Revealing Nature’s Cellulase Diversity: The Digestion Mechanism of
            <i>Caldicellulosiruptor bescii</i>
            CelA

Brunecky, Roman; Alahuhta, Markus; Xu, Qi; Donohoe, Bryon S.; Crowley, Michael F.; Kataeva, Irina; Yang, Soo Jin; Resch, Michael G.; Adams, Michael W. W.; Лунин, В.В.; Himmel, Michael E.; Bomble, Yannick J.

doi:10.1126/science.1244273

Cited by 263 publications

(331 citation statements)

References 34 publications

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“…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.…”

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confidence: 96%

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

Brunecky

Alahuhta

et al. 2014

Science

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*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 ...

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

Brunecky

Alahuhta

et al. 2014

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“…In some cases a multivalent effect has been observed, where single cellulase enzymes contain multiple CBMs to increase association with target substrates (49,50). The presence of multiple CBMs may also be deleterious for enzymatic function depending on the substrate and the enzymatic mechanism, as seen with cellulosomes on pretreated biomass (51).…”

Section: Molecularmentioning

confidence: 99%

Predicting Enzyme Adsorption to Lignin Films by Calculating Enzyme Surface Hydrophobicity

Sammond

Yarbrough

Mansfield

et al. 2014

Journal of Biological Chemistry

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Background:Lignin is a plant cell wall polymer that inhibits enzymatic saccharification of polysaccharides for the production of biofuel. Results: The adsorption of enzymes to lignin surfaces correlates to solvent-exposed hydrophobic clusters. Conclusion: Hydrophobicity, not surface charge, identifies proteins that preferentially adsorb to lignin. Significance: The method could be used to design improved cellulase cocktails to lower the cost of biofuel production.

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“…In many cases, the enzymes used by these organisms are multimodular, consisting of one or more catalytic domains of various function (2)(3)(4)(5)(6)(7)(8) linked to a carbohydrate-binding module (CBM) that targets plant cell wall polysaccharides through specific recognition mechanisms (9). To date, 67 families of CBMs have been discovered (10), and many of these families contain members important in biomass depolymerization.…”

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confidence: 99%

Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

Chen

Drake

Resch

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The majority of biological turnover of lignocellulosic biomass in nature is conducted by fungi, which commonly use Family 1 carbohydrate-binding modules (CBMs) for targeting enzymes to cellulose. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation are examined on the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase at three glycosylation sites. To enable this work, a procedure to synthesize glycosylated Family 1 CBMs was developed. Subsequently, a library of 20 CBMs was synthesized with mono-, di-, or trisaccharides at each site for comparison of binding affinity, proteolytic stability, and thermostability. The results show that, although CBM mannosylation does not induce major conformational changes, it can increase the thermolysin cleavage resistance up to 50-fold depending on the number of mannose units on the CBM and the attachment site. O-Mannosylation also increases the thermostability of CBM glycoforms up to 16°C, and a mannose disaccharide at Ser3 seems to have the largest themostabilizing effect. Interestingly, the glycoforms with small glycans at each site displayed higher binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of three positions conferred the highest affinity enhancement of 7.4-fold. Overall, by combining chemical glycoprotein synthesis and functional studies, we show that specific glycosylation events confer multiple beneficial properties on Family 1 CBMs. chemical synthesis | cellulase | biofuels | protein engineering

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Revealing Nature’s Cellulase Diversity: The Digestion Mechanism of Caldicellulosiruptor bescii CelA

Cited by 263 publications

References 34 publications

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

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

Predicting Enzyme Adsorption to Lignin Films by Calculating Enzyme Surface Hydrophobicity

Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

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