BIOTECHNOLOGY TECHNIQUES Volme 7 No.5 (May 1993) pp.345-350 Received as revised 19th March

USE OF SORBOSE TO ENHANCE CELLOBIASE ACTIVITY IN A Trichodenna reesei CELLULASE SYSTEM PRODUCED ON WHEAT HYDROLYSATE

Shu Chen* and Morris Wayman Department of Chemical Engineering and Applied Chemistry University of Toronto, Toronto, Ontario, Canada M5S lA4

SUMMARY

Several methods were investigated for increasing cellobiase activity of T. reesei cellulases produced using an acid-hydrolyzed wheat flour. The use of sorbose in cellulase production is preferred to mixed cultures with Aspergillus niger. Using 13.5 g/L wheat hydrolysate and 1.0 g/L sorbose, the culture of T. reesei produced 3.72 IU/mL of filter paper activity (FPA) and 0.53 IUimL of cellobiase activity. The addition of sorbose resulted in 89% increase in cellobiase activity and 47% increase in FPA.

INTRODUCTION

Effective hydrolysis of crystalline cellulose to glucose requires the synergistic action of at least three classes of cellulolytic enzymes, endoglucanase, exoglucanase (cellobiohydrolase) and P-glucosidase (cellobiase). Mutants of Trichodermu reesei are

the best known hyper-producers of cellulases. However, the cellulase preparations from this fungus are usually deficient in j3-glucosidase, resulting in cellobiose accumulation which in turn inhibits the cellobiohydrolase and endoglucanase enzymes, and thereby reduces the rate and degree of enzymatic hydrolysis of cellulose (Ryu and Mandels, 1980; Enari and Niku-Paavola, 1987). The present “routine” approach to solve this problem is to add j3-glucosidase (Sternberg et al., 1977; Stockton et al., 1991) produced separately. However, a separate process to produce p-glucosidase results in an increase in enzyme production costs. The development of a method to increase P-glucosidase activity in a T. reesei cellulase system would make the

production of fermentable sugars from lignocellulosics a more likely economical

process.

The /3-glucosidases from T. reesei do not appear to be sufficiently well characterized and are commonly considered to be highly active on both aryl P-D-

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glucoside and cellobiose. The name most frequently seen in the literature is p- glucosidase which is assayed using p-nitrophenyl-P-D-glucoside as the substrate. However, Esterbauer et al. (1991) have found that their Trichudema cellulase preparations are much more active on aryl-8-D-glucoside than on cellobiose. We have found that some of our T. reesei cellulase preparations are more active on cellobiose than on aryl-P-D-glucoside. Therefore, it is reasonable to accept that there are two distinct types of P-glucosidase: one is highly active mainly on aryl-B-D-glucoside while the other possesses high activity mainly towards cellobiose. The enzyme discussed in this paper is referred to as cellobiase which catalyses the hydrolysis of

cellobiose to glucose.

The objective of this research is to provide a method of production of cellulase with sufficient cellobiase that addition of supplementary cellobiase is not necessary for efficient saccharification and fermentation. This paper describes a comparative study of different methods to increase cellobiase activity of T. reesei cellulases produced using a wheat-derived carbon source.

MATERIALS AND METHODS

Microorganisms: Trichodema reesei Rut-C30 and Aspergillus niger were ATCC 56765 and ATCC 6275 respectively; another strain of Aspergillus niger was provided by Dr. B. Davis (Sheffield City Polytechnic). The fungi were maintained on potato dextrose agar (Difco) slants at 4°C and subcultured every month.

Celluhe production: The procedures used for inoculum preparation and cellulase production were as described previously (Chen and Wayman, 1991). The carbon source used for cellulase production was 13.5 g/L (based on total reducing sugars) of acid hydrolysed whole soft wheat flour (ASW6) which was prepared by hydrolyzing a wheat flour slurry at a concentration of 25 % dry solids with 0.6 M HCl in an autoclave at 121°C for 2 h (Wayman and Chen, 1992). The initial pH value for cellulase production was 5.5 and was not adjusted during fermentation; it was controlled by 0.1 M KH2P0, in the culture medium. All the fermentations were carried out in 250 mL Erlenmeyer flasks containing 55 mL medium for 6 days at 28”C, 150 rpm. At the termination of cellulase production, the pH value of the fermentation medium was adjusted to 4.8 with 10% HCl or 10% NaOH solution. The cellulase solution was separated from the mycelium by filtration using Gooch crucibles with medium porosity, and used for saccharification or analysis.

Saccharification: The cellulase preparations from 6-day cultures were evaluated by their 48 h saccharification performance on shredded waste office paper (approximately 0.3 x 2.0 cm) in a 10% suspension as described previously (Chen and Wayman, 1991).

Analytical techniques: Cellulase filter paper activity (IU/mL) and cellobiase activity (IU/mL) were determined by the methods of the International Union of Pure

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and Applied Chemistry (IUPAC) (Ghose, 1987). Protein in cellulase preparations was precipitated by 10% trichloroacetic acid (TCA) solution and quantified by the calorimetric method of Lowry. Analysis of the office paper was conducted according to the method of Wayman and Parekh (1990). Monosaccharides and disaccharides were analyzed by HPLC (Sugar Analyser 1, Waters) with an Aminex HPX-87P column using water at 65°C as an eluent, refractive index detector and 16 g/L n- propanol as the internal standard. Mycelial dry weights were measured by filtering 30 mL samples through Gooch crucibles with medium porosity, washing with three volumes of distilled water, and drying in a vacuum oven at 80°C for 24 h to constant weight (Allen and Roche, 1989).

RESULTS AND DISCUSSION

From our previous study, it was found that acid hydrolyzed whole wheat flour was a very effective substrate for the production of T. reesei cellulases and that the cellulases so produced contained approximately 0.1 IU of cellobiase activity for each IU of filter paper activity (Wayman and Chen, 1992). The cellulases were effective in saccharifying waste office paper, but the saccharification efficiency could be further increased if the cellobiase activity were higher. In order to increase cellobiase activity

of T. reesei cellulases induced by the wheat hydrolysate, different methods were investigated in this study with the results as shown in Table 1 and Figure 1. The control means that cellulase production was carried out using the wheat hydrolysate by T. reesei or A. niger under the standard conditions used in this research without additional additives or treatments.

The use of chemical additives during cellulase production may be a convenient method to enhance cellobiase activity if a right chemical can be found. Two chemicals, sorbose and amygdalin, were tested in this study. In our previous study, it was found that sorbose greatly enhanced cellulase production when it was applied to a newspaper-based cellulase production process but did not enhance cellulase production significantly when applied to a starch-based cellulase production process (Chen and Wayman, 1991 and 1992). This means that the effect of sorbose on cellulase production is carbon source dependent. In our study, it was also found that the time of adding sorbose was important and that it was appropriate to add sorbose at the fourth day of cellulase production. In this study where cellulase production was based on an acid- hydrolyzed wheat, the addition of sorbose to the cellulase production medium after 96 hours of cultivation resulted in substantial increase in cellulase and cellobiase production. As can be seen in Table 1, using 13.5 g/L wheat hydrolysate and 1 g/L sorbose, the culture of T. reesei produced 3.72 IU/mL of filter paper activity and 0.53 IU/mL of cellobiase activity. The specific activity of the cellulases was 1.0 IU for each mg of protein, higher than the common value of 0.6-0.7 IU/mg (Ryu and

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Mandels, 1980). The saccharification efficiency of the culture filtrate against office

paper in a 10% suspension was 83% of the theoretical value. When the culture filtrate was diluted to 50% of its original strength and then used for saccharification, more than 62% saccharification efficiency was obtained. These values represent about a 47% increase in FPA, 89% increase in cellobiase activity and 23 % or 62% increase in specific activity in terms of filter paper activity or cellobiase activity respectively compared with using wheat hydrolysate alone.

Table 1: Evaluation of different methods for increasing cellobiase

Method Cell Protein FPA Cello- SE* mass biase WQ WI (lU/mL) (lU/mL) 60)

Control 1 (T. reesei ) Control 2 (A. niger ATCC 6275) Control 3 (A. niger, Sheffield) Addition of 1.0 g/L sorbose (T. reesei) Addition of 2.0 g/L amyg- dalin (T. reesei) Cell wall break (T. reesei) Mixed culture 1 (T. reesei + A. niger ATCC 6275) Mixed culture 2 (T. reesei + A. niger, Sheffield)

5.56 3.10 2.52 0.28 78.0 9.75 0.25 nd 0.22 6.6

nd 0.05

4.40 3.62 3.72 0.53 83.0

5.72 1.0 nd 0.09 48.1

nd 7.40

3.59 nd 0.33 nd 1.29 1.75 0.46 69.1

nd 0.92 1.18 0.47 59.6

0.02 0.03 6.6

Note: SE*: saccharification efficiency; nd: not determined.

Amygdalin was reported to be an inducer of P-glucosidase production by fungi of the Aspergillus genus (Stemberg et al., 1977) and was integrated with the medium for j3-glucosidase production by A. phoenicis (Duff et al., 1985). It was expected that amygdalin might have similar enhancement effect on cellobiase production by T. reesei. However, it turned out to be a strong inhibitor of cellulase and cellobiase production by T. reesei in this study.

P-Glucosidase is mainly cell wall bound, and its release to fermentation media results from lytic enzymes during conidiogenesis and from cell lysis (Acebal et al., 1988; Jackson and Talburt, 1988). It was found that the cellulase preparation without filtration was more effective than that with filtration (Wayman et al., 1992). To

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release possibly cell wall-bound cellobiase, the mycelia of T. reesei cultivated on the wheat hydrolysate after 6 days were broken mechanically at O°C by a sonicator and a

homogenizer. As expected more cellobiase activity was released from the cells. However, the increase in cellobiase activity due to cell wall breakage was only 18%, indicating that the majority of the cellobiase was released into the medium at the end of cultivation.

-II-

O-f= Cant1 Cont2 Cont3 Amyg. Mi: Mix2 Sorb* Sorb**

Es3 Glucose

m Cellobiose

Xylose

Total sugars

Methods to increase cellobiase

Figure 1: Saccharification performance of the cellulases produced with different methods to increase cellobiase (Table 1). Sorb”, sorbose 1giL added during cellulase production; sorb”“, filtrate of sorb* diluted with water 1:l.

Duff et al. (1985) showed that cellulase produced through a mixed culture of

T. rewei and A. phoenicis, a /3-glucosidase producer, had increased P-glucosidase activity and improved hydrolysis potential. The two strains of A. niger used in this study, especially the one from Sheffield City Polytechnic (Davis, 1990), were expected to be good cellobiase producers. Cellulases produced by mixed cultures in this study exhibited a significant increase in celiobiase activity but a dramatic decrease in FPA or the saccharification ability of the culture filtrate. This could be due to the cultural conditions which were set up for T. reesei and were not suitable for the mixed culture fermentation. According to cell concentrations at the end of the cellulase production, the conditions used in this study favoured the growth of A. niger rather

than its enzyme production. More research is required to find out the optimal

conditions for the mixed culture fermentation.

According to the above results, the use of sorbose in cellulase production on acid hydrolyzed wheat flour to enhance cellobiase activity as well as overall cellulase

activity in the cellulase system is a preferred method. A further increase in cellulase activity may be possible by breaking the cell wall of T. reesei cultivated on wheat hydrolysate and sorbose or by using the whole cellulase production broth for saccharification. Saccharification would be improved by simultaneous saccharification and fermentation, and addition of supplementary cellobiase is not necessary when

sorbose is added during cellulase production, as in this study.

ACKNOWLEDGMENT

Financial support by the Natural Sciences and Engineering Research Council (NSERC) of Canada and the Ontario Ministry of the Environment is gratefully acknowledged.

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