Search

The kinetic theory of enzymes that modify insoluble substrates is still underdeveloped, despite the prevalence of this type of reaction both in vivo and industrial applications. Here, we present a steady-state kinetic approach to investigate inhibition occurring at the solid–liquid interface. We propose to conduct experiments under enzyme excess (E0 ≫ S0), i.e. the opposite limit compared with the conventional Michaelis–Menten framework. This inverse condition is practical for insoluble substrates and elucidates how the inhibitor reduces enzyme activity through binding to the substrate. We claim that this type of inhibition is common for interfacial enzyme reactions because substrate accessibility is low, and we show that it can be analyzed by experiments and rate equations that are analogous to the conventional approach, except that the roles of enzyme and substrate have been swapped. To illustrate the approach, we investigated the major cellulases from Trichoderma reesei (Cel6A and Cel7A) acting on insoluble cellulose. As model inhibitors, we used catalytically inactive variants of Cel6A and Cel7A. We made so-called inverse Michaelis–Menten curves at different concentrations of inhibitors and found that a new rate equation accounted well for the data. In most cases, we found a mixed type of surface-site inhibition mechanism, and this probably reflected that the inhibitor both competed with the enzyme for the productive binding-sites (competitive inhibition) and hampered the processive movement on the surface (uncompetitive inhibition). These results give new insights into the complex interplay of Cel7A and Cel6A on cellulose and the approach may be applicable to other heterogeneous enzyme reactions.

The enzymes from hyperthermophilic microorganisms populating volcanic sites represent interesting cases of protein adaptation and biotransformations under conditions where conventional enzymes quickly denature. The difficulties in cultivating extremophiles severely limit access to this class of biocatalysts. To circumvent this problem, we embarked on the exploration of the biodiversity of the solfatara Pisciarelli, Agnano (Naples, Italy), to discover hyperthermophilic carbohydrate-active enzymes (CAZymes) and to characterize the entire set of such enzymes in this environment (CAZome). Here, we report the results of the metagenomic analysis of two mud/water pools that greatly differ in both temperature and pH (T = 85 °C and pH 5.5; T = 92 °C and pH 1.5, for Pool1 and Pool2, respectively). DNA deep sequencing and following in silico analysis led to 14 934 and 17 652 complete ORFs in Pool1 and Pool2, respectively. They exclusively belonged to archaeal cells and viruses with great genera variance within the phylum Crenarchaeota, which reflected the difference in temperature and pH of the two Pools. Surprisingly, 30% and 62% of all of the reads obtained from Pool1 and 2, respectively, had no match in nucleotide databanks. Genes associated with carbohydrate metabolism were 15% and 16% of the total in the two Pools, with 278 and 308 putative CAZymes in Pool1 and 2, corresponding to ~ 2.0% of all ORFs. Biochemical characterization of two CAZymes of a previously unknown archaeon revealed a novel subfamily GH5_19 β-mannanase/β-1,3-glucanase whose hemicellulose specificity correlates with the vegetation surrounding the sampling site, and a novel NAD+ -dependent GH109 with a previously unreported β-N-acetylglucosaminide/β-glucoside specificity. DATABASES: The sequencing reads are available in the NCBI Sequence Read Archive (SRA) database under the accession numbers SRR7545549 (Pool1) and SRR7545550 (Pool2). The sequences of GH5_Pool2 and GH109_Pool2 are available in GenBank database under the accession numbers MK869723 and MK86972, respectively. The environmental data relative to Pool1 and Pool2 (NCBI BioProject PRJNA481947) are available in the Biosamples database under the accession numbers SAMN09692669 (Pool1) and SAMN09692670 (Pool2).

Glycoside hydrolase family 7 (GH7) cellulases are some of the most efficient degraders of cellulose, making them particularly relevant for industries seeking to produce renewable fuels from lignocellulosic biomass. The secretome of the cellulolytic model fungus Trichoderma reesei contains two GH7s, termed TrCel7A and TrCel7B. Despite having high structural and sequence similarities, the two enzymes are functionally quite different. TrCel7A is an exolytic, processive cellobiohydrolase (CBH), with high activity on crystalline cellulose, whereas TrCel7B is an endoglucanase (EG) with a preference for more amorphous cellulose. At the structural level, these functional differences are usually ascribed to the flexible loops that cover the substrate-binding areas. TrCel7A has an extensive tunnel created by eight peripheral loops, and the absence of four of these loops in TrCel7B makes its catalytic domain a more open cleft. To investigate the structure-function relationships of these loops, here we produced and kinetically characterized several variants in which four loops unique to TrCel7A were individually deleted to resemble the arrangement in the TrCel7B structure. Analysis of a range of kinetic parameters consistently indicated that the B2 loop, covering the substrate-binding subsites -3 and -4 in TrCel7A, was a key determinant for the difference in CBH- or EG-like behavior between TrCel7A and TrCel7B. Conversely, the B3 and B4 loops, located closer to the catalytic site in TrCel7A, were less important for these activities. We surmise that these results could be useful both in further mechanistic investigations and for guiding engineering efforts of this industrially important enzyme family.

Enzyme reactions, both in Nature and technical applications, commonly occur at the interface of immiscible phases. Nevertheless, stringent descriptions of interfacial enzyme catalysis remain sparse, and this is partly due to a shortage of coherent experimental data to guide and assess such work. We have produced and kinetically characterized 83 cellulases, which revealed a conspicuous linear free energy relationship (LFER) between the strength of substrate binding and the activation barrier. This common scaling occurred despite the investigated enzymes were structurally and mechanistically diverse. We suggest that the scaling reflects basic physical restrictions of the hydrolytic process and that evolutionary selection have condensed cellulase phenotypes near the line. One consequence of the LFER is that the activity of a cellulase can be estimated from substrate binding strength, irrespectively of structural and mechanistic details, and this appears promising forin silicoselection and design within this industrially important group of enzymes. On a more general note, the LFER may identify a link to inorganic heterogeneous catalysis, and hence open for the implementation of approaches from this field within interfacial enzymology.

Thermostable cellulases from glycoside hydrolase family 7 (GH7) are the main components of enzymatic mixtures for industrial saccharification of lignocellulose. Activity improvement of these enzymes via rational design is a promising strategy to alleviate the industrial costs, but it requires detailed structural knowledge. While substantial biochemical and structural data are available for GH7 cellobiohydrolases, endoglucanases are more elusive and only few structures have been solved so far. Here, we report a new crystal structure and biochemical characterization of a thermostable endoglucanase from the thermophilic ascomycete Rasamsonia emersonii, ReCel7B. The enzyme was compared with the homologous endoglucanase from the mesophilic model ascomycete Trichoderma reesei (TrCel7B), which unlike ReCel7B possesses an additional carbohydrate-binding module (CBM). With a temperature optimum of 80 °C, ReCel7B displayed a number of differences in activity and ability to synergize with cellobiohydrolases compared to TrCel7B. We improved both binding and kinetics in a chimeric variant of ReCel7B and a CBM, while we observe the opposite effect when the CBM was removed in TrCel7B. The crystal structure of ReCel7B was determined at 2.48 A resolution, with Rwork and Rfree factors of 0.182 and 0.206, respectively. Structural analyses revealed that ReCel7B has increased rigidity in a number of peripheral loops compared to TrCel7B and fewer aromatics in the substrate-binding cleft. An increased number of glycosylations were identified in ReCel7B, and we propose a stabilizing mechanism for one of the glycans. Global structure-function interpretations of ReCel7B highlight the differences in temperature stability, turnover, binding, and cellulose accessibility in GH7 endoglucanases. DATABASE: Structural data are available in RCSB Protein Data Bank database under the accession number 6SU8. ENZYMES: ReCel7B, endoglucanase (EC3.2.1.4) from Rasamsonia emersonii; ReCel7A, cellobiohydrolase (EC3.2.1.176) from Rasamsonia emersonii; TrCel7B, endoglucanase (EC3.2.1.4) from Trichoderma reesei; TrCel7A, cellobiohydrolase (EC3.2.1.176) from Trichoderma reesei.

Glycoside hydrolase family 7 (GH7) cellulases are some of the most efficient degraders of cellulose, making them particularly relevant for industries seeking to produce renewable fuels from lignocellulosic biomass. The secretome of the cellulolytic model fungus Trichoderma reesei contains two GH7s, termed TrCel7A and TrCel7B. Despite having high structural and sequence similarities, the two enzymes are functionally quite different. TrCel7A is an exolytic, processive cellobiohydrolase (CBH), with high activity on crystalline cellulose, whereas TrCel7B is an endoglucanase (EG) with a preference for more amorphous cellulose. At the structural level, these functional differences are usually ascribed to the flexible loops that cover the substrate-binding areas. TrCel7A has an extensive tunnel created by eight peripheral loops, and the absence of four of these loops in TrCel7B makes its catalytic domain a more open cleft. To investigate the structure–function relationships of these loops, here we produced and kinetically characterized several variants in which four loops unique to TrCel7A were individually deleted to resemble the arrangement in the TrCel7B structure. Analysis of a range of kinetic parameters consistently indicated that the B2 loop, covering the substrate-binding subsites −3 and −4 in TrCel7A, was a key determinant for the difference in CBH- or EG-like behavior between TrCel7A and TrCel7B. Conversely, the B3 and B4 loops, located closer to the catalytic site in TrCel7A, were less important for these activities. We surmise that these results could be useful both in further mechanistic investigations and for guiding engineering efforts of this industrially important enzyme family.

We have measured activity and substrate affinity of the thermostable cellobiohydrolase, Cel7A, from Rasamsonia emersonii over a broad range of temperatures. For the wild type enzyme, which does not have a Carbohydrate Binding Module (CBM), higher temperature only led to moderately increased activity against cellulose, and we ascribed this to a pronounced, temperature induced desorption of enzyme from the substrate surface. We also tested a "high affinity" variant of R. emersonii Cel7A with a linker and CBM from a related enzyme. At room temperature, the activity of the variant was similar to the wild type, but the variant was more accelerated by temperature and about two-fold faster around 70 °C. This better thermoactivation of the high-affinity variant could not be linked to differences in stability or the catalytic process, but coincided with less desorption as temperature increased. Based on these observations and earlier reports on moderate thermoactivation of cellulases, we suggest that better cellulolytic activity at industrially relevant temperatures may be attained by engineering improved substrate affinity into enzymes that already possess good thermostability.

Lack of access to certain types of oligosaccharides is a severe bottleneck for advances inglycosciences. The transglycosylation activity of retaining glycoside hydrolases (GH) has been used to provide oligosaccharides. The main drawbacks of those enzymes are the competing hydrolysis reaction and the fact that the products are also substrates, thus needing a kinetic control of the reaction. Several approaches have been developed to overcome these, including mechanism modifications (e.g. glycosynthases, chemical rescue), functional screening and data mining to find natural transglycosidases, directed evolution and targeted mutagenesis. Here we focused on N-acetyl hexosaminidases from family GH20 that catalyse removal or additionof GlcNAc and GalNAc. Despite sharing a substrate-assisted mechanism with GH85, for which several glycosynthases have been created, no successful GH20 glycosynthase has been reported. Thus, we turned to discovery and characterization of new GH20s and performing a systematic mutagenesis study. Several new GH20s of bacterial origin were isolated and described by functional screening and data mining, including transglycosidases able to synthesize lacto-N-triose, a valuableoligosaccharide, as well as genuine hydrolases. Mutational analysis of all residues within the catalytic domain which were unchanged in >99% of 371 aligned GH20 sequences was pursued. Indeed, it has been shown that targeting conserved residues increases the likelihood of finding advantageous mutations. Furthermore, it allows for transfer of successful mutations to other GH20s to find new efficient transglycosidases. Notably, even though most conserved residues occur within the first and second shell of substrateinteraction, 9 residues inside the (β/α)8 barrel pointing toward the active site are also conserved. To the best of our knowledge, such residues were not studied, although one of them mutated by directed evolution of a GH29 enzyme improved the transglycosylation yield and was transferable to other GH29 members. Here transglycosylation yields of mutants in first shell, second shell and other residues within the (β/α)8 barrel will be compared for GH20.