Exo‐exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo‐lytic character of the enzymes

Abstract

Synergy between cellulolytic enzymes is essential in both natural and industrial breakdown of biomass. In addition to synergy between endo- and exo-lytic enzymes, a lesser known but equally conspicuous synergy occurs among exo-acting, processive cellobiohydrolases (CBHs) such as Cel7A and Cel6A from Hypocrea jecorina. We studied this system using microcrystalline cellulose as substrate and found a degree of synergy between 1.3 and 2.2 depending on the experimental conditions. Synergy between enzyme variants without the carbohydrate binding module (CBM) and its linker was strongly reduced compared to the wild types. One plausible interpretation of this is that exo-exo synergy depends on the targeting role of the CBM. Many earlier works have proposed that exo-exo synergy was caused by an auxiliary endo-lytic activity of Cel6A. However, biochemical data from different assays suggested that the endo-lytic activity of both Cel6A and Cel7A were 10 -10 times lower than the common endoglucanase, Cel7B, from the same organism. Moreover, the endo-lytic activity of Cel7A was 2-3-fold higher than for Cel6A, and we suggest that endo-like activity of Cel6A cannot be the main cause for the observed synergy. Rather, we suggest the exo-exo synergy found here depends on different specificities of the enzymes possibly governed by their CBMs. Biotechnol. Bioeng. 2017;114: 1639-1647. © 2017 Wiley Periodicals, Inc.

Direct kinetic comparison of the two cellobiohydrolases Cel6A and Cel7A from Hypocrea jecorina

“…Many cellulose degrading fungi have secretomes that are dominated by mixtures of cellobiohydrolases (CBHs) from respectively Glucoside Hydrolase family 6 and 7. CBHs from both families are processive and primarily exo-lytic enzymes [4-6, 42, 50, 51] although they both show a small auxiliary endo-activity [14, 42,52,53]. They are structurally rather different and utilize respectively the retaining (GH7) and inverting (GH6) hydrolytic mechanism.…”

“…The distinctive reduction in specific activity even for very low coverage and degree of conversion suggests that high Avicel conversion will be hard to attain even in prolonged reactions with these mono-component enzymes. This, in turn, may be related to the pronounced synergy of Cel6A and Cel7A (so-called exo-exo synergy), which probably reflects specificity for different types of surface structures and hence the ability of one enzyme to exhume good attack sites for the other [42,43], but we will not pursue this topic further in the current work. .…”

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

“…The CBD has been proposed to have multiple roles in the hydrolysis of cellulose (e.g., increase the concentration of cellulase close to the substrate) to target the CD to specific sites on the substrate and to disrupt the crystalline structure of the substrate. Furthermore, it has been shown that CBD increases the adsorption surface, which increases the activity [13] and thermostability of cellulases [14]. Several studies have been conducted in order to understand cellulase adsorption to cellulose and the role of CBDs [15,16].…”

“…Thereby, substrates from different sources or with different pretreatment also require distinct cellulase formulations [64]. To date, most studies have focused on understanding the synergic effect between natural producing cellulases or commercially available cocktails [13,65]. Few In the "cocktail" formulation, the ratios and combinations of cellulases greatly affect the hydrolysis efficiency.…”

Insight into the Underlying Synergy between Exo–Lytic Cellulases

“…According to the study of Trudeau et al, substantial cooperation between exo-lytic enzymes, i.e., CBHI and cellobiohydrolase II (CBHII or Cel6A) also exists [14], whereas the mechanism is poorly understood [18][19][20][21]. The rate of glycoside hydrolase family 7 (GH7) CBHI leaving cellulose after catalysis, namely dissociation rate is known to be the rate-limiting step when employed alone [22][23][24][25][26][27][28][29].…”