Biodetoxification of toxins generated from lignocellulose pretreatment using a newly isolated fungus, Amorphotheca resinae ZN1, and the consequent ethanol fermentation

Background

Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings.

Results

When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-β1,4-xylanase (EX3) and a lower proportion of endo-β1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, β-mannanase, amyloglucosidase, α-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-β1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-β1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and β-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h.

Conclusion

The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.     

Enhancement of Synthetic Trichoderma-Based Enzyme Mixtures for Biomass Conversion with an Alternative Family 5 Glycosyl Hydrolase from Sporotrichum thermophile

Enzymatic conversion of lignocellulosic materials to fermentable sugars is a limiting step in the production of biofuels from biomass. We show here that combining enzymes from different microbial sources is one way to identify superior enzymes. Extracts of the thermophilic fungus Sporotrichum thermophile (synonym Myceliophthora thermophila) gave synergistic release of glucose (Glc) and xylose (Xyl) from pretreated corn stover when combined with an 8-component synthetic cocktail of enzymes from Trichoderma reesei. The S. thermophile extracts were fractionated and an enhancing factor identified as endo-β1,4- glucanase (StCel5A or EG2) of subfamily 5 of Glycosyl Hydrolase family 5 (GH5_5). In multi-component optimization experiments using a standard set of enzymes and either StCel5A or the ortholog from T. reesei (TrCel5A), reactions containing StCel5A yielded more Glc and Xyl. In a five-component optimization experiment (i.e., varying four core enzymes and the source of Cel5A), the optimal proportions for TrCel5A vs. StCel5A were similar for Glc yields, but markedly different for Xyl yields. Both enzymes were active on lichenan, glucomannan, and oat β-glucan; however, StCel5A but not TrCel5A was also active on β1,4-mannan, two types of galactomannan, and β1,4-xylan. Phylogenetically, fungal enzymes in GH5_5 sorted into two clades, with StCel5A and TrCel5A belonging to different clades. Structural differences with the potential to account for the differences in performance were deduced based on the known structure of TrCel5A and a homology-based model of StCel5A, including a loop near the active site of TrCel5A and the presence of four additional Trp residues in the active cleft of StCel5A. The results indicate that superior biomass-degrading enzymes can be identified by exploring taxonomic diversity combined with assays in the context of realistic enzyme combinations and realistic substrates. Substrate range may be a key factor contributing to superior performance within GH5_5.     

Alkaline peroxide pretreatment of corn stover: effects of biomass, peroxide, and enzyme loading and composition on yields of glucose and xylose

Background

Pretreatment is a critical step in the conversion of lignocellulose to fermentable sugars. Although many pretreatment processes are currently under investigation, none of them are entirely satisfactory in regard to effectiveness, cost, or environmental impact. The use of hydrogen peroxide at pH 11.5 (alkaline hydrogen peroxide (AHP)) was shown by Gould and coworkers to be an effective pretreatment of grass stovers and other plant materials in the context of animal nutrition and ethanol production. Our earlier experiments indicated that AHP performed well when compared against two other alkaline pretreatments. Here, we explored several key parameters to test the potential of AHP for further improvement relevant to lignocellulosic ethanol production.

Results

The effects of biomass loading, hydrogen peroxide loading, residence time, and pH control were tested in combination with subsequent digestion with a commercial enzyme preparation, optimized mixtures of four commercial enzymes, or optimized synthetic mixtures of pure enzymes. AHP pretreatment was performed at room temperature (23°C) and atmospheric pressure, and after AHP pretreatment the biomass was neutralized with HCl but not washed before enzyme digestion. Standard enzyme digestion conditions were 0.2% glucan loading, 15 mg protein/g glucan, and 48 h digestion at 50°C. Higher pretreatment biomass loadings (10% to 20%) gave higher monomeric glucose (Glc) and xylose (Xyl) yields than the 2% loading used in earlier studies. An H₂O₂ loading of 0.25 g/g biomass was almost as effective as 0.5 g/g, but 0.125 g/g was significantly less effective. Optimized mixtures of four commercial enzymes substantially increased post-AHP-pretreatment enzymatic hydrolysis yields at all H₂O₂ concentrations compared to any single commercial enzyme. At a pretreatment biomass loading of 10% and an H₂O₂ loading of 0.5 g/g biomass, an optimized commercial mixture at total protein loadings of 8 or 15 mg/g glucan gave monomeric Glc yields of 83% or 95%, respectively. Yields of Glc and Xyl after pretreatment at a low hydrogen peroxide loading (0.125 g H₂O₂/g biomass) could be improved by extending the pretreatment residence time to 48 h and readjusting the pH to 11.5 every 6 h during the pretreatment. A Glc yield of 77% was obtained using a pretreatment of 15% biomass loading, 0.125 g H₂O₂/g biomass, and 48 h with pH adjustment, followed by digestion with an optimized commercial enzyme mixture at an enzyme loading of 15 mg protein/g glucan.

Conclusions

Alkaline peroxide is an effective pretreatment for corn stover. Particular advantages are the use of reagents with low environmental impact and avoidance of special reaction chambers. Reasonable yields of monomeric Glc can be obtained at an H₂O₂ concentration one-quarter of that used in previous AHP research. Additional improvements in the AHP process, such as peroxide stabilization, peroxide recycling, and improved pH control, could lead to further improvements in AHP pretreatment.     

Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5–15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei.     

The O-glycosylated linker from the Trichoderma reesei Family 7 cellulase is a flexible, disordered protein

Fungi and bacteria secrete glycoprotein cocktails to deconstruct cellulose. Cellulose-degrading enzymes (cellulases) are often modular, with catalytic domains for cellulose hydrolysis and carbohydrate-binding modules connected by linkers rich in serine and threonine with O-glycosylation. Few studies have probed the role that the linker and O-glycans play in catalysis. Since different expression and growth conditions produce different glycosylation patterns that affect enzyme activity, the structure-function relationships that glycosylation imparts to linkers are relevant for understanding cellulase mechanisms. Here, the linker of the Trichoderma reesei Family 7 cellobiohydrolase (Cel7A) is examined by simulation. Our results suggest that the Cel7A linker is an intrinsically disordered protein with and without glycosylation. Contrary to the predominant view, the O-glycosylation does not change the stiffness of the linker, as measured by the relative fluctuations in the end-to-end distance; rather, it provides a 16 Å extension, thus expanding the operating range of Cel7A. We explain observations from previous biochemical experiments in the light of results obtained here, and compare the Cel7A linker with linkers from other cellulases with sequence-based tools to predict disorder. This preliminary screen indicates that linkers from Family 7 enzymes from other genera and other cellulases within T. reesei may not be as disordered, warranting further study.     

Synthetic enzyme mixtures for biomass deconstruction: Production and optimization of a core set

The high cost of enzymes is a major bottleneck preventing the development of an economically viable lignocellulosic ethanol industry. Commercial enzyme cocktails for the conversion of plant biomass to fermentable sugars are complex mixtures containing more than 80 proteins of suboptimal activities and relative proportions. As a step toward the development of a more efficient enzyme cocktail for biomass conversion, we have developed a platform, called GENPLAT, that uses robotic liquid handling and statistically valid experimental design to analyze synthetic enzyme mixtures. Commercial enzymes (Accellerase 1000 +/? Multifect Xylanase, and Spezyme CP +/? Novozyme 188) were used to test the system and serve as comparative benchmarks. Using ammonia‐fiber expansion (AFEX) pretreated corn stover ground to 0.5 mm and a glucan loading of 0.2%, an enzyme loading of 15 mg protein/g glucan, and 48 h digestion at 50°C, commercial enzymes released 53% and 41% of the available glucose and xylose, respectively. Mixtures of three, five, and six pure enzymes of Trichoderma species, expressed in Pichia pastoris, were systematically optimized. Statistical models were developed for the optimization of glucose alone, xylose alone, and the average of glucose + xylose for two digestion durations, 24 and 48 h. The resulting models were statistically significant (P < 0.0001) and indicated an optimum composition for glucose release (values for optimized xylose release are in parentheses) of 29% (5%) cellobiohydrolase 1, 5% (14%) cellobiohydrolase 2, 25% (25%) endo‐β1,4‐glucanase 1, 14% (5%) β‐glucosidase, 22% (34%) endo‐β1,4‐xylanase 3, and 5% (17%) β‐xylosidase in 48 h at a protein loading of 15 mg/g glucan. Comparison of two AFEX‐treated corn stover preparations ground to different particle sizes indicated that particle size (100 vs. 500 µm) makes a large difference in total digestibility. The assay platform and the optimized “core” set together provide a starting point for the rapid testing and optimization of alternate core enzymes from other microbial and recombinant sources as well as for the testing of “accessory” proteins for development of superior enzyme mixtures for biomass conversion. Biotechnol. Bioeng. 2010;106: 707–720. © 2010 Wiley Periodicals, Inc.     

Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin

The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs.     

Cellulase–lignin interactions—The role of carbohydrate-binding module and pH in non-productive binding

Non-productive cellulase adsorption onto lignin is a major inhibitory mechanism preventing enzymatic hydrolysis of lignocellulosic feedstocks. Therefore, understanding of enzyme–lignin interactions is essential for the development of enzyme mixtures and processes for lignocellulose hydrolysis. We have studied cellulase–lignin interactions using model enzymes, Melanocarpus albomyces Cel45A endoglucanase (MaCel45A) and its fusions with native and mutated carbohydrate-binding modules (CBMs) from Trichoderma reesei Cel7A. Binding of MaCel45A to lignin was dependent on pH in the presence and absence of the CBM; at high pH, less enzyme bound to isolated lignins. Potentiometric titration of the lignin preparations showed that negatively charged groups were present in the lignin samples and that negative charge in the samples was increased with increasing pH. The results suggest that electrostatic interactions contributed to non-productive enzyme adsorption: Reduced enzyme binding at high pH was presumably due to repulsive electrostatic interactions between the enzymes and lignin. The CBM increased binding of MaCel45A to the isolated lignins only at high pH. Hydrophobic interactions are probably involved in CBM binding to lignin, because the same aromatic amino acids that are essential in CBM–cellulose interaction were also shown to contribute to lignin-binding.     

Binding characteristics of Trichoderma reesei cellulases on untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated lignocellulosic biomass

Studying the binding properties of cellulases to lignocellulosic substrates is critical to achieving a fundamental understanding of plant cell wall saccharification. Lignin auto-fluorescence and degradation products formed during pretreatment impede accurate quantification of individual glycosyl hydrolases (GH) binding to pretreated cell walls. A high-throughput fast protein liquid chromatography (HT-FPLC)-based method has been developed to quantify cellobiohydrolase I (CBH I or Cel7A), cellobiohydrolase II (CBH II or Cel6A), and endoglucanase I (EG I or Cel7B) present in hydrolyzates of untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated corn stover (CS). This method can accurately quantify individual enzymes present in complex binary and ternary protein mixtures without interference from plant cell wall-derived components. The binding isotherms for CBH I, CBH II, and EG I were obtained after incubation for 2 h at 4 °C. Both AFEX and dilute acid pretreatment resulted in increased cellulase binding compared with untreated CS. Cooperative binding of CBH I and/or CBH II in the presence of EG I was observed only for AFEX treated CS. Competitive binding between enzymes was found for certain other enzyme-substrate combinations over the protein loading range tested (i.e., 25-450 mg/g glucan). Langmuir single-site adsorption model was fitted to the binding isotherm data to estimate total available binding sites E(bm) (mg/g glucan) and association constant K(a) (L/mg). Our results clearly demonstrate that the characteristics of cellulase binding depend not only on the enzyme GH family but also on the type of pretreatment method employed.     

Cellulase and Xylanase Production by the Mexican Strain <Emphasis Type="Italic">Talaromyces stollii</Emphasis> LV186 and Its Application in the Saccharification of Pretreated Corn and Sorghum Stover

A Mexican strain of Talaromyces stollii LV186 was isolated from decaying pretreated corn stover. The production of cellulase and xylanase enzyme cocktails was evaluated with corn and sorghum stover used as inducers in a mineral medium. The volumetric and specific activities of T. stollii LV186 were compared with the values produced by Trichoderma reesei ATCC 26921 in a time-course experiment. After the submerged culture and a posterior ultrafiltration stage, the enzyme complexes were evaluated over acid-pretreated corn or sorghum stover in baffled flasks under controlled temperature and agitation conditions, and hydrolysis levels of 30 and 39 % of the theoretical maximum were obtained after only 72-h reactions, for each substrate. A side-by-side comparison showed a better ratio of endoglucanase to cellobiohydrolase to β-glucosidase and of xylanase to β-xylosidase enzymes in T. stollii than in T. reesei ATCC 26921. Furthermore, the hydrolysis of pretreated corn and sorghum stover achieved by T. stollii is significantly higher compared with that of a commercial cocktail from T. reesei ATCC 26921 (Celluclast). Therefore, the T. stollii LV186 strain is a good candidate for the hydrolysis of complex lignocellulose substrates. To the authors’ knowledge, this study is the first to describe the cellulolytic and hemicellulolytic activities produced by a T. stollii strain.     

Lignocellulolytic characterization and comparative secretome analysis of a Trichoderma erinaceum strain isolated from decaying sugarcane straw

The fungus Trichoderma reesei is employed in the production of most enzyme cocktails used by the lignocellulosic biofuels industry today. Despite significant improvements, the cost of the required enzyme preparations remains high, representing a major obstacle for the industrial production of these alternative fuels. In this study, a new Trichoderma erinaceum strain was isolated from decaying sugarcane straw. The enzyme cocktail secreted by the new isolate during growth in pretreated sugarcane straw-containing medium presented higher specific activities of β-glucosidase, endoxylanase, β-xylosidase and α-galactosidase than the cocktail of a wild T. reesei strain and yielded more glucose in the hydrolysis of pretreated sugarcane straw. A proteomic analysis of the two strains' secretomes identified a total of 86 proteins, of which 48 were exclusive to T. erinaceum, 35 were exclusive to T. reesei and only 3 were common to both strains. The secretome of T. erinaceum also displayed a higher number of carbohydrate-active enzymes than that of T. reesei (37 and 27 enzymes, respectively). Altogether, these results reveal the significant potential of the T. erinaceum species for the production of lignocellulases, both as a possible source of enzymes for the supplementation of industrial cocktails and as a candidate chassis for enzyme production.     

Implications of cellobiohydrolase glycosylation for use in biomass conversion

The cellulase producing ascomycete, Trichoderma reesei (Hypocrea jecorina), is known to secrete a range of enzymes important for ethanol production from lignocellulosic biomass. It is also widely used for the commercial scale production of industrial enzymes because of its ability to produce high titers of heterologous proteins. During the secretion process, a number of post-translational events can occur, however, that impact protein function and stability. Another ascomycete, Aspergillus niger var. awamori, is also known to produce large quantities of heterologous proteins for industry. In this study, T. reesei Cel7A, a cellobiohydrolase, was expressed in A. niger var. awamori and subjected to detailed biophysical characterization. The purified recombinant enzyme contains six times the amount of N-linked glycan than the enzyme purified from a commercial T. reesei enzyme preparation. The activities of the two enzyme forms were compared using bacterial (microcrystalline) and phosphoric acid swollen (amorphous) cellulose as substrates. This comparison suggested that the increased level of N-glycosylation of the recombinant Cel7A (rCel7A) resulted in reduced activity and increased non-productive binding on cellulose. When treated with the N-glycosidase PNGaseF, the molecular weight of the recombinant enzyme approached that of the commercial enzyme and the activity on cellulose was improved.     

Enhanced lignocellulosic biomass hydrolysis by oxidative lytic polysaccharide monooxygenases (LPMOs) GH61 from Gloeophyllum trabeum

Lignocellulose is a renewable resource that is extremely abundant, and the complete enzymatic hydrolysis of lignocellulose requires a cocktail containing a variety of enzyme groups that act synergistically. The hydrolysis efficiency can be improved by introducing glycoside hydrolase 61 (GH61), a new enzyme that belongs to the auxiliary activity family 9 (AA9). GH61was isolated from Gloeophyllum trabeum and cleaves the glycosidic bonds on the cellulose surface via oxidation of various carbons. In this study, we investigated the properties of GH61. GtGH61 alone did not exhibit any notable activity, but the synergistic activity of GtGH61 with xylanase (GtXyl10G) or cellulase (GtCel5B) showed efficient bioconversion rates of 56 and 174% in pretreated kenaf (Hibiscus cannabinus L.) and oak (Quercus spp.), respectively. Furthermore, the GtGH61 activity was strongly accelerated in the presence of cobalt Co²⁺. Enzyme cocktails (GtXyl10G, GtCel5B, and GtGH61) increased the amount of sugar released by 7 and 6% for pretreated oak and kenaf, respectively, and the addition of Co²⁺ stimulated bioconversion by 12 and 11% in pretreated oak and kenaf, respectively.     

Processivity, Substrate Binding, and Mechanism of Cellulose Hydrolysis by Thermobifida fusca Cel9A

Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(−1) O1. R378, a residue interacting with Glc(+1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(−2) to Glc(−4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(−2) to Glc(−4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.     

Liquid chromatography combined with mass spectrometry for the investigation of endoglucanase selectivity on carboxymethyl cellulose

Endoglucanases are useful tools in the chemical structure analysis of cellulose derivatives. However, knowledge on the endoglucanase selectivity, which is of central importance for data interpretation, is still limited. In this study, new reverse-phase liquid chromatography mass spectrometry (LC–MS) methods were developed to investigate the selectivity of the endoglucanases Cel5A, Cel7B, Cel45A, and Cel74A from the filamentous fungus Trichoderma reesei. The aim was to improve the identification of the regioisomers in the complex mixtures that are obtained after enzymatic hydrolysis. Reduction followed by per-O-methylation was performed in order to improve the separation in reverse-phase LC, increase MS sensitivity, and to facilitate structure analysis by MS/MS of O-carboxymethyl glucose and cellooligosaccharides. The cellulose selective enzymes that were investigated displayed interesting differences in enzyme selectivity on CMC substrates.     

A 42,000-Da protein in rabbit tissues and in a glycogen synthase preparation cross-reacts with antibodies to glycogenin

Rabbit skeletal muscle glycogen previously has been shown to be covalently bound to a 40,000-Da protein ("glycogenin") via a novel glucosyl-tyrosine linkage [I.R. Rodriguez and W.J. Whelan (1985) Biochem. Biophys. Res. Commun. 132, 829-836]. Antibodies raised against rabbit skeletal muscle glycogenin cross-react with a similar protein present in rabbit heart and liver glycogens, as well as with a 42,000-Da "acceptor protein" present in high-speed supernatants of rabbit muscle, heart, retina, and liver. This 42,000-Da protein incorporates [U-14C]Glc when an ammonium sulfate fraction prepared from the tissue supernatants is incubated with UDP-[U-14C]Glc. The [U-14C]Glc incorporated can be removed quantitatively by treatment with amylolytic enzymes, indicating that the [U-14C]Glc incorporation represents elongation of a preexisting glucan attached to the acceptor protein. Furthermore, a commercial preparation of rabbit skeletal muscle glycogen synthase contains this 42,000-Da protein. We propose that the 42,000-Da protein represents the free form of glycogenin in tissues, with its covalently attached glucan chain(s) providing a "primed" elongation site for glycogen synthesis.     

Asn64-glycosylation affects Hypocrea jecorina (syn. Trichoderma reesei) cellobiohydrolase Cel7A activity expressed in Pichia pastoris

In this study, four N-glycosylation sites, Asn45, Asn64, Asn270 and Asn384 of Hypocrea jecorina (syn. Trichoderma reesei) Cel7A (family 7 cellobiohydrolase I) were replaced by serines using site-directed mutagenesis. These four mutants and wild type H. jecorina Cel7A gene were transformed into P. pastoris, and the recombinant enzymes were purified and analyzed. The enzymatic activities of recombinant Cel7A (rCel7A), and mutants N45S, N270S and N384S were very low while mutant N64S displayed about seven times higher activity than that of rCel7A, and about 10% of the wild-type Cel7A activity from H. jecorina. The results indicate that N-glycosylation of Asn64 had an effect on the activity of the Cel7A enzyme expressed in P. pastoris, and that glycosylation at this site would be only a subordinate reason for the low activity of the recombinant enzyme.     

Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF,recycling of hydrolytic enzymes and yeast,and recombinant cellulase-producing Aspergillus niger

Bioethanol and enzymes were produced from fiber sludges through sequential microbial cultivations. After a first simultaneous saccharification and fermentation (SSF) with yeast, the bioethanol concentrations of sulfate and sulfite fiber sludges were 45.6 and 64.7 g/L, respectively. The second SSF, which included fresh fiber sludges and recycled yeast and enzymes from the first SSF, resulted in ethanol concentrations of 38.3 g/L for sulfate fiber sludge and 24.4 g/L for sulfite fiber sludge. Aspergillus niger carrying the endoglucanase-encoding Cel7B gene of Trichoderma reesei was grown in the spent fiber sludge hydrolysates. The cellulase activities obtained with spent hydrolysates of sulfate and sulfite fiber sludges were 2,700 and 2,900 nkat/mL, respectively. The high cellulase activities produced by using stillage and the significant ethanol concentrations produced in the second SSF suggest that onsite enzyme production and recycling of enzyme are realistic concepts that warrant further attention.     

Different effects of carbohydrate binding modules on the viscoelasticity of nanocellulose gels

Many cellulose degrading and modifying enzymes have distinct parts called carbohydrate binding modules (CBMs). The CBMs have been shown to increase the concentration of enzymes on the insoluble substrate and thereby enhance catalytic activity. It has been suggested that CBMs also have a role in disrupting or dispersing the insoluble cellulose substrate, but dispute remains and explicit evidence of such a mechanism is lacking. We produced the isolated CBMs from two major cellulases (Cel6A and Cel7A) from Trichoderma reesei as recombinant proteins in Escherichia coli. We then studied the viscoelastic properties of native unmodified cellulose nanofibrils (CNF) in combination with the highly purified CBMs to detect possible functional effects of the CBMs on the CNF. The two CBMs showed clearly different effects on the viscoelastic properties of CNF. The difference in effects is noteworthy, yet it was not possible to conclude for example disruptive effects. We discuss here the alternative explanations for viscoelastic effects on CNF caused by CBMs, including the effect of ionic cosolutes.     

Processivity,Synergism, and Substrate Specificity of Thermobifida fusca Cel6B

A relationship between processivity and synergism has not been reported for cellulases, although both characteristics are very important for hydrolysis of insoluble substrates. Mutation of two residues located in the active site tunnel of Thermobifida fusca exocellulase Cel6B increased processivity on filter paper. Surprisingly, mixtures of the Cel6B mutant enzymes and T. fusca endocellulase Cel5A did not show increased synergism or processivity, and the mutant enzyme which had the highest processivity gave the poorest synergism. This study suggests that improving exocellulase processivity might be not an effective strategy for producing improved cellulase mixtures for biomass conversion. The inverse relationship between the activities of many of the mutant enzymes with bacterial microcrystalline cellulose and their activities with carboxymethyl cellulose indicated that there are differences in the mechanisms of hydrolysis for these substrates, supporting the possibility of engineering Cel6B to target selected substrates.Cellulose is a linear homopolymer of β-1,4-linked anhydrous glucosyl residues with a degree of polymerization (DP) of up to 15,000 (5). Adjacent glucose residues in cellulose are oriented at an angle of 180° to each other, making cellobiose the basic unit of cellulose structure (5). The β-1,4-glycosidic bonds of cellulose are enzymatically hydrolyzed by three classes of cellulases. Endocellulases (EC 3.2.1.4) cleave cellulose chains internally, generating products of variable length with new chain ends, while exocellulases, also called cellobiohydrolases (EC 3.2.1.91), act from one end of a cellulose chain and processively cleave off cellobiose as the main product. The third class is the processive endocellulases, which can be produced by bacteria (2, 20).Processivity and synergism are important properties of cellulases, particularly for hydrolysis of crystalline substrates. Processivity indicates how far a cellulase molecule proceeds and hydrolyzes a substrate chain before there is dissociation. Processivity can be measured indirectly by determining the ratio of soluble products to insoluble products in filter paper assays (14, 19, 39). Although this approach might not discriminate exocellulases from highly processive endocellulases (12), it is very helpful for comparing mutants of the same enzyme (19). The processivity of some glycoside hydrolases also can be determined from the ratio of dimers to monomers in the hydrolysate (13).Four types of synergism have been demonstrated in cellulase systems: synergism between endocellulases and exocellulases, synergism between reducing- and nonreducing-end-directed exocellulases, synergism between processive endocellulases and endo- or exocellulases, and synergism between β-glucosidases and other cellulases (3). Synergism is dependent on a number of factors, including the physicochemical properties of the substrate and the ratio of the individual enzymes (10).Great effort has been focused on improving enzymatic hydrolysis of cellulases in biomass (24). However, studying biomass is difficult due to its complexity; instead, nearly pure cellulose, amorphous cellulose, or carboxymethyl cellulose (CMC) are commonly used as substrates (22).Random mutagenesis approaches and rational protein design have been used to study cellulose hydrolysis (18), to improve the activity of catalytic domains and carbohydrate-binding modules (19), and to thermostabilize cellulases (9). Increased knowledge of cellulase structures and improvements in modeling software (1) have facilitated rational protein design. The structures of five glycoside hydrolase family 6 cellulases from four microorganisms, Trichoderma reesei (23), Thermobifida fusca (26), Humicola insolens (6, 29), and Mycobacterium tuberculosis (30), have been determined. Structural analysis showed that the active sites of the exocellulases are enclosed by two long loops forming a tunnel, while the endocellulases have an open active site groove. Movement of one of these loops is important for enzymatic activity (6, 35, 37).In nature, as well as for industrial applications, mixtures of cellulase are required; therefore, a better strategy for designing individual enzymes to improve the activity of mixtures is critical. In this study, we used Cel6B, a nonreducing-end-directed, inverting exocellulase from Thermobifida fusca, a thermophilic soil bacterium, as a model cellulase to investigate the impact of improved exocellulases in mixtures with endocellulases since T. fusca Cel6B is important for achieving the maximum activity of synergistic mixtures (35). Cel6B activity is similar to that of the fungal T. reesei exocellulase Cel6A, but Cel6B has higher thermostability and a much broader pH optimum (36). Six noncatalytic residues in the active site tunnel of T. fusca exocellulase Cel6B were mutated to obtain insight into the role of these residues in processivity and substrate specificity. Two mutant enzymes that showed higher activity with filter paper and processivity were investigated further for production of oligosaccharides and synergism to analyze the relationship between processivity and synergism.