Australasian Biotechnology (backfiles)
AusBiotech
ISSN: 1036-7128
1998
Australasian Biotechnology, Vol.8, No. 6, December 1998

Saccharification of Paper Materials by Mixtures of Cellulase from Penicillium Funiculosum and Aspergillus Niger

Jacobus P.H. van Wyk and Alfred M. Mogale, Department of Chemistry and Biochemistry, Medical University of Southern Africa, South Africa

Code Number:AU98043

Paper materials were treated with cellulase from Penicillium funiculosum and Aspergillus niger to hydrolyze there cellulose component and mixtures of these enzymes were applied to increase the extent of degradation. Cellulases were prepared at equal concentrations and a ratio mixture of 1 : 1 resulted in the strongest increase of saccharification relative to the individual action of both enzymes on all substrates. Office paper resulted in the highest increase of saccharification at this enzyme combination relative to the action of P. funiculosum cellulase on it. P. funiculosum cellulase showed the strongest hydrolysis on all substrates except with office paper that exhibited a higher suscep-tibility for cellulase from A. niger.

Introduction

The recycling of waste is topical not only to conserve the environment but it could also be applied to develop alternative and renewable energy resources. Used paper materials constitute a major component of organic based waste dumped annually. The polymer cellulose is a major structural component of paper materials with its monomeric glucose units linked by means of b-1,4-glycosidic bonds. Cellulose is susceptible for acid- (Ullal et al, 1984) and cellulase catalyzed (Van Wyk and Botha, 1997) hydrolysis producing reducing sugars such as glucose, but its association with hemi-cellulose and lignin results in a relative high resistance toward these ways of saccharification. The hydrolysis of cellulose is a complex process and attempts to improve it, receive currently attention (Baker et al, 1998). Cellulases are mostly manifested in fungi (Persson et al, 1991) and bacteria (Wood et al, 1986) and mixtures of cellulases from P. funiculosum and A. niger were used to increase degradation of cellulose present in foolscap paper, filter paper, news paper, office paper and micro-crystalline cellulose.

Materials and Methods

Cellulose Materials

Foolscap paper, filter paper (Whatman no 1), newspaper, office paper were prepared as pieces of 0.5 cm x 0.5 cm and 20 mg used during an incubation as well as powdered microcrystalline cellulose (MCC) from Merck.

Enzymes and Incubations

Commercial cellulases from P. funiculosum and A. niger were obtained from Sigma and prepared separately at a concentration 0.7 mg/ml in 0.05 M Na-citrate buffer pH 4.0. All cellulose materials were incubated with both enzymes individually and at ratios of P. funiculosum : A. niger, 1:3, 1:1, 3:1. Cellulose materials were mixed with 1 ml of the enzyme solutions and incubated for 2h at 550C followed by determination of the total reducing sugars produced. All incubations were performed in duplicate.

Determination of total reducing sugars and protein concentration

Reducing sugars were determined with the DNS method (Miller et al, 1960) using glucose as standard. For protein determinations BSA (fraction V, Sigma) was used as a standard (Lowry et al, 1951).

Results and Discussion

Figure 1 reflects the saccharification of all paper materials and MCC by cellulases from P. funiculosum and A. niger as well as mixtures thereof. Foolscap paper showed the strongest susceptible for hydrolysis by cellulase from P. funiculosum followed by filter paper, MCC and newspaper. Office paper exhibited the highest resistance towards degradation by this enzyme whilst it showed a relative high susceptibility toward degradation by cellulase from A. niger. Foolscap paper also exhibited the highest susceptibility towards hydrolysis by cellulase from A. niger. Cellulase from P. funiculosum exhibited however a higher activity on all cellulose materials except with office paper as substrate.

Cellulase is an enzyme consisting of different components (Lee and Fan, 1980) responsible for different steps during hydrolysis of cellulose that also contains structural sections with varying suscep-tibilities for enzymatic catalyzed hydrolysis (Fan et al, 1980). Cellulases from different sources exhibit different compositions and a lack of specific components could be supplemented from other sources making the mixing of cellulases an important procedure for effective hydrolysis of cellulose materials.

    Figure 1Formation of total sugars during saccharification of paper materials and microcrystalline cellolose by individual action and mixtures of celluloses from P.funiculosum and A. niger.

To increase the degree of saccharification both cellulases were mixed and incubated with all cellulosic materials (Figure 1). An equal mixture (1:1) resulted in the strongest increase in saccharification with all substrates. Similar tendencies with cellulases from P. funiculosum and Trichoderma reesei on these cellulose materials were observed (Van Wyk, 1998). The increase in total reducing sugar production at optimum cellulase ratio relative to individual cellulase action is reflected in Table 1. The highest increase in saccharification was observed with office paper, relative to the action of cellulase from P. funiculosum (1640 % increase) on it and with newspaper relative to the action A. niger cellulase (871% increase).

Due to the lost in structural qualities used paper materials can be recycled only a number of times before dumping thereof becomes obvious. It is thus important to research the paper component of organic based waste as a potential resource of bioenergy.

Table 1. Increase (%) of total reducing sugar production from paper materials and micro-crystalline cellulose at the optimum ratio mixture of cellulase from P. funiculosum and A. niger relative to the individual cellulase action.

Substrate Increase (%) in total reducing sugar production relative to individual cellulase action
Penicillium funiculosum Aspergillus niger
Foolscap paper 9 70
Filter paper 12 66
Office paper 1640 30
News paper 126 871
CMC 43 320

References

Baker, J.O., Ehrman, C.I., Adney, W.S., Thomas, S.R. & Himmel, M.E. (1998) Hydrolysis of cellulose using ternary mixtures of purified cellulases. Appl. Biochem. Biotechnol. 70, 395-403.

Fan, L.T., Lee, Y. & Gharpuray, M.M. (1980) The nature of lignocellulosics and their pretreatments for enzymatic hydrolysis. Adv. Biochem. Eng. 17, 156_187.

Lee, Y. & Fan, L.T. (1980) Properties and mode of action of cellulases. Adv. Biochem. Eng. 17, 101_130.

Lowry, O.H., Rosebrough, N.J., Farr, N.J. & Randall, R.J. (1951) Protein measurements with the folin phenol reagent. J. Biol. Chem. 193, 265-215.

Miller,G.L., Blum, R., Glennon. W.E. & Burton, A.L. (1960) Measurements of carboxymethylcellulase activity. Anal./ Biochem. 2, 127-132.

Persson, I, Tjernld, F. & Hegerdal, B. (1991) Fungal cellulolytic enzyme production: A review. Process Biochem. 26, 65-74.

Ullal, V.G., Matharasan, R, & Grossmann, E.D. (1984) New insights into high solids acid hydrolysis of biomass. Bioeng. Symp. 14, 69-93.

Van Wyk, J.P.H. (1998) Saccharification of paper products by cellulase from Penicillium funiculosum and Trichoderma reesei. Biomass & Bioenergy, In press.

Van Wyk, J.P.H. & Botha, A.C. (1997) Hydrolysis of cellulose materials during successive treatment with cellulase from Penicillium funiculosum. Biotechnol. Lett. 19(7), 687-689.

Wood, T.M., Wilson, C.A., McCrae, S.I. & Joblin, K.N. (1986) A highly active extracellular cellulase from the anaerobic rumen fungus Neocallimastix frontalis. FEMS Microbiol Lett. 34, 37-40.

Copyright 1998 Australian Biotechnology Association Ltd.


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