Properties and mode of action of cellulase

Abstract

Native cellulose undergoing an attack by cellulase exhibits extensive changes in physical properties prior to producing a measurable quantity of reducing sugar. These changes include fragmentation, swelling, considerable loss in tensile strength, transverse cracking, and lowering of the degree of polymerization. Fragmentation and swelling of cellulose fibers are characteristic of purified C _x enzyme action rather than C ₁, action.

Reese and his co-workers (1950) first suggested a mechanism for the enzymatic breakdown of cellulose which involves a C ₁ component. They postulated that the conversion of native cellulase was a two-step process: C ₁ “activates” or desaggregates the cellulose chains, and the enzyme classified as C _x then carries out the hydrolysis reaction. Early research mostly related to studies of the C _x component. Since 1964, an extensive search for the C ₁ component has been carried out, and substantial evidence was found to support the existence of a C ₁-like components. Several investigators have supported the concept that the C ₁ component has a nonhydrolytic function. However, based on the results of recent fractionation studies, it has been suggested that the (C ₁−C _x) concept be re-evaluated and that the mechanism of cellulase action be reformulated. The evidence cited by the various authors to support the claim that C ₁ is a cellobiohydrolase appears to be convincing. Nevertheless, there remain striking differences in the extent to which highly ordered cellulose is hydrolyzed by the various C ₁-type components. The enzyme system of Trichoderma sp. has been examined more extensively than other enzyme systems. The mode of action of each component of this cellulase is shown graphically in Fig. 3 and can be summarized as follows:

Endo-β-1,4-glucanase. This contains several components with varying degrees of randomness. One of these may be the enzyme that acts on crystalline cellulose; however, it acts randomly, mainly on CMC, phosphoric acid-swollen cellulose, and cellodextrin. This component does not act on cellobiose. The main products are cellobiose and cellotriose.

Exo-β-1,4-glucanases. This is present in several forms. β-l,4-Glucan glucohydrolase removes a single glucose unit from the nonreducing end of the chain. This enzyme acts on Walseth, CMC, and cellodextrin chains of four to seven units produced by the action of the endo-glucanase but attacks insoluble cellulose with difficulty. This component has rarely been reported. β-1,4-Glucan cellobiohydrolase (CBH) removes a cellobiose unit from the nonreducing ends of the chain. The CBH is currently being equated with the classical C₁ enzyme by many investigators. This component has the greatest affinity for cellulose; it can not attack CMC and acts very slowly on H₃PO₄-swollen cellulose. Although it is unable to attack crystalline cellulose to any significant extent, it can degrade cellulose substrates by successively removing cellobiose residues from the chain ends. When CBH is recombined with C_x and β-glucosidase. it plays a major role in hydrolysis of cotton or Avicel-like crystalline cellulose.

β-Glucosidase. This hydrolyzes cellobiose and short chain cellooligosaccharides to glucose but has no effect on cellulose. While it rapidly hydrolyzes cellobiose and cellotriose, its rate of attack decreases markedly with an increasing degree of polymerization. This is in contrast to exo-β-1,4 glucanas, which acts preferentially on longer cellooligosaccharides.

The physical properties of each cellulase component, such as the molecular weight, the diffusion coefficient, the sedimentation constant, and the molecular size and shape, have been described herein. The molecular weight of the C ₁ component from Trichoderma sp. lies in the range of 53,000 to 62,000. The endo-β-l,4-glucanases show considerable variation in molecular weights, ranging from 5,300 to 55,000. Exo-glucanase and β-glucosidase are reported to have molecular weights of 50,000 to 76,000. The molecular weight of β-1,4-glucan cellobiohydrolase was measured at about 42,000. If the cellulase molecules are spherical, their size would range from about 25 to 80 Å in diameter with an average of 60 Å. If the enzymes are ellipsoids with an axial ratio of about 6, their sizes would range from about 15 to 40 Å in width and from 80 to 250 Å in length, giving rise to an average size of 35 Å×200 Å.