Cellulase system

Production of Trichoderma reesei Cellulase System with High Hydrofytic Potential by Solid-State Fermentation... 

A literature survey indicated that very little work has been done to produce an optimal cellulase system as described above. Here, we used solid-state fermentation (SSF) to achieve this objective. SSF processes, such as the "koji" process, have been used extensively for amylase production on wheat bran in Japan its application was extended to cellulase production on wheat bran and Ugnocellulosic materials by Toyama (13), Since then, wheat bran has become an important substrate for producing various products by SSF (14-20), In this study, we tested various lignocellulosic substrates for the production of cellulase and )3-glucosidase from T, reesei QMY-1 by SSF. 

Pretreatment of Substrate. Several different lignocelluloses were pretreated with NaOH. This pretreatment partially solubilizes the hemicelluloses and lignin and swells the cellulose so that the organism can utilize it for its growth and for production of a cellulase system in SSF. The treated lignocelluloses were not washed. The NaOH treatment is done with a minimum amount of water so that, after the addition of nutrient solution and inoculum, the moisture content is less than 80% wt/wt and there is no free water in the medium. More water was added to make suspensions of different lignocellulosic substrates of the desired concentration (1% or 5%) for liquid-state (submerged) fermentation (LSF). 

Extraction of the cellulase system. The culture of SSF from each flask (originally 5 g of substrate) was mixed well with more water to bring the final weight of the mixture (mycelium plus unutilized lignin, cellulose, and hemicelluloses) to 100 g. Tween 80 was added at a rate of 0.1%. The mixture was shaken for 0.5 h and centrifuged. The supernatant was used for enzyme determination. We estimated that about 7% to 10% cellulases remained adsorbed on the residues (mycelium and unutilized cellulose, hemicelluloses, and lignin) when the residues were suspended in water and Tween 80 as before and the supernatant was tested for cellulase titer. 

Our first detailed analysis of the cellulase system produced on wheat straw in SSF indicated that the activities of various enzymes were as follows (lU/ml) FP cellulase, 8.6 -glucosidase, 10.6 and xylanase, 190-480. The FP cellulase -glucosidase ratio was 1 1.23 (21), These results encouraged us to compare the composition of the cellulase systems produced in SSF and LSF on different substrates. 

Table V. Composition of Cellulase System Produced on Pro-cell with Trichoderma reesei QMY-1 in SSF...

Comparison of Composition of Cellulase Systems of Various Fungi Produced in SSF... 

Hydrolytic Potential of Cellulase Systems Produced with Different Substrates... 

Hydrolysis of delignified wheat straw up to 90% was obtained in 96 hours with the cellulase system produced in SSF with wheat straw. It took only 72 hours to obtain over 90% hydrolysis of wheat straw with cellulase system produced with SSF on Pro-cell (Table VII). It is interesting to note that the quantity of cellobiose in the hydrolysate obtained with the cellulase system produced on Pro-cell was higher than that of the cellulase system produced on wheat straw. This is consistent with the observation that cellulase system produced on Pro-cell had a lower ratio of FP cellulase )3-glucosidase (1 0.77) as compared to that produced on wheat straw (1 1.2). However, this cellulase system had a faster hydrolysis rate it took only 72 hours to obtain over 90% hydrolysis. This might be related to cotton hydrolyzing activity of this cellulase system (Table V). 

Cellulase system from washed steam-exploded wood (LSF, Fed-batch) 1 1.48 —- — — 40.00 36.00 ... 

R. flavefaciens cells, during growth in pure culture, released cellulase, endoglucanase, and xylanase into the culture fluid. This microorganism hydrolyzed cellulose to yield only cellobiose as a product 49). It had been reported that a ceUobiose phosphorylase and glucokinase were present in R flavefaciens (8). The Ruminococcus cellulase system was repressed by disaccharides such as cellobiose, sucrose, and lactose 50). 

As mentioned earlier, Cx enzymes exist in multiple forms in most cellulase systems. As a rule they vary in the degree of randomness of their attack on CM-cellulose, and they may be distinguished by plotting... 

Component enzymes of the cellulase system have been purified from several microbial species (1-13), among which mutants of the imperfect fungus Trichoderma provide the highest levels of extracellular enzyme activity (14). From this organism have been purified / -glucosi-dases (EC 3.2.1.21), endo-l,4-/ -D-glucanases (EC 3.2.1.4) and 1,4-/ -d-... 

Figure 1. Cooperative action of enzymes of the cellulase system in hydrolytically converting cellulose to glucose...

Since we had been able to purify the enzymes of the cellulase system of T. viride, the purification and identification of the T. reesei system was attempted. It was of interest to compare the enzymes produced during growth on cellulose with those produced in response to sophorose. The... 

The stimulation of the synthesis of the cellulase system of T. reesei QM 9414 by sophorose was established as shown by the results of experiments summarized in Table IV. Other than sophorose, of the glycosides and oligosaccharides tested, only lactose caused even a limited production of the enzymes of the cellulase system. Lactose is not as closely related structurally to sophorose as is, for example, the disaccharide laminaribiose it is more closely related structurally to cellobiose, which, despite being the major product of cellulose breakdown, does not promote enzyme production under the conditions of this experiment. It was noted that both intra- and extracellular constitutive enzyme levels produced by cells growing on glucose (or by resting cells without inducer, Table IV) are less than 0.5% of the fully induced levels and thus are negligible. 

Table IV. Effect of Several Carbohydrates on the Production of the Enzymes of the Cellulase System by T. reesei...

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