Cellulases differentiation

SAXS measurements with CBH II indicated a very similar tertiary structure for both CBH I and CBH II, in spite of a different domain arrangement (to be published). Discrete differences in the tail parts could, however, be noticed. The maximum diameter of CBH II (21.5 nm) was higher than in CBH I this might be due to duplication of the glycosylated part in the former case. Thus, the functional differentiation of these cellulases can be reflected by structural differences. 

Chromophoric substrates were also used as tools in the study of the binding of several cellulase components to their natural substrates (such as Avicel). This is illustrated here in the investigation of the synergy in binding of CBH I and CBH II from Trichoderma reesei onto Avicel. The enzymes were differentiated with CNPL (see above), which was a substrate only for CBH I (core I). Thus, the amount of CBH II adsorbed when a mixture of both enzymes was added, either simultaneously or sequentionally, to Avicel was calculated from the amount of CBH I bound (activity measurements with CNPL) subtracted from the values for total protein binding (280 nm absorbance reading). The results obtained from these experiments are summarized as follows ... 

Another method which may become a useful technique for selective inactivation of cellulases in enzyme mixtures is the use of selective heat inactivation. While establishing the thermostability properties of crude xylanases from a fungal strain Y-94, Mitsuishi et al. (80) observed differential heat labilities of the cellulase and xylanase activities in the culture filtrate. After an incubation period of 20 minutes at 65°C, the xylanase activity was reduced by 5-10% whereas the Avicelase and /3-glucosidase activities were reduced by 100% and 60%, respectively. We have observed a similar temperature dependency of xylanase and cellulase activities in T. auranti-acus. As indicated in Figure 2, treatment of the culture filtrate at 70°C for 20 minutes resulted in less than a 5% loss in xylanase activity whereas cellulase activities were reduced by 40-50%. A similar effect has also been observed for the xylanases and cellulase enzymes produced in culture filtrates from T. harzianum (93). Further work in the area of heat treatments may improve the effectiveness of cellulase inactivation. Since the cellulase activities of some enzyme preparations can be more rapidly inactivated on... 

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.

From a critical study of the metabolism of poly (A) in auxin-treated pea epicotyl, Verma and Maclachlan (73) showed that discreet classes of poly (A) (presumably part of mRNA s) are differentially associated with free and membrane-bound polysomes. The induction of specific mRNA s, the decline in the rate of synthesis of mRNA s, the polysome content per cell, and the formation of cellulase were all related to the membrane-bound polysomes. Although the rate of in vivo enzyme synthesis is... 

Baker, J. O., Tatsumoto, K., Grohmann, K., Woodward, I, Wichert, J. M., Shoemaker, S. R, and Himmel, M. E. 1992. Thermal denaturation of Trichoderma reesei Cellulases Studied by Differential Scanning Calorimetry and Tryptophan Fluorescence. Appl. Biochem. Biotechnol., 34/35, 217-231. 

Bartley, T. D., Murphy-Holland, K., and Eveleigh, D. E., A method for the detection and differentiation of cellulase components in polyacrylamide gels. Anal Biochem 1984, 140 (1), 157-61. 

Biely, P., Detection and differentiation of cellulases and xylanases. Abstracts of Papers of the American Chemical Society 1988, 195, 202-CELL. 

Enzymes are an increasingly important component in detergent formulations, both in terms of effectiveness and as a means of product differentiation. The primary types of detergent enzymes are protease, amylase, lipase, and cellulase all are derived from bacterial and fungal sources. Enzyme activity is subject to various forms of chemical and physical degradation, problems that are particularly acute in liquid formulations, where the enzymes cannot be physically isolated from the harmful effects of surfactants and bleaches. 

The principal features of a mathematical model described for the enzymatic hydrolysis and fermentation of cellulose by Trichoderma reesei are the assumption of two forms of cellulose (crystalline and amorphous), two sugars (cellobiose and D-glucose), and two enzymes (cellulase and j3-D-glucosidase). An inducer-repressor-messenger RNA mechanism is used to predict enzyme formation, and pH effects are included. The model consists of 12 ordinary differential equations for 12 state variables and contains 38 parameters. The parameters were estimated from four sets of experimental data by optimization. The results appear satisfactory, and the computer programs permit simulation of a variety of system changes. 

The ( >PAsc - Avicel difference varied among cellulases, indicating differential cellulose accessibility of these cellulases, even when they were close structural/functional analogs. For instance, T. reesei CBH-I had a < i on PASC 3 times larger than that on Avicel, but H. insolens CBH-I had a i > on PASC 26 times larger dian that on Avicel. 

Unlike processive T. reesei CBH-I s preference for non-reducing cellulose chain ends, non-processive T. reesei EG-I is believed capable of randomly act along the cellulose chain (2,13,15). Under oiu conditions, 1 1 T. reesei CBH-I and EG-I mix had a < ) of 0.082 for PASC, while 15 1 or 17 1 mix had a ( ) of 0.067. For Avicel, 1 17. reesei CBH-I and EG-I mix had a < ) of 0.015. These ( ) seemed to correspond to die ( > sums of the two cellulases, indicating different productive adsorption regions for them. However, because the ( ) of T. reesei EG-I was 15 times smaller than tiiat of T. reesei CBH-I, our experiment could not conclusively differentiate a trae siun that was 15% higher tiian ( )(CBH-I) from a ( (CBH-I) with 10% experinental error. 

Biely P (1990) Artificial substrate for cellulolytic glycanases and their use for the differentiation of Trichoderma enzymes. In Kubicek CP, Eveleigh DE, Esterbauer H, Sten W, Kubicek-Pranz EM (eds) Trichoderma Cellulases. Biochemistry, Genetics, Physiology and Applications. Royal Chem Soc Cambridge p 30... 

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