Response to Comment on “Revealing Nature’s Cellulase Diversity: The Digestion Mechanism of Caldicellulosiruptor bescii CelA”

Abstract

*Gusakov critiques our methodology for comparing the cellulolytic activity of the bacterial cellulase CelA with the fungal cellulase Cel7A. We address his concerns by clarifying some misconceptions, carefully referencing the literature, and justifying our approach to point out that the results from our study still stand. G usakov (1) argues that the pH used in our study (2) for the enzymatic assays of Cel7A was not within the range of optimal pH values for this enzyme and that the substrate loading data were missing. He provides various references that we believe are hard to interpret, because they do not show curves representing activity versus pH. However, we have found such data in a paper by Boer et al. (3), according to which Trichoderma reesei Cel7A at pH 5.5 retains 95 to 96% of the activity it had at pH 5.0 [see figure 5 in (3)]. Nevertheless, we realized that there was a typographical error in the supplementary material (SM) of our paper, stating that our assays were performed at pH 5.5; the Cel7A assays were in fact run at pH 5.0, and the SM has been corrected. Regarding the substrate loading, the data in figure 1 in our paper were measured using 10 mg glucan/ml [1% (w/v)] of buffer for the Avicel experiments and comparable loadings for the other substrates. The original SM specified that the mass loading of the substrate was 1% for carboxymethyl cellulose (CMC), Avicel, and oat spelt xylan but did not explicitly clarify that it was similar for all the assays.Gusakov also argues that the experiments to compare the activity of CelA/b-glucosidase with Cel7A/b-glucosidase should have been conducted instead of referencing published work. We feel that the articles we referenced for this discussion were sufficient in this regard because they evaluated the effects of b-glucosidases on the performance of Cel7A using a wide range of b-glucosidase loadings (4-6). Additionally, our manuscript focused on the characterization of CelA, and the comparison to Cel7A was only conducted without the presence of b-glucosidases. Given that our Cel7A assays were actually conducted at pH 5.0, we feel that the addition of b-glucosidase could not provide more than a 30% improvement.Gusakov also argues that our results would be less dramatic if other mixtures or individual cellobiohydrolases, thought to be more efficient than those produced by T. reesei, were to have been used instead of Cel7A. However, the point of our paper was to compare single enzymes, not mixtures of enzymes, and in doing so, we selected the most commonly used single enzyme in commercial preparations, T. reesei Cel7A. We chose to compare purified enzymes because the variability in microbial broths can be considerable.Additionally, Gusakov mentions a patent showing the existence of a cellobiohydrolase I (CBH I) more active than the enzyme from T. reesei. This patent compares T. reesei CBH I and Penicillium funiculosum CBH I on a timeto-target basis-in other words, time to achieve a certain level of conversion (7). If we compare enzyme activities in terms of ...

Conversion of Paper Mill Waste Substrates to Sugars: Cooperative Research and Development Final Report, CRADA Number CRD-16-00643

“…20 is added to both samples CelA becomes more competitive but still does not outperform Ctec2. The Tween 20 surfactant presumably helps the CelA based mix avoid inhibition by lignin and presumably other compounds that present that may have an inhibitory effect on CelA as has been reported previously (1)(2)(3)(4). The effect does not appear to be as pronounced for the Ctec2 formulation in this case, but a benefit is still observed.…”

“…We studied both a relatively clean carboard waste (S3) and a mixed paper waste substrate. For the enzymes systems utilized we used a commercial Ctec2 formulation, and a experimental hyperthermophilic enzyme mixture based around the CelA enzyme (1)(2)(3)(4). The results indicated that the experimental CelA based formulation was clearly superior to Ctec2 when converting the clean carboard waste, but only equivalent to Ctec2 when converting the mixed paper waste.…”

The Multi Domain Caldicellulosiruptor bescii CelA Cellulase Excels at the Hydrolysis of Crystalline Cellulose

“…A hydrophobic affinity purified C. bescii broth enriched in CelA was utilized as described in refs 35 , 42 for the CelA mix data reported here, we emphasize that this is not the raw C. bescii exproteome, rather it is enriched in the hydrophobic components such as CelA that represents a the majority of the activity of the broth 2 . For the experiments requiring pure CelA, CelA was tagged with 6xHis tag, and expressed in C. bescii .…”