Characterization of xylanase production by a local isolate of Penicillium janthinellum

Characterization of xylanase production by a local isolate of Penicillium janthinellum Adriane M. F. Milagres, Lynda S. Lacis and Rolf Alexander Prade Center f o r Bioteehnology and Chemistry, Foundation ]'or Industrial Technology, Lorena, Brazil

A strain ofP. janthinellum was isolated Iocallyfi'om plant materialJound in a termite colony. Comparison with T. reesei revealed high xylanolytic activity. This activity was inducible by xylan, sugar cane bagasse, or xylose. Induction by xylan was repressed by the presence of xylose or glucose. A temperature of 40°C and a pH of 5.5 were optimal conditions for xylanase activity (98 units ml-I). Unlike man), filamentous fungi, only very low or no endocelhdolytic activio, could be detected in the presence qf various potential inducers, including xylan and celhdose.

Keywords:Xylanase; sugar cane bagasse; P. janthinelhm7

Introduction E n z y m e s involved in lignocellulosic degradation in sita continue to be of interest for their potential application to processes which utilize lignocellulosic substrates. 1-3 Xylanases and the microorganisms which produce them could potentially be applied to the production of hydrolysate f r o m agro-industrial wastes,~'4 to nutritional i m p r o v e m e n t of lignocellulosic feeds, 5-7 and to the processing of food, 8•9 agrofiber, 9'1° and pulp. ~1-15 In the case of agrofiber or pulp processing, e n z y m e preparations with low cellulase activity are desirable. 4'~6 Production of xylanase from a xylanase gene cloned into a noncellulolytic host is one means of achieving cellulase-free preparations. To date, however, e n z y m e activities a p p e a r to be lower than those resulting from fungal cultures, 4'lva~ and r e c o v e r y from bacterial cells presents more of a p r o b l e m in d o w n s t r e a m processing, 19,2° especially if the e n z y m e is not secreted, as is often the case. Is'2~ Thus bacterial and fungal systems should both continue to be explored further until one, if either, d e m o n s t r a t e s a clear advantage. 22

Address reprint requests to Dr. Milagres at the Center for Biotechnology and Chemistry, Foundation for Industrial Technology, Lorena, SP, 12600, Brazil The current address of Lynda S. Lacis is the Department of Genetics, University of Melbourne, Parkville 3052, Australia The current address of Rolf Alexander Prade is the Department of Genetics, University of Georgia, Athens, Georgia 30602 Received 19 March 1992; revised 19 July 1992

248

Enzyme Microb. Technol., 1993, vol. 15, March

Local microbial isolates were screened for high xylanase activity. One isolate, identified as P. janthinellum and described below, produced high xylanase activities, yet low cellulase activities, under various conditions. Although this has not been previously reported for this species, it is not unique a m o n g fungi in this regard, since M. albomyees showed the same characteristic in n o n h y d r o l y z e cellulose. 4° P. janthinellum shows one of the highest xylanase/cellulase ratios of fungi that have been studied. 2~

Materials and methods Media The basic medium contained glucose (20 g 1 n), yeast extract (2.5 g 1 n), and a salts solution (2% v/v). 24 The salts solution contained (per liter): trisodium citrate, 125 g; KH2PO 4 • 5H20, 250 g; N H 4 N O 3 , 100 g; MgSO4 • 7H20, 100 g; CaC12 • 2H20, 50 g; citric acid, 0.25 g; ZnSO 4 - 7H20, 0.25 g; Fe(NH4)2SO 4 • 5H20, 50 mg; CuSO 4 • 5H20, 15 rag; MnSO4 • 2H20, 2 rag; Na2MoO 2 • 2H20, 2 rag. The complete medium contained yeast extract (l g 1 i), peptone (1 g 1- ~), glucose (0.5 g 1-1), and agar (20 g 1 1). I s o l a t i o n a n d initial s c r e e n i n g o f f u n g i Samples of (1) plant material found in termite colonies (source of FM isolates), (2) termites (FC isolates), (3) ants (FF isolates), or (4) sugar cane bagasse were macerated and suspended in distilled water• Suspensions

cc; 1993

Butterworth-Heinemann

Penicillium janthinellum xylanase: A. M. F. Milagres et al. were inoculated on agar plates containing complete medium, supplemented with either xylan or carboxymethylcellulose (CMC) (10 g 1-~). After a 7-day incubation at 30°C, colonies that showed a clearing of a radius of at least 1 cm were isolated on agar plates. The glycanolytic activities produced by these isolates in liquid medium were assessed after inoculating spores into 125-ml Erlenmeyer flasks containing 25 ml of complete medium supplemented with 10 g 1 1 of glucose, xylose, xylan, or CMC, and incubating flasks for 96 h on a rotary shaker (150 rev rain ~) at 30°C.

Preparation of spore inocula Conidial spores were harvested from 7- to 10-day-old agar slants by suspending spores in water and filtering through gauze into Erlenmeyer flasks.

Determination of the effect of various carbon substrates upon xylanase production Spores were inoculated into 125-ml flasks containing 25-ml volumes of basic medium at a concentration of 10 7 m1-1. Flasks were incubated for 48 h at 30°C in a rotary shaker at 150 rev min- J. Spent culture medium was removed by suction. The remaining mycelial pellets were washed twice with water and resuspended in medium containing carbon-free basic medium supplemented with various carbon substrates (10 g I-~). Xylanase activities were measured 24 h later. The effect of xylose and glucose upon induction by xylan was determined by incubating flasks for 32 h after the point of medium replacement with xylan (10 g 1-J) and then adding glucose or xylose (10 g 1-1). Xylanase activities were determined over the next 24 or 48 h.

Measurement of enzyme activities Xylanase activities were routinely determined by incubating 0.5 ml of diluted culture filtrate with 0.5 ml of a xylan suspension (10 g 1-1) in 0.1 M phosphate buffer (pH 6.0) for 15 min at 30°C. The released reducing equivalents were measured by a colorimetric assay based on that of Miller, 25 using a xylose solution as a standard reference. Units of activity were expressed as micromoles of reducing equivalents released per minute. Avicelase, FPase, CMCase, and amylase activities were determined in an analogous manner, except that a 60-min incubation period and a glucose standard reference were used.

Characterization of xylanase activity The optimum temperature for activity was determined as above, except that the assay was carried out at 20, 25, 30, 35, 40, 50, 60, or 70°C. Temperature stability was assessed by preincubating enzyme solutions for 4 h at various temperatures prior to the addition of substrate and the determination of activity at 40°C. The optimum pH for xylanase activity was determined as in the standard assay, except that the buffer was in either 0.1 M phosphate buffer (pH 6.5-8.0) or 0.1 M

phthalate buffer (pH 4.0-6.0). pH stability was determined by preincubating the enzyme solution in one of the above buffers for 4 h prior to the addition of substrate and the determination of activity.

Results

Initial screen of isolates In situ samples of organic matter were diluted and inoculated on agar media containing xylan or CMC. After a 7-day incubation at 30°C, 30 putative fungal isolates had produced clear zones on plates. The isolates were then screened by measuring glycanolytic activities in liquid culture. The four most xylanolytic isolates showed xylanase activities comparable to that of T. reesei QM9414 (Table 1), which is regarded as a high producer of hemicellulolytic enzymes. 16.26 Two of these isolates, FC-4 and FC-6, were eliminated from further study because they, like the T. reesei strains, also produced significant CMCase activities. Of the two remaining isolates, FM-5 was of greater interest because it produced high xylanase levels when grown on either xylose or xylan. It thus differed from isolate FF-5, which produced significant xylanase activity only when grown on xylan. The production of xylanase from xylose-containing media by FM-5 was most likely not simply a response to carbon starvation following exhaustion of the xylose, because xylanase activity was also not produced from the glucose-containing medium, which, as the biomass yields suggest, would have been similarly exhausted of carbohydrate. Morphological characteristics of isolate FM-5 included the production of blue-green spore-forming colonies after 3 days of growth on basic medium agar. The spores appeared bright and spherical under a light microscope, and their inoculation into liquid medium in Erlenmeyer flasks, and agitation on a rotary shaker for 2 days, produced relatively homogeneous, smooth, spherical, compact pellets. Agriculture Canada identified the fungus as Penicillium janthinellum, and the cultures were deposited both there and at the University of Pernambuco, Brazil. P. janthinellum has been isolated previously in Japan and Indiafl 7-29 The present result is the first example of high xylanase/cellulase ratios for this species. Only high cellulolytic activities have been reported previously. 3°

Characterization of extracellular glycanolytic activities It was also of interest to test the ability of various polysaccharides to induce glycanolytic activities in a manner independent of their ability to support growth. Thus, after allowing growth in glucose-containing media for 48 h, spent growth medium was replaced with fresh media containing various substrates, and the mycelia were incubated for a further 24 h before enzyme activities were measured. These conditions were chosen to maximize xylan-induced xylanase activity from glucose-grown mycelia. 31By the end of the 48-h growth

Enzyme Microb. Technol., 1993, vol. 15, March

249

Papers Table 1

Selected fungal isolates from a screen of glycanolytic activities in various growth media Glycanolytic activities in filtrate (#mol min 1 ml 1)

Dry

Carbon source

Strain FM-5

-

Glucose CMC Xylose Xylan FF-5

FC-4

T, r e e s e i

QM9414

T. r e e s e i

CMCase

Avicelase

0.9 10.4 0.9 9.8 6.2

0.1 0.4 0.0 21.4 44.1

0.00 0.00 0.00 0.00 0.04

0.01 0.00 0.00 0.10 0.11

-

1.0

9.9 0.9 10.0 7.1

0.0 0.0 0.0 0.4 40.4

0.00 0.00 0.00 0.03 0.01

0.01 0.02 0.12 0.0t 0.02

Glucose CMC Xylose Xylan

0.6 5.2 0.6 6.0 2.6

0.1 0.1 0.4 0.1 44.9

0.00 0.00 0.01 0.02 0.36

0.01 0.00 0.01 0.00 0.00

Glucose CMC Xylose Xylan

0.5 6.0 0.6 7.0 3.6

0.1 0,2 0.2 0.5 44.9

0.00 0.00 0.00 0.00 0.15

0.02 0.00 0.22 0.02 0.03

-

0.4 5.4 1.0 5.3 2.0

0.0 0.0 2.2 0.5 41.8

0.00 0.00 0.16 0.07 0.66

0.08 0.01 0.17 0.03 0.14

0.5 7.5 1.2 8.5 2.9

0.0 0.3 1.0 3.4 89.3

0.00 0.00 0.31 0.16 0.90

0,01 0.02 0.21 0.01 0.04

Glucose CMC Xylose Xylan -

RUT C-30

Xylanase

Glucose CMC Xylose Xylan -

FC-6

weight (g I ~1)

Glucose CMC Xylose Xylan

Cultures were g r o w n for 96 h with the indicated carbohydrate provided at a concentration of 10 g I FM, FF and FC designate the source of isolate, as described in Materials and methods

phase, most of the original 20 g l t glucose had been catabolized, yielding 10 g 1 ~ of mycelia (dry weight). As in the initial screening results, (1) xylan was the tested substrate most capable of inducing xylanolytic activities, and (2) little or no cellulolytic activity was detectable (Table 2). Also of interest was the ability of sugar cane bagasse to induce high xylanase activities. This was attributed primarily to the hemicellulose portion of the substrate, since cellulosic substrates generally did not induce high xylanase activities. The exception to this was filter paper, which induced significant xylanolytic activity but which might have contained some hemicellulose components. 32

Screening of various carbon substrates for induction or repression More extensive screening of potential xylanase inducers showed that the abilities of arabinose, lactose, and sucrose to induce xylanase activity were similar to that of xylose, and that xylan was the best inducer tested (Table 3). As in Table 1, the activity apparently

250

Enzyme Microb. Technol., 1993, vol. 15, March

induced by xylose does not appear to be a response to carbon starvation, because the sugar-free control showed insignificant levels of xylanase. All tested sources of carbon, when included with xylan in media, were capable of reducing the level of xylanase activity induced by xylan alone. This likely reflected a delay in the induction by xylan until the alternative carbon source was exhausted, since the addition of glucose or xylose to cultures already producing xylanase inhibited its further net production (Fi~,ure 1). The enzyme itself was presumably not inhibited by the addition of sugars, because up to 1.5% xylose had no effect upon assayed xylanase activity. 3~ Furthermore, inhibition was clearly reversible in the case of the glucose additional trial, and likely corresponded to exhaustion of glucose from the medium.

Characterization of xylanase activity The optimal pH and temperature for xylanase activity were determined to be pH 5.5 and 40°C (Figure 2), and

Penicillium janthinellum xylanase: A. M. F. Milagres et al. Table 2

Glycanolytic activities induced by various complex carbohydrates Glycanolytic activities in culture filtrate (/xmol min -1 ml -~)

Carbon source

Xylanase

CMCase

Avicelase

FPase

Amylase

0.6 98.5 3.4 2.1 11.1 0.4 38.2

0.04 0.03 0.00 0.00 0.00 0.00 0.00

0.12 0.03 0.03 0.05 0.18 0.03 0.08

0.07 0.17 0.02 0.01 0.01 0.01 0.04

0.10 0.11 0.01 0.01 0,02 0.12 0.04

None Xylan CMC Avicel Filter paper Starch Bagasse

Pregrown mycelia were incubated in media containing the indicated carbon source for 24 h. Carbon sources included Sigma oat spelts xylan, fluka CMC, avicel, and filter paper; and local mill-run sugar cane bagasse, 325 mesh

Table 3 Induction and/or repression of xylanase activities by various carbon sources Filtrate xylanase activities (/xmol rain -1 ml 1) Carbon source None Xylan Glucose Xylose Arabinose Fructose Cellobiose Lactose Sucrose Sorbitol Xylitol Glycerol Glutamic acid Casamino acids

C source only

C source + glucose

Xylan + C source

0.1 58.2 0.2 4.5 5.8 0.3 0.0 3.6 3.0 0.1 0.1 0.1 0.1 0.2

0.2 19.6 0.9 0.2 0.1 0.2 0.1 0.9 1.4 0.2 0.0 0.0 0.0

58.2 19.6 14.1 12.9 16.4 4.7 10.0 7.2 18.5 16.7 12.3 16.7 8.2

All carbon sources as well as glucose and xylan were provided in a concentration of 10 g 1-1

the optima for stability were determined to be pH 6 and 30-40°C (Figure 3).

Effect o f xylan or xylose concentration The optimum concentration of xylan or xylose for inducing xylanase production by mycelia previously grown on glucose was 10 g 1-1. Concentrations both lower and higher than this value resulted in lower enzyme activities (Figure 4).

Discussion The results described an indigenous Penicillium species of potential application to xylanolytic processes. That the fungus already produces xylanase activities at high levels was indicated by its production of levels which were half or the same as that produced by two Trichoderma reesei strains known for their high extra-

-!3o zo

i ,° --I

~-

20

60

20

60

X

TIME (HOURS) Figure 1 Effect of xylose or glucose addition upon the accumulation of xylanase activity in culture filtrates. After 32 h, glucose ( t ) or xylose (A) was added to a concentration of 10 g 1-1 to some flasks, and no addition was made to control flasks (©). Incubation and subsequent sampling of all flasks was continued until 60 or 80 h

cellular glycanolytic activities. Even higher levels of productivity might yet be achieved through mutation and selection programs, which have yielded hypercellulolytic strains of Trichoderma reesei 33and hyperxylanolytic strains of Aspergillus niger. 26 The pH and temperature characteristics of the P. janthinellum xylanase were typical of xylanases isolated from filamentous mesophilic fungi. ] The xylanase would therefore likely be best suited for operations carried out in close to ambient temperatures. Other xylanases have been described that might be preferred for higher-temperature operations. 34-37 The strain is attractive for agrofiber and pulp-based processes because of its low cellulolytic activities. Unlike some other fungal systems, 23,32,35,38detectable levels of CMCase activity could not be induced by typical inducing compounds such as CMC, filter paper, or AviEnzyme Microb. Technol., 1993, vol. 15, March

251

Papers cel. Furthermore, whether in the presence ofa hemicellulosic substrate or of a cellulosic substrate, the activity ratios of xylanase to FPase or of xylanase to endocellulase are high when considered against a recent compilation of literature values. 23 Since celhlolytic activity appears to be at least partly under separate control from xylanase activity, further optimization of this ratio might be possible. The maximum achievable xylanase to endocellulase ratio may or may not be sufficient to avoid significant deterioration of paper-making properties. However, if such were the case, further purification of the xylanase to a point where degradation by celhlase became negligible might be achievable. This has already been demonstrated with a T. h a r z i a n u m xylanase.14'39 The initial investigation of the possibility of using locally available substrates for enzyme production was promising in that sugar cane bagasse induction yielded

"T.. li

i )"J X

I

10

XYLAN

ZO

OR XYLOSE ( g . l - l l

Figure 4 Effect of xylan (©) or xylose (e) concentration upon xylanase activity measured in culture filtrates after 24 h

| Io

I

a; q

4.0

.J X

6,0 pH

>.

&O TEMPERATURE ("C)

Figure 2 Determination of temperature and pH optima for xylanase activity. Details given in the text

!,o 6O

it 4.0

6.0 pH

' , 2.0 4.0 6,0 TEMPERATURE ('C)

Figure 3 Determination of temperature and pH optima for xylanase stability. Culture filtrate samples were incubated under the indicated conditions for 4 h prior to activity measurement

252

Enzyme Microb. Technol., 1993, vol. 15, March

xylanase to a level greater than one-third that induced by xylan alone. Hemicellulose hydrolysate of sugar cane bagasse, which would be available as a product of bagasse pretreatment, might also be a suitable substrate for xylanase production. From the results of F i g u r e 1, one could anticipate that there could be an initial utilization of xylose and other low-molecular-weight sugars for cell biomass production and then a subsequent induction by higher-molecular-weight otigomers for xylanase production. Of note is the observation by Senior et al. 23 that 0.5% xylan and 0.5% hemicellulose hydrolysate resulted in higher xylanase activity than did 1.0% xylan alone, possibly because hydrolysate was a better growth substrate than xylan. Also promising here was the observation with xylose as the sole carbon source ( T a b l e s 1 and 3). Such induction in a fungal system is not a unique observation, 22'3638'4° but it is not always the case for xylan-inducible xylanases. That the concentration of xylan or xylose yielding the maximum xylanase activities was 10 g 1-1 is of some concern, since the use of more concentrated hydrolysates would sometimes be desirable. Senior et al. 23 observed an analogous maximum at 2.5 g 1 1 with T. harzianurn, even though extracellular protein concentrations continued to increase linearly with xylan concentrations up to the maximum tested concentration of 30 g 1-1. They suggested that inhibition of xylanase production beyond concentrations of 2.5 g I i might result from catabolite repression by hydrolysis products or monosaccharide/disaccharide impurities in the substrate. This being the case, some improvement might be expected through a mutation and selection program, since the selection of mutants resistant to carbon catabolite repression has been

Penicillium janthinellum xylanase: A. M. F. Milagres et al. achieved in other fungal systems, including the T. reesei cellulase system) 3

References 1

2 3 4 5 6 7 8 9 10 11 12 13 14 15

16

17

Eriksson, K.-E. L., Blanchetter, R. A. and Ander P. Microbial and Enzymatic Degradation of Wood and Wood Components. Springer-Verlag, New York, 1990 Enzyme Systems for Lignocellulose Degradation (Coughlan, M. P., ed.) Elsevier Applied Science, New York, 1989 Parisi, F. Adv. Biochem. Eng./Biotechnol. 1989, 38, 53-87 Wong, K. K. Y., Tan, L. U. L. and Saddler, J. N. Microbiol. Rev. 1988, 52, 305-317 Giovanozzi-Sermanni, G., Bertoni, G. and Porri, A. in Enzyme Systems for Lignocellulose Degradation (Coughlan, M. P. ed.) Elsevier Applied Science, New York, 1989, pp. 371-382 Jorgensen, O. B. and Cowan, D. in Enzyme Systems for Lignocellulose Degradation (Coughlan, M. P., ed.) Elsevier Applied Science, New York, 1989, pp. 347-356 Van der Meer, J. M. and Ketelaar, R. in Enzyme Systems for Lignocellulose Degradation (Cougblan, M. P., ed.) Elsevier Applied Science, New York, 1989, pp. 383-396 Woodward, J. in Topics in Enzyme and Fermentation Biotechnology (Wiseman, A., ed.) John Wiley and Sons, Rexdale, Canada, pp. 9-30 Sharma, H. S. S. Appl. Microbiol. Biotechnol. 1987, 26, 358-362 Biely, P. Trends Biotechnol. 1985, 3, 286-290 Noe, P. J. Wood Chem. Technol. 1986, 6, 167-184 Roberts, J. C., McCarthy, A. J., Flynn, N. J. and Broda, P. Enzyme Microb. Technol. 1990, 12, 210-213 Jurasek, L. and Paice, M. G. Biomass 1988, 15, 103-108 Senior, D. J., Mayers, P. R., Miller, D., Sutcliffe, R., Tan, L. U. L. and Saddler, J. N. Biotechnol. Lett. 1988, 10,907-912 Kantelinen, A., Viikari, L., Linko, M., Ratto, M., Ranva, M. and Sundquist, J. in 1988 Int. Pulp Bleaching Congress, Tappi Proceedings, pp. 1-9 Linko, M., Poutanen, K. and Viikari, L. in Enzyme Systems forLignocellulose Degradation (Coughlan, M. P., ed.) Elsevier Applied Science, New York, pp. 331-346 Paice, M. G., Bernier, R. and Jurasek, L. Biotechnol. Bioeng. 1988, 32, 235-239

18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

34 35 36 37 38 39 40

Luthi, E., Jasmat, N. B. and Bergquist, P. L. Appl. Environ. Microbiol. 1990, 56, 2677-2683 Whittington, PN. Appl. Biochem. Biotechnol. 1990, 23, 91-121 Bajpai, P., Gera, R. K. and Bajpai, P. K. J. Ferment. Bioeng. 1991, 71, 284-285 Yang, R. C. A., Mackenzie, C. R., Bilous, D. and Narang, S. A. Appl. Environ. Microbiol. 1989, 55, 568-572 Leathers, T. D., Detroy, R. W. and Bothast, R. J. Biotechnol. Lett. 1986, 8, 867-872 Senior, D. J., Mayers, P. R. and Saddler, J. L. Appl. Microb. Biotechnol. 1989, 32, 137-142 Vogel, H. J. Microb. Gen. Bull. 1956, 13, 42 Miller, G. L. Anal. Chem. 1959, 31, 426 Biswas, S. R., Jana, S. C., Mishra, A. K. and Nanda, D. Biotechnol. Bioeng. 1990, 35, 244 Takenishi, S. and Tsujisaka, Y. J. Ferment. Technol. 1973, 51, 458-468 Srinivasan, M. C., Rama Rao, R., Deshmukh, S. S. and Sahasrabudhe, N. A. Enzyme Microb. Technol. 1983, 5, 269-272 Mandal, S. K. and Chatterjee, S. P. Folia Microbiol. 1986, 31, 15-18 Rao, M., Mishra, C., Gaikward, S. and Srinivasan, M. C. Biotechnol. Bioeng. 1986, 28, 129-132 Milagres, A. M. F. M.Sc. thesis 1988, University of Viqosa, Viqosa, Brazil Hrmova, M., Biely, P. and Vrsanska, M. Arch. Microbiol. 1986, 144, 307-311 Montenecourt, B. S., Nhlapo, S. D., Trimino-Vazquez, H., Cuskey, S., Schamhart, D. H. J. and Eveleigh, D. E. in Trends in the Biology of Fermentation for Fuels and Chemicals (Hollaender, A., ed.) Plenum Press, New York, 1981, pp. 33-35 Anand, L., Krishnamurthy, S. and Vithayathil, P. J. Arch. Biochem. Biophys. 1990, 276, 546-553 Yu, E. K. C., Tan, L. U. L., Chan, K.-H., Deschatelets, L. and Saddler, J. N. Enzyme Microb. Technol. 1987, 9, 16-24 Mountfort, D. O. and Asher, R. A. Appl. Environ. Microbiol. 1989, 55, 1016 Ganju, R. K., Vithayathil, P. J. and Murthy, S. K. Can. J. Microbiol. 1989, 35, 836-842 Hrmova, M., Biely, P. and Vrsanska, M. Enzyme Microb. Technol. 1989, 11, 610-616 Tan, L. U. L., Yu, E. K. C., Louis-Seize, G. W. and Saddler, J. N. Biotechnol. Bioeng. 1987, 30, 96-100 Maheshwari, R. and Kamalam, P. T. J. Gen. Microbiol. 1985, 131, 3017

Enzyme Microb. Technol., 1993, vol. 15, March

253