Agricultural Wastes 18 (1986) 283-288

Production of Hemicellulytic Enzymes by Fungi

Abdel-Mohsen S. Ismail, Mohamed A. Abdel-Naby & Ahmed F. Abdel-Fattah

Microbiology Research Group, Laboratory of Natural Products, National Research Centre, Dokki, Cairo, Egypt

(Received 17 December 1985; accepted 9 April 1986)

A B S T R A C T

Production of extracellular hemicellulases by ten fungal isolates under different cultural conditions, using wheat straw hemicellulose B as the sole carbon source in the culture medium, was investigated. Aspergillus terreus 603 in 7-day-old surface culture was the most potent producer. Hemicellulose concentration of lOg per litre of culture medium was the most effective for production of hemicellulase. An initial pH value of the culture medium of 6.0 gave the highest enzyme activity. Sodium nitrate was the most suitable nitrogen source, with 0"1% dipotassium hydrogen phosphate added to the medium.

IN T RO D U CT ION

The bioconversion of plant polysaccharides to fermentable sugars by microbial enzymes has gained importance in recent years. Xylans represent about a third of the total carbohydrate content of plant biomass (Timell, 1967). Hydrolysis of hemicellulose residues with acids leads to the formation of undesirable products. On the other hand, hemicellulases can hydrolyse specific hemicelluloses to their monomer sugars, which can be converted by various microorganisms to products of high economic value (Rosenberg, 1980). Emphasis has therefore been recently focused on the search for microbial hemicellulases suitable for saccharifying hemicelluloses (Yasuda & Shimabukuro, 1980; Gosh et al., 1980; Ogushi et al., 1980; Frederick e t

283 Agricultural Wastes 0141-4607/86/$03-50 © Elsevier Applied Science Publishers Ltd, England, 1986. Printed in Great Britain

284 Abdel-Mohsen S. lsmail, Mohamed A. Abdel-Naby, Ahmed F. Abdel-Fattah

al., 1981; Wankhede et al., 1981; Imazato, 1982; Comtat, 1983; Chung et al., 1983; Oreshchenko et al., 1983).

The present paper reports on the production of hemicellulytic enzymes by local fungi to determine the suitability of the fungal cultures as hemicellulase producers.

METHODS

Micro-organisms

The fungi were obtained from the fungal collection of the Centre of Cultures of the National Research Centre, Cairo, Egypt, and from the United States Department of Agriculture, Research Service, Northern Regional Research Center, Peoria, Illinois 61604, USA (NRRC).

Culture media

Medium 1 contained (g per litre): wheat straw hemicellulose, 15; sodium nitrate, 2; K2HPO4, 1; MgSO 4 . 7H20, 0.5; KC1, 0-5; FeSO 4 . 7H20, 0.01; distilled water. Media 2, 3, 4, 5 and 6 had the same composition as medium 1 but sodium nitrate was replaced by the following equivalent-nitrogen sources: ammonium sulphate, 1.55g; ammonium phosphate, 1.16g; soybean meal, 6.97 g; corn steep powder, 6.32 g; milk whey liquor containing 0.329g Kjeldahl nitrogen. These media were adjusted to pH 7.0 before autoclaving.

Wheat straw, rice straw and onion skins

Samples of wheat straw, rice straw and pigmented onion skins were analysed for hemicellulose and cellulose. These samples were collected from the market in the summer of 1980. Before use, they were freed from foreign substances and milled. Determination of cellulose and hemicellulose was by the method adopted by Mansour (1963). Wheat straw hemicellulose B was prepared according to the method reported by C h e n & Anderson (1980). Acid hydrolysis of wheat straw hemicellulose was according to the method of Fischer & D6rfel (1955).

Cultivation

Cultures were grown on Dox's agar plates incubated at 30°C for 7 days. Liquid cultures were grown in 250-ml Erlenmeyer flasks, each containing

Fungal hemicellulase 285

50 ml of sterile medium. Two discs, each of 1 cm diameter, were cut from the 7-day-old culture plates for inoculating each flask. The culture flasks were incubated at 30°C using the surface and shaken culture techniques. The culture medium from each flask was filtered to separate the mycelium from the filtrate. The latter was then centrifuged in a refrigerated centrifuge. The clear culture filtrate was dialysed against 0.05M acetate buffer, pH 4-99.

Protein determination

The protein content of the enzyme preparation was determined by a micro- Kjeldahl method and by the method of Lowry et al. (1951).

Assay for cellulytic activity

This was done by measuring the decrease in the viscosity of buffered sodium carboxymethylcellulose. The enzyme solution was added to 1.0% Na-CMC solution and 0"2M acetate buffer, pH 4.99 (1:3:1, by volume) and the reaction mixture was incubated at 30°C for 15 min. Thereafter, 5 ml of the reaction mixture were pipetted into an Ostwald viscometer in a water bath at 30°C and the time of run was recorded. In control tests, heated enzyme solutions were employed.

Assay for hemicellulytic activity

The reaction mixture consisted of 0.1% wheat straw hemicellulose suspen- sion (2.5ml), 0.05u acetate buffer, pH4.99 (2.5ml) and 1.0ml enzyme solution and the mixture was incubated at 45°C. After 20 minutes incubation, the pentoses released due to the hemicellulytic activity were determined by the method reported by Neish (1952) and based on those described by Nelson (1944) and Somogyi (1945). A heated enzyme solution was used as control. One enzyme unit was defined as the amount of enzyme that released 1.0/~g xylose from hemicellulose per minute under assay conditions.

RESULTS AND DISCUSSION

Hemicellulose and cellulose comprised 33-1, 41-1; 29.3, 51.1 and 24.2, 47.2% of wheat straw, rice straw and pigmented onion skins, respectively. Wheat straw was therefore selected for the preparation of hemicellulose. The hemicellulose B isolated from wheat straw contained (%): xylose,

286 Abdel-Mohsen S. Ismail, Mohamed A. Abdel-Naby, Ahmed F. Abdel-Fattah

76"93; arabinose, 13.39; glucose, 1.9; uronic acid, 6-18; ash, 0.927 (analysed according to the method of Fischer & D6rfel, 1955).

Ten fungal isolates were investigated for their abilities to produce extracellular hemicellulases during different incubation periods, using wheat straw hemicellulose B as the sole carbon source in the culture medium. No correlation seemed to exist between the mycelial growth, the protein level of the culture filtrate and the extracellular hemicellulase activity. With most of the fungal cultures investigated, hemicellulase activity was better in surface than shaken cultures (Table 1).

T A B L E 1 Hemicellulase Activity (Unit per mg of protein) of the Filtrates of the Fungal Cultures in

Medium I a

Micro-organism Type of culture and incubation period (days)

Sur[aee Shaken

4 7 I0 4 7 10

Aspergillusfumigatus 449 49.40 54.80 22.60 36"10 26 .30 16"50 A. fumigatus 545 12.30 12.50 11.60 00-00 4.60 0"74 A. niger NRRL 559 18.15 74.91 69-35 6'24 68 .23 64"88 A. terreus 603 70'87 76 .02 44"83 58"61 64 '70 27"83 A. terreus 627 14'25 26.21 28' 10 14'00 19"70 25" 11 Penicilliumfuniculosum 460 20'70 20.78 64'21 4'86 9'35 13.47 Phoma sp. 552 10"03 9.43 14.60 14.60 13-48 14'84 Triehoderma viride 250 9"01 39"60 38"60 21.37 40 .94 32'20 T. viride 253 24'26 29 .70 29-26 19'95 23 '97 22.12 T. viride 264 51"51 58 .14 48"00 49.10 45.94 41-60

a See 'Methods'.

All the culture filtrates of the fungal cultures showed slight carboxy- methylcellulase activity. Of the ten fungal isolates, Asperg i l t u s terreus 603 in surface culture produced active hemicellulase associated with feeble carboxymethylcellulase activity. That fungal isolate was, therefore, used in the succeeding experiments.

Some cultural conditions affecting the productivity of hemicellulase by Asperg i l lu s terreus 603, in surface culture for 7 days, were investigated. Of the nitrogen sources tested (see 'Methods') , sodium nitrate gave the highest specific hemicellulase activity (Table 2). With other media the specific activities lay between 50-5 and 63.8 units.

In medium 1, hemicellulase activity declined as hemicellulose concen- tration increased. The maximum and minimum values are shown in Table

Fungal hemicellulase 287

TABLE 2 Representative Results Showing the Effects of Some Cultural Conditions on the Production

of Hemicellulase by Aspergillus terreus 603 in Medium 1 a

p14 of culture filtrate

Dry Protein content of weight of myeelium M y c e H u m Culture

(g per 50 ml (rag per 50 ml filtrate of medium) of medium) (rag per ml)

Specific hemi-

cellulase activity

( Unit per mg of protein)

Sodium nitrate: (2 g per litre) 7-50 0.570 182.0 0.420 86.90

Hemicellulose (g per litre):

10 7"20 0-522 188"0 0.380 82.32 30 7"50 1"012 342"0 0.815 21.36

Initial pH of medium:

3.0 3.80 0.449 107.30 0-379 24-80 6.0 6.80 0.784 290.00 0.413 87-47

K2HPO 4 (%): 0.05 7-98 0.591 181.00 0,399. 84.20 0.10 7.90 0'612 188-04 0.400 86.83 0.20 7.19 0-530 161 "30 0.440 70.70

a See 'Methods'. Surface culture.

2. The higher concentrat ions of hemicellulose were used in the product ion of mycelium and proteins other than hemicellulase.

An initial pH value of the culture medium of 6.0 gave the highest hemicellulase activity (Table 2). Initial pH values below 6.0 had an adverse effect on enzyme productivity. However, Richards & Shambe (1976) reported that initial pH values had little influence on the final yield of Cephalosporium sacchari. The productivity of the fungal hemicellulase was highest with addition of 0" 1% dipotassium hydrogen phosphate (Table 2). The decline in enzyme activity was more pronounced at higher than at lower phosphate levels.

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Chung, K. C., Kawai, K. & Eguchi, Y. (1983). Production of polysaccharide decomposing enzymes from Eucalyptus leaves by Trichoderma viride. Hakko Kogaku Kaishi, 61, 85.

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Comtat, J. (1983). Isolation, properties and POstulated role of some of the xylanases from basidiomycete Sporotrichum dimorphosporum. Carbohyd. Res., 118, 215.

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