Comparison of H2O2-producing enzymes in selected white rot fungi

ELSEVIER

FEMS Microbiology

Letters 139 ( 1996) 2 15-22

I

Comparison of H ,O,-producing enzymes in selected white rot fungi Jiong Zhao, Bernard J.H. Janse

*

Deparhnent of Microbiology, Uni~~ersityof Stellenbosch, Stellenbosch 7600, South Africa Received

18 December

1995; revised 8 April 1996; accepted 8 April 1996

Abstract Using fungi grown on synthetic agar medium, we evaluated and compared the concentration of various H,O,-producing enzymes. Our results showed that oxidase production in solid medium was better than that found in liquid medium and as high as that detected in wood samples. High yields of oxidases made it possible to compare different oxidases in the same culture extracts and under different conditions. Our results also indicated that H,O, production is ubiquitous in the white rot fungi tested and that enzyme levels are influenced by the substrate composition. Keywords: Glucose oxidase; Glyoxal oxidase; Cellobiose

oxidase; White rot fungus

1. Introduction Hydrogen

peroxide

(H,O,)

has been

implicated

in lignin- and cellulose-degrading systems of both white and brown rot fungi [l]. In brown rot fungi, H,O, is thought to play a role in the initial attack of cellulose by mediating the production of hydroxyl radicals. In white rot fungi, two peroxidases, viz. manganese and lignin peroxidase, are known to play important roles in lignin degradation and both require H,O, as co-substrates [2]. To date, a number of oxidases have been suggested as possible donors of H,O, during white rot decay of lignocellulose. These enzymes include glucose I-oxidase [3], glucose 2-oxidase [4-61, glyoxal oxidase (GLOX) [7,8], cellobiose oxidase (CbO) [2], veratryl alcohol oxi-

* Corresponding author. [email protected]

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+ 27 (21) 808 3611;

0378-1097/96/$12.00 0 1996 Federation PII SO378-1097(96)00144-9

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Microbiological

dase [l], methanol oxidase [l], and fatty-acyl coenzyme oxidase [l]. Of these enzymes, sugar-oxidising enzymes and GLOX have been most intensively studied as they are reportedly produced in lignocellulolytic cultures [4,7]. Though many papers have been published about oxidase enzymes, it is uncertain which enzyme is the major source of H,O, in lignocellulolytic cultures of white rot fungi. Glucose I-oxidase, which catalyses the formation of glucuronic acid from glucose, was suggested to be the major source of H,O, in Phanerochaete chlysosporium [3]. However, this enzyme was only detected intracellularly and it has previously been suggested that H,O,-producing oxidases should be extracellular or at least have a subcellular location so that H,O, may be freely released either to function in conjunction with peroxidases, or to act directly on the lignocellulose substrate [5]. Kersten and Kirk [7] described a GLOX in ligninolytic cultures of P. chrysosporium. GLOX activity was also Societies. All rights reserved

found in supplemented oak sawdust culture extracts of other wood rot fungi [9]. This enzyme was also thought to be an important source of H ?02, though it was suspected that the concentration of glyoxal or methyl glyoxal in the culture fluid of P. t.h~~sr)spt>rium may be too low to support enzyme activity [3,8]. Volt and Eriksson [4] purified and partially characterised a glucose 2-oxidase from P. chtyosporium. This enzyme oxidised glucose at the C-2 carbon and was also called a pyranose oxidase (POD). Ultrastructural and immunocytochemical studies showed that both intra- and extra-cellular POD activity was found in Liquid and solid cultures and it was suggested that glucose 2-oxidase is the major source of HzO, during wood degradation by P. chtyosporium, Trametes r~ersicolor. and Oudemansiello mucida [5,6]. Recently, the cellobiose-oxidising enzyme. CbO, has gained interest due to its specific role both in lignin and cellulose degradation in nature [2]. To produce high yields of oxidases from white rot fungi, fungal strains were usually cultivated in chemically defined liquid medium, under conditions previously determined suitable for lignin mineralization by P. chrysosporium. However. oxidase activities were low, even under optimised conditions. De Jong et al. [IO] used liquid media to screen for the presence of various oxidases present in 67 isolates of wood rot fungi. Only few of the strains tested had up to 3 mu/ml of GLOX activity. whilst most of them had no detectable oxidase activity. Pelaez et al. [ 1 l] screened 90 strains of 68 basidiomycete species for the presence of aryl alcohol oxidase. In shaking cultures, low oxidase activity (up to 4 mu/ml> was detected in a few of the strains tested. Using an optimised liquid medium, with daily flushing of 100% O,, Kersten [8] obtained GLOX activity of up to 32 mu/ml. When wood was used as substrate, Orth et al. [9] only detected GLOX activity in six of the 11 wood-degrading fungi tested, contrasted with this, manganese peroxidase activity was found in all studied strains. However, Daniel et al. [6] found up to 149 mu/g POD activity in wood blocks inoculated with P. chqwsporium after four months. Although many individual oxidases have been identified in ligninolytic cultures, they have been studied in isolation and limited knowledge exists about the presence of different oxidases in the same

culture extract. An understanding of the levels of different oxidases in the same extract may contribute greatly to our knowledge regarding the role of individual oxidases in lignin degradation. However. the low levels of oxidase activity present in liquid cultures have prevented direct comparison of oxidases in the same culture extract. Using white rot fungi grown on synthetic agar medium, we evaluated the concentration of various HzO,-producing enzymes. Enzyme activities detected in agar medium were much higher than those detected in liquid medium and comparable to those found in wood. High yields of oxidases made it possible to compare different oxidases in the same culture extracts and under different conditions.

2. Materials

and methods

2. 1. Organisms hind culture wnditifms P. chtyosporium strains BKM-F- 1767, Phlehia radiata MJL- 1 19%sp, L- 13 127~sp, Ceriporiopsis suhr~ermispora FP-9003 1-sp and T. r~ersicolnr R IO5 were obtained from the Forest Products Laboratory, Madison, WI, USA. Pleurotus sajor-caju 27. and Lentinus tigrim4s 2 were obtained from the Department of Biology. The Chinese University of Hong Kong: Aspergil1u.s niger ATCC 90196 and Trichodrrmrt reesei QM 6a were obtained from American Type Culture Collection. P. chry’sosporium KKPI 0, a Phanerochaete sp. strain, and a Merulills sp. strain were from our culture collection (Department of Microbiology, University of Stellenbosch, South Africa) [ 121. All fungi were routinely maintained on 2% (w/v) malt extract agar slants. A synthetic medium (DMS), with dimethylsuccinic acid as buffer, was used in this study [ 131. If not specified otherwise, glucose was added to a concentration of 10.0 g 1~ ’ Nitrogen, in the form of NH,NO, and I.-asparagine, was added to a final concentration of 2.6 mM N and 26 mM N for low nitrogen (LN) and high nitrogen (HN) medium [ 131. The final pH was adjusted to 4.5 with 2.0 M KOH. In solid medium. agar (Biolab, South Africa) was added to a final concentration of 12 g I ’ . 20 ml sterilised agar medium was added per 90 mm petri dish. To study the effect of oxygen on enzyme

J. Zhao, B.J.H. Janse/

FEMS Microbiology Letters 139 (1996) 215-221

production, petri dishes were incubated in a sealed incubator and were flushed with 99.5% oxygen for 10 min daily. Petri dishes incubated under air were used as controls. Shallow stationary cultures were incubated in 250 ml rubber-stoppered flasks containing 30 ml liquid DMS medium and were also flushed with 99.5% oxygen for 10 min daily [81. Cultures incubated in air were used as controls. Oxygen concentration in flasks and incubators over 24-hour period after flushing was determined using an oxygen meter (HI9 143, HANNA). Conidiospores from an &day-old culture of P. chrysosporium were used to inoculate liquid media (1 .O X lo6 conidiospores per ml final concentration). The inoculum of the other fungi was prepared from 7- to 14-day-old mycelia mats (grown on DMS LN agar medium). A volume of ca. 10 ml from these plates was added to 50 ml DMS liquid media and blended for 30 s in a Waring blender. 2 ml of this mycelia homogenate was used as inoculum. A 7 mm diameter plug from a 7-14-day-old culture served as inoculum for DMS agar plates. Phanerochaete strains were incubated at 37°C and other fungi were incubated at 30°C.

217

solid medium, whereas enzyme activity in liquid medium was expressed as units per ml supematant. For comparison, enzyme activity in solid medium was also expressed as units per ml of solid medium using a density of 1.01 g ml-’ solid medium. The o-dianisidine method [3] and MBTH-DMAB method [4] were also initially used to detect H,O,. Protein concentrations were estimated by the method of Lowry et al. [14] and using bovine serum albumin (Sigma) as standard.

3. Results and discussion Three assay methods have been reported to detect different H,O,-producing enzymes in white rot fungi: 250

,

.

.

.

.

.

.

,

2.2. Enzyme assays To determine accumulated enzyme activity in agar, plugs with mycelia were added to 50 mM dimethylsuccinate buffer (pH 6.0) at a ratio of 50 mg plug per ml buffer. Extracellular enzymes were eluted from the plugs for 30 min. The plug and mycelium were then removed by centrifugation (2000 X g, 10 min) and the supematant was used for various enzyme assays. Boiled agar plugs (10 min) served as controls. To detect oxidase activity in submerged cultures, culture supematant was used directly in the reaction mixtures. The production of H,O, was assayed using the modified phenol red method [7]. The reaction mixture included: 50 mM dimethylsuccinate buffer (pH 6.0) 0.1 g 1-l phenol red, 3.6 U of horseradish peroxidase, 0.1 M of the respective sugar or 0.01 M methylglyoxal, and oxidase sample. One unit of oxidase was defined as 1 pmol of H,O, formed per min and calculated from the increase in absorbance at 610 nm using a molar extinction coefficient of 2.2 X lo4 M- ’ cm-’ [7]. In solid medium, enzyme activity was expressed as units per gram of

Time

(days)

Fig. 1. Glucose oxidase and glyoxal oxidase activity of P. chrysosporium BKM-F-1767 (open symbols) and Phanerochaete sp. (filled symbols) grown in LN agar medium (A) and in LN liquid medium (B) (both under oxygen). Symbols: glucose oxidase activity in liquid medium (0, 0) and in agar medium ( 0, n 1; GLOX activity in liquid medium ( v , v ) and in agar medium ( A , A). Results are given as the means of three replicates. Vertical bars represent the standard deviation of the mean.

218

J. Zhao. B.J.H. Jansr/

FEMS Micwhiolo,~~

the phenol red method [7], the o-dianisidine method [3], and the MBTH-DMAB method [4]. In a calibration test with known concentration of H202 solution, a good correlation was shown to exist between the theoretical and experimental determination of H .O, when the phenol red assay was used. The coefficient of variation (the standard deviation divided by the mean) in these determinations was less than IO%, indicating the usefulness of this method (data not shown). In contrast, the o-dianisidine method was less sensitive and less effective in detecting H,O, when compared to both the other methods. Though the MBTH-DMAB method was more sensitive than the phenol red assay, no HZOZ could be detected in the presence of methylglyoxal (data not shown). Our results, therefore, indicated that of the three methods tested, the phenol red method was the best method to determine H,O, production and was used in this study. Oxidase activities of P. chtyosporium BKM-F 1767 and Phanerochaete sp. were compared after cultures were grown in liquid medium and on solid agar plates (Fig. 1). Both strains showed high oxidase activities in a preliminary screening. In DMS LN liquid medium under OZ. glucose oxidase activity in strain BKM-F- 1767 was detected on day 2 and

Table I Effect of different agar media on H.O,-producing Medium and condition

DMS (LN) (in air) DMS (LN) (in oxygen) DMS (HN) (in air) DMS (HN) (in oxygen) DMS (LN) + 0.2 mg/ml Poly R-478 (in air) DMS (LN) + 2 mM VA (in oxygen) DMS (LN) + I % cellulose (in air) DMS (HN) + I% cellulose (in air) DMS (LN) + I % cellulose (in oxygen) DMS (HN) + I % CMC (in air) DMS (HN) + I % xylan (in air) DMS (HN) + 1% xylan (in oxygen)

13’) (IYY6) 215-221

peak activity (1.7 mU/ml) was detected on the third day. GLOX activity in the same culture was also detected on day 2 and peak activity on day 3 (0.8 mu/ml) (Fig. 1). In Phanerochaete sp., peak activity (0.8 mu/ml) of glucose oxidase was detected on the third day in liquid medium. However, GLOX activity was detected on day 2 and then increased to 3.4 mu/ml on day 5. A similar pattern of enzyme production was followed by both strains when grown on DMS LN agar medium under 0, although the oxidase concentrations were significantly higher than those produced in liquid cultures. The peak activity of glucose oxidase and GLOX in strain BKM-F- 1767 was 75 mu/ml and 33 mu/ml, respectively (measured on day 4). For Phanerochaete sp., peak glucose oxidase activity was 200 mu/ml (measured on day 4) and that of GLOX, 220 mu/ml (measured on day 5). In strain BKM-F-1767, glucose oxidase activity was always higher than the GLOX activity, whereas, in Phanerochaete sp., the GLOX activity was initially lower than glucose oxidase (for the first three days in liquid medium and the first four days in agar medium), but later exceeded the glucose oxidase activity. In both cultures, oxygen concentration was maintained at above 95% for the 24-h period after flushing. In air, however, LN or HN liquid

enzyme activity in strains BKM-F-1767

Maximum HzOz-producing BKMF-

Lrttrrs

enzyme activity (U g-’

I767

and Phanerochaetr

sp. A

of solid media) with different substrates Phanrrochuete

sp.

glucose

methylglyoxal

Xylo\C

cellobiose

glucose

methylglyoxal

xylose

cellobiose

0.054 0.074 0.086 0.106 0.064

(4) h (4) (6) (6) (4)

0.013 0.033 0.019 0.026 0.016

(4) (4) (6) (6) (3)

0.018 0.026 0.020 0.022 0.017

(4) (4) (6) (6) (4)

0.006 (4) NA L NA NA NA

0.095 (4) 0.196(4) 0. I30 (6) 0.210 (6) 0. IO5 (4)

0.026 (4) 0.219(5) 0.065 (6) 0.229 (6) 0.065 (4)

0.023 (4) 0.031 (4) 0.05 1 (6) 0.059 (6) 0.05 I (4)

0.008 (4) NA NA NA NA

0.053 0.030 0.046 0.035 0.007 0.030 0.041

(5) (5) (7) (5) (7) (6) (6)

0.005 0.017 0.044 0.022 0.005 0.017 0.025

(5) (5) (7) (5) (7) (6) (6)

NA 0.026 0.034 0.027 0.004 0.027 0.039

(5) (7) (5) (7) (6) (6)

NA 0.016 (5) 0.025 (7) 0.018 (5) NA NA NA

0.014 0.077 0.089 0.030 0.003 0.046 0.058

0.009 (7) 0.042 (5) 0.136 (7) 0.1 IO (5) 0.01 I (7) 0.175 (6) 0.279 (6)

NA 0.051 0.089 0.009 0.004 0.064 0. I16

NA 0.023 (5) 0.037 (5) 0.015 (5) NA NA NA

a The results are the mean of triplicates and the coefficient of variations in these determinations h Numbers in parentheses indicate the time of maximum activity (in days). I‘ Not assayed.

(7) (5) (5) (5) (7) (6) (6)

was less than 10%.

(5) (5) (5) (7) (6) (6)

J. Zhao, B.J.H. Janse/FEMS

Microbiology Letters 139 (1996) 215-221

media did not produce detectable oxidase, which was consistent with other reports [4,7]. In contrast, high levels of oxidases were produced in solid medium under air (Table 1). A comparison of extracellular protein concentrations of cultures grown in liquid and on solid medium indicated that no significant differences were apparent (data not shown). Therefore, our results indicated that oxidase production on solid medium was much higher than that found in liquid medium of the same composition and is comparable to that usually found in wood extracts [6]. Cultures on agar plates, however, grow much faster than those in wood. High levels of oxidases made it possible to compare different oxidases from the same culture extract. The production of oxidase by BKM-F-1767 and Phanerochuere sp. on different solid media, was determined (Table 1). Four substrates, n-glucose (glucose oxidase activity), methylglyoxal (GLOX activity), D-xylose and cellobiose (CbO activity), were used separately in the enzyme assays and tbe effect of nitrogen level, Poly R-478, veratryl alcohol (VA), cellulose, carboxymethylcellulose (CMC) and xylan, on oxidase production, was determined. In strain BKM-F-1767, cultivated in DMS LN medium under air, peak glucose oxidase activity appeared at day 4 (54 mu/g>. Though the peaks of other oxidases in this strain also appeared at day 4, their activities were lower than that detected for glucose oxidase. The presence of oxygen stimulated glucose oxidase and GLOX production in this strain, as did the presence of high levels of nitrogen (Table 1). The results obtained for strain Phanerochaete sp. were similar to those obtained for strain BKM-F- 1767 in LN and HN medium under air. However, in contrast to strain BKM-F-1767, the increase in GLOX production in both LN and I-IN media in the presence of O,, was more pronounced. Rothschild et al. [15] found no GLOX activity in high nitrogen liquid medium, whereas Highley [16] reported that low concentrations of nitrogen in an agar medium had little effect on H,O,-production in some white rot fungi. Our results indicated that high nitrogen levels stimulated the production of H,O,-producing enzymes in both strains studied. This effect may, however, be due to increased biomass production [ 1,151. It was reported that Poly-B411 increased H,O, production in some wood rot strains [16]. In our

219

study, Poly R-478, a polymeric dye similar in structure to Poly B-4 11, was added to the agar medium at a final concentration of 0.2 g 1-l. Our results (Table 1) showed that Poly R-478 slightly increased the oxidase levels in both strains. The addition of VA (used to stabilise lignin peroxidase) was reported to result in decreased GLOX activity in BKM-F-1767 in liquid medium [17]. Consistent with this finding, our results also show that VA inhibited GLOX activity in both strains, but had no effect on glucose oxidase production in strain BKM-F-1767 (Table 1). When glucose was replaced by cellulose, CbO activity increased in both strains with a concomitant decrease in glucose oxidase activity (Table 1). However, CbO activity was always lower than the glucose ox&se activity. Due to its poor oxygen reducing abilities it has recently been suggested that CbO be renamed as a cellobiose dehydrogenase [ 181. This may explain the low levels of H,O, produced by CbO in the medium with cellulose. The addition of CMC, instead of glucose, resulted in low oxidase activities in both strains and all the enzymes tested, thereby indicating that this substrate (or monomers from it> cannot induce oxidase production (Table 1). When glucose was replaced by xylan, glucose oxidase activity in both strains was repressed (Table 1). The GLOX activity of Phanerochaete sp. was induced in the presence of xylan under air or oxygen. Under the same conditions, GLOX activity of strain BKM-F-1767 was increased. These data indicated that the level of H,O,-producing enzymes in both strains are influenced by the carbon source. Both glucose I-oxidase and glucose 2-oxidase have been reported to be present in P. chrysosporium [3,4]. To distinguish between these two enzymes in the same culture extracts, use can be made of their different substrate specificity [3,4,19]. Glucose 1-oxidase is highly specific for n-glucose and has a low afftnity for D-xylose, an important wood sugar [3]. Glucose 1-oxidase from Aspergillus and Penicillium has been shown to have only 2% of its activity when D-xylose was used as a substrate [3]. In P. chrysosporium glucose 1-oxidase activity in Dxylose was only 13% when compared to o-glucose [18]. In contrast, glucose 2-oxidase exhibits activity against various sugar substrates. Relative activity of glucose 2-ox&se from P. chlysosporium and Peniophoru gigantea, with o-xylose as substrate, was

J. Zhao. B.J.H. Janse/ F&MS Microbiology Letters 139 (I9961 215-221

220

shown to be 37% [4] and 50% [ 191, respectively. In this study, our inability to detect any extracellular activity when using the D-glucose in the o-dianisidine method, together with the relatively high activity in reactions supplemented with xylose, would support the data that glucose 2-oxidase is the predominant glucose oxidase in both strains. The production of oxidases by selected white rot fungi in low and high nitrogen medium under air or oxygen was measured (Table 2). As A. niger has been used to produce glucose oxidase commercially [20], and as this enzyme is also produced by the soft rot strain T. reesei, we assayed the oxidase levels in A. niger ATCC 90196 and 7’. reesei QM 6a. The level of glucose oxidase in both strains was lower than that detected in P. chtysosporium BKM-F- 1767 (data not shown). Two P. chrysosporium isolates from our culture collection, KKPlO and BG5, as well as T. uersicolor R105, showed a similar pattern to BKM-F-1767 regarding glucose oxidase and GLOX production. Interestingly, L. tigrinus 2 showed more GLOX activity than glucose oxidase activity. This is in contrast to the other studied strains and suggested that L. tigrinus 2 had a regulation pattern for oxidase production similar to Phanerochaete sp.. Strains P. radiata 13127 and C. subvermispora 90031 showed low glucose oxidase and Table 2 H?O,-producing

enzymes produced

Strain

Ceriporiopsis subvermispora m-9003 I -sp Merulius sp. Lentinus tigrinus 2 Phanerocharte chtyosporium BG5 P. chtyvsosporium KKP 10 Phlebia radiata MJL-I 198s~ P. radiuta L-13127~sp Pleurotus sajor-caju 27 Trametes cersicolor R-105

GLOX activities under all experimental conditions tested. Furthermore, oxygen inhibited the growth of the former strain, which resulted in poor oxidase production (Table 2). Our results also indicated that glucose oxidase and GLOX enzyme activity in strains Merulius sp. and P. sajor-caju 27 are independent of nitrogen level and oxygen concentration, thereby indicating a different regulatory mechanism when compared to the other strains studied. Some unexpected results came from P. radiata 1198. In LN medium, no significant difference in oxidase levels were detected in air or oxygen. High nitrogen inhibited the production of glucose oxidase and GLOX (Table 2). To our knowledge this is the first report comparing the levels of H,O,-producing enzymes in white rot fungi. Results presented here indicate that although H,O, production is ubiquitous in the white rot fungi studied, great variations exist in the levels of oxidases produced by these fungi in different media and under different incubation conditions. Consistently higher glucose oxidase levels in white rot fungi support the importance of these enzymes in the production of H,O,, although the role of other H,O,-producing enzymes in lignin degradation cannot be excluded. The variations in oxidase regulation by these fungi, indicates that oxidases should be

by selected white rot fungi grown under different conditions

Maximum H .,O,-producing _.

enzyme activity (U g-

DMS(LN) (in air)

DMS(HNJ (in air)

a

’ of solid media) with different substrates DMS(LNJ (in oxygen)

DMS(HN) (in oxygen)

glucose

methylglyoxal

glucose

methylglyoxal

glucose

methylglyoxal

glucose

methylglyoxal

0.006 (6) h

0.003 (6)

0.007 (6)

0.005 (6)

0.006 (6)

0.005 (6)

0.007 (6)

0.005 (6))

0.013 (9) 0.018 (5) 0.053 (5)

0.014 (9) 0.036 03) 0.041 (5)

0.016 (9) 0.020 (5 ) 0.067 (6)

0.014 (9) 0.054 (8) 0.023 (6)

0.015 (9) 0.026 (5) 0.062 (5)

0.011 (9) 0.037 (8) 0.013 (5)

0.017 (9) 0.029 (5) 0.065 (6)

0.016 (9) 0.037 (8) 0.010 (6)

0.059 0.030 0.003 0.007 0.043

0.044 0.020 0.003 0.006 0.026

0.095 0.003 0.002 0.009 0.092

0.046 0.002 0.002 0.008 0.042

0.052 0.032 NA 0.006 0.048

0.017 0.013 NA 0.008 0.037

0. I I7 0.003 NA 0.011 0.012

0.014 0.002 NA 0.010 0.003

(5) (7) (8) (8) (8)

(5) (7) (81 (8) (8)

(6) (7) (8) 03) (8)

(6) (7) 03) (8) (8)

(5) (7) (8) (8)

a The results are the mean of triplicates and the coefficient of variations in these determinations b Numbers in parentheses indicate the time of maximum activity (in days). ’ Not assayed.

(5) (7) (8) (8)

was less than 10%

(6) (7) (8) (8)

(6) (7) (8) 03)

J. Zhao. B.J.H. Janse/

FEMS Microbiology Letters 139 (1996) 215-221

studied simultaneously before any conclusions about the contribution of individual oxidases to lignin degradation are made. The diversity and complexity of the oxidases present in white rot fungi probably contribute to their ability to adapt to the presence of different lignocellulolytic material.

Acknowledgements We thank Florian Bauer for his critical review of the manuscript. We are grateful to D. Cullen, T. Jeffries, and the Forest Products Laboratory for providing us with the following fungal strains: P. chrysosporium BKM-F- 1767, P. rudiatu MJL- 1198sp; P. rudiata L-13127-sp; C. subvermisporu Fp9003 1-sp and T. uersicolor R105. We are also grateful to S.T. Chang for strains P. sujor-cuju 27, and L. tigrinus 2, and to W.H. van Zyl for strains T. reesei QM 6a and A. niger ATCC 90196. This research was supported by Mondi Kraft, a division of Mondi Ltd.

References rt1 Ander, P. f 1994) The cellobiose-oxidizing

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