Cellulase cultural conditions

Wheat straw. Wheat straw ground to 20 mesh was treated with 2% NaOH solution (wt/vol) in 1 2 (solidiliquid) ratio at 121 C for 0.5 h (i.e., 4 g NaOH/100 g wheat straw). Trichoderma reesei QMY-1 was grown on pretreated wheat straw in SSF as well as in LSF under otherwise identical culture conditions. The SSF was carried out with full nutrient concentrations in one set and with one-half nutrient concentrations in the other set to evaluate the possible deleterious effects of elevated osmotic pressure. T reesei QMY-1 produced FP cellulase of 8.6 lU/ml (430 lU/g cellulose or 172 lU/g substrate) in 22 days. This showed that the organism was able to tolerate the high salt concentrations required in the SSF. In contrast, when the nutrients were supplied in one-half concentration, FP cellulase activity dropped to 6.7 lU/ml (335 lU/g cellulose or 134 lU/g substrate). However, the maximum enzyme activity was obtained one week earlier (14 days) than that obtained with full salt concentrations (Table I). 

Both the quantity and properties of cellulases produced by microorganisms depend on the culture conditions. Commonly, cellulases are produced by culture of the organism either (a) in a liquid medium, which may be stationary, shaken, or submerged with aeration, or (b) by a Koji process on a solid substrate such as wheat bran (7). The complexity of the crude cellulosic carbon source usually leads to the production of a mixture of hydrolytic enzymes which may include amylases, proteases, chitinases, etc., in addition to the cellulases. Separation of proteins from culture filtrates by high resolution techniques such as chromatography, electrophoresis, or electrofocusing often reveals a number of enzyme species which may differ in specificity toward cellulosic substrates. These forms may represent ... 

For the last few years, we have investigated the cellulase components of Pseudomonas fluorescens var. cellulosa in special regard to the physiological relation of their synthesis and localization to the cultural conditions. This pseudomonad is an aerobic mesophilic cellulose-decomposing bacterium isolated earlier by Ueda et al. (39) from field soil. Some of the enzymatic properties of cellulases obtained from it have previously been reported (28). In the present review, the results of our recent studies (42) are described and discussed together with the related works of other authors. [In these studies, activities of cellulase and aryl / -glucosidase were assayed by the same methods as described in a recent paper (31), and that of amylase by the blue value method modified by Fuwa (10)]. 

Moreover, it is worthwhile to note that cellulase components A and B always occurred in pairs in all fractions of the cell under the cultural conditions to cause an exo-type synthesis of cellulase. The component C was, on the contrary, produced under the cultural conditions to cause both exo- and endo-type syntheses. These facts suggest not only the possible relationship between the components A and B with regard to their formation, but also the possible formation of the component C being independent of the other two. 

Ahamed A, Vermette P. (2008a). Culture-based strategies to enhance cellulase enzyme production from Trichoderma reesei RUT-C30 in bioreactor culture conditions. Biochem Eng J, 40, 399 07. 

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. 

BSame culture filtrate of cellulase T applied under identical conditions 14 days later. 

In batch culture, Eq. (10) determines the rate of growth. For soluble substrates, during most of the culture, S> and therefore u = Wmax From Fig. 4, there is little protein produced at this rapid growth condition. Batch culture of readily-metabolized substrates is therefore not used to make cellulase with Trichoderma. 

Electrophoretic properties of typical cellulase preparations, an extracellular cellulase from a culture on 0.5% cellulose and a cell-bound cellulase from that on 0.5% cellobiose, were compared in respect to their behavior in zone electrophoresis on cellulose acetate film. As shown in Figure 2, the former was separated into two components, A (fast moving to the cathode) and B (almost no moving). With the latter, a single component was detected under the same conditions. This fast moving component was in approximate agreement with component A in regard to its mobility, but as will be mentioned later, there was considerable difference in substrate specificity and other properties. Therefore, it seems to be a different component, and is referred to as component C. 

Since the inhibition of cellulase synthesis in 0.5% cellobiose culture seems to be caused by so-called catabolite repression as reported for the formation of many other hydrolases, the same results would be expected for other metabolizable sugars, which will show an enhanced cellulase synthesis depending on the supply conditions. Most of the sugars tested... 

Figure 8. Activity—PS curves of cellulase II for cellotriose, cellotetraose ana cellopentaose (13). Cellulase II C2 component obtained from a culture filtrate of Irpex lacteus by starch-zone electrophoresis. Conditions Plastic tray 2 X 5 X 45 cm., veronal buffer, pH 8.7, p 0.1, 24 hr., 5.6 V/cm., 2.0-3.0 mA./cm.2...

Trembling aspen wood (Populus tremuloides Michx.) was reduced to a powder in a Hurricane Pulverizer. Samples were prepared from extracted pulverized wood by ball-milling for 10 days in a rotating ceramic mill equipped with an air-conditioning unit which prevented the temperature of the contents of the mill from rising above ambient. Digestion of the samples with cellulase was done with a mixture of Schizophyllum commune and Trichoderma reesei culture filtrates at 30°C for 3 days. 

Table 1. Concentrations of individual cellulases in the culture fluid upon cultivation of T. reesei QM 9414 under different conditions ...

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