Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose.

Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single-molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well-characterized exo-cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a Ki of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate-binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme's velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the "back door" product release site to slow activity and to the "front door" substrate-binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy.

Regulatory function of the novel transcription factor CxrC in Penicillium oxalicum.

Numerous transcription factors (TFs) in ascomycete fungi play crucial roles in cellular processes; however, how most of them function is poorly understood. Here, we identified and characterized a novel TF, CxrC (POX01387), acting downstream of the key TF CxrA, which is essential for plant-biomass-degrading-enzyme (PBDE) production in Penicillium oxalicum. Deletion of cxrC in P. oxalicum significantly affected the production of PBDEs, as well as mycelial growth and conidiospore production. CxrA directly repressed the expression of cxrC after about 12 hr following switch to Avicel culture. CxrC bound the promoters of major PBDE genes and genes involved in conidiospore development. CxrC was found to bind the TSSGTYR core sequence (S: C and G; Y: T and C; R: G and A) of the important cellulase genes cbh1 and eg1. Both N- and C-terminal peptides of CxrC and the CxrC phosphorylation were found to mediate its homodimerization. The conserved motif LPSVRSLLTP (65-74) in CxrC was found to be required for regulating cellulase production. This study reveals novel mechanisms of TF-mediated regulation of the expression of PBDE genes and genes involved in cellular processes in an ascomycete fungus.

Impact of phenolic compound as activators or inhibitors on the enzymatic hydrolysis of cellulose.

The influence of phenolic compounds on the enzymatic hydrolysis of cellulose was studied in depth using spectrophotometric techniques, adsorption analysis and Scanning Electron Microscopy (SEM). In this paper for the first time, both possible interactions between phenolic compounds and the enzyme or the substrate were investigated, with the use of various phenolic compounds, cellulase from T. reesei, and Avicel as cellulose source. Three classes of phenolic compounds have been identified, based on their effect on the hydrolysis of cellulose: inhibitors (quercetin, kaempferol, trans-cinnamic acid, luteolin, ellagic acid), non-inhibitors (p-coumaric acid, rutin, caffeic acid), and activators (ferulic acid, syringic acid, sinapic acid, vanillic acid). Secondly, since various structures of phenolic compounds were tested, a structure - action comprehensive correlation was possible leading to the conclusion that an -OCH3 group was necessary for the activating effect. Finally, based on the adsorption spectra and unique SEM images, a different way of adsorption (either on the enzyme or on the substrate) was noticed, depending on the activating or inhibiting action of the phenolic compound.

Comparative study on the properties of lignin isolated from different pretreated sugarcane bagasse and its inhibitory effects on enzymatic hydrolysis.

Five sugarcane bagasse lignin samples, namely, dilute sulfuric acid (DSAL), sodium hydroxide (SHL), ethanol (EL), hot liquid water (HLWL)-pretreated residual solids, and raw material (cellulolytic enzyme lignin, CEL), were extracted. Comparative studies on the physicochemical properties of isolated lignin, nonproductive adsorption of cellulase by lignin, and its effect on enzymatic hydrolysis was performed. Results showed that the molecular weight and homogeneity of lignin remarkably decreased after pretreatment compared with CEL. Lignin with low negative zeta potential, high phenolic hydroxyl group content and hydrophobicity exhibited strong nonproductive adsorption performance to cellulase. This phenomenon was positively correlated with it's inhibitory effect on enzymatic hydrolysis. Compared with the control (without lignin), the Avicel conversion rate (40 mg lignin/200 mg Avicel) decreased by 10.74%, 9.28%, 8.73%, 4.22%, and 2.80% after digestion of Avicel for 72 h with the presence of EL, SHL, CEL, HLWL, and DSAL, respectively.

Preventing fungal growth on heritage paper with antifungal and cellulase inhibiting magnesium oxide nanoparticles.

Microorganisms such as bacteria, fungi, algae and moulds are highly proficient at colonizing artistic and architectural heritage. The irreparable damage they cause to unique artefacts results in immeasurable cultural and societal losses to our shared cultural heritage, which represent an important social and economic resource for Europe. With the overall aim of preventing fungal deterioration of paper artefacts, we report the use of magnesium oxide nanoparticles (MgO NPs) of average diameter 12 nm as potent antifungal agents against fungi commonly found colonising paper heritage: A. niger, C. cladosporioides and T. reesei. Dispersions of MgO NPs on original 18th century paper samples from the Archives of the Spanish Royal Botanic Garden were effective at preventing fungal colonisation without altering the appearance of the paper artefacts. Importantly, MgO NPs also inhibit cellulase activity in the filamentous fungi T. resei and A. niger, two of the principle biodeteriogens of cellulosic materials. In addition, our report provides three simple new procedures for studying the fungal colonisation prevention properties of nanomaterials on paper samples. Overall this opens the door to the use of colourless, low-cost, and scalable nanomaterials for preventing biodeterioration in cellulose-based artefacts.

Effect of fipronil on biogas production performance during anaerobic digestion of chicken manure and corn straw.

Fipronil is a broad-spectrum insecticide that has a good control effect on pests of commercial poultry. Although many studies have reported the environmental fate of fipronil, the influence of residual fipronil in poultry waste on biogas production has not been further explored yet. In this article, an experimental comparative study on anaerobic digestion (AD) of chicken manure (CM) and corn straw (CS) with different fipronil concentrations (FCs) was carried at 8% of total solid (TS) and mid-temperature (35 ± 1)°C. The results showed that fipronil had a significant effect on biogas production during AD of CM and CS. When the FC is at a low level (≤10 mg·kg-1), the biogas production rate is increased and the digestion period was shortened, while higher FC (≥ 20 mg·kg-1) showed an inhibitory effect. During the monitoring of enzyme activity, low FC showed no significant effect on cellulase and saccharase, but the urease activity increased in the early stage. High FC showed inhibition of activity of cellulase and urease, but the saccharase activity was significantly inhibited until FC reached 40 mg·kg-1. This study also confirms that the environment in anaerobic digester is favorable for the degradation of fipronil, and its half-life is about 15.83 days.

Efficacy of a novel sequential enzymatic hydrolysis of lignocellulosic biomass and inhibition characteristics of monosugars.

Efficient production of sugar monomers from lignocellulose is often hampered by serious bottle-necks in biomass hydrolysis. The present study reveals that ultra-sonication assisted pretreatment following autoclaving, termed as combined pretreatment, can lead to more efficient delignification of lignocellulosic biomass and an open, deformed polysaccharide matrix, found favorable for subsequent enzymatic hydrolysis, is formed. The pattern of inhibition for the enzymatic hydrolysis reaction on combined-pretreated saw dust is identified. Two main inhibition models (competitive and noncompetitive) are proposed and a better fit of experimental values with the theoretical values for the competitive inhibition model validates the proposition that in the present experiment, glucose inhibits the enzymes competitively. Additionally, accuracy of the inhibitory kinetics based models is estimated over a series of enzyme and substrate concentrations.

RNA interference of endoglucanases in the formosan subterranean termite Coptotermes formosanus shiraki (Blattodea: Rhinotermitidae) by dsRNA injection or ingestion.

Termites obtain energy and nutrition from wood and wood-related materials by utilizing endogenous and symbiotic cellulases. Endoglucanase is one of the key cellulases in cellulose digestion. Previous studies have shown that the inhibition of the cellulase enzyme system would be a plausible approach for termite control. In the present study, we studied the effect of RNAi on termites by targeting a conserved region of five endoglucanase genes from Coptotermes formosanus (CfEGs). Both dsRNA injection and oral delivery resulted in significant gene silencing of CfEGs and consequently led to mortality, reduced enzyme activity, and reduced weight compared to control worker termites. An injection dose of 150 ng and a feeding dose of 2 µg/cm2 provided for the best RNAi efficiency. dsCfEG was further combined with flufenoxuron, an insect growth regulator used to manage/suppress subterranean termites, and when fed to workers, caused a lower enzyme activity compared to the dsCfEG- or flufenoxuron-only treatment. The weight loss (∼0.598 mg) and mortality (∼28%) observed in the combined dsCfEG and flufenoxuron treatment differed significantly from those observed in the flufenoxuron-only treatment (∼0.208 mg and ∼16%, respectively). Although the effects of these dsCfEG treatments on mortality were insufficient to serve as termiticides, dsCfEGs could be used in combination with other treatments to increase efficacy. This study provides a research basis for the use of RNAi in termiticides.

Effect of Particle Size on the Kinetics of Enzymatic Hydrolysis of Microcrystalline Cotton Cellulose: a Modeling and Simulation Study.

As the bioconversion of cellulosic substrate to fuels is essential to suppress the dependence on conventional fossil fuels, development of new improved bioprocess engineering techniques are requisite for fulfilling the rising demand of biofuels throughout the world. For this purpose, the effect of particle size on enzymatic hydrolysis of cotton cellulose has been explored in great detail. The model simulations for the enzymatic hydrolysis of microcrystalline cotton cellulose of different concentrations (0.25-20 mg/ml) were performed for the average particle size ranging from 0.78 to 25.52 µm. A highest glucose yield (99.8%) was observed for the smallest particle size of 0.78 µm in 50 h of enzymatic hydrolysis. Effect of inhibition (competitive and non-competitive) on glucose yield was analyzed through the incorporation of product inhibition in the kinetic model. The extent of cleaving of 1-4 glycosidic bonds by cellulase was quantified by degree of polymerization (DP) of cotton polymers which also indicates that faster scission of bonds can be observed under competitive inhibition and, hence, more glucose yield. The model simulations shows that particle size reduction may be useful for reducing the long residence time required for the hydrolysis step in the bioconversion of cellulose to ethanol.

Mechanism of Competitive Inhibition and Destabilization of Acidothermus cellulolyticus Endoglucanase 1 by Ionic Liquids.

The ability of ionic liquids (ILs) to solubilize cellulose has sparked interest in their use for enzymatic biomass processing. However, this potential is yet to be realized, primarily because ILs inactivate requisite cellulases by mechanisms that are yet to be identified. We used a combination of enzymology, circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular dynamics (MD) methods to investigate the molecular basis for the inactivation of the endocellulase 1 (E1) from Acidothermus cellulolyticus by the imidazolium IL 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). Enzymatic studies revealed that [BMIM][Cl] inactivates E1 in a biphasic manner that involves rapid, reversible inhibition, followed by slow, irreversible deactivation. Backbone NMR signals of the 40.5 kDa E1 were assigned by triple resonance NMR methods, enabling monitoring of residue-specific perturbations. 1H-15N NMR titration experiments revealed that [BMIM][Cl] binds reversibly to the E1 active site, indicating that reversible deactivation is due to competitive inhibition of substrate binding. Prolonged incubation with [BMIM][Cl] led to substantial global changes in the 1H-15N heteronuclear single quantum coherence NMR and CD spectra of E1 indicative of protein denaturation. Notably, weak interactions between [BMIM][Cl] and residues at the termini of several helices were also observed, which, together with MD simulations, suggest that E1 denaturation is promoted by [BMIM][Cl]-induced destabilization of helix capping structures. In addition to identifying determinants of E1 inactivation, our findings establish a molecular framework for engineering cellulases with improved IL compatibility.

Lignin-derived inhibition of monocomponent cellulases and a xylanase in the hydrolysis of lignocellulosics.

Non-productive enzyme binding onto lignin is the major inhibitory mechanism, which reduces hydrolysis rates and yields and prevents efficient enzyme recycling in the hydrolysis of lignocellulosics. The detailed mechanisms of binding are still poorly understood. Enzyme-lignin interactions were investigated by comparing the structural properties and binding behaviour of fungal monocomponent enzymes, cellobiohydrolases TrCel7A and TrCel6A, endoglucanases TrCel7B and TrCel5A, a xylanase TrXyn11 and a ß-glucosidase AnCel3A, onto lignins isolated from steam pretreated spruce and wheat straw. The enzymes exhibited decreasing affinity onto lignin model films in the following order: TrCel7B>TrCel6A>TrCel5A>AnCel3A>TrCel7A>TrXyn11. As analysed in Avicel hydrolysis, TrCel6A and TrCel7B were most inhibited by lignin isolated from pretreated spruce. This could be partially explained by adsorption of the enzyme onto the lignin surface. Enzyme properties, such as enzyme surface charge, thermal stability or surface hydrophobicity could not alone explain the adsorption behaviour.

A paralogous decoy protects Phytophthora sojae apoplastic effector PsXEG1 from a host inhibitor.

The extracellular space (apoplast) of plant tissue represents a critical battleground between plants and attacking microbes. Here we show that a pathogen-secreted apoplastic xyloglucan-specific endoglucanase, PsXEG1, is a focus of this struggle in the Phytophthora sojae-soybean interaction. We show that soybean produces an apoplastic glucanase inhibitor protein, GmGIP1, that binds to PsXEG1 to block its contribution to virulence. P. sojae, however, secretes a paralogous PsXEG1-like protein, PsXLP1, that has lost enzyme activity but binds to GmGIP1 more tightly than does PsXEG1, thus freeing PsXEG1 to support P. sojae infection. The gene pair encoding PsXEG1 and PsXLP1 is conserved in many Phytophthora species, and the P. parasitica orthologs PpXEG1 and PpXLP1 have similar functions. Thus, this apoplastic decoy strategy may be widely used in Phytophthora pathosystems.

A nematicidal tannin from Punica granatum L. rind and its physiological effect on pine wood nematode (Bursaphelenchus xylophilus).

The ethanol extract of Punica granatum L. rind was tested to show significant nematicidal activity against pine wood nematode. Three nematicidal compounds were obtained from the ethanol extract by bioassay-guided fractionation and identified as punicalagin 1, punicalin 2, and corilagin 3 by mass and nuclear magnetic resonance spectral data analysis. Punicalagin 1 was most active against PWN among the purified compounds with the LC50 value of 307.08µM in 72h. According to the enzyme assays in vitro, punicalagin 1 could inhibit the activity of acetylcholinesterase, amylase and cellulase from PWN with IC50 value of 0.60mM, 0.96mM and 1.24mM, respectively. The morphological structures of PWNs treated by punicalagin 1 were greatly changed. These physiological effects of punicalagin 1 on PWN may helpful to elucidate its nematicidal mechanism.

Structure-Function Analysis of a Mixed-linkage ß-Glucanase/Xyloglucanase from the Key Ruminal Bacteroidetes Prevotella bryantii B(1)4.

The recent classification of glycoside hydrolase family 5 (GH5) members into subfamilies enhances the prediction of substrate specificity by phylogenetic analysis. However, the small number of well characterized members is a current limitation to understanding the molecular basis of the diverse specificity observed across individual GH5 subfamilies. GH5 subfamily 4 (GH5_4) is one of the largest, with known activities comprising (carboxymethyl)cellulases, mixed-linkage endo-glucanases, and endo-xyloglucanases. Through detailed structure-function analysis, we have revisited the characterization of a classic GH5_4 carboxymethylcellulase, PbGH5A (also known as Orf4, carboxymethylcellulase, and Cel5A), from the symbiotic rumen Bacteroidetes Prevotella bryantii B14. We demonstrate that carboxymethylcellulose and phosphoric acid-swollen cellulose are in fact relatively poor substrates for PbGH5A, which instead exhibits clear primary specificity for the plant storage and cell wall polysaccharide, mixed-linkage ß-glucan. Significant activity toward the plant cell wall polysaccharide xyloglucan was also observed. Determination of PbGH5A crystal structures in the apo-form and in complex with (xylo)glucan oligosaccharides and an active-site affinity label, together with detailed kinetic analysis using a variety of well defined oligosaccharide substrates, revealed the structural determinants of polysaccharide substrate specificity. In particular, this analysis highlighted the PbGH5A active-site motifs that engender predominant mixed-linkage endo-glucanase activity vis à vis predominant endo-xyloglucanases in GH5_4. However the detailed phylogenetic analysis of GH5_4 members did not delineate particular clades of enzymes sharing these sequence motifs; the phylogeny was instead dominated by bacterial taxonomy. Nonetheless, our results provide key enzyme functional and structural reference data for future bioinformatics analyses of (meta)genomes to elucidate the biology of complex gut ecosystems.

Two nematicidal furocoumarins from Ficus carica L. leaves and their physiological effects on pine wood nematode (Bursaphelenchus xylophilus).

The ethanol extract of the Ficus carica L. leaves was tested to show strong nematicidal activity against pine wood nematode (PWN), Bursaphelenchus xylophilus, causing 90.93% corrected mortality within 72 h at 1.0 mg/mL. From the ethyl acetate soluble fraction of the F. carica L. leaves extract, the main nematicidal constituents were obtained by bioassay-guided isolation and identified as linear furocoumarins bergapten (1) and psoralen (2) by mass and NMR spectral data analysis. Bergapten and psoralen had significant nematicidal activity against PWN with the LC50 values of 97.08 aKSnd 115.03  µ g/mL within 72 h, respectively. The two furocoumarins could inhibit the activities of amylase, cellulase and acetylcholinesterase (AchE) from PWN. The morphologies of PWNs changed much after they were treated by bergapten and psoralen. The physiological effects of bergapten and psoralen on PWN might provide helpful clues to elucidate their nematicidal mechanisms.

Phenols and lignin: Key players in reducing enzymatic hydrolysis yields of steam-pretreated biomass in presence of laccase.

Phenols are known as inhibitors for cellulases and fermentative microorganisms in bioethanol production processes. The addition of laccases removes the phenolic compounds and subsequently reduces the lag phase of the fermentative microorganism. However, the application of laccases diminishes glucose release during the enzymatic hydrolysis. In this study a model cellulosic substrate (Sigmacell) together with lignin extract, whole steam-pretreated wheat straw (slurry) and its water insoluble solid fraction (WIS) were subjected to enzymatic hydrolysis to evaluate the effects of laccase treatment in presence of lignin and phenols. The presence of laccase in enzymatic hydrolysis of Sigmacell with lignin extract reduced glucose yield by 37% compared with assays without laccase. Furthermore, this reduction was even more marked in presence of phenols (55% reduction). Interestingly, when hydrolyzing WIS, the addition of phenols coupled with laccase treatment did not show a reduction when compared with only laccase addition. This fact suggests the key role of lignin in the hydrolysis inhibition since in WIS the ratio cellulase per gram of lignin was much lower than in Sigmacell experiments. Finally, the lower cellobiose and xylose recoveries point out that phenolic oligomers formed by laccase oxidation play important roles in the inhibition of endoglucanases, cellobiohydrolases and xylanases. To conclude, the proportion of lignin and the composition of phenols are key players in the inhibition of cellulases when the enzymatic hydrolysis is combined with laccases detoxification.

Lignocellulosic hydrolysate inhibitors selectively inhibit/deactivate cellulase performance.

In this study, we monitored the inhibition and deactivation effects of various compounds associated with lignocellulosic hydrolysates on individual and combinations of cellulases. Tannic acid representing polymeric lignin residues strongly inhibited cellobiohydrolase 1 (CBH1) and ß-glucosidase 1 (BGL1), but had a moderate inhibitory effect on endoglucanase 2 (EG2). Individual monomeric lignin residues had little or no inhibitory effect on hydrolytic enzymes. However, coniferyl aldehyde and syringaldehyde substantially decreased the activity of CBH1 and deactivated BGL1. Acetic and formic acids also showed strong inhibition of BGL1 but not CBH1 and EG2, whereas tannic, acetic and formic acid strongly inhibited a combination of CBH1 and EG2 during Avicel hydrolysis. Diminishing enzymatic hydrolysis is largely a function of inhibitor concentration and the enzyme-inhibitor relationship, rather than contact time during the hydrolysis process (i.e. deactivation). This suggests that decreased rates of hydrolysis during the enzymatic depolymerisation of lignocellulosic hydrolysates may be imparted by other factors related to substrate crystallinity and accessibility.

Characterization of a novel endoglucanase from Ganoderma lucidum.

We evaluated the production and characterization of endoglucanase from Ganoderma lucidum using different lignocellulose biomasses. We purified a novel carboxymethyl cellulose (CMC) hydrolyzing endoglucanase from the white-rot fungus G. lucidum when the medium was supplemented with 1% (w/v) wheat bran. Endoglucanase was purified 12.5-fold via ammonium sulfate fractionation, Sephadex G-100, and Q-Sepharose column chromatography with a final yield of 15%. SDS-PAGE analysis revealed that the endoglucanase had a molecular mass of 64.0 kDa. The optimal activity of purified endoglucanase was at pH 5.0 and 35 °C, though it was stable between pH 4.0-7.0 and temperatures of 30-60 °C. The purified enzyme was specific to CMC as a suitable substrate. The metal ions Hg(2+), Fe(2+), and Cr(2+) inhibited enzyme activity, while Ca(2+), Mg(2+), and Mn(2+) enhanced enzyme activity. The endoglucanase showed high activity and stability in the presence of different surfactants and non-polar hydrophobic organic solvents. This endoglucanase is tolerant to high temperature, metal ions, surfactants, and solvents, suggesting that it is appropriate for use in biomass conversion for biofuel production under harsh environmental conditions.

Inhibitory effect of vanillin on cellulase activity in hydrolysis of cellulosic biomass.

Pretreatment of lignocellulosic material produces a wide variety of inhibitory compounds, which strongly inhibit the following enzymatic hydrolysis of cellulosic biomass. Vanillin is a kind of phenolics derived from degradation of lignin. The effect of vanillin on cellulase activity for the hydrolysis of cellulose was investigated in detail. The results clearly showed that vanillin can reversibly and non-competitively inhibit the cellulase activity at appropriate concentrations and the value of IC50 was estimated to be 30 g/L. The inhibition kinetics of cellulase by vanillin was studied using HCH-1 model and inhibition constants were determined. Moreover, investigation of three compounds with similar structure of vanillin on cellulase activity demonstrated that aldehyde group and phenolic hydroxyl groups of vanillin had inhibitory effect on cellulase. These results provide valuable and detailed information for understanding the inhibition of lignin derived phenolics on cellulase.

Stability of endoglucanases from mesophilic fungus and thermophilic bacterium in acidified polyols.

Recent developments in chemical pretreatments of lignocellulosic biomass using polyols as co-solvents (e.g., glycerol and ethylene glycol) at temperatures less than 100°C may allow the effective use of thermostable and non-thermostable cellulases in situ during the saccharification process. The potential of biomass saccharifying enzymes, endoglucanases (EG) from a thermophilic bacterium (Thermotoga maritima) and a mesophilic fungus (Trichoderma longibrachiatum), to retain their activity in aqueous buffer, acidified glycerol, and acidified ethylene glycol used as co-solvents at pretreatment temperatures at or below 100°C were examined. The results show that despite its origin, T. longibrachiatum EG (Tl-EG) retained 75% of its activity after exposure to 100°C for 5 min in aqueous buffer while T. maritima EG (Tm-EG) retained only 5% activity. However, at 90°C both enzymes retained over 87% of their activity. In acidified (0.1% (w/w) H2SO4) glycerol, Tl-EG retained similar activity (80%) to that obtained in glycerol alone, while Tm-EG retained only 35%. With acidified ethylene glycol under these conditions, both Tl-EG and Tm-EG retained 36% of their activity. The results therefore show that Tl-EG is more stable in both acidified glycerol and ethylene glycol than Tm-EG. A preliminary kinetic study showed that pure glycerol improved the thermal stability of Tl-EG but destabilized Tm-EG, relative to the buffer solution. The half-lives of both Tl-EG and Tm-EG are 4.5 min in acidified glycerol, indicating that the effectiveness of these enzymes under typical pretreatment times of greater than 15 min will be considerably diminished. Attempts have been made to explain the differences in the results obtained between the two enzymes.