Cellulase production

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. 

When wheat straw was fermented in LSF, the FP cellulase level reached 6 lU/ml (300 lU/g cellulose or 120 lU/g substrate) (Table I) by day 11, decreasing thereafter. This showed that SSF was better than LSF for cellulase production when using wheat straw. 

Table I. Cellulase Production on Wheat Straw with Trichoderma reesd QMY-1...

A maximum FP cellulase of 6.3 lU/ml (191 lU/g cellulose or 126 lU/g substrate) was obtained on NaOH-treated CTMP after 20 days in SSF. On untreated CTMP, the FP cellulase remained about 5 lU/ml from 20 to 26 days of fermentation and then increased to 7.2 at 30 days of fermentation (Table III). This indicated that CTMP was a good substrate for cellulase production in SSF even without the mild NaOH treatment. 

Bacteria represent a promising source for the production of industrial enzymes. Bacterial cellulases are an especialfy interesting case in point. Many thermophilic bacterial species produce cellulases that are stable and active at high temperature, resistant to proteolytic attack, and stable to mechanical and chemical denaturation. However, cellulase productivities in bacteria are notoriously low compared to other microbial sources. In this paper bacterial enzyme production systems will be discussed with a focus on comparisons of the productivities of known bacterial cellulase producers. In an attempt to draw conclusions concerning the regulation of cellulase synthesis in bacterial systems, a tentative model for regulation in Acidothennus cellulofyticus has been developed. 

RunUnococcus albus and Ruminococcus flavefadens. These bacteria are important cellulose-degraders found in the rumen of cattle and sheep (2). Most isolated strains ferment cellulose and xylan and all ferment cellobiose. Fermentation of glucose and some other carbohydrates depends on the particular strain. R flavefadens and B. succinogenes can ferment the highly ordered crystalline cellulosic su trates but R albus cannot. No evidence has been found for extracellular cellulase production by R albus, but Ohmiya et al. purified cellobiosidase from this culture 17). Laboratory growth of R albus has been conducted at pH 7.0 and 37 C. 

Langsford et al. reported that Cellulomonas fimi culture supernatants contained cellulase and proteinase activities, for which there appeared to be a relationship. Glucose repressed the synthesis of both activities and cellulose induced both 60), Adding cellulose to Cellulomonas sp. (NRCC 2406) cultures stimulated growth and improved production of cellulases 61). Optimum conditions for growth and cellulase production were pH 6.5 and 30 C. The addition of glucose in the presence of cellulose inhibited growth. Several species of Cellulomonas have cellobiose phosphorylase. 

Much effort has been expended over the years on increasing enzyme production of T. reesei by isolation of high yielding mutants and optimizing media and fermentation conditions. Strains have been isolated that produce 2-6 times the cellulase productivity of the parent wild strain (QM 6a) in batch culture. The mutants produce higher levels of cellulase protein but the specie activity of the enzymes and the proportions of the individual components (ca. 30% endo- -glucanase, 70% o- -glucanase, and less 1% cellobiase) are similar to those of the parent. 

Table II. Cellulase Production by Some Mesophilic and Thermophilic Microorganisms...

Table HI. Comparison of Cellulase Production Parameters Obtained Using Various Substance Addition with Parameters Obtained Using Solka Floe Only...

The fourth group of effectors are compounds that inhibit cellulase production and/or cell growth. They are L-arabinose, D-mannose, lactose, and IPTGal. 

Acidothermus cellulolyticus cellulase activity, 334,335/,341,342/ cellulase production parameters, 341,343r cellulase synthesis regulation, model development, 341-346 concentration vs. rate of cellulase synthesis, 344,345/... 

Figure 7. Pilot plant process for cellulase production. Adapted from Ref. 20.

Tuerker and Mavituna immobilized Trichoderma reesei within the open porous networks of reticulated polyurethane foam matrices. Growth pattern, glucose consumption, and cellulase production were compared with those of freely suspended cells. The method of immobilization was simple and had no detrimental effect on cell activity. Hundreds of similar projects could be cited. Not all rated the use of polymethane as the preferred technique. If a statistical analysis were conducted on aU the immobilization literature, we are sure that no single technique would be dominant. However, the combination of ease of immobilization, cost of materials, flow-through properties, control of flux rate through the immobilizing membrane, high surface-to-volume ratio, and other factors make polymethane a viable substratum for the continuous production of proteins. 

The two compounds which stimulate cellulase production, sophorose and lactose, obviously do not promote amylase production. This is especially evident in the case of sophorose, since the reduction in amylase... 

It had been reported that T. reesei secreted protease into culture media during cellulase production (21). We have found that in young culture filtrates of T. reesei, the protease activity is very low, but that... 

T. reesei, a saprophytic fungus, is capable of utilizing a variety of carbohydrates. Yet, only a few carbohydrates induce cellulase production. Inducers include cellulose, cellulose derivatives, lactose, and sophorose (31,32). Mandels and Reese (31,32) studied the inducibility of various sugars and found that sophorose is an excellent cellulase inducer in T. reesei while having little effect in other fungi or bacteria. On further examination they found that trace amounts of sophorose present in glucose caused the apparent ability of glucose to be a cellulase inducer in T. reesei. 

One possible explanation is that the mere contact of the cell surface on the insoluble inducer is sufficient to trigger cellulase production. Another explanation is that cellololytic microorganisms have trace quantities of constitutive cellulases which are continuously released. A third possibility is that the cells are able to synthesize cellulases under starvation conditions. 

Preliminary experiments done in our laboratory showed that antibodies specific for cellobiohydrolase failed to cross react with either purified cellobiase, purified endoglucanase, or crude endoglucanase. These results, together with data reported in the literature, which show that endoglucanase and cellobiohydrolase have different physical structures, indicate that the three cellulases could be transcribed and translated by different genomes. In this context, then, the question arises as to whether cellulase production is regulated by a common regulatory circuit or by different circuits. 

Figure 3. Cellulase production in shake flasks by Rut-C30 and QM 9414 under repressed and nonrepressed conditions. Antibiotic disk as substrate (43). ( — ) Rut-C30,1% cellulose, (O—O) Rut-C30,1% cellulose, 5%...

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