Synergistic effect of cellulo-xylanolytic and laccase enzyme consortia for improved deinking of waste papers.

This study reports the cellulo-xylanolytic cocktail production from Hypocrea lixii GGRK4 using multi-objective genetic algorithm-artificial neural network tool, resulting in 8.32 ± 1.07 IU/mL, 51.53 ± 3.78 IU/mL activity of CMCase and xylanase, respectively with more than 85 % residual activity at 60 °C and pH 6.0. Interestingly, metal ions viz. K+ and Ca2+ stimulated the enzyme activity, whereas Fe2+ and Cu2+ reduced the activity. Significant amounts of hydrophobic compounds, chromophores, and phenolics were released after wastepapers deinking. The deinking efficiency of 73.60 ± 2.45 % and 38.60 ± 1.34 % was obtained for photocopier paper and newspaper, respectively, whereas brightness of 89.90 ± 2.10 % ISO and 44.90 ± 1.63 % ISO was reported for both types of waste papers. The physical strength of deinked photocopier paper and newspapers, i.e., tensile index (3.10 and 0.50 %), tearing index (7.10 and 4.83 %), and burst factor (8.61) were enhanced whereas double fold property was decreased proving wastepaper reusability. This consortium showed effective and significant enzymatic deinking efficiency for recycled wastepapers.

Dual role of a dedicated GAPDH in the biosynthesis of volatile and non-volatile metabolites- novel insights into the regulation of secondary metabolism in Trichoderma virens.

Trichoderma virens produces viridin/viridiol, heptelidic (koningic) acid, several volatile sesquiterpenes and gliotoxin (Q strains) or gliovirin (P strains). We earlier reported that deletion of the terpene cyclase vir4 and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH, designated as vGPD) associated with the "vir" cluster abrogated the biosynthesis of several volatile sesquiterpene metabolites. Here we show that, the deletion of this GAPDH also impairs the biosynthesis of heptelidic acid (a non-volatile sesquiterpene), viridin (steroid) and gliovirin (non-ribosomal peptide), indicating regulation of non-volatile metabolite biosynthesis by this GAPDH that is associated with a secondary metabolism gene cluster. To gain further insights into the details of this novel form of regulation, we identified the terpene cyclase gene responsible for heptelidic acid biosynthesis (hereafter designated as has1) and prove that the expression of this gene is regulated by vGPD. Interestingly, deletion of has1 impaired biosynthesis of heptelidic acid (HA), viridin and gliovirin, but not of volatile sesquiterpenes. Deletion of the vir cluster associated terpene cyclase gene (vir4), located next to the vGPD gene, did not impair biosynthesis of HA, viridin or gliovirin. We thus unveil a novel circuitry of regulation of secondary metabolism where an HA-tolerant GAPDH isoform (vGPD) regulates HA biosynthesis through the transcriptional regulation of the HA-synthase gene (which is not part of the "vir" cluster). Interestingly, impairment of HA biosynthesis leads to the down-regulation of biosynthesis of other non-volatile secondary metabolites, but not of volatile secondary metabolites. We thus provide evidence that the "vir" cluster associated, HA-tolerant GAPDH in T. virens participates in the biosynthesis of volatile sesquiterpenes as a biosynthetic enzyme, and regulates the production of non-volatile metabolites via regulation of HA biosynthesis. The orthologue of the "vir" cluster in Aspergillus oryzae was earlier reported to synthesize HA by another group. Our study thus proves that the same gene cluster can code for unrelated metabolites in different species.

Structural Insight into the Substrate Scope of Viperin and Viperin-like Enzymes from Three Domains of Life.

Viperin is a member of the radical S-adenosylmethionine superfamily and has been shown to restrict the replication of a wide range of RNA and DNA viruses. We recently demonstrated that human viperin (HsVip) catalyzes the conversion of CTP to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP or ddh-synthase), which acts as a chain terminator for virally encoded RNA-dependent RNA polymerases from several flaviviruses. Viperin homologues also exist in non-chordate eukaryotes (e.g., Cnidaria and Mollusca), numerous fungi, and members of the archaeal and eubacterial domains. Recently, it was reported that non-chordate and non-eukaryotic viperin-like homologues are also ddh-synthases and generate a diverse range of ddhNTPs, including the newly discovered ddhUTP and ddhGTP. Herein, we expand on the catalytic mechanism of mammalian, fungal, bacterial, and archaeal viperin-like enzymes with a combination of X-ray crystallography and enzymology. We demonstrate that, like mammalian viperins, these recently discovered viperin-like enzymes operate through the same mechanism and can be classified as ddh-synthases. Furthermore, we define the unique chemical and physical determinants supporting ddh-synthase activity and nucleotide selectivity, including the crystallographic characterization of a fungal viperin-like enzyme that utilizes UTP as a substrate and a cnidaria viperin-like enzyme that utilizes CTP as a substrate. Together, these results support the evolutionary conservation of the ddh-synthase activity and its broad phylogenetic role in innate antiviral immunity.

Strain improvement of Trichoderma spp. through two-step protoplast fusion for cellulase production enhancement.

Fungal protoplast fusion is an approach to introduce novel characteristics into industrially important strains. Cellulases, essential enzymes with a wide range of biotechnological applications, are produced by many species of the filamentous fungi Trichoderma. In this study, a collection of 60 natural isolates were screened for Avicel and carboxymethyl cellulose degradation, and two cellulase producers of Trichoderma virens and Trichoderma harzianum were used for protoplast fusion. One of the resulting hybrids with improved cellulase activity, C1-3, was fused with the hyperproducer Trichoderma reesei Rut-C30. A new selected hybrid, F7, was increased in cellulase activity 1.8 and 5 times in comparison with Rut-C30 and C1-3, respectively. The increases in enzyme activity correlated with an upregulation of the cellulolytic genes cbh1, cbh2, egl3, and bgl1 in the parents. The amount of mRNA of cbh1 and cbh2 in F7 resembled that of Rut-C30 while the bgl1 mRNA level was similar to that of C1-3. AFLP (amplified fragment length polymorphism) fingerprinting and GC-MS (gas chromatography - mass spectrometry) analysis represented variations in parental strains and fusants. In conclusion, the results demonstrate that a 3-interspecific hybrid strain was isolated, with improved characteristics for cellulase degradation and showing genetic polymorphisms and differences in the volatile profile, suggesting reorganizations at the genetic level.

Expression of a heptelidic acid-insensitive recombinant GAPDH from Trichoderma virens, and its biochemical and biophysical characterization.

Trichoderma virens genome harbors two isoforms of GAPDH, one (gGPD) involved in glycolysis and the other one (vGPD) in secondary metabolism. vGPD is expressed as part of the "vir" cluster responsible for the biosynthesis of volatile sesquiterpenes. The secondary metabolism-associated GAPDH is tolerant to the anti-cancer metabolite heptelidic acid (HA), produced by T. virens. Characterizing the HA-tolerant form of GAPDH, thus has implications in cancer therapy. In order to get insight into the mechanism of HA-tolerance of vGPD, we have purified recombinant form of this protein. The protein displays biochemical and biophysical characteristics analogous to the gGPD isoform. It exists as a tetramer with Tm of about 56.5 °C, and displays phosphorylation enzyme activity with Km and Kcat of 0.38 mM and 2.55 sec-1, respectively. The protein weakly binds to the sequence upstream of the vir4 gene that codes for the core enzyme (a terpene cyclase) of the "vir" cluster. The EMSA analysis indicates that vGPD may not act as a transcription factor driving the "vir" cluster, at least not by directly binding to the promoter region. We also succeeded in obtaining small crystals of this protein. We have constructed structural models of vGPD and gGPD of T. virens. In silico constrained docking analysis reveals weaker binding of heptelidic acid in vGPD, compared to gGPD protein.

TBRG-1 a Ras-like protein in Trichoderma virens involved in conidiation, development, secondary metabolism, mycoparasitism, and biocontrol unveils a new family of Ras-GTPases.

Ras-GTPases are nucleotide hydrolases involved in key cellular processes. In fungi, Ras-GTPases regulate conidiation, development, virulence, and interactions with other fungi or plants. Trichoderma spp. are filamentous saprophytic fungi, widely distributed along all latitudes, characterized by their rapid growth and metabolic diversity. Many species of this genus interact with other fungi, animals or plants. Furthermore, these fungi are used as biocontrol agents due to their ability to antagonize phytopathogenic fungi and oomycetes, through competence, antibiosis, and parasitism. However, the genetic and molecular regulation of these processes is scarcely described in these fungi. In this work, we investigated the role of the gene tbrg-1 product (GenBank accession number XP_013956100; JGI ID: Tv_70852) of T. virens during its interaction with other fungi and plants. Sequence analyses predicted that TBRG-1 bears the characteristic domains of Ras-GTPases; however, its size (1011 aa) is 3- to 4-times bigger compared with classical GTPases. Interestingly, phylogenetic analyses grouped the TBRG-1 protein with hypothetical proteins of similar sizes, sharing conserved regions; whereas other known Ras-GTPases were perfectly grouped with their respective families. These facts led us to classify TBRG-1 into a new family of Ras-GTPases, the Big Ras-GTPases (BRG). Therefore, the gene was named tbrg-1 (TrichodermaBigRas-GTPase-1). Quantification of conidia and scanning electron microscopy showed that the mutants-lacking tbrg-1 produced less conidia, as well as a delayed conidiophore development compared to the wild-type (wt). Moreover, a deregulation of conidiation-related genes (con-10, con-13, and stuA) was observed in tbrg-1-lacking strains, which indicates that TBRG-1 is necessary for proper conidiophore and conidia development. Furthermore, the lack of tbrg-1 affected positively the antagonistic capability of T. virens against the phytopathogens Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum, which was consistent with the expression patterns of mycoparasitism-related genes, sp1 and cht1, that code for a protease and for a chitinase, respectively. Furthermore, the antibiosis effect of mycelium-free culture filtrates of Δtbrg-1 against R. solani was considerably enhanced. The expression of secondary metabolism-related genes, particularly gliP, showed an upregulation in Δtbrg-1, which paralleled an increase in gliotoxin production as compared to the wt. These results indicate that TBRG-1 plays a negative role in secondary metabolism and antagonism. Unexpectedly, the biocontrol activity of Δtbrg-1 was ineffective to protect the tomato seeds and seedlings against R. solani. On the contrary, Δtbrg-1 behaved like a plant pathogen, indicating that TBRG-1 is probably implicated in the recognition process for establishing a beneficial relationship with plants.

Improved enzymatic hydrolysis of hardwood and cellulase stability by biomass kraft lignin-based polyoxyethylene ether.

Water-soluble kraft lignin-based polyoxyethylene ether (KL-PEG), synthesized from the black liquor of kraft pulping and PEG, was used to improve the enzymatic hydrolysis efficiency of dilute acid pretreated (DA-pretreated) Eucalyptus hardwood and cellulase stability. The physicochemical properties of KL-PEG polymer such as solubility, surface tension, charge and aggregation behavior in the solution were first studied. KL-PEG could enhance the enzymatic hydrolysis of Avicel and DA-pretreated Eucalyptus from 63.6% and 58.3% to 78.5% and 93.8%, respectively. The enzymatic activity of cellulase after the enzymatic hydrolysis of Avicel and DA-pretreated Eucalyptus for 72 h remained approximately 84% and 44% in the presence of KL-PEG polymer. KL-PEG could improve the stability and longevity of the cellulase, facilitate the recovery and save the amount of cellulase. The efficient utilization of the pulping black liquor lignin was of great significance to alleviate the pressure brought by the shortage of petrochemical resources, and build an energy-saving and low-carbon society.

Kinetic and molecular dynamics study of inhibition and transglycosylation in Hypocrea jecorina family 3 ß-glucosidases.

ß-Glucosidases enhance enzymatic biomass conversion by relieving cellobiose inhibition of endoglucanases and cellobiohydrolases. However, the susceptibility of these enzymes to inhibition and transglycosylation at high glucose or cellobiose concentrations severely limits their activity and, consequently, the overall efficiency of enzyme mixtures. We determined the impact of these two processes on the hydrolytic activity of the industrially relevant family 3 ß-glucosidases from Hypocrea jecorina, HjCel3A and HjCel3B, and investigated the underlying molecular mechanisms through kinetic studies, binding free energy calculations, and molecular dynamics (MD) simulations. HjCel3B had a 7-fold higher specificity for cellobiose than HjCel3A but greater tendency for glucose inhibition. Energy decomposition analysis indicated that cellobiose has relatively weak electrostatic interactions with binding site residues, allowing it to be easily displaced by glucose and free to inhibit other hydrolytic enzymes. HjCel3A is, thus, preferable as an industrial ß-glucosidase despite its lower activity caused by transglycosylation. This competing pathway to hydrolysis arises from binding of glucose or cellobiose at the product site after formation of the glycosyl-enzyme intermediate. MD simulations revealed that binding is facilitated by hydrophobic interactions with Trp-37, Phe-260, and Tyr-443. Targeting these aromatic residues for mutation to reduce substrate affinity at the product site would therefore potentially mitigate transglycosidic activity. Engineering improved variants of HjCel3A and other structurally similar ß-glucosidases would have a significant economic effect on enzymatic biomass conversion in terms of yield and production cost as the process can be consequently conducted at higher substrate loadings.

Enantioselective Binding of Propranolol and Analogues Thereof to Cellobiohydrolase Cel7A.

At the catalytic site for the hydrolysis of cellulose the enzyme cellobiohydrolase Cel7A binds the enantiomers of the adrenergic beta-blocker propranolol with different selectivity. Methyl-to-hydroxymethyl group modifications of propranolol, which result in higher affinity and improved selectivity, were herein studied by 1 H,1 H and 1 H,13 C scalar spin-spin coupling constants as well as utilizing the nuclear Overhauser effect (NOE) in conjunction with molecular dynamics simulations of the ligands per se, which showed the presence of all-antiperiplanar conformations, except for the one containing a vicinal oxygen-oxygen arrangement governed by the gauche effect. For the ligand-protein complexes investigated by NMR spectroscopy using, inter alia, transferred NOESY and saturation-transfer difference (STD) NMR experiments the S-isomers were shown to bind with a higher affinity and a conformation similar to that preferred in solution, in contrast to the R-isomer. The fact that the S-form of the propranolol enantiomer is pre-arranged for binding to the protein is also observed for a crystal structure of dihydroxy-(S)-propranolol and Cel7A presented herein. Whereas the binding of propranolol is entropy driven, the complexation with the dihydroxy analogue is anticipated to be favored also by an enthalpic term, such as for its enantiomer, that is, dihydroxy-(R)-propranolol, because hydrogen-bond donation replaces the corresponding bonding from hydroxyl groups in glucosyl residues of the natural substrate. In addition to a favorable entropy component, albeit lesser in magnitude, this represents an effect of enthalpy-to-entropy compensation in ligand-protein interactions.

Heterologous Expression and Characterization of an Acidic GH11 Family Xylanase from Hypocrea orientalis.

A gene encoding glycoside hydrolase family 11 xylanase (HoXyn11B) from Hypocrea orientalis EU7-22 was expressed in Pichia pastoris with a high activity (413 IU/ml). HoXyn11B was partly N-glycosylated and appeared two protein bands (19-29 kDa) on SDS-PAGE. The recombinant enzyme exhibited optimal activity at pH 4.5 and 55 °C, and retained more than 90% of the original activity after incubation at 50 °C for 60 min. The determined apparent K m and V max values using beechwood xylan were 10.43 mg/ml and 3246.75 IU/mg, respectively. The modes of action of recombinant HoXyn11B on xylo-oligosaccharides (XOSs) and beechwood xylan were investigated by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which indicated that the modes of action of HoXyn11B are different from HoXyn11A since it is able to release a significant amount of xylose from various substrates. This study provides an opportunity to better understand the hydrolysis mechanisms of xylan by xylanases from Trichoderma.

Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution.

Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 °C increase in Tm and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 °C. The X-ray crystal structure of the catalytic domain was determined at 2.1 Å resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites.

The influence of different linker modifications on the catalytic activity and cellulose affinity of cellobiohydrolase Cel7A from Hypocrea jecorina.

Various cellulases consist of a catalytic domain connected to a carbohydrate-binding module (CBM) by a flexible linker peptide. The linker if often strongly O-glycosylated and typically has a length of 20-50 amino acid residues. Functional roles, other than connecting the two folded domains, of the linker and its glycans, have been widely discussed, but experimental evidence remains sparse. One of the most studied cellulose degrading enzymes is the multi-domain cellobiohydrolase Cel7A from Hypocrea jecorina. Here, we designed variants of Cel7A with mutations in the linker region to elucidate the role of the linker. We found that moderate modification of the linker could result in significant changes in substrate affinity and catalytic efficacy. These changes were quite different for different linker variants. Thus, deletion of six residues near the catalytic domain had essentially no effects on enzyme function. Conversely, a substitution of four glycosylation sites near the middle of the linker reduced substrate affinity and increased maximal turnover. The observation of weaker binding provides some support of recent suggestions that linker glycans may be directly involved in substrate interactions. However, a variant with several inserted glycosylation sites near the CBM also showed lower affinity for the substrate compared to the wild-type, and we suggest that substrate interactions of the glycans depend on their exact location as well as other factors such as changes in structure and dynamics of the linker peptide.

High-resolution structure of a lytic polysaccharide monooxygenase from Hypocrea jecorina reveals a predicted linker as an integral part of the catalytic domain.

For decades, the enzymes of the fungus Hypocrea jecorina have served as a model system for the breakdown of cellulose. Three-dimensional structures for almost all H. jecorina cellulose-degrading enzymes are available, except for HjLPMO9A, belonging to the AA9 family of lytic polysaccharide monooxygenases (LPMOs). These enzymes enhance the hydrolytic activity of cellulases and are essential for cost-efficient conversion of lignocellulosic biomass. Here, using structural and spectroscopic analyses, we found that native HjLPMO9A contains a catalytic domain and a family-1 carbohydrate-binding module (CBM1) connected via a linker sequence. A C terminally truncated variant of HjLPMO9A containing 21 residues of the predicted linker was expressed at levels sufficient for analysis. Here, using structural, spectroscopic, and biochemical analyses, we found that this truncated variant exhibited reduced binding to and activity on cellulose compared with the full-length enzyme. Importantly, a 0.95-Å resolution X-ray structure of truncated HjLPMO9A revealed that the linker forms an integral part of the catalytic domain structure, covering a hydrophobic patch on the catalytic AA9 module. We noted that the oxidized catalytic center contains a Cu(II) coordinated by two His ligands, one of which has a His-brace in which the His-1 terminal amine group also coordinates to a copper. The final equatorial position of the Cu(II) is occupied by a water-derived ligand. The spectroscopic characteristics of the truncated variant were not measurably different from those of full-length HjLPMO9A, indicating that the presence of the CBM1 module increases the affinity of HjLPMO9A for cellulose binding, but does not affect the active site.

A quenched-flow system for measuring heterogeneous enzyme kinetics with sub-second time resolution.

Even though many enzyme processes occur at the interface of an insoluble substrate, these reactions are generally much less studied than homogenous enzyme reactions in the aqueous bulk. Interfacial (or heterogeneous) enzyme reactions involve several reaction steps, and the established experimental approach to elucidate multi-step reactions is transient (or pre steady-state) kinetics. A key requirement for pre steady-state measurements is good time resolution, and while this has been amply achieved in different commercial instruments, they are generally not applicable to precipitating suspensions of insoluble substrate. Perhaps for this reason, transient kinetics has rarely been reported for heterogeneous enzyme reactions. Here, we describe a quenched-flow system using peristaltic pumps and stirred substrate suspensions with a dead time below 100ms. The general performance was verified by alkali catalyzed hydrolysis of 2,4-dinitrophenyl acetate (DNPA), and the applicability to heterogeneous reactions was documented by two cellulases (Cel7A and Cel7B) acting on suspensions of microcrystalline cellulose (Avicel) at different loads up to 15g/l. The results showed distinctive differences between the two enzymes. In particular, we found that endo-lytic Cel7B combined very quickly with the substrate and reached the maximal activity within the dead-time of the instrument. Conversely, exo-lytic Cel7A showed a much slower initiation with maximal activity after 5-8s and a 10-fold lower turnover. We suggest that the instrument may provide an important tool in attempts to elucidate the mechanism of cellulases and other enzymes' action on insoluble substrate.

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

Cellulose degrading fungi such as Hypocrea jecorina secrete several cellulases including the two cellobiohydrolases (CBHs) Cel6A and Cel7A. The two CBHs differ in catalytic mechanism, attack different ends, belong to different families, but are both processive multi-domain enzymes that are essential in the hydrolysis of cellulose. Here we present a direct kinetic comparison of these two enzymes acting on insoluble cellulose. We used both continuous- and end-point assays under either enzyme- or substrate excess, and found distinct kinetic differences between the two CBHs. Cel6A was catalytically superior with a maximal rate over four times higher than Cel7A. Conversely, the ability of Cel6A to attack diverse structures on the cellulose surface was inferior to Cel7A. This latter difference was pronounced as the density of attack sites for Cel7A was almost an order of magnitude higher compared to Cel6A. We conclude that Cel6A is a fast but selective enzyme and that Cel7A is slower, but promiscuous. One consequence of this is that Cel6A is more effective when substrate is plentiful, while Cel7A excels when substrate is limiting. These diverse kinetic properties of Cel6A and Cel7A might elucidate why both cellobiohydrolases are prominent in cellulolytic degrading fungi.

Inactivation kinetics and conformation change of Hypocrea orientalis ß-glucosidase with guanidine hydrochloride.

The relationship between unfolding and inactivation of Hypocrea orientalis ß-glucosidase has been investigated for the first time. The secretion of ß-glucosidase from H. orientalis is induced by raw cassava residues. The enzyme was 75 kD without glycosylation. Guanidine hydrochloride (GuHCl) could reversibly inactivate the enzyme with an estimated IC50 value of 0.4 M. The inactivation kinetics model by GuHCl has been established and the microscopic inactivation rate constants are determined. The values of forward inactivation rate constants of free enzyme are found to be larger than that of substrate-enzyme complex suggesting the enzyme could be protected by substrate during denaturation. Conformational change of the enzyme during denaturation is observed as the intrinsic fluorescence emission peaks appeared red-shift (334-354 nm) with intensity decreased following increase of GuHCl concentrations. Inactivation extent is found to be greater than conformation change of the whole enzyme, indicating that the active site of H. orientalis ß-glucosidase might be a more flexible region than the whole enzyme.

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

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 103 -104 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.

Endo/exo-synergism of cellulases increases with substrate conversion.

Synergy between cellulolytic enzymes is important for their industrial utilization, and numerous studies have addressed the problem of how to optimize the composition of enzyme cocktails with respect to this. The degree of synergy (DS) may change with substrate conversion, and some studies have suggested a maximum in DS early in the process. Here, we systematically investigated interrelationships of DS and conversion in a model system covering a wide range of experimental conditions. The results did not reveal any correlation between DS and contact time, but when plotted against the degree of substrate conversion we saw a systematic increase in DS. We suggest that this is linked to a decreasing reactivity of the substrate. Hence, synergy became increasingly important as the recalcitrance of the remaining substrate grew. Such conversion dependent changes in DS appear to be important both in mechanistic studies and attempts to find industrial enzymes blends with optimal synergy. Biotechnol. Bioeng. 2017;114: 696-700. © 2016 Wiley Periodicals, Inc.

Anomeric Selectivity and Product Profile of a Processive Cellulase.

Cellobiohydrolases (CBHs) make up an important group of enzymes for both natural carbon cycling and industrial deconstruction of lignocellulosic biomass. The consecutive hydrolysis of one cellulose strand relies on an intricate pattern of enzyme-substrate interactions in the long, tunnel-shaped binding site of the CBH. In this work, we have investigated the initial complexation mode with cellulose of the most thoroughly studied CBH, Cel7A from Hypocrea jecorina (HjCel7A). We found that HjCel7A predominantly produces glucose when it initiates a processive run on insoluble microcrystalline cellulose, confirming the validity of an even and odd product ratio as an estimate of processivity. Moreover, the glucose released from cellulose was predominantly α-glucose. A link between the initial binding mode of the enzyme and the reducing end configuration was investigated by inhibition studies with the two anomers of cellobiose. A clear preference for ß-cellobiose in product binding site +2 was observed for HjCel7A, but not the homologous endoglucanase, HjCe7B. Possible relationships between this anomeric preference in the product site and the prevalence of odd-numbered initial-cut products are discussed, and a correlation between processivity and anomer selectivity is proposed.

Structural and functional studies of the glycoside hydrolase family 3 ß-glucosidase Cel3A from the moderately thermophilic fungus Rasamsonia emersonii.

The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. ß-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the ß-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the ß-glucosidase from H. jecorina (HjCel3A) with the ß-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 ß-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.