Positive role of non-catalytic proteins on mitigating inhibitory effects of lignin and enhancing cellulase activity in enzymatic hydrolysis: Application, mechanism, and prospective.

Fermentable sugar production from lignocellulosic biomass has received considerable attention and has been dramatic progress recently. However, due to low enzymatic hydrolysis (EH) yields and rates, a high dosage of the costly enzyme is required, which is a bottleneck for commercial applications. Over the last decades, various strategies have been developed to reduce cellulase enzyme costs. The progress of the non-catalytic additive proteins in mitigating inhibition in EH is discussed in detail in this review. The low efficiency of EH is mostly due to soluble lignin compounds, insoluble lignin, and harsh thermal and mechanical conditions of the EH process. Adding non-catalytic proteins into the EH is considered a simple and efficient approach to boost hydrolysis yield. This review discussed the multiple mechanical steps involved in the EH process. The effect of physicochemical properties of modified lignin on EH and its interaction with cellulase and cellulose are identified and discussed, which include hydrogen bonding, hydrophobic, electrostatic, and cation-π interactions, as well as physical barriers. Moreover, the effects of different conditions of EH that lead to cellulase deactivation by thermal and mechanical mechanisms are also explained. Finally, recent advances in the development, potential mechanisms, and economic feasibility of non-catalytic proteins on EH are evaluated and perspectives are presented.

Characterization of a novel GH2 family α-L-arabinofuranosidase from hyperthermophilic bacterium Thermotoga thermarum.

The 2,367-bp ORF of TtAFase from Thermotoga thermarum DSM 5069 encodes a calculated 90-kDa α-L-arabinofuranosidase (TtAFase), which does not belonging to any reported glycosyl hydrolase families α-L-arabinofuranosidases in the database and represents a novel one of glycosyl hydrolase family 2. The purified recombinant TtAFase produced in Escherichia coli BL21 (DE3) had optimum activity at pH 5.5 and at 80 °C. It was stable up to 80 °C and from pH 4.5-8.5. Kinetic experiments at 80 °C with p-nitrophenyl α-L-arabinofuranoside as a substrate gave a K m of 0.77 mM, V max of 2.3 µmol mg(-1) min(-1) and k cat of 4.5 s(-1). The enzyme had no apparent requirement of metal ions for activity, and its activity was significantly inhibited by Cu(2+) or Zn(2+).

Advances and perspectives on mass transfer and enzymatic hydrolysis in the enzyme-mediated lignocellulosic biorefinery: A review.

Enzymatic hydrolysis is a critical process for the cellulase-mediated lignocellulosic biorefinery to produce sugar syrups that can be converted into a whole range of biofuels and biochemicals. Such a process operating at high-solid loadings (i.e., scarcely any free water or roughly ≥ 15% solids, w/w) is considered more economically feasible, as it can generate a high sugar concentration at low operation and capital costs. However, this approach remains restricted and incurs "high-solid effects", ultimately causing the lower hydrolysis yields with increasing solid loadings. The lack of available water leads to a highly viscous system with impaired mixing that exhibits strong transfer resistance and reaction limitation imposed on enzyme action. Evidently, high-solid enzymatic hydrolysis involves multi-scale mass transfer and multi-phase enzyme reaction, and thus requires a synergistic perspective of transfer and biotransformation to assess the interactions among water, biomass components, and cellulase enzymes. Porous particle characteristics of biomass and its interface properties determine the water form and distribution state surrounding the particles, which are summarized in this review aiming to identify the water-driven multi-scale/multi-phase bioprocesses. Further aided by the cognition of rheological behavior of biomass slurry, solute transfer theories, and enzyme kinetics, the coupling effects of flow-transfer-reaction are revealed under high-solid conditions. Based on the above basic features, this review lucidly explains the causes of high-solid hydrolysis hindrances, highlights the mismatched issues between transfer and reaction, and more importantly, presents the advanced strategies for transfer and reaction enhancements from the viewpoint of process optimization, reactor design, as well as enzyme/auxiliary additive customization.

Advances in organosolv modified components occurring during the organosolv pretreatment of lignocellulosic biomass.

The valorization of organosolv pretreatment (OP) is a required approach to the industrialization of the current enzyme-mediated lignocellulosic biorefinery. Recent literature has demonstrated that the solvolysis happening in the OP can modify the soluble components into value-added active compounds, namely organosolv modified lignin (OML) and organosolv modified sugars (OMSs), in addition to protecting them against excessive degradation. Among them, the OML is coincidental with the "lignin-first" strategy that should render a highly reactive lignin enriched with ß-O-4 linkages and less condensed structure by organosolv grafting, which is desirable for the transformation into phenolic compounds. The OMSs are valuable glycosidic compounds mainly synthesized by trans-glycosylation, which can find potential applications in cosmetics, foods, and healthcare. Therefore, a state-of-the-art OP holds a big promise of lowering the process cost by the valorization of these active compounds. Recent advances in organosolv modified components are reviewed, and perspectives are made for addressing future challenges.

Surfactants facilitated glycerol organosolv pretreatment of lignocellulosic biomass by structural modification for co-production of fermentable sugars and highly reactive lignin.

This study reported that surfactants could facilitate the organosolv pretreatment of lignocellulosic biomass (LCB) to produce fermentable sugars and highly active lignin. Under the optimized conditions, the surfactant-assisted glycerol organosolv (saGO) pretreatment achieved 80.7% delignification with a retention of 93.4% cellulose and 83.0% hemicellulose. The saGO pretreated substrate exhibited an excellent enzymatic hydrolyzability, achieving 93% of glucose yield from the enzymatic hydrolysis at 48 h. Structural analysis showed that the saGO lignin contained rich ß-O-4 bondings with less repolymerization and lower phenolic hydroxyl groups, thus forming highly reactive lignin fragments. The analysis evidenced that the surfactant graft the lignin by structural modification, which was responsible for the excellent substrate hydrolyzability. The co-production of fermentable sugars and organosolv lignin almost recovered a gross energy (87.2%) from LCB. Overall, the saGO pretreatment holds a lot of promise for launching a novel pathway towards lignocellulosic fractionation and lignin valorization.

Metabolic Engineering for the Comprehensive Utilization of N,N-Dimethylformamide-Containing Wastewater.

N, N-dimethylformamide is frequently present in industrial wastewater and is environmentally detrimental. The current study aims to assess the utilization and biodegradation of N, N-dimethylformamide-containing wastewater to lessen the associated environmental load. Results show that addition of wastewater containing N, N-dimethylformamide to Trichoderma reesei fermentation media enhances cellulase production and facilitates cellulose hydrolysis. However, N, N-dimethylformamide is a cellulase enhancer that is not degraded during cellulase production in T. reesei fermentation and is retained in the N, N-dimethylformamide-enhanced cellulase solution. Indeed, the cellulosic sugar solution generated via lignocellulose hydrolysis with N, N-dimethylformamide-enhanced cellulase retains N, N-dimethylformamide. We further identified three core enzyme modules─N, N-dimethylformamidase, dimethylamine dehydrogenase, and methylamine dehydrogenase enzyme─which were inserted into Escherichia coli to develop metabolically engineered strains. These strains degraded N, N-dimethylformamide and produced succinate using N, N-dimethylformamide-enhanced cellulosic sugar as the substrate. The platform described here can be applied to effectively convert waste into valuable bioproducts.

Effect of residual extractable lignin on acetone-butanol-ethanol production in SHF and SSF processes.

BACKGROUND: Lignin plays an important role in biochemical conversion of biomass to biofuels. A significant amount of lignin is precipitated on the surface of pretreated substrates after organosolv pretreatment. The effect of this residual lignin on enzymatic hydrolysis has been well understood, however, their effect on subsequent ABE fermentation is still unknown. RESULTS: To determine the effect of residual extractable lignin on acetone-butanol-ethanol (ABE) fermentation in separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes, we compared ABE production from ethanol-washed and unwashed substrates. The ethanol organosolv pretreated loblolly pine (OPLP) was used as the substrate. It was observed that butanol production from OPLP-UW (unwashed) and OPLP-W (washed) reached 8.16 and 1.69 g/L, respectively, in SHF. The results showed that ABE production in SHF from OPLP-UW prevents an "acid crash" as compared the OPLP-W. In SSF process, the "acid crash" occurred for both OPLP-W and OPLP-UW. The inhibitory extractable lignin intensified the "acid crash" for OPLP-UW and resulted in less ABE production than OPLP-W. The addition of detoxified prehydrolysates in SSF processes shortened the fermentation time and could potentially prevent the "acid crash". CONCLUSIONS: The results suggested that the residual extractable lignin in high sugar concentration could help ABE production by lowering the metabolic rate and preventing "acid crash" in SHF processes. However, it became unfavorable in SSF due to its inhibition of both enzymatic hydrolysis and ABE fermentation with low initial sugar concentration. It is essential to remove extractable lignin of substrates for ABE production in SSF processes. Also, a higher initial sugar concentration is needed to prevent the "acid crash" in SSF processes.

Factors associated with a successful external cephalic version in the early ECV trial.

OBJECTIVES: The objective of this research was to determine factors that were associated with a successful external cephalic version (ECV) procedure. METHODS: We undertook a secondary analysis of data from a randomized controlled trial, The Early External Cephalic Version (Pilot) Trial. In this secondary analysis, we included data for the subset of 178 women who had an ECV as part of the pilot trial (123 nulliparous women with any breech presentation and 55 multiparous women with a frank breech presentation only). Using this dataset, we began with two separate univariate analyses, one of characteristics that could be determined before undertaking a procedure, and the other of factors associated with the ECV procedure itself. Variables that had a P value of < or = 0.1 in the univariate analyses were included in two separate logistic regression models, one for preprocedural and one for procedural factors, using a backward elimination approach. RESULTS: Multiparity and a non-engaged presenting part were significant preprocedural predictors of ECV success. Procedural factors predictive of ECV success included lower reported maternal pain scores during the procedure, a single attempt at ECV, and a more mobile fetus. CONCLUSION: Non-engagement of the presenting part was the only modifiable factor predicting ECV success that was identified in this analysis, and it supports the hypothesis that beginning the ECV procedure earlier in pregnancy, prior to engagement, may have merit. The Early ECV 2 Trial is in progress and will further test this hypothesis.

Effect of overliming and activated carbon detoxification on inhibitors removal and butanol fermentation of poplar prehydrolysates.

BACKGROUND: Biomass prehydrolysates from dilute acid pretreatment contain a considerable amount of fermentable sugars for biofuels production. However, carbonyl degradation compounds present severe toxicity to fermentation microbes. Furans (such as furfural and hydroxymethylfurfural), aliphatic acids (such as acetic acid, formic acid and levulinic acid) and phenolic compounds (such as vanillin and syringaldehyde) have been suggested to be the main inhibitors in biomass prehydrolysates. However, no single compound has been determined as the dominant toxic inhibitor. The effects of various detoxification methods on inhibitors removal have not been fully understood. RESULTS: The effects of overliming and activated carbon (AC) detoxification on the removal of inhibitors and butanol fermentation of the poplar prehydrolysates were investigated. Gas chromatography-mass spectrometry (GC/MS) was used to identify and quantify 46 carbonyl compounds as potential inhibitors. It was observed that overliming and AC treatment alone did not make the prehydrolysates fermentable with Clostridium saccharobutylicum. The sequential overliming and AC resulted in a remarkable fermentability and a high butanol yield at 0.22 g g-1 sugar. The inhibitor removal in the prehydrolysates treated by overliming and AC was also examined by GC/MS. Overliming removed 75.6% of furan derivatives and 68.1% of aromatic monomers. In comparison, AC (5.0% w/v) removed 77.9% of furan derivatives and 98.6% of aromatic monomers. In addition, overliming removed much more 2,5-furandicarboxyaldehyde, 5-ethylfuran-2-carbaldehyde and 2,5-hexanedione than AC did. On the contrary, AC could remove considerably more phenolic acids than overliming. In the sequential detoxification, both dialdehydes/diketones and phenolic acids were extensively removed. This could be the main reason why the sequential detoxification enabled a remarkable ABE fermentation for the prehydrolysates. CONCLUSIONS: This study indicated that the effect of overliming and AC treatment on inhibitors removal was related to their chemical structures. Overliming removed more dialdehydes and diketones than AC treatment, while AC removed more phenolic acids than overliming. Sequential overliming and AC treatment were required to make the prehydrolysates fermentable with C. saccharobutylicum. The study also suggested different detoxification method was needed for ABE fermentation of the prehydrolysate as compared to ethanol fermentation.

Synergistic effects of pH and organosolv lignin addition on the enzymatic hydrolysis of organosolv-pretreated loblolly pine.

The effect of ethanol organosolv lignin (EOL) on enzymatic hydrolysis was examined at pH 4.8-6.0. The addition of EOL prepared from sweetgum enhanced the enzymatic hydrolysis of organosolv-pretreated loblolly pine (OPLP) by 38.8% and 88.0% at pH 4.8 and 5.6, respectively. The addition of EOL prepared from loblolly pine inhibited the enzymatic hydrolysis of OPLP at pH 4.8 but improved it by 43.0% at pH 5.6. This suggests that the addition of EOL and increase in pH act synergistically to improve the enzymatic hydrolysis of OPLP. The effect of EOL addition on cellulase adsorption onto residual lignins was examined. The results revealed that increasing the pH intensified the suppression of non-productive binding between enzymes and residual lignins by EOL. The potential stabilization effects of EOL on enzymes can contribute to the improvement of enzymatic hydrolysis with EOL at higher pH.

Effect of fermentation stillage of food waste on bioelectricity production and microbial community structure in microbial fuel cells.

A single-chamber microbial fuel cell (MFC) was used in this study to treat recycled stillage obtained from food waste ethanol fermentation. Corresponding substrates inside the system were evaluated by fluorescence spectra, and microbial communities were also investigated. Results demonstrated that output voltage and current, respectively, reached 0.29 V and 1.4 mA with an external resistance of 200 Ω. Corresponding total organic carbon and chemical oxygen demand removal efficiency reached more than 50% and 70%, respectively. Results of fluorescence spectra demonstrated that tryptophan-like aromatic, soluble microbial by-product-like and humic acid-like substances accumulated and were not easily degraded. Microbial community analysis by high-throughput sequence indicated that Advenella and Moheibacter occupied the highest proportion among all genera at the anode instead of Geobacter. These results may be due to complicated accumulated stillage, and potential tetracyclines possibly influenced microbial communities. Details on how stillage affects MFC operation should be further studied, and a solution on relieving effects should be established.

Detoxification of Organosolv-Pretreated Pine Prehydrolysates with Anion Resin and Cysteine for Butanol Fermentation.

Bioconversion of lignocellulose to biofuels suffers from the degradation compounds formed during pretreatment and acid hydrolysis. In order to achieve an efficient biomass to biofuel conversion, detoxification is often required before enzymatic hydrolysis and microbial fermentation. Prehydrolysates from ethanol organosolv-pretreated pine wood were used as substrates in butanol fermentation in this study. Six detoxification approaches were studied and compared, including overliming, anion exchange resin, nonionic resin, laccase, activated carbon, and cysteine. It was observed that detoxification by anion exchange resin was the most effective method. The final butanol yield after anion exchange resin treatment was comparable to the control group, but the fermentation was delayed for 72 h. The addition of Ca(OH)2 was found to alleviate this delay and improve the fermentation efficiency. The combination of Ca(OH)2 and anion exchange resin resulted in completion of fermentation within 72 h and acetone-butanol-ethanol (ABE) production of 11.11 g/L, corresponding to a yield of 0.21 g/g sugar. The cysteine detoxification also resulted in good detoxification performance, but promoted fermentation towards acid production (8.90 g/L). The effect of salt on ABE fermentation was assessed and the possible role of Ca(OH)2 was to remove the salts in the prehydrolysates by precipitation.

Concise review on ethanol production from food waste: development and sustainability.

The development of sustainable bioethanol fuel production from food waste has increasingly become an attractive topic. Food waste is recognized as the most available and costless feedstock. Therefore, ethanol production has been adopted as cost-efficient and an ecological way for FW disposal. This paper reviewed the microorganisms utilized for ethanol fermentation, the effect of enzymatic hydrolysis on ethanol concentration, optimization of accurate process parameters, and recycling of huge volumes of stillage for ethanol production towards reducing any incurred environmental burdens and minimizing the cost. The statistical tools which may enhance the process efficiency had been presented. Also, the perspective and the future development were introduced. All these aimed to fully utilize the food waste and also reduce the cost for side-product in this process; proper operation conditions and the control methods for stillage recycling were considered as the methods to improve ethanol fermentation from food waste.

Evaluating the distribution of cellulases and the recycling of free cellulases during the hydrolysis of lignocellulosic substrates.

The recycling of cellulase enzymes is one potential strategy for reducing the cost of the enzymatic hydrolysis step during the bioconversion of lignocellulosics to ethanol. To determine the influence of lignin on the post-hydrolysis distribution of cellulase enzymes between the liquid and solid phases, the hydrolysis of Avicel was compared to an organosolv-pretreated Douglas fir substrate with a lignin content of 3.0%. After a 12 h hydrolysis reaction on Avicel, 90% of the added cellulases (including beta-glucosidases) remained "free" in the liquid phase compared to only 65% in the case of the hydrolysis of the organosolv-pretreated Douglas fir substrate. The readsorption of free cellulases by supplementing the hydrolysis reaction with fresh substrate was explored as a potential means of recovering the free cellulases that remain in the liquid phase after hydrolysis. The Langmuir adsorption isotherm was used to develop a model predicting that 82% of the free cellulases could be recovered via readsorption onto fresh substrates during the hydrolysis of an ethanol-pretreated mixed softwood substrate with a lignin content of 6%. Recoverable free cellulase values of 85% and 88% based on cellulase activity and protein content, respectively, were obtained after experimental verification of the model. The readsorption of free cellulases onto fresh lignocellulosic substrates was shown to be an effective method for free enzyme recovery.

Recycling cellulases during the hydrolysis of steam exploded and ethanol pretreated Lodgepole pine.

Recycling of cellulases is one way of reducing the high cost of enzymes during the bioconversion process. The effects of surfactant addition on enzymatic hydrolysis and the potential recycling of cellulases were studied during the hydrolysis of steam exploded Lodgepole pine (SELP) and ethanol pretreated Lodgepole pine (EPLP). Three cellulase preparations (Celluclast, Spezyme CP, and MSUBC) were evaluated to determine their hydrolysis efficiencies over multiple rounds of recycling. The surfactant, Tween 80, significantly increased the yield from 63% to 86% during the hydrolysis of the SELP substrate. The addition of surfactant to the hydrolysis of the EPLP substrate increased the free enzymes in the supernatant from 71% of the initial protein to 96%. Based on the Langmuir adsorption constants, cellulases (Celluclast and Spezyme CP) from Trichoderma reesei showed a higher affinity (3.48 mL/mg and 3.17 mL/mg) for the EPLP substrate than did the Penicillium enzyme (0.62 mg/mg). The Trichoderma reesei enzyme was used in four successive rounds of enzyme recycling using surfactant addition and readsorption onto fresh substrates during the hydrolysis of EPLP. In contrast, the Penicillium-derived enzyme preparation (MSUBC) could only be recycled once. When the same recycling strategy was carried out using the SELP substrate, the hydrolysis yield declined during each enzyme recycling round. These results suggested that the higher lignin content of the SELP substrate, and the low affinity of cellulases for the SELP substrate limited enzyme recycling by readsorption onto fresh substrates.

Stimulation and inhibition of enzymatic hydrolysis by organosolv lignins as determined by zeta potential and hydrophobicity.

BACKGROUND: Lignin typically inhibits enzymatic hydrolysis of cellulosic biomass, but certain organosolv lignins or lignosulfonates enhance enzymatic hydrolysis. The hydrophobic and electrostatic interactions between lignin and cellulases play critical roles in the enzymatic hydrolysis process. However, how to incorporate these two interactions into the consideration of lignin effects has not been investigated. RESULTS: We examined the physicochemical properties and the structures of ethanol organosolv lignins (EOL) from hardwood and softwood and ascertained the association between lignin properties and their inhibitory and stimulatory effects on enzymatic hydrolysis. The zeta potential and hydrophobicity of EOL lignin samples, isolated from organosolv pretreatment of cottonwood (CW), black willow (BW), aspen (AS), eucalyptus (EH), and loblolly pine (LP), were determined and correlated with their effects on enzymatic hydrolysis of Avicel. EOLs from CW, BW, and AS improved the 72 h hydrolysis yield by 8-12%, while EOLs from EH and LP decreased the 72 h hydrolysis yield by 6 and 16%, respectively. The results showed a strong correlation between the 72 h hydrolysis yield with hydrophobicity and zeta potential. The correlation indicated that the hydrophobicity of EOL had a negative effect and the negative zeta potential of EOL had a positive effect. HSQC NMR spectra showed that ß-O-4 linkages in lignin react with ethanol to form an α-ethoxylated ß-O-4' substructure (A') during organosolv pretreatment. Considerable amounts of C2,6-H2,6 correlation in p-hydroxybenzoate (PB) units were observed for EOL-CW, EOL-BW, and EOL-AS, but not for EOL-EH and EOL-LP. CONCLUSIONS: This study revealed that the effect of lignin on enzymatic hydrolysis is a function of both hydrophobic interactions and electrostatic repulsions. The lignin inhibition is controlled by lignin hydrophobicity and the lignin stimulation is governed by the negative zeta potential. The net effect of lignin depends on the combined influence of hydrophobicity and zeta potential. This study has potential implications in biomass pretreatment for the reduction of lignin inhibition by increasing lignin negative zeta potential and decreasing hydrophobicity.

Acetone-butanol-ethanol production from Kraft paper mill sludge by simultaneous saccharification and fermentation.

Paper mill sludge (PS), a solid waste from pulp and paper industry, was investigated as a feedstock for acetone-butanol-ethanol (ABE) production by simultaneous saccharification and fermentation (SSF). ABE fermentation of paper sludge by Clostridium acetobutylicum required partial removal of ash in PS to enhance its enzymatic digestibility. Enzymatic hydrolysis was found to be a rate-limiting step in the SSF. A total of 16.4-18.0g/L of ABE solvents were produced in the SSF of de-ashed PS with solid loading of 6.3-7.4% and enzyme loading of 10-15FPU/g-glucan, and the final solvent yield reached 0.27g/g sugars. No pretreatment and pH control were needed in ABE fermentation of paper sludge, which makes it an attractive feedstock for butanol production. The results suggested utilization of paper sludge should not only consider the benefits of buffering effect of CaCO3 in fermentation, but also take into account its inhibitory effect on enzymatic hydrolysis.

Heterologous expression of codon optimized Trichoderma reesei Cel6A in Pichia pastoris.

The Cel6A deficiency has become one of the limiting factors for cellulose saccharification in biochemical conversion of cellulosic biomass to fuels and chemicals. The work attempted to use codon optimization to enhance Trichoderma reesei Cel6A expression in Pichia pastoris. Two recombinants P. pastoris GS115 containing AOX1 and GAP promotors were successfully constructed, respectively. The optimal temperatures and pHs of the expressed Cel6A from two recombinants were consistent with each other, were also in the extremely similar range to that reported on the native Cel6A from T. reesei. Based on the shake flask fermentation, AOX1 promotor enabled the recombinant to produce 265U/L and 300mg/L of the Cel6A enzyme, and the GAP promotor resulted in 145U/L and 200mg/L. High cell density fed batch (HCDFB) fermentation significantly improved the enzyme titer (1100U/L) and protein yield (2.0g/L) for the recombinant with AOX1 promotor. Results have showed that the AOX1 promotor is more suitable than the GAP for the Cel6A expression in P. pastoris. And the HCDFB cultivation is a favorable way to express the Cel6A highly in the methanol inducible yeast.

Weak lignin-binding enzymes: a novel approach to improve activity of cellulases for hydrolysis of lignocellulosics.

Economic barriers preventing commercialization of lignocellulose-to-ethanol bioconversion processes include the high cost of hydrolytic enzymes. One strategy for cost reduction is to improve the specific activities of cellulases by genetic engineering. However, screening for improved activity typically uses "ideal" cellulosic substrates, and results are not necessarily applicable to more realistic substrates such as pretreated hardwoods and softwoods. For lignocellulosic substrates, nonproductive binding and inactivation of enzymes by the lignin component appear to be important factors limiting catalytic efficiency. A better understanding of these factors could allow engineering of cellulases with improved activity based on reduced enzyme-lignin interaction ("weak lignin-binding cellulases"). To prove this concept, we have shown that naturally occurring cellulases with similar catalytic activity on a model cellulosic substrate can differ significantly in their affinities for lignin. Moreover, although cellulose-binding domains (CBDs) are hydrophobic and probably participate in lignin binding, we show that cellulases lacking CBDs also have a high affinity for lignin, indicating the presence of lignin-binding sites on the catalytic domain.