Purification of cellulase

The latest summary of separation and purification of cellulases and related enzymes is written by Hashimoto and Nisizawa 17). Their list... 

Here we describe the purification of cellulases from S. cellulophilum, the existence of multi-enzyme components and their relationship. 

Fig. 1. Purification of cellulase and 3-glucosidase by Toyo-pearl TSK-HW55 column chromatography. The active fractions in D A Sephadex A-50 chromatography were charged after concentration. 31> 32, CII, cm, CIV and CV indicated in the figure were pooled and concentrated and further purified by preparative polyacrylamide gel electrophoresis.

Fig. 2. Purification of cellulase CIV by preparative polyacrylamide gel electrophoresis. The cellulase CIV fraction from HW-55 chromatography was charged. After electrophoresis at pH 8, the enzyme was eluted as described in Methods and the activities on CMC and PNPG in each fraction were determined.

However, we have observed that values obtained with crude extracts were only qualitative. Often, they did not accurately estimate the quantities of the individual enzymes present. Inhibitors were typically present that caused the underestimation of certain enzymes ie.g., ligninases Table II) and that could potentially mask less dominant enzymes. Also, certain polysaccharidases e.g., hemicellulases) were often overestimated due to the action of non-specific or synergistic enzymes e.g., other hemicellulases or cellulases) (9,14), This artifact resulted in low apparent recovery of a given activity and only moderate increases in specific activity upon purification of the major corresponding enzyme present, in spite of the fact that SDS polyacrylamide gels indicated good recovery and substantial removal of contaminants (14),... 

Compound (44 g, NHAc form) (Scheme 14) was found to be a competitive inhibitor for CBHI cellulase (family 7) from Trichoderma reesei, when 4-methyl-umbelliferyl / -lactoside was used as substrate. Therefore (44 g, NHj form) was coupled to CH-Sepharose 4B, and the affinity gel was very effective for the purification of cellobiohydrolases from a crude commercial cellulolytic extract of T. reesei [40c]. Using the same approach aryl 1,4-dithioxylobioside and l,4,4 -trithioxylotrioside (44 h, NH2 form) were coupled to CH-Sepharose 4B to give affinity gels which were used for the purification of xylanases [40a,b]. 

The minor content of impurities found in the starch slurry are connected to the starch granules themselves. To facilitate the purification of the starch during filtration, cellulases, pentosanases, glucanases, proteases, and pectinases are sometimes used. Wheat starch is known to form precipitates or hazes that are difficult to filter. Arabinoxylan, pentosanes, andlysophospholipids are claimed to be responsible for this problem (73). 

Fractionation and Purification of Ex-1 Cellulase Component from Driselase. Driselase powder (50g) was extracted with several aliquots of water and the precipitate formed upon salting out with ammonium sulfate (on a saturation between 20% and 80%) was fractionated on a DEAE-Sephadex A-50 column. Each fraction was tested for -glucosi-dase, xylanase, CMCase, Avicelase activities, and protein content. The elution patterns are shown in Figures 1 and 2. 

Biosynthesis, Purification, and Mode of Action of Cellulases of Trichoderma reesei... 

Figure 1. Elution profiles of cellulase activity from Sephadex G-100 gel chromatographs of crude extracts of auxin-treated pea apices. BS cellulose activity has an elution volume corresponding to a molecular weight of 20,000. BI cellulase activity dissolves in 1M NaCl and elutes with a molecular weight of 70,000. These values correspond to those observed for purified cellulases (3), indicating that the enzymes were not altered in molecular weight during purification, and could be effectively separated by differential extraction.

Cellobiase. Figure 2 outlines the general purification steps used to isolate a pure cellobiase (named for its function in the cellulase complex) and three forms of the hydrocellulase. In purifying cellobiase it was expedient to replace the adsorption or affinity column with a batch separation on DEAE-Sephadex A-50 and to complete the purification with cation exchange chromatography on SP-Sephadex (49, 50). Table IV is a summary of the purification of the cellobiase and co-purification of... 

Two years later Selby and Maitland (17) resolved the T. viride system into three components which they called Ci, CM-cellulase, and cellobiase. The principal advance was the purification of the Ci to the point that it had little effect on cotton. This permitted a large increase in total solubilization as the result of recombination of individually ineffective components. In a similar fashion in 1969 Wood (54) reported resolution of the Fusarium solani complex into three components which he called Ci, Cx, and / -glucosidase. 

In 1972 Ogawa and Toyama (56) purified three components— A-I-a, A-I-b, and A-II-1—which were adsorbed on a gauze column during purification from Cellulase Onozuka P1500, a commercial preparation of T. viride cellulase. These three components had molecular weights of 32,000, 48,000, and 48,000 as determined by gel filtration and contained 7-16% carbohydrate. Each is reported to carry out the random hydrolysis of CM-cellulose and to degrade hydrocellulose (Avicel) and cellooligosaccharides except for cellobiose. The order of reactivity toward either cotton or Avicel was A-II-1 > A-I-b > A-I-a. The proteins adsorbed on cellulose comprised 38% of the total cellulase protein. 

Gong C-S, Ladisch MR, Tsao GT (1977) Cellobiase from Trichoderma viride-. purification, properties,kinetics, and mechanism. Biotechnology and Bioengineering 19 959-981 Gong C-S, Ladisch MR, Tsao GT (1979) Biosynthesis, purification and mode of action of cellulase of Trichoderma reesei-. hydrolysis of cellulose mechanisms of enzymatic and acid catalysis. Advances in Chemistry Series vol. 181, American Chemical Society, Washington, DC, pp 261-287... 

The modem technique for separation and purification of proteins has in all essentials been applied to cellulases. The methods which will be further discussed here are the following ... 

Specific Adsorption on Different Cellulose Columns. Purification of cellulolytic enzymes, including the Ci enzyme, has been carried out by Li et al. (32) by specific adsorption. By passing the crude enzyme through a column of Avicel the Ci fraction was retained by the column while 95% of the cellulase activity could be eluted. The cellulase fraction was then further purified by passing a column of alkali-swollen cellulose where the cellulase was retained. The enzyme could, however, be eluted by buffer in a high yield from the column. The adsorption on an alkali-swollen cellulose column was then repeated. The yield of enzyme obtained with the specific adsorption method is surprisingly high if compared with the yield on Sephadex and on polyacrylamide gel columns. 

In this study, we will explore whether an objective function, relating enzyme activity to cost, can be developed to establish the cost function of cellulase and other enzymes. We will determine the cost increase in cmde cellulase (in terms of activity per mass) after processing used to enhance the purity and concentration of this protein. We shall start with a more generalized objective function comprised of measurable purification process responses to create our cost model and reduce that model to the specific cost function for this study. A previously developed objective function quantifies the tradeoff between maximizing the enzyme concentration in a separation process such as a foam fiactionation process and minimizing the loss of enzyme mass and enzyme activity in that process [1] is shown below. 

Nilesh A., Kamat, M., Arvind, L. (2004). Expanded bed affinity purification of bacterial a-amylase and Cellulase on composite substrate analogue-Cellulose matrices. Process Biochemistry, 39, 565-570. 

---