Gene integration and expression and extracellular secretion of Erwinia chrysanthemi endoglucanase CelY (celY) and CelZ (celZ) in ethanologenic Klebsiella oxytoca P2

The development of methods to reduce costs associated with the solubilization of cellulose is essential for the utilization of lignocellulose as a renewable feedstock for fuels and chemicals. One promising approach is the genetic engineering of ethanol-producing microorganisms that also produce cellulase enzymes during fermentation. By starting with an ethanologenic derivative (strain P2) of Klebsiella oxytoca M5A1 with the native ability to metabolize cellobiose, the need for supplemental beta-glucosidase was previously eliminated. In the current study, this approach has been extended by adding genes encoding endoglucanase activities. Genes celY and celZ from Erwinia chrysanthemi have been functionally integrated into the chromosome of P2 using surrogate promoters from Zymomonas mobilis for expression. Both were secreted into the extracellular milieu, producing more than 20,000 endoglucanase units (carboxymethyl cellulase activity) per liter of fermentation broth. During the fermentation of crystalline cellulose with low levels of commercial cellulases of fungal origin, these new strains produced up to 22% more ethanol than unmodified P2. Most of the beneficial contribution was attributed to CelY rather than to CelZ. These results suggest that fungal enzymes with substrate profiles resembling CelY (preference for long-chain polymers and lack of activity on soluble cello-oligosaccharides of two to five glucosyl residues) may be limiting in commercial cellulase preparations.     

Cellulase production from <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> sp. NO3 isolated from the sea squirt <Emphasis Type="Italic">Halocynthia rorentzi</Emphasis>

Pseudoalteromonas sp. NO3 was isolated from the hemolymph of diseased sea squirts (Halocynthia rorentzi) with symptoms of soft tunic syndrome. The strain was found to produce an extracellular cellulase (CelY) that consisted of a 1,476 bp open reading frame encoding 491 amino acid residues with an approximate molecular mass of 52 kDa. Homologies of the deduced amino acid sequence of celY with the products of the celA, celX, celG and cel5Z genes were 92.6, 93.3, 92.6, and 59.1%, respectively. Additionally, CelY had 50–80% remnant catalytic activity at temperatures of 10–20°C. Highest carboxymethyl cellulose (CMC) hydrolysis was observed at pH 8.0 and 40°C. CMC activity was determined by zymogram active staining and different degraded product profiles for CelY were obtained when cellotetraose, cellopentaose, and CMC were used as substrates. This study identified a transglycosylation activity in CelY that allows the enzyme to digest G4 to G2 and G3 without the production of G1.     

Purification, characterization, and synergistic action of endoglucanases from Fusarium lini

Five endoglucanases (1,4-beta-D-glucan-glucanohydrolase, EC 3.2.1.4) were isolated from Fusarium lini. Endo I and II were purified by preparative gel electrophoresis and Endo III, IV, and V were purified in a single-step procedure involving preparative flat-bed isoelectric focusing. All the endoglucanases were homogenous on disk gel electrophoresis and analytical isoelectric focusing in polyacrylamide gel. The pi values were between 6 and 6.6 for Endo III, IV, and V; for Endo I, the pi value was 8. The molecular weights of the enzymes were between 4 x 10(4) and 6.5 x 10(4). The K(m) values for endoglucanases using carboxymethyl cellulose (CM-cellulose) as the substrate were 2-12 mg/mL. The specificity of the enzymes was restricted to beta-1, 4-linkages. All the enzymes showed activity towards D-xylan. The endoglucanases had high viscosity reducing activity with CM-cellulose. Striking synergism was observed for the hydrolysis of CM-cellulose by endoglucanases. Endo II, IV, and V attacked cellopentaose and cellotetraose more readily than cellotriose. Endo II and V hydrolyzed cellotriose, cellotetraose, and cellopentaose, yielding a mixture of cellobiose with a trace amount of glucose; endo IV produced only cellobiose.     

A new approach for studying correlations between the chemical structure and the rheological properties in carboxymethyl cellulose

Two model sodium carboxymethyl celluloses (CMC) with similar monomer composition but with significant differences in the viscoelastic properties, that could not be assigned to variations in the average molar mass or molar mass distribution, were investigated with respect to the fraction of nonsubstituted cellulose segments in the polymers. The CMCs were hydrolyzed by a purified highly selective endoglucanase. The average molar mass and molar mass distribution of the enzyme products, as measured by size-exclusion chromatography with online multi-angle light scattering and refractive index detection (SEC/MALS/RI), revealed that the enzyme-catalyzed hydrolysis was more effective on one of the CMCs. To investigate whether this was due to a higher fraction of nonsubstituted cellulose segments in the polymer, the concentrations of nonsubstituted enzyme products, e.g., cellotetraose and cellopentaose, were measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). It was concluded that the two CMCs displayed significant differences in the fraction of nonsubstituted cellulose segments. Furthermore, the CMC with the strongest attractive intermolecular interactions, according to rheometry, also contained the highest fraction of nonsubstituted cellulose segments.     

Gene Integration and Expression and Extracellular Secretion of Erwinia chrysanthemi Endoglucanase CelY (celY) and CelZ (celZ) in Ethanologenic Klebsiella oxytoca P2

The development of methods to reduce costs associated with the solubilization of cellulose is essential for the utilization of lignocellulose as a renewable feedstock for fuels and chemicals. One promising approach is the genetic engineering of ethanol-producing microorganisms that also produce cellulase enzymes during fermentation. By starting with an ethanologenic derivative (strain P2) of Klebsiella oxytoca M5A1 with the native ability to metabolize cellobiose, the need for supplemental β-glucosidase was previously eliminated. In the current study, this approach has been extended by adding genes encoding endoglucanase activities. Genes celY and celZ from Erwinia chrysanthemi have been functionally integrated into the chromosome of P2 using surrogate promoters from Zymomonas mobilis for expression. Both were secreted into the extracellular milieu, producing more than 20,000 endoglucanase units (carboxymethyl cellulase activity) per liter of fermentation broth. During the fermentation of crystalline cellulose with low levels of commercial cellulases of fungal origin, these new strains produced up to 22% more ethanol than unmodified P2. Most of the beneficial contribution was attributed to CelY rather than to CelZ. These results suggest that fungal enzymes with substrate profiles resembling CelY (preference for long-chain polymers and lack of activity on soluble cello-oligosaccharides of two to five glucosyl residues) may be limiting in commercial cellulase preparations.     

Expression and Purification of Cellulase Xf818 from Xylella fastidiosa in Escherichia coli

Xylella fastidiosa was the first plant pathogen whose complete genome sequence was available. X. fastidiosa causes citrus variegated chlorosis, but the physiological basis of the disease in unknown. Through comparative sequence analysis, several putative plant cell wall–degrading enzymes were identified on the X. fastidiosa genome. We have cloned Xf818, a putative endoglucanase ORF, into expression vectors pET20b and pET28b, and purified a recombinant form of Xf818 containing a His₆ tag. Through biochemical assays, we have characterized the endoglucanase activity of this protein. The best conditions for hydrolysis over carboxymethyl cellulose (CMC) were on pH 5.2 at 65°C. Xf818 hydrolyzed CMC, acid swollen cellulose, Avicel, birch wood, oat spels xylans, and the oligosaccharides cellotetraose and cellopentaose. Xf818 carried out transglycosylation and had a functional cellulose-binding domain.     

Degradation of cellulose by the major endoglucanase produced from the brown-rot fungus Fomitopsis pinicola

An endoglucanase that is able to degrade both crystalline and amorphous cellulose was purified from the culture filtrates of the brown-rot fungus Fomitopsis pinicola grown on cellulose. An apparent molecular weight of the purified enzyme was approximately 32 kDa by SDS-PAGE analysis. The enzyme was purified 11-fold with a specific activity of 944 U/mg protein against CMC. The partial amino acid sequences of the purified endoglucanase had high homology with endo-beta-1,4-glucanase of glycosyl hydrolase family 5 from other fungi. The K(m) and K(cat)values for CMC were 12 mg CMC/ml and 670/s, respectively. The purified EG hydrolyzed both cellotetraose (G4) and cellopentaose (G5), but did not degrade either cellobiose (G2) or cellotriose (G3).     

Kinetic characterization of a glycoside hydrolase family 44 xyloglucanase/endoglucanase from Ruminococcus flavefaciens FD-1

Two forms of Ruminococcus flavefaciens FD-1 endoglucanase B, a member of glycoside hydrolase family 44, one with only a catalytic domain and the other with a catalytic domain and a carbohydrate binding domain (CBM), were produced. Both forms hydrolyzed cellotetraose, cellopentaose, cellohexaose, carboxymethylcellulose (CMC), birchwood and larchwood xylan, xyloglucan, lichenan, and Avicel but not cellobiose, cellotriose, mannan, or pullulan. Addition of the CBM increased catalytic efficiencies on both CMC and birchwood xylan but not on xyloglucan, and it decreased rates of cellopentaose and cellohexaose hydrolysis. Catalytic efficiencies were much higher on xyloglucan than on other polysaccharides. Hydrolysis rates increased with increasing cellooligosaccharide chain length. Cellotetraose hydrolysis yielded only cellotriose and glucose. Hydrolysis of cellopentaose gave large amounts of cellotetraose and glucose, somewhat more of the former than of the latter, and much smaller amounts of cellobiose and cellotriose. Cellohexaose hydrolysis yielded much more cellotetraose than cellobiose and small amounts of glucose and cellotriose, along with a low and transient amount of cellopentaose.     

Effect ofMyrothecium sp. cellulase on different types of cellulose and cellulosic materials

Three types of cellulase preparations were applied to different types of cellulose and cellulosic materials. The action of these types of cellulase on cellulose powder was increased with the increase of enzyme concentration. Both carboxymethyl cellulose (CMC) and sodium carboxymethyl cellulose (Na-CMC) released high amounts of reducing sugar as affected by cellulase application. Different types of paper pulp were moderately hydrolyzed, while agricultural wastes were slightly hydrolyzed. Vegetable and fruits cellulose were equally hydrolyzed but at low rate. Pretreatment of cellulose or cellulosic materials by grinding or by swelling with phosphoric acid gave rise to increased hydrolysis by the enzyme. Cellobiose was detected chromatographically as an intermediate product of hydrolysis of both cellulose and carboxymethyl cellulose with glucose.     

Purification and properties of an endo-1,4-beta-glucanase from Clostridium josui.

An enzyme active against carboxymethyl cellulose (CMC) was purified from the stationary-phase-culture supernatant of Clostridium josui grown in a medium containing ball-milled cellulose. The purification in the presence of 6 M urea yielded homogeneous enzyme after an approximately 50-fold increase in specific activity and a 13% yield. The enzyme had a molecular mass of 45 kilodaltons. The optimal temperature and pH of the enzyme against CMC were 60 degrees C and 6.8, respectively. The enzyme hydrolyzed cellotetraose, cellopentaose, and cellohexaose to cellobiose and cellotriose but did not hydrolyze cellobiose or cellotriose. A microcrystalline cellulose, Avicel, was also hydrolyzed significantly, but the extent of hydrolysis was remarkably less than that of CMC. On the basis of these results, the enzyme purified here is one of the endo-1,4-beta-glucanases. The N-terminal amino acid sequence of the enzyme is Tyr-Asp-Ala-Ser-Leu-Lys-Pro-Asn-Leu-Gln-Ile-Pro-Gln-Lys-Asn-Ile-Pro-Asn- Asn-Asp-Ala-Val-Asn-Ile-Lys.     

Purification and properties of endo-(1→4)-β-d-glucanase from Ruminococcus albus

An enzyme active against O-(carboxymethyl)cellulose (CMC) was purified from a synthetic medium containing ball-milled cellulose wherein Ruminococcus albus had been cultivated for 70 h. After 570-fold purification, a homogeneous enzyme was obtained in a yield of 3%. The enzyme degraded CMC (molecular weight, 180,000; degree of substitution, 0.6) to a smaller polymer having a molecular weight of ∼20,000, and generated a small proportion of glucose, but negligible proportions of such cello-saccharides as cellobiose, cellotriose, cellotetraose, or cellopentaose. The fact that the enzyme could produce water-insoluble fragments was discovered by dissolving substrate and products in Cadoxen solution. No water-soluble cello-oligomers were detected by thin-layer chromatography after degradation of water-insoluble cellulose by the purified enzyme. Therefore, the enzyme was classified as an endo-(1→4)-β-d-glucanase.     

Isolation of four major subunits from Clostridium thermocellum cellulosome and their synergism in the hydrolysis of crystalline cellulose

The cellulosome of Clostridium thermocellum, purified by affinity chromatography, was dissociated under mild conditions and separated by SDS-PAGE. Two major p-nitrophenylcellobiosidases (PNPCases I and II) corresponding to the S₅ (103 kDa) and S₈ (78 kDa) subunits and one major carboxymethylcellulase (CMCase) coinciding with the S₁₁ (60.5 kDa) subunit were isolated and characterized using carboxymethylcellulose (CMC), H₃PO₄-swollen cellulose and cello-oligosaccharides. Both PNPCases showed little effect on the viscosity of CMC and released twice as much total sugar as reducing sugar from H₃PO₄-swollen cellulose. The CMCase released ten times more total sugar than reducing sugar from H₃PO₄-swollen cellulose and reduced the viscosity of CMC rapidly. None of these enzymes was active on cellotriose. Both PNPCases released cellobiose from cellotetraose, and cellobiose and cellotriose from cellopentaose. In contrast, CMCase was active only on cellopentaose and released mainly glucose. Use of MeUmb(Glc)_n revealed that both PNPCases cleaved preferentially either the second or fourth linkage from the non-reducing end while the CMCase was specific for the internal glycosidic bonds. Thus, the PNPCases and CMCase behaved as typical exo- and endoglucanases, respectively. When tested individually, all three enzymes degraded Avicel only to a small extent. A 1.5–2.0-fold increase in sugar release was observed when CMCase was combined with either PNPCase I, II or both. Combining S₁ with either PNPCase II or CMCase resulted in fourfold synergism in the hydrolysis of Avicel. Synergism was sevenfold when all three enzymes were combined with S₁.     

Site-directed mutation of noncatalytic residues of Thermobifida fusca exocellulase Cel6B.

Fifteen mutant genes in six loop residues and eight mutant genes in five conserved noncatalytic active site residues of Thermobifida fusca Cel6B were constructed, cloned and expressed in Escherichia coli or Streptomyces lividans. The mutant enzymes were assayed for catalytic activity on carboxymethyl cellulose (CMC), swollen cellulose (SC), filter paper (FP), and bacterial microcrystalline cellulose (BMCC) as well as cellotetraose, cellopentaose, and 2, 4-dinitrophenyl-beta-D-cellobioside. They were also assayed for ligand binding, enzyme processivity, thermostability, and cellobiose feedback inhibition. Two double Cys mutations that formed disulfide bonds across two tunnel forming loops were found to significantly weaken binding to ligands, lower all activities, and processivity, demonstrating that the movement of these loops is important but not essential for Cel6B function. Two single mutant enzymes, G234S and G284P, had higher activity on SC and FP, and the double mutant enzyme had threefold and twofold higher activity on these substrates, respectively. However, synergism with endocellulase T. fusca Cel5A was not increased with these mutant enzymes. All mutant enzymes with lower activity on filter paper, BMCC, and SC had lower processivity. This trend was not true for CMC, suggesting that processivity in Cel6B is a key factor in the hydrolysis of insoluble and crystalline cellulose. Three mutations (E495D, H326A and W329C) located near putative glycosyl substrate subsites -2, +1 and +2, were found to significantly increase resistance to cellobiose feedback inhibition. Both the A229V and L230C mutations specifically decreased activity on BMCC, suggesting that BMCC hydrolysis has a different rate limiting step than the other substrates. Most of the mutant enzymes had reduced thermostability although Cel6B G234S maintained wild-type thermostability. The properties of the different mutant enzymes provide insight into the catalytic mechanism of Cel6B.     

Active-site mutations which change the substrate specificity of the Clostridium stercorarium cellulase CelZ implications for synergism.

CelZ from the cellulolytic thermophile Clostridium stercorarium has been described as a 'monomeric' cellulase able to effect both the endoglucanolytic hydrolysis of internal glycosidic linkages and the exoglucanolytic degradation from the chain ends in a processive mode of action. The putative catalytic residues of this family 9 cellulase, Asp84 and Glu447 located within the N-terminal domain of the modular protein, were replaced by site-directed mutagenesis. A minimized CelZ derivative (CelZC') comprising the catalytic domain and the adjacent cellulose-binding domain (CBD) family IIIc domain C' was used as target for mutagenesis. Six mutant enzymes and the unmodified CelZC' protein were purified to homogeneity and compared with respect to thermoactivity, substrate specificity, product profile and synergism. CD studies revealed that no major changes to the overall structure of the proteins had occurred. Replacement of either one or both catalytic residues completely eliminated the ability of CelZ to attack insoluble Avicel preparations indicative of the exo-activity, whereas the endo-activity measured via hydrolysis of CM-cellulose was retained upon substitution of the catalytic base Asp84. Thus, endo-active CelZ mutants defective in the exo-activity were available for co-operativity studies with the C. stercorarium exoglucanase CelY. Synergism was found to be dependent on the endo-activity of CelZ. Mutants Asp84Gly and Asp84Glu were able to enhance the degradation of crystalline cellulose significantly, although no products could be released from this substrate by individual action of the mutants.     

Characterization of a family 45 glycosyl hydrolase from Fibrobacter succinogenes S85

Fibrobacter succinogenes is one of the most active cellulolytic bacteria ever isolated from the rumen, but enzymes from F. succinogenes capable of hydrolyzing native (insoluble) cellulose at a rapid rate have not been identified. However, the genome sequence of F. succinogenes is now available, and it was hoped that this information would yield new insights into the mechanism of cellulose digestion. The genome has a single family 45 beta-glucanase gene, and some of the enzymes in this family have good activity against native cellulose. The gene encoding the family 45 glycosyl hydrolase from F. succinogenes S85 was cloned into Escherichia coli JM109(DE3) using pMAL-c2 as a vector. Recombinant E. coli cells produced a soluble fusion protein (MAL-F45) that was purified on a maltose affinity column and characterized. MAL-F45 was most active on carboxymethylcellulose between pH 6 and 7 and it hydrolyzed cellopentaose and cellohexaose but not cellotetraose. It also cleaved p-nitrophenyl-cellopentose into cellotriose and p-nitrophenyl-cellobiose. MAL-F45 produced cellobiose, cellotriose and cellotetraose from acid swollen cellulose and bacterial cellulose, but the rate of this hydrolysis was much too low to explain the rate of cellulose digestion by growing cultures. Because the F. succinogenes S85 genome lacks dockerin and cohesin sequences, does not encode any known processive cellulases, and most of its endoglucanase genes do not encode carbohydrate binding modules, it appears that F. succinogenes has a novel mechanism of cellulose degradation.     

Expression, purification and characterization of two thermostable endoglucanases cloned from a lignocellulosic decomposing fungi Aspergillus fumigatus Z5 isolated from compost

Two genes encoding endoglucanase, designated as egl2 and egl3, were cloned from a lignocellulosic decomposing fungus Aspergillus fumigatus Z5 and were successfully expressed in Pichia pastoris X33. The deduced amino acid sequences encoded by egl2 and egl3 showed strong similarity with the sequence of glycoside hydrolase family 5. SDS-PAGE and western blot assays indicated that the recombinant enzymes were secreted into the culture medium and the zymogram analysis confirmed that both recombinant enzymes had endoglucanase activity. Several biochemical properties of the two recombinant enzymes were studied: Egl2 and Egl3 showed optimal activity at pH 5.0 and 4.0, respectively, and at 50 and 60°C, respectively. Egl2 and Egl3 showed good pH stability in the range of 4-7, and both enzymes demonstrated good thermostability ranging from 30 to 60°C. The K(m) and V(max) values using carboxymethyl cellulose (CMC, soluble cellulose, polymerized by β-1, 4-linked glucose residues) as the substrate at optimal conditions were determined. The activities of the enzymes on a variety of cello-oligosaccharide substrates were investigated, and Egl2 can hydrolyze cellotetraose and cellopentaose but not cellobiose and cellotriose, whereas Egl3 can hydrolyze all cello-oligosaccharides, except cellobiose.     

Characterization and substrate specificity of an endo-beta-1,4-D-glucanase I (Avicelase I) from an extracellular multienzyme complex of Bacillus circulans.

An endo-1,4-beta-D-glucanase I (Avicelase I; EC 3.2.1.4) was purified to homogeneity from an extracellular celluloxylanosome of Bacillus circulans F-2. The purification in the presence of 6 M urea yielded homogeneous enzyme. The enzyme had a monomeric structure, its relative molecular mass being 75 kDa as determined by gel filtration and 82 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI was 5.4, and the N-terminal amino acid sequence was ASNIGGWVGGNESGFEFG. The optimal pH was 4.5, and the enzyme was stable at pH 4 to 10. The enzyme has a temperature optimum of 50 degrees C, it was stable at 55 degrees C for 46 h, and it retains approximately 20% of its activity after 30 min at 80 degrees C. It showed high-level activity towards carboxymethyl cellulose (CMC) as well as p-nitrophenyl-beta-D-cellobioside, 4-methylumbelliferyl cellobioside, xylan, Avicel, filter paper, and some cello-oligosaccharides. Km values for birch xylan, CMC, and Avicel were 4.8, 7.2, and 87.0 mg/ml, respectively, while Vmax values were 256, 210, and 8.6 mumol x min-1 x mg-1, respectively. Cellotetraose was preferentially cleaved into cellobiose (G2) plus G2, and cellopentaose was cleaved into G2 plus cellotriose (G3), while cellohexaose was cleaved into cellotetraose plus G2 and to a lesser extent G3 plus G3. G3 was not cleaved at all. G2 was the main product of Avicel hydrolysis. Xylotetraose (X4) and xylobiose (X2) were mainly produced by the enzyme hydrolysis of xylan. G2 inhibited the activity of carboxymethyl cellulase and Avicelase, whereas Mg2+ stimulated it. The enzyme was completely inactivated by Hg2+, and it was inhibited by a thiol-blocking reagent. Hydrolysis of CMC took place, with a rapid decrease in viscosity but a slow liberation of reducing sugars. On the basis of these results, it appeared that the cellulase should be regarded as endo-type cellulase, although it hydrolyzed Avicel.     

Importance of cellulase cocktails favoring hydrolysis of cellulose

Depolymerization of lignocellulosic biomass is catalyzed by groups of enzymes whose action is influenced by substrate features and the composition of cellulase preparation. Cellulases contain a mixture of variety of enzymes, whose proportions dictate the saccharification of biomass. In the current study, four cellulase preparation varying in their composition were used to hydrolyze two types of alkali-treated biomass (aqueous ammonia-treated rice straw and sodium hydroxide-treated rice straw) to study the effect on catalytic rate, saccharification yields, and sugar release profile. We found that substrate features affected the extent of saccharification but had minimal effect on the sugar release pattern. In addition, complete hydrolysis to glucose was observed with enzyme preparation having at least a cellobiase units (CBU)/carboxymethyl cellulose (CMC) ratio (>0.15), while a modified enzyme ratio can be used for oligosaccharide synthesis. Thus, cellulase preparation with defined ratios of the three main enzymes can improve the saccharification which is of utmost importance in defining the success of lignocellulose-based economies.     

Gas Chromatographic Determination of Optical Purity of a Chiral Derivatization Reagent,N-Acyl-l-prolyl Chloride

Two endo-type cellulases, tentatively called carboxymethyl cellulases (CMCases) I and II, were purified by gel filtration, ion-exchange chromatography, affinity chromatography, and chromato-focusing from a culture supernatant of Penicillium purpurogenum. Their homogeneity was verified by analytical polyacrylamide gel electrophoresis. The molecular weights of CMCases I and II, estimated by gel filtration, were 72,000 and 50,000, respectively. CMCases I and II contained about 12% and 8% carbohydrate, and had isoelectric points of 4.3 and 3.9, respectively. CMCase I produced cellobiose, glucose, and a trace amount of cellotriose from H₃PO₄-swollen cellulose and Avicel (microcrystalline cellulose), while CMCase II produced cellobiose and cellotriose with a small amount of glucose and cellotetraose. The products from reduced cellopentaose by both enzymes were released predominantly in the β-configuration. CMCase II appeared to act in more random fashion than I against carboxymethyl cellulose. These results suggest that both enzymes attack insoluble cellulose randomly, although there are some differences in the mode of hydrolytic action.