Cellulases concentration

Cellulase and all chemicals used in this work were obtained from Sigma. Hydrolysis experiments were conducted by adding a fixed amount of 2 x 2 mm oflSce paper to flasks containing cellulase in 0.05 M acetate buffer (pH = 4.8). The flasks were placed in an incubator-shaker maintained at 50 °C and 100 rpm. A Box-Behnken design was used to assess the influence of four factors on the extent of sugar production. The four factors examined were (i) reaction time (h), (ii) enzyme to paper mass ratio (%), (iii) amount of surfactant added (Tween 80, g/L), and (iv) paper pretreatment condition (phosphoric add concentration, g/L), as shown in Table 1. Each factor is coded according to the equation... 

Fig. 1 Time couise of lactic acid yield and its concentration in SSF with or without pretreatment using 0.1 mol/l FICI with heating at 121 1 for 30 min. Famentation conditions 3TC, pH=5.0, initial load of bean curd refiise(BCR) 10 g, cellulase amount=l gin ILsuspension.

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). 

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). 

Wheat straw. FP cellulase per unit volume in LSF was very low at 1% wheat straw. It doubled when the wheat straw concentration was raised to 5%. But the FP cellulase per unit weight of wheat straw produced at 5% concentration was reduced to almost one-half that obtained on 1%. Similar observations were made with the )3-glucosidase and xylanase activities (Table IV). The low productivity of all the enzymes could be the result of an O2 transfer problem in the thick fermentation medium in the LSF. 

The most important advantage of SSF was that the ratio of FP cellulase )3-glucosidase obtained was 1 0.8, which is very close to the recommended ratio of 1 1 (10-12), On the other hand, a very low FP cellulase )3-glucosidase ratio (1 0.26 and 1 0.32) was obtained in LSF whether the wheat straw concentration was 1% or 5%. 

Since protein adsorption to an anion exchange resin is reversible and does not constitute a classical immobilization, the ability of the resins to retain activity under various conditions must be determined. Macrosorb KAX DEAE bound -D-glucosidase was tested with solutions of primary interest for their final application. Several batch washes of a 1% w/v slurry were required to ensure complete equilibrium elution for a given concentration, as determined from the absence of pNPG units in subsequent washes. Several salt solutions of typical fermentation media components were tested. These included 3 mM to 50 mM solutions of MgSO, KHgPO, NaQ, and sodium acetate. Also, incubations with cellulase solutions were tested to determine if the proteins present in a cellulose hydrolysis would displace the -D-glucosidase. Both of these displacement studies were carried out at 22°C and 40 C. 

The classification of cellulase sequences into domains allows the further recognition of distinct sequences that characterize each family. To make these sequences evident, one must use multiple sequence alignments of the members of each individual family. We have concentrated on the Microhispora bispora endoglucanase, and present some initial multiple sequence alignments of the catalytic and binding domain families of which it is a member. 

Figure 2. Time course of cellulase activity accumulation in A. cellulofyticus cultures containing 15 g/L Solka Floe and various concentrations of glucose-l-phosphate.

---