KINETICS OF THE HYDROLYSIS OF CELLODEXTRINS BY SELECTED CELLULASE COMPONENTS

ANTONIO SILVA PINTO DE NAZARE PEREIRA, Purdue University

Abstract

The intrinsic properties of cellulolytic enzymes are not fully defined. Purified enzymes and soluble substrates are required to characterize cellulases. In this work, cellodextrins, water soluble fragments of cellulose, have been hydrolyzed by purified components of the cellulolytic complex of Trichoderma reesei QM9414 produced under controlled fermentations. The cellulolytic complex consists of cellobiohydrolase (EC 3.2.1.91), endocellulase (EC 3.2.1.4), and cellobiase (EC 3.2.1.21). At least cellobiohydrolase and endocellulase are needed for hydrolysis of insoluble substrates. Because fractionation results in loss of measurable activity using standard methods, the purification was also monitored by a highly sensitive aqueous high pressure liquid chromatography. The method developed in the course of this work used styrene-divinyl benzene cation exchange resins, in the H$\sp+$ form, to detect less than 150 $\mu$M of glucose or 10 $\mu$M of cellohexaose. Cellobiohydrolase and endocellulase, free of competing activities, have been recovered. Cellobiohydrolase, the main enzyme, is unable to hydrolyze cellobiose. It slowly hydrolyzes cellotriose (G$\sb3).$ Cellopentaose (G$\sb5)$ is hydrolyzed to cellobiose and cellotriose. Cellotetraose (G$\sb4)$ and cellohexaose give cellobiose and cellotriose as products. These results are consistent with at least five subsites in the active site of the enzyme. Time course hydrolysis reveals significant inhibition by cellobiose (K$\sb1$ = 1-10$\mu$M) and high affinity for substrate (K$\sb{\rm m}$ = 70 $\mu$M for G$\sb3$; K$\sb{\rm m}$ =0.1 $\mu$M for G$\sb4$; K$\sb{\rm m}$ ca. 1.0 $\mu$M for G$\sb5$) The substrate affinity and specific activity of cellobiohydrolase on cellodextrins is similar to the activity of $\beta$-amylase on maltooligosaccharides. The major difference between the enzymes is that cellobiohydrolase is much more inhibited by the products of hydrolysis. Combined endocellulase and cellobiohydrolase act on cellotetraose with a slight increase of the rate of hydrolysis. The dramatic effects of the combined activity of both enzymes in the hydrolysis of insoluble substrates are thus not observed on soluble cellulose fragments. The results suggest that pretreatment of cellulose to below a soluble form is a key parameter for practical processes since higher rate of hydrolysis and less dependence on synergistic activity should result. Ultimately, product inhibition becomes the major limiting factor; as little as 1-10 $\mu$M of cellobiose will be significant to reduce cellobiohydrolase activity by 50%. Both modifications of enzyme characteristics and development of an effective cellulolytic pretreatment is needed to obtain hydrolysis rates similar to amylases.

Degree

Ph.D.

Subject Area

Food science

COinS