7JiJL) t (Table I) -=- h GO)~~f~(I ~m~JlE13)j~O)Jl~BJ 7JL

F 7)v j T - --C (En-I) C 11 SA gt iH~ 7J 6 ~ G

I HPLC ~ ffl v (1J07Jlt5t~+jampJ ~ dd c O)-f- ~m

~JE13O)M-C(I ~M CMC ~JjWHs~-c ~~t I) J

tJ1li -c5J-fiJT 6iJ - 0) M -C 11 fUf F~J iJ3f I) J~JliXEr-J

2 7Jt I) 7 - ~1j[ -c5t~ijo v 6 -=- iJb 1J -J t

(Fig I) i t - 0) gjLk11 1 0 A ~ c)-- t - A ~ 1 0 C

t -A 1 0 -- I) t -A 1 0 T -- 7 t -A 15tJJ+ L t

(Fig 3 (A) ) C - ~ 1J ) 7T I) 71v 0 - A tHE 9~

-c1 D A ~ -tj-t A ~ IEjfit i ampJZ 2 -tt -c h t c - ~

v -J tIv~UJ 2 htt I) JJJ1JamprtIlrFs9 c c b Ii~z t

(Fig 3 (B)) t)I1JG -O)ir1f5i-MI ampijii~-C

06772 I) _ 8 ~T6 ctRfi2i1 v6iJ tJ1lijiit ~~tJ b t~ -cv 6 PJ~~I1iJ~D~ 2 i1t

298 1 Appl Glycosci Vol 51 NO4 (2004)

M sodium acetate buffer (pH 50) in a total volume of 04 mL After incubation at 30T for 24 h reducing sugars produced per mL of the reaction mixture were determined by the method of SomogyiOI-Nelson) One unit was deshyfined as the amount of enzyme producing 1 p mol of glushycose per min

Saccharification activity on water-insoluble cellulose The reaction mixtures contained 01 mL of a substrate

(l wt Avicel 01 wt HA HC or 025wt BC) 01 mL of enzyme solution and 02 mL of 005 1 sodium acetate buffer pH 50 Incubation was conducted at 30

v C with

mechanical shaking (l00 strokesmin) for 24 h and the mixtures were filtered through a glass filter Reducing sugars produced were measured in the same way as the above One unit was defined as the amount of enzyme producing 1 umol of glucose per min

p -Nitrophenyl f3 -glucoside hydrolyzing activity A reaction mixture contained 01 mL of 5 ffiJV1 of pNPG 01 mL of enzyme solution and 02 mL of 005 M sodium acetate buffer pH 50 After incubation at 30degC for 24 h 10 mL of 10wt NaC03 and 20 mL of distilled water were added to the mixture The amount of p-nitrophenol liberated was measured at 420 nm One unit was defined as the amount of enzyme producing I umol of pshy

nitrophenol per min Hydrolyzing activity on aBR -HEe The reaction

mixture contained 035 mL of 10wt OBR-HEC solushytion 035 mL of enzyme solution and 07 mL of 005 M

sodium acetate buffer pH 50 After incubation at 30degC for 24 h 04 mL of the reaction mixture was mixed with 12 mL of acetone The precipitated substrate was reshymoved by centrifugation and the absorbance of the supershynatant was measured at 550 nm One unit was defined as the amount of enzyme changing the absorbance (005h)

Viscometry of degradation of CMC and XG The acshytivity was assayed viscometrically in a viscometer at 30degC with 1 TIL of enzyme solution and 4 mL of 005 M soshydium acetate buffer (pH 50) and 10 mL of 10wt CMC or 05wt XG One unit of activity was defined as the amount of enzyme required to cause a 10 decrease in relative viscosity for 24 h under these conditions

Molecular weight (Mw) distribution of CMC and XG The molecular weight distributions of CMC and XG

were determined using HPLC (801 JASCO Tokyo Jashypan) They were detected using a RI monitor (410 Washyters USA) equipped with a G 3000 PWXL column (78 X 300 mm Tosoh Tokyo Japan) The mobile phase is disshytilled water at the flow rate of 08 mLmin MOlecular weight was also determined using Shodex STANDARD P-82 (Hayashibara Biochemical Laboratories Japan) as the standard carbohydrate

Thin -layer chromatography (TLC) The reaction mixture consisted of 01 mL of enzyme solution 02 mL of 5 mgmL cellohexaose or 5 mgmL cellohexaose to which was added 1 mgmL bacterial cellulose and 01 mL of 005 M sodium acetate buffer at pH 50 After inshycubation for appropriate periods 30uL aJiljuots of reacshytion mixture and authentic sugars (ccllo-oligosaccharides) solution were spotted individually Analytical TLC was performed with silica-gel 60 (05 mm thickness Merck) in the solvent system of chloroform-methanol-water (90

65 15) The resolved sugars were detected by heating the plate at 120degC for 10 min after spraying with 30 sulfushyric acid

RESULTS

Enzyme activity in crude enzyme preparation The protein content of the crude enzyme preparation

(AEG) was determined to be 143 mgmL Table 1 sumshymarizes the hydrolyzing activity of AEG against watershysoluble and insoluble substrates AEG hydrolyzed watershysoluble cellulose but did not hydrolyze water-insoluble cellulose AEG also containing j1-glucosidase as p NPG was degraded On the other hand En-1 hydrolyzed watershysoluble and insoluble cellulose as reported previously2 I

Hydrolysis of CMC by AEG and En-I The time courses of hydrolysis of CMC were compared

with typical endo-glucanase En-I from I laeteus (Fig I) En-1 hydrolyzed CMC rapidly at the initial stage and deshycreased the viscosity of CMC following the typical curve of endo cellulase On the other hand AEG decreased the viscosity very slowly The molecular weight distribution of CMC was determined using HPLC (Fig 2 Table 2) It was estimated at about 180000 initially and decreased to 300 after treatment with En-l for 24 h This suggests that the main products produced by En-l were celJoshyoligosaccharides On the other hand the elution profile of CMC treated with AEG revealed that the original peak of CMC shifted to three clear peaks showing low molecular weights Their molecular weights were estimated as 180000 50000 and 5000 respectively It is suggested this enzyme cleaved specific glycosidic bonds of CMC and then stopped From these results the mode of action of AEG was completely different from En-I

Table 1 Specific activities (SP) of AEG against various subshystrates

Substrate (water soluble)

SP (X 10- Umg)

Substrate (water insoluble)

SP (X 10- Umg)

CMC 066 AviceJ 0

XG 04 HPO-Avicel 0

OBR-HEC

pNPG

068

106

HPO-cotton

BC

0

0

08 ~

E ~

06 ~

AEGC v 04 0

ii 02

En-lf 0

0 [ 2 Time (h)

Fig 1 Viscosity changes of CMC during incubation with AEG and En-I

The activity was assayed viscometrically at 30v

C with an enzyme solution and 10wt CMC One unit of activity was defined as the amount of enzyme required to cause a 100lt decrease in relative visshycosity for 24 h under these conditions


AEG En-I

Oh

~~~)~ ~I reg

JC ~=J[ o 75 150 o 75 150





Mw AEG

o h 48 h Oh

En-l

48 h

(1)180000

0)50000

0)5000

300

60

40

55

25

20

60

40 18

82





04

~02 o () U)

gt AEG

o o 24 48 72 96

Time (h)



o 75 150 o 75 150





Mw AEG

o h 48 h

En-I

oh 48 h

(1)9000000 70 70

0)380000

Q) I20000

30 67

30

30 15vo

70

60000 3 15


DISCUSSION



(A) (8) _--_------

Gl

G2

G3

G4

G5

G6 S 0 05 3 6 12 24 S S 0 3 6 12 24 48 S

Time (h) Time (h)



bull















































17











































181+1IgMm14tIH (380-8553 ampf)mS~ 4-17-])


Texas at Austin







-c~~0-J~c-~ CMc~~VO~~LT~1~









t -A 1 0 -- I) t -A 1 0 T -- 7 t -A 15tJJ+ L t

(Fig 3 (A) ) C - ~ 1J ) 7T I) 71v 0 - A tHE 9~






AEG En-I

Oh

~~~)~ ~I reg

JC ~=J[ o 75 150 o 75 150





Mw AEG

o h 48 h Oh

En-l

48 h

(1)180000

0)50000

0)5000

300

60

40

55

25

20

60

40 18

82





04

~02 o () U)

gt AEG

o o 24 48 72 96

Time (h)



o 75 150 o 75 150





Mw AEG

o h 48 h

En-I

oh 48 h

(1)9000000 70 70

0)380000

Q) I20000

30 67

30

30 15vo

70

60000 3 15


DISCUSSION



(A) (8) _--_------

Gl

G2

G3

G4

G5

G6 S 0 05 3 6 12 24 S S 0 3 6 12 24 48 S

Time (h) Time (h)



bull















































17











































181+1IgMm14tIH (380-8553 ampf)mS~ 4-17-])


Texas at Austin







-c~~0-J~c-~ CMc~~VO~~LT~1~









t -A 1 0 -- I) t -A 1 0 T -- 7 t -A 15tJJ+ L t

(Fig 3 (A) ) C - ~ 1J ) 7T I) 71v 0 - A tHE 9~






(A) (8) _--_------

Gl

G2

G3

G4

G5

G6 S 0 05 3 6 12 24 S S 0 3 6 12 24 48 S

Time (h) Time (h)



bull















































17











































181+1IgMm14tIH (380-8553 ampf)mS~ 4-17-])


Texas at Austin







-c~~0-J~c-~ CMc~~VO~~LT~1~









t -A 1 0 -- I) t -A 1 0 T -- 7 t -A 15tJJ+ L t

(Fig 3 (A) ) C - ~ 1J ) 7T I) 71v 0 - A tHE 9~






























181+1IgMm14tIH (380-8553 ampf)mS~ 4-17-])


Texas at Austin







-c~~0-J~c-~ CMc~~VO~~LT~1~









t -A 1 0 -- I) t -A 1 0 T -- 7 t -A 15tJJ+ L t

(Fig 3 (A) ) C - ~ 1J ) 7T I) 71v 0 - A tHE 9~





hydrolysis of water-soluble and water-insoluble cellulosic substrates by endo-,b

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