Xianzhen Li~ Yunzhan Huang I Degui Xu I DongpingXiao t Fengxie Jin I Peiji Gao. Institute of Microbiology, Shandong University, Jinan 250100, China.
BIOTECHNOLOGY L E T T E R S Volume 18 No.2 (February 1996) p.205-210 Received as revised 2nd January.
CELLOBIOSE-OXIDIZING ENZYME FROM A NEWLY ISOLATED C E L I , U L O L Y T I C B A C T E R I U M C'),tophaga sp. LX-7 Xianzhen Li~ Yunzhan Huang I Degui Xu I DongpingXiao t Fengxie Jin I Peiji Gao Institute of Microbiology, Shandong University, Jinan 250100, China i Department of Food Technology, Dalian College of Light Industry, Dalian 116001, China
SUMARY The cellobiose oxidizing enzyme of the newly isolated cellulolytic bacterium Cytophaga sp. I.,X-7 was produced extracellularly when grown on cellulose or other saccharides, which was previously noted only in fungi. The enzyme could use not only cellobiose, maltose, glucose and other saccharides but also cellulose as substrates, and use dichlorophenol indophenol and oxygen as electron acceptors. INTRODUCTION The degradation of cellulose by cellulolytic microorganisms occurs mainly through the concerted action of several hydrolytic enzymes ~Vood and McCrae, 1979), though occasionally additional nonhydrolytic enzymes, e.g. oxidative enzymes have also been reported to be operative ~ri'ksson et al., 1974; Highley, 1980). Cellobiose oxidizing enzymes, capable of utilizing a wide variety of electron acceptors, have been detected in many fungi. In 1974, Westermark and Eriksson (1974a, b) reported that white-rot fungi Polyporus versicolor and
Phanerochacte chrysosporium could produce extracellular celiobiose:quinone oxidoreductase (CBQ) when grown on cellulosic substrates, which used lignin or its degradation products as electron acceptors. Vaheri (1982) also found an oxidative system to be involved in the degradation of cellulose by Trichoderma reeset. A similar oxi,tative enzyme, cellobiose dehydrogenase, but which does not
* The present address: Dr Xianzhen Li, Department of Food Technology, Dalian College of Light Industry, 2 Baoding Street, Dallian I ] 6001, PR. China
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react with quinones, has been detected innon-lignolytic Monilia sp. 0-~kkor, 1980), and ceUulolytic but non-lignolytic fungus Sporotrichum thermophile (Coudray et al., 1982). Subsequently Eriksson et al. (1974) fotmd that the presence of oxygen can increase the rate of cellulose breakdown in extracts from wood-rotting fungi. Since cellobiose dehydrogenase does not utilize oxygen they concluded that a separate oxidative enzyme must be involved in cellulose digestion, and then cellobioso oxidase (CBO) has been discoveried and purified, which oxidizes cellobiose or higher cellodextrin reducing ends to their corresponding lactone as probable imediate products using molecular oxygen (Aye~ et al., 1978; Eriksson, 1978). Since Eriksson et al (1974) fast discovered oxidative enzymes to be involved in the cellulose degradation process, substantial research into the biotechnologieal utilization of lignocellulosie materials has focused on white-rot fungi and the enzyme mechanisms they use for degradation of wood and wood components (Eriksson et al., 1990). However, the oxidative system in bacteria was almost neglected, and is currently not known. The cellulase system of Cytophaga has been investigated by several workers (Walker and Warren, 1938; Fahreaus, 1947; Chang and Thayer, 1977). Walker and Warren (1938) isolated a strain of
Cytopphaga hutchinsonii which was able to eat through filter paper suspended in a mineral salts medium and found that the degradation was enhvalced by an increased oxygen supply. Regretfully, they did not make research tbr detecting whether there is an oxidative system involved in Cytophaga. We recently isolated a new cellulolytic bacterium Cytophaga, designated as LX-7, from the campus of Shandong University at Jinan, China, which can wholly degrade cellulose, but when grown on cellulose almost no detectable reducing sugars are released into the medium. This observation prompted us to examine if an oxidative system oceured in Cytophaga, and found that strain LX-7 did produce a cellobiose oxidizing enzyme. To our knowledge, this is the fast description of a cellobiose oxidizing enzyme produced by bacterium Cytophaga sp. LX-7.
MATERIALS AND METHODS Bacterial strain and culture conditions
Cellulolytio bacteria were enriched on mineral salts [(%, w/v): MgSO4 • 7}½0, 0.02; KNO3, 0.075; KzHPO4, 0.05; FoSO 4 • 7I-IzO, 0.002; CaCIz • 2HzO, 0.004 and Peptone, 0.2] with 1.5% (w/v) agar overlapped a piece of filter paper by stamping file soil on the filter paper with the fiat end of a glass
206
rod. After incubated for 5-6 days, the areas where filter paper was liquefied were suspended in 2 ml sterile physiological saline, and purified by dilution plating on cellodextrin agarose plates (the mineral salts medium with 0.1% eellodextrin and 0.5% agarose). The isolate was gram negative, yellow pigmented and nonflagellated but able to glide on the surface of the agar media. The strain was identified as a new strain of Cytophaga based on the key to the genus Cytophaga described by Larkin (1989) and Mitchell et al. (1969), and designated as Cytophagasp. LX-7. The organism was cultured in mineral salts medium containing 0.5% cellulose powder CFll (Whatman) at 30 C. Cell-free filtrates was obtained by centrifugation at 10OO0rpm for 20 rain. The supematant was used for detem~ination of cellulase or oxidative enzyaue.
Assays Reducing sugar was detennined with 3,5-dinitrosalicylic acid reagent (Miller, 1959) and total sugars was determined by the method of Dubois et al.(1956). Cellobiose oxidizing activity was measured as described by Renganathan et al. (! 990). Cellulase for CMC-saecharifying activity was determined using 0.5% carboxymethylcellulose (CIVIC) in O.1 M phosphate-citrate buffer (pH 6.6) at 40°C (Mandels et al., 1976). Cellulase for CMC-liquifying activity was assayed viscometrically. After 19.8 ml of 0.5% CMCin O.1 M phosphate-citrate buffer (pH6.6) was preheatred in an Rotational Viseometer NDJ-I (Shanghai Balances Instrement Factory, China) at 40°C for 10 rain, the enzyme reaction was started by adding 0.2 ml of appropriately diluted enzyme followed by immediate mixing. The viscosity of CIVIC was determined at intervals of 3 rain from the addition of enzyme. One unit of cellulase for CMCliquefying activity was defined as the amount of enzyme which produced a net decrease in Viscosity of 1 centi-poise (cp).
RESULTS AND DISCUSSION
Production of ceiloblose oxidizing enzyme by Cytophaga sp. LX-7 The ability of various substrates to support cellobiose oxidizing enzyme production is shown in Table 1. Cellulosic substrates were the strongest inducers of the enzyme, while CMC was less effective inducer. The activities were obtained when grown on cellobiose and maltose, which was different from that previously reported (Dekker, 1980; Rengznathan et al., 1990), and glucose also induced cellobiose oxidizing enzyme but the activity was lower than that grown on cellulose. Table 1 The production of cellobiose oxidizing enzyme by Cytophaga sp. LX-7 grown on various carbon sources
Carbon sources Cellobiose oxidizing activity .....(U/ml) ' Cdlobiose 15.81 Maltose 15.30 Sucrose 11.73 Glucose 11.06
Carbon sources Cellobiose oxidizing activity ,
Whatman CF11 Avicel oc-Cellulose CMC
fO/ml) 15.40 15.40 17.52 2.13
The growth of Cytophaga sp. LX-7 in minimal medium supplemented with a-cellulose or Avicel as the sole carbon source suggested that strain LX-7 is a celhdolytic bacterium, since such substrates
207
were considered to be specially recalcitrant to the hydrolysis, requiring a complete sot of cellulolytie activities for solubilization (Mullings, 1985). Time course experiments showed that cellulosic substmte was digested thoroughly after 100-110 h of culture, while the higher level of eellulase w~,s reached at aound 136 h. A representative example of such experments is given in figure 1. Cellobiose oxidizing enzyme was released in the culture medium during growth on cellulose pmallel with the bacterial growth. The results showed that no reducing sugars could be detected in the media of cellulose cultures. Berg et al. (1972) believed that the cellulase system appears to produce no more free sugars than what is immediately consumed by the bacteria, the concentration of which is maintained at too low level to be detected. However, the present results confirmed that the cellobiose oxidiTi_ngactivity produced by Cytophagasp. LX-7 occurred m medium over the process of cellulose degradation. Presumably oxidation may lead to the formation of non-reducing sugars. Cellulose may be oxidized directly by cellobiose oxidizing enzyme to introduce earboxyl groups, causing disturbance to intmchain and interchain hydrogen bounds, and further cause greater disruption and can lead to cleavage of a cellulose chain (Eriksson et al., 1974; Ayers et al., 1978).
Altematively oxidation by ceilobiose
~~ IOF 12
conversion of cellobiose into celiobionic acid
,o
(Ayers et al., 1978). It is not yet
§ g
° 4
possible to say whether the non-accumulation
~
,, 0
cellulose
of reducing sugars is due to the oxidation by the
eellobiose
oxidizing
enzyme
//~X ~ - - ~ l O14 i140
,o
40
BO
120 180
,
0
Ttm, (),)
when
Cytophagagrownon cellulose. Similar results were obtained when grown in medium with Avicel or cellulose powder CF 11.
208
Figure 1 Production of cellobiose oxidizing enzyme by Cytophaga sp. LX-7 grown on (xcellulose. • , CMC-liquefying activity; m, CMC-saccharifying activity; A , Cellobiose oxidizing activity.
Characterization
of celloblose oxidizing enzyme
Several mono- and disaceharides and cellulose were tested as potential substrates for the cellobiose oxidizing enzyme of Cytophaga sp. LX-7. Table 2 lists all of the saccharides and cellulose which have been found to be oxidized with DCPIP as oxidizing agents. Contrary to the cellobiose oxidizing enzyme of
Sclerotium rolfsii (Sadana and Patil, 1985) and Sporotrichum pulverulentum (Morpeth,
1985), the crude enzyme tested in this study could oxidize not only cellobiose but also other monoand disaccharides. Among the substrates listed in table 2, maltose, cellobiose and glucose were the most effective. Coudray et al. (Coudray et al., 1982) also observed the reaction with glucose and maltose while oxidation was minimal. The oxidation of cellulose by the cellobiose oxidizing enzyme is similiar to that by cellobiose dehydrogenase ofMobilia (Dekker, 1980), but the enzyme produced by strain LX also oxidized CMC effectively. Table 2 Substrate ' specificities of cellobiose oxidizin~ enz)nne. Substrate Substrate Relative.. activ!t), 4%) Xytose Cellobiose 100 Avicel Maltose 105.48 Whatman CF 11 Glucose 98.15 Sucrose 80.71 or-Cellulose CMC Mannose 54.40
Relative activity (%) 59.76 58.21 62.66 55.56 64.88
The cellobios¢ oxidizing activity of the • Tlmo ( , , , i n )
enzyme was compared using molecular oxygen 0
and DCPIP as the electron accepter (figure 2).
~u,
The enzyme activity was followed by decrease
I:1 0
O.S
B J
4
J
'
8
|
'
0
I
~'
I
10 ~
l|
0,4
t 0.2
II
in absorbance at 600 ran due to reduction of
O.ll
o
DCPIP, or by decrease in concentration of
O.Z O.1
e
reducing sugar due to the oxidation of
0.0
cellobiose. Both decrease were linear with
1 0
0.0 1
S
S
4,
• TJtae (h)
• time, however, the ability of cellobiose Figure 2 Cellobiose oxidation by cellobiose oxidizing enzyme with IX~.t)IP ( • ) or oxygen ([]) as electron acceptors.
oxidizing enzyme to utilize DCPIP as electron accepter was stronger than flint use oxygen for
209
oxidation of eellobiose. Similiarly, Morpeth and Jones (1986) also found that eellobiosc oxidizing enzyme can us0 oxygen as an electron aoceptor, although at a very low rate. A preliminary physieo-chemical characterization of crude cellobiose oxidizing enzyme showed that the pI-Ioptimum was 6.0, and the temperature optimum was 40 C. Since unfractionated extracollular proteins were used in these experiments, Only initial catalytic properties was studied, and why the enzyme is able to oxidize maltose and glucose is unknown. Further experiments with homogenous enzyme will identify the celiobiose oxidizing enzyme and clarify the relation of cellobiose oxidizing enzyme to cellulose degradation. ACKNOWLb.TDGEMENTS This work was supported by The National Natural Sciences Foundation of China. REFFERENCES
Ayers, A.R., Ayerz, S.B. and Eriksson, K.E. (1978). Eur. J. Biochent 90, 171-181. Berg B., Hofsten, B.V. and Pettersson, G. (1972). J. AppL BacL 35, 201-214. Chang W.T.H. and Thayer, D.W. (1977). Cart J. MicrobioL 23, 1285-1292. Coudray, M.R., Canevaseini, G. and Meier, H. (1982). Biochem. J. 203, 217-284. Dekker, R.F.H. (1980). J. Gert MicrobioL 120, 309-316. Dubois, M., Gilten, K., Hamilton, J.K., Rebirs, P.A. and Smith, F. (1956), AnaL Chent 28, 350-356. Eriksson, K.E. (1978). BiotechnoL Bioeng. 20, 317-332. Eriksson, K.E., Pettersson, B. and Westermark, U. (1974). FEBS LetL 49, 282-285. Eriksson K,E.L., Blanchette, R.A. and Ander, P. (1990). Microbial and Enzymatic Degradation of Wood and Wood Components. pp.407, Berlin and Heidelberg: Springer-verlag. Fahreaus, G. (1947). Symb. Bet Ups., 78-130. Highley, T.L .(1980). AppL EnvirorL MicrobioL 40, 1145-1147. Larkin, J.M. (1989). Nonphotosynethetie, nonfruiting gliding bacteria. In: Bergey's Mmmal of Systematic Bccteriology, Holt, J.G., Bryant, M.P. and Staley, J.T. eds. vol. 3. pp. 2010-2050, London: Williams and Wilkins. Mandels, M., Andretti, R. and Roche, C. (1976). BiotechnoL Bioeng. Syrup, 6, 21-23. Miller, G.L. (1959). AnaL Cherg 31, 1426-1428. Mitchell, T.G., Hendrie, M.S. and Shewan, J.M. (1969). J. AppL BacL 32, 40-50. Morpeth, F.F. (1985). Biochent J. 228, 557-564. Morpeth, F.F. and Jones, G.D. (1986). BiochenL d. 236, 221-226. Mulling,s, R. (1985). Enzyme Microb. TechnoL 7, 586-591. Renganathan, V., Usha, S.N. and Lindenburg, F. (1990). AppL MicrobioI. Biotechnol. 32, 609-613. Sadana, J.C. and Patti, R.V. (1985). J. Gert MicrobioL 131, 1917-1923. Vaheri, M.P. (1982). J. Biohent 4, 356-363. Walker, E. and Warren, F.L. (1938). Biochent ,,1.32, 34-43. Westermark, V. and Erikszon, K.E. (1974a). Acta. Chert Scand. I328, 204-208. Westermark, V. and Eriksson, K.E. (1974b). Acta. Chent Scand B28, 209-214. Wood, T.M. and McCrae, S.I. (1979). Adv. in Chent Series. 181, 181-209.
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