Biochemistry and Genetics of Actinomycete Cellulases

Abstract

The order Actinomycetales includes a number of genera that contain species that actively degrade cellulose and these include both mesophilic and facultative thermophilic species. Cellulases produced by strains from two of the genera containing thermophilic organisms have been studied extensively: Microbispora bispora and Thermomonospora fusca. Fractionation of M. bispora cellulases has identified six different enzymes, all of which were purified to near homogeneity and partially characterized. Two of these enzymes appear to be exocellulases and gave synergism with each other and with the endocellulases. The structural genes of five M. bispora cellulases have been cloned and one was sequenced. Fractionation of T. fusca cellulases has identified five different enzymes, all of which were purified to near homogeneity and partially characterized. One of the T. fusca enzymes gives synergism in the hydrolysis of crystalline cellulose with several T. fusca endocellulases and with Trichoderma reesei CBHI but not with T. reesei CBHII. Each T. fusca cellulase contains distinct catalytic and cellulose binding domains. The structural genes of four of the T. fusca endoglucanases have been cloned and sequenced, while three cellulase genes have been cloned from “T. curvata”. The T. fusca cellulase genes are expressed at a low level in Escherichia coli, but at a high level in Streptomyces lividans. Sequence comparisons have shown that there are no significant amino acid homologies between any of the catalytic domains of the four T. fusca cellulases, but each of them shows extensive homology to several other cellulases and fits in one of the five existing cellulase gene families. There have been extensive studies of the regulation of the synthesis of these cellulases and a number of regulatory mutants have been isolated. This work has shown that the different T. fusca cellulases are coordinately regulated over a 100-fold range by two independent controls; induction by cellobiose and repression by any good carbon source.     

Compatible ionic liquid-cellulases system for hydrolysis of lignocellulosic biomass

Ionic liquids (ILs) have been increasingly recognized as novel solvents for dissolution and pretreatment of cellulose. However, cellulases are inactivated in the presence of ILs, even when present at low concentrations. To more fully exploit the benefits of ILs it is critical to develop a compatible IL‐cellulases system in which the IL is able to effectively solubilize and activate the lignocellulosic biomass, and the cellulases possess high stability and activity. In this study, we investigated the stability and activity of a commercially available cellulases mixture in the presence of different concentrations of 1‐ethyl‐3‐methylimidazolium acetate ([Emim][OAc]). A mixture of cellulases and β‐glucosidase (Celluclast1.5L, from Trichoderma reesei, and Novozyme188, from Aspergillus niger, respectively) retained 77% and 65% of its original activity after being pre‐incubated in 15% and 20% (w/v) IL solutions, respectively, at 50°C for 3 h. The cellulases mixture also retained high activity in 15% [Emim][OAc] to hydrolyze Avicel, a model substrate for cellulose analysis, with conversion efficiency of approximately 91%. Notably, the presence of different amounts of yellow poplar lignin did not interfere significantly with the enzymatic hydrolysis of Avicel. Using this IL‐cellulase system (15% [Emim][OAc]), the saccharification of yellow poplar biomass was also significantly improved (33%) compared to the untreated control (3%) during the first hour of enzymatic hydrolysis. Together, these findings provide compelling evidence that [Emim][OAc] was compatible with the cellulase mixture, and this compatible IL‐cellulases system is promising for efficient activation and hydrolysis of native biomass to produce biofuels and co‐products from the individual biomass components. Bioeng. 2011; 108:1042–1048. © 2010 Wiley Periodicals, Inc.     

Application of cellulases from Acrophialophora nainiana and Penicillium echinulatum in textile processing of cellulosic fibres

New cellulases from the fungi Acrophialophora nainiana and Penicillium echinulatum were used in the finishing of knitted cotton fabrics (biopolishing) and compared with the well established enzymes from Trichoderma reesei. Both cellulases reduced the pilling tendency with a lower weight loss than T. reesei cellulases. Cellulases from P. echinulatum were also studied in stonewashing of denim fabrics to obtain the fashionable aged look in indigo dyed jeans ware and were found to remove more colour from denim fabrics and produce less indigo dye redeposition (back-staining) than commercial acid or neutral cellulases under the test conditions. Efficiency was found to be influenced by pH during textile processing and the substrate used for the production of cellulases. Cellulases produced by P. echinulatum grown on cellulose showed better stonewashing results (higher colour removal and less back-staining) than cellulases produced on sugar cane bagasse. The substrate used during enzyme production of P. echinulatum cellulases seems to have a significant influence on cellulose composition, which affects textile processing results.     

Structure of hydrolases: lipases and cellulases

Knowledge of lipase mechanisms has increased significantly during the past year. The structural characterization of the opening mechanism of the active site of lipases, as first described for Rhizomucor miehei lipase, has now been extended to the pancreatic lipase-colipase system, and to the Geotrichum candidum/Candida rugosa lipases. In the latter two lipase families, lid opening is far more complicated than for R. miehei lipase. Resolution of the structure of cutinase, an esterase with lipase activity, and determination of the sequence of guinea pig pancreatic lipase showed that these lipases have no lid. The fact that both enzymes are not activated at the interface shows the importance of the lid in the latter phenomenon. On the basis of sequence analysis, cellulases have been divided into different families. Structural determinations of some members of a few of these families confirm that they have different folds. The active sites of these cellulases always seem to contain acidic catalytic groups. The relative spatial position of these groups and their accessibility varies considerably among the cellulases for which structural determinations have been made.     

<Emphasis Type="Italic">Thermobifida fusca</Emphasis> exoglucanase Cel6B is incompatible with the cellulosomal mode in contrast to endoglucanase Cel6A

Cellulosomes are efficient cellulose-degradation systems produced by selected anaerobic bacteria. This multi-enzyme complex is assembled from a group of cellulases attached to a protein scaffold termed scaffoldin, mediated by a high-affinity protein–protein interaction between the enzyme-borne dockerin module and the cohesin module of the scaffoldin. The enzymatic complex is attached as a whole to the cellulosic substrate via a cellulose-binding module (CBM) on the scaffoldin subunit. In previous works, we have employed a synthetic biology approach to convert several of the free cellulases of the aerobic bacterium, Thermobifida fusca, into the cellulosomal mode by replacing each of the enzymes’ CBM with a dockerin. Here we show that although family six enzymes are not a part of any known cellulosomal system, the two family six enzymes of the T. fusca system (endoglucanase Cel6A and exoglucanase Cel6B) can be converted to work as cellulosomal enzymes. Indeed, the chimaeric dockerin-containing family six endoglucanase worked well as a cellulosomal enzyme, and proved to be more efficient than the parent enzyme when present in designer cellulosomes. In stark contrast, the chimaeric family six exoglucanase was markedly less efficient than the wild-type enzyme when mixed with other T. fusca cellulases, thus indicating its incompatibility with the cellulosomal mode of action.     

Endogenous origin of endo-β-1,4-glucanase in common woodlouse Porcellio scaber (Crustacea, Isopoda)

Because endogenous cellulases have been observed in arthropods, the potential ability to produce cellulose degrading enzymes was examined in the terrestrial isopod Porcellio scaber, an important decomposer of decayed plant material. cDNA fragments encoding portions of two novel endo-β-1,4-glucanase amino acid sequences were amplified by RT-PCR, and the amino acid sequences predicted were affiliated to endo-β-1,4-glucanases from other arthropods, where they cluster with endo-β-1,4-glucanases of decapod crustaceans. Hybridization in situ reveals the hepatopancreas to be the primary site of gene expression and provides direct evidence of the endogenous origin of endo-β-1,4-glucanase in P. scaber. Conservation of catalytically important amino acid residues suggests that both sequences translate into functional cellulases. Cellulolytic activity was detected in hepatopancreatic extract after separation by SDS-PAGE, which included CMC as substrate. This is the first evidence of endogenous cellulases in peracarid crustaceans and gives strong support for the involvement of isopod endo-β-1,4-glucanases in the degradation of cellulose in their diet.     

Wetland environmental bioreactor system contributes to the decomposition of cellulose

Recently, numerous species of aquatic invertebrates inhabiting wetlands have been shown to possess endogenous cellulase, following the discovery that termites have cellulase genes encoded in their own genome rather than relying on symbiotic bacteria for decomposing cellulose. Wetlands have been empirically shown to play an important role in the decomposition of land‐originating hard‐to‐degrade polysaccharides such as cellulose. However, the mechanism that connects the cellulase producer and the wetlands remains unknown, which makes it very difficult to evaluate the ecological function of wetlands. Here we found that a macrobenthic bivalve, Corbicula japonica, secretes its cellulase to the wetland sediment. Secreted cellulases are immobilized in the components of the sediment. Moreover, adding cellulose or glucose to C. japonica could trigger its cellulase secretion level. These findings suggest a novel wetland cellulose decomposition mechanism. The decomposition ability of wetlands was previously ascribed only to microbes and/or invertebrates that contain cellulases. Our findings suggest that benthic animals supply wetlands with their enzymes as decomposition agents, while wetland sediments serve as immobilization scaffolds for the enzymes. This system, which was named by us an “environmental bioreactor system,” could provide a key function in wetlands.     

Trichoderma reesei cellobiohydrolase II is associated with the outer membrane when overexpressed in Escherichia coli

Cellulose degradation is essential for the future production of many advanced biofuels. Cellulases from the filamentous fungus Trichoderma reesei are among the most efficient enzymes for the hydrolysis of cellulosic materials. One of the cellulases from T. reesei, cellobiohydrolase II (CBH2), was studied because of its industrial relevance and proven enzymatic activity. Using both crude and rigorous membrane fractionation methods we show that full length T. reesei CBH2 is exclusively localized to the outer membrane when expressed recombinantly in Escherichia coli. Even fusing signal sequence-free maltose-binding protein to the N-terminus of CBH2, which has been shown to increase solubility of other proteins, did not prevent the outer membrane localization of CBH2. These results highlight the difficulties in producing fungal cellulases in bacterial hosts and provide a stepping stone for future cellulase engineering efforts.     

Trichoderma reesei cellulases in the bleaching of kraft pulp

In this work the effects of individual purified cellulases of Trichoderma reesei were studied in the enzyme-aided bleaching of kraft pulps. The cellobiohydrolases I and II, when used alone, had no positive effect on the bleachability of kraft pulps. The endoglucanase I (EG I), however, acted on pulp similarly to xylanases and with an enzyme dosage of 0.1 mg/g a clear increase in pulp brightness could be observed. Due to the unspecificity of this enzyme, the viscosity of the pulp was simultaneously decreased. Of the cellulases, EG II was clearly most detrimental in reducing the pulp viscosity. Hence, the action of purified cellulases of T. reesei on pulp as a substrate differs profoundly, and all cellulases are not detrimental to the pulp properties. Correspondence to: J. Buchert     

Biochemistry and genetics of actinomycete cellulases.

The order Actinomycetales includes a number of genera that contain species that actively degrade cellulose and these include both mesophilic and facultative thermophilic species. Cellulases produced by strains from two of the genera containing thermophilic organisms have been studied extensively: Microbispora bispora and Thermomonospora fusca. Fractionation of M. bispora cellulases has identified six different enzymes, all of which were purified to near homogeneity and partially characterized. Two of these enzymes appear to be exocellulases and gave synergism with each other and with the endocellulases. The structural genes of five M. bispora cellulases have been cloned and one was sequenced. Fractionation of T. fusca cellulases has identified five different enzymes, all of which were purified to near homogeneity and partially characterized. One of the T. fusca enzymes gives synergism in the hydrolysis of crystalline cellulose with several T. fusca endocellulases and with Trichoderma reesei CBHI but not with T. reesei CBHII. Each T. fusca cellulase contains distinct catalytic and cellulose binding domains. The structural genes of four of the T. fusca endoglucanases have been cloned and sequenced, while three cellulase genes have been cloned from "T. curvata". The T. fusca cellulase genes are expressed at a low level in Escherichia soli, but at a high level in Streptomyces lividans. Sequence comparisons have shown that there are no significant amino acid homologies between any of the catalytic domains of the four T. fusca cellulases, but each of them shows extensive homology to several other cellulases and fits in one of the five existing cellulase gene families. There have been extensive studies of the regulation of the synthesis of these cellulases and a number of regulatory mutants have been isolated. This work has shown that the different T. fusca cellulases are coordinately regulated over a 100-fold range by two independent controls; induction by cellobiose and repression by any good carbon source.     

Thermobifida fusca cellulases exhibit limited surface diffusion on bacterial micro‐crystalline cellulose

Elucidation of cellulase–cellulose interactions is key to modeling biomass deconstruction and in understanding the processes that lead to cellulase inactivation. Here, fluorescence recovery after photobleaching and single molecule tracking (SMT) experiments are used to assess the surface diffusion of Thermobifida fusca cellulases on bacterial micro‐crystalline cellulose. Our results show that cellulases exhibit limited surface diffusion when bound to crystalline cellulose and that a large fraction of the cellulases remain immobile even at temperatures optimal for catalysis. A comparison of our experimental results to Monte Carlo (MC) simulations, which use published diffusion coefficients to model cellulase displacements, shows that even those enzymes that are mobile on the cellulose surface exhibit significantly slower diffusive motions than previously reported. In addition, it is observed that the enzymes that show significant displacements exhibit complex, non‐steady surface motions, which suggest that cellulose–bound cellulases exist in molecular states with different diffusive characteristics. These results challenge the notion that cellulases can freely diffuse over cellulose surfaces without catalyzing bond cleavage. Biotechnol. Bioeng. 2013; 110: 47–56. © 2012 Wiley Periodicals, Inc.     

Simultaneous release of recombinant cellulases introduced by coexpressing colicin E7 lysis in <Emphasis Type="Italic">Escherichia coli</Emphasis>

With the increasingly serious global environment problem caused by slather use of fossil fuels, numerous efforts have been made in developing renewable alternatives. Converting lignocellulose to bioenergy has accomplished by cellulases but the production is limited, therefore the recombinant expression platforms in E. coli have been established. Since E. coli is partly restricted by its inability to secrete enzymes to extracellular membrane, we constructed a synthetic circuit to coexpress cellulases and colicin E7 lysis to solve this problem. The pBAD-RBS-lysis-TT (BBa_K117010) sequence from iGEM was added to form a chimeric plasmid based on pET system, which harboring cellulases from Bacillus subtilis or Thermobifida fusca. The presence of arabinose for the promoter pBAD, leading to induce lysis and further breakdown of the host membrane to release cellulase to extracellular membrane. The extracellular activities increase to 48.1 and 55.5% under lysis gene functioned on Cel5 and CD1, respectively. This is the first attempt to show that cellulases have successfully expressed and released to extracellular membrane without cumbersome steps. Finally, this synthetic circuit simplifies and achieves the simultaneous release and heterologous expression of the cellulases as well as obtain a long-term stable enzyme in E. coli system.     

A GHF7 CELLULASE FROM THE PROTIST SYMBIONT COMMUNITY OF Reticulitermes flavipes ENABLES MORE EFFICIENT LIGNOCELLULOSE PROCESSING BY HOST ENZYMES

Termites and their gut microbial symbionts efficiently degrade lignocellulose into fermentable monosaccharides. This study examined three glycosyl hydrolase family 7 (GHF7) cellulases from protist symbionts of the termite Reticulitermes flavipes. We tested the hypotheses that three GHF7 cellulases (GHF7‐3, GHF7‐5, and GHF7‐6) can function synergistically with three host digestive enzymes and a fungal cellulase preparation. Full‐length cDNA sequences of the three GHF7s were assembled and their protist origins confirmed through a combination of quantitative PCR and cellobiohydrolase (CBH) activity assays. Recombinant versions of the three GHF7s were generated using a baculovirus‐insect expression system and their activity toward several model substrates compared with and without metallic cofactors. GHF7‐3 was the most active of the three cellulases; it exhibited a combination of CBH, endoglucanase (EGase), and β‐glucosidase activities that were optimal around pH 7 and 30°C, and enhanced by calcium chloride and zinc sulfate. Lignocellulose saccharification assays were then done using various combinations of the three GHF7s along with a host EGase (Cell‐1), beta‐glucosidase (β‐glu), and laccase (LacA). GHF7‐3 was the only GHF7 to enhance glucose release by Cell‐1 and β‐glu. Finally, GHF7‐3, Cell‐1, and β‐glu were individually tested with a commercial fungal cellulase preparation in lignocellulose saccharification assays, but only β‐glu appreciably enhanced glucose release. Our hypothesis that protist GHF7 cellulases are capable of synergistic interactions with host termite digestive enzymes is supported only in the case of GHF7‐3. These findings suggest that not all protist cellulases will enhance saccharification by cocktails of other termite or fungal lignocellulases.     

Functional display of fungal cellulases from <Emphasis Type="Italic">Trichoderma </Emphasis><Emphasis Type="Italic">reesei</Emphasis> on phage M13

To test whether the phage display technology could be applied in cellulase engineering, phagemids harboring the genes encoding the mature forms of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) from filamentous fungus Trichoderma reesei were constructed, respectively. CBH I and EG I fused to the phage coat protein encoded by the g3 gene were expressed and displayed on phage M13. The phage-bound cellulases retained their activities as determined by hydrolysis of the corresponding substrates, Also, their binding abilities to insoluble cellulose substrate were confirmed by an ELISA method. Overall, these results demonstrate that cellulases can be displayed on phage surface while maintaining their biological function, thus providing an alternative for directed evolution and high-throughput screening for improved cellulases.     

The resistance of cellulases and xylanases to proteolytic inactivation

The sensitivity of a range of cellulases and xylanases to proteolytic inactivation was investigated. The xylanases, all the Clostridium thermocellum cellulases and cellulase E from Pseudomonas fluorescens subsp. cellulosa exhibited no decrease in catalytic activity during a 3-h incubation with proteinases of the small intestine. Under these conditions, the control Escherichia coli enzymes analysed had half-lives of 4.3–13.5 min. The addition of substrate significantly decreased the sensitivity of proteinase-labile enzymes to inactivation. The significance of these data in relation to the use of cellulases and xylanases for improving animal nutrition is discussed.     

Comparative secretome analyses of two <Emphasis Type="Italic">Trichoderma reesei</Emphasis> RUT-C30 and CL847 hypersecretory strains

Background  

Due to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly been researched in various fields of white biotechnology, especially in biofuel production from lignocellulosic biomass. The commercial enzyme mixtures produced at industrial scales are not well characterized, and their proteinaceous components are poorly identified and quantified. The development of proteomic methods has made it possible to comprehensively overview the enzymes involved in lignocellulosic biomass degradation which are secreted under various environmental conditions.     

Recycle of cellulases and the use of lignocellulosic residue for enzyme production after hydrolysis of steam-pretreated aspenwood

Summary Various modes of substrate and enzyme addition were used to hydrolyze a 10% concentration (w/v) of steam-exploded, water-and-alkali extracted aspenwood withTrichoderma harzianum E58 cellulases. Although cellulose conversion was high (94–100%), enzyme recovery was low in all cases. Low enzyme recovery was due to a combination of thermal inactivation and adsorption of the cellulases onto the lignocellulosic residue. Enzyme recycle was not feasible as the activity of the recovered cellulases towards crystalline cellulose was low. However, the residual material from enzyme hydrolysis was a suitable carbon source for cellulase enzyme production byT. harzianum based on enzyme yield and hydrolytic potential. These residues could only be used up to a 1% substrate concentration, since at higher substrate loadings cellulase production was reduced, likely because of lignin inhibitors.     

Termite gut symbiotic archaezoa are becoming living metabolic fossils

Over the course of several million years, the eukaryotic gut symbionts of lower termites have become adapted to a cellulolytic environment. Up to now it has been believed that they produce nutriments using their own cellulolytic enzymes for the benefit of their termite host. However, we have now isolated two endoglucanases with similar apparent molecular masses of approximately 36 kDa from the not yet culturable symbiotic Archaezoa living in the hindgut of the most primitive Australian termite, Mastotermes darwiniensis. The N-terminal sequences of these cellulases exhibited significant homology to cellulases of termite origin, which belong to glycosyl hydrolase family 9. The corresponding genes were detected not in the mRNA pool of the flagellates but in the salivary glands of M. darwiniensis. This showed that cellulases isolated from the flagellate cells originated from the termite host. By use of a PCR-based approach, DNAs encoding cellulases belonging to glycosyl hydrolase family 45 were obtained from micromanipulated nuclei of the flagellates Koruga bonita and Deltotrichonympha nana. These results indicated that the intestinal flagellates of M. darwiniensis take up the termite's cellulases from gut contents. K. bonita and D. nana possess at least their own endoglucanase genes, which are still expressed, but without significant enzyme activity in the nutritive vacuole. These findings give the impression that the gut Archaezoa are heading toward a secondary loss of their own endoglucanases and that they use exclusively termite cellulases.     

The first evidence that a single cellulase can be essential for cellulose degradation in a cellulolytic microorganism

Cellulose is the most abundant carbon source in nature but it is very difficult to degrade because of its insolubility, quasi‐crystalline structure and its presence in plant cell walls in a matrix with other polymers that limit access to the cellulose surface. Most cellulose in soils is degraded by cellulolytic microorganisms that use a number of different approaches to overcome the recalcitrance of cellulose in plant cell walls. All of these approaches involve multiple cellulases and, since cellulose is insoluble and microorganisms cannot ingest particles, the cellulases are present outside of the cell although they can be attached to its outer surface. An impressive article by Tolonen et al. in this issue of Molecular Microbiology shows that deletion of the single family 9 cellulase gene in Clostridium phytofermentans prevents growth on cellulose although the mutant strain grows perfectly well on glucose and its other cellulase genes are transcribed normally. These results show for the first time that a single cellulase can be essential for cellulose degradation by an organism despite the presence of several other cellulases. It will be interesting to learn the detailed mechanism that C. phytofermentans uses to degrade cellulose.