Substrates cellulase

For T. viride cellulase substrate system a temperature of 50 °C. and a pH of 4.5-5.5 are reported (3) to be optimal. Over this pH range, loss of enzyme activity may be caused by adsorption on adsorbents such as Fullers earth or on substrates. Enzyme alone does not appear to lose activity at the temperature and pH of the reaction (50°C., pH 4.8-5.0) mixture. Rates of hydrolysis increase with enzyme and substrate con-... 

Cellulase enzyme complexes consist of three major types of proteins that synergistically catalyze the breakdown of a cellulosic substrate. Because the enzymes are strictly substrate-specific in their action, any change in the structure or accessibility of the substrate can have a considerable influence on the course of the hydrolysis reaction. A pretreatment method based on exposing cellulosic substrate to phosphoric acid solution [9] and addition of the nonionic... 

Dry bean curd refuse was used as the substrate in the lactic acid fermentation with simultaneous saccharification (SSF). The dry bean curd refuse was preliminarily sieved under a mesh size of 250 II m. It contained 12.3% water, 4.0% ash, 0.8% lipid, 29.3% protein, 53.6% carbohydrate, respectively, in weight basis. The cellulase derived from Aspergilltis niger with an enzymatic activity of 25,000 units/g (Tokyo Kasei Industry Inc.) was employed as the saccharification enzyme. 

Table 1 shows the dry weight of substrate, and amounts of HCl aqueous solution for pretreatment, cellulase and suspension broth for the lactic acid fermentation with ESS. The initially supplied amount of bean curd refuse in dry weight basis was changed from 10 to 150 g to examine the influence of substrate loading. The amount of cellulase was increased against initial substrate loading. And also, the amoimt of 0.1 mol/1 HCl was increased against... 

They may produce extracellular enzymes, which attack the substrate without the need for transport into the cell, for example, cellulase, DNAse, or gelatinase. 

In the treatment of cellulose pulps one essential criterion for a suitable enzyme preparation is that its cellulase activity should be as low as possible, or preferably absent completely. As even extremely low cellulase activities may ruin pulp quality, Trichoderma enzyme preparations are unlikely to be suitable for these applications. Many bacterial and fungal enzymes with low cellulase activity have been shown to be suitable for treatment of pulps 14, 15, 16,17), Regulation of the often synchronous production of cellulolytic and hemicellulolytic enzymes in micro-organisms is not well understood, and is further complicated by substrate cross-specificity of these enzymes. Enzymes with both endoglucanase and xylanase activity have been reported for bacteria 18, 19) and fungi 20, 21, 22), In addition to selection of strain and... 

Figure 1. Enzyme activities in crude extracts made from cultures of different ages detected with substrates selected for cellulases, hemicellulases, amylases, and pectinases. Cultures were grown at 22 C. Black bars indicate activity for 105-day-old cultures given a 12-day-long cold shock at 5 C starting on day 90. (Reproduced with permission from ref. 8. Copyright 1985 American Society for Microbiology.)...

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. 

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). 

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. 

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. 

Fermentation time (days) FP cellulase (lU/ml) FP cellulase (lU/g cellulose) FP cellulase (lU/g substrate)... 

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. 

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