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.     

Endoglucanase activities and growth of marine‐derived fungi isolated from the sponge Haliclona simulans

Aims: The conversion of cheap cellulosic biomass to more easily fermentable sugars requires the use of costly cellulases. We have isolated a series of marine sponge‐derived fungi and screened these for cellulolytic activity to determine the potential of this unique environmental niche as a source of novel cellulase activities. Methods and Results: Fungi were isolated from the marine sponge Haliclona simulans. Phylogenetic analysis of these and other fungi previously isolated from H. simulans showed fungi from three phyla with very few duplicate species. Cellulase activities were determined using plate‐based assays using different media and sea water concentrations while extracellular cellulase activities were determined using 3,5‐dinitrosalicylic acid (DNSA)‐based assays. Total and specific cellulase activities were determined using a range of incubation temperatures and compared to those for the cellulase overproducing mutant Hypocrea jecorina QM9414. Several of the strains assayed produced total or relative endoglucanase activities that were higher than H. jecorina, particularly at lower reaction temperatures. Conclusions: Marine sponges harbour diverse fungal species and these fungi are a good source of endoglucanase activities. Analysis of the extracellular endoglucanase activities revealed that some of the marine‐derived fungi produced high endoglucanase activities that were especially active at lower temperatures. Significance and Impact of the Study: Marine‐derived fungi associated with coastal marine sponges are a novel source of highly active endoglucanases with significant activity at low temperatures and could be a source of novel cellulase activities.     

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.     

Expression of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae

Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full‐length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum (TcEG1), and evaluated the activity of the transgenic product (rTcEG1). Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde‐3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β‐1,4‐endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments.     

Large-Scale Analyses of Glycosylation in Cellulases

Cellulases are important glycosyl hydrolases (GHs) that hydrolyze cellulose polymers into smaller oligosaccharides by breaking the cellulose β (1→4) bonds,and they are widely used to produce cellulosic ethanol from the plant biomass.N-linked and O-linked glycosylations were proposed to impact the catalytic efficiency,cellulose binding affinity and the stability of cellulases based on observations of individual cellulases.As far as we know,there has not been any systematic analysis of the distributions of N-...     

The heterotrimeric G‐protein beta subunit Gpb1 controls hyphal growth under low oxygen conditions through the protein kinase A pathway and is essential for virulence in the fungus Mucor circinelloides

Mucor circinelloides, a dimorphic opportunistic pathogen, expresses three heterotrimeric G‐protein beta subunits (Gpb1, Gpb2 and Gpb3). The Gpb1‐encoding gene is up‐regulated during mycelial growth compared with that in the spore or yeast stage. gpb1 deletion mutation analysis revealed its relevance for an adequate development during the dimorphic transition and for hyphal growth under low oxygen concentrations. Infection assays in mice indicated a phenotype with considerably reduced virulence and tissue invasiveness in the deletion mutants (Δgpb1) and decreased host inflammatory response. This finding could be attributed to the reduced filamentous growth in animal tissues compared with that of the wild‐type strain. Mutation in a regulatory subunit of cAMP‐dependent protein kinase A (PKA) subunit (PkaR1) resulted in similar phenotypes to Δgpb1. The defects exhibited by the Δgpb1 strain were genetically suppressed by pkaR1 overexpression, indicating that the PKA pathway is controlled by Gpb1 in M. circinelloides. Moreover, during growth under low oxygen levels, cAMP levels were much higher in the Δgpb1 than in the wild‐type strain, but similar to those in the ΔpkaR1 strain. These findings reveal that M. circinelloides possesses a signal transduction pathway through which the Gpb1 heterotrimeric G subunit and PkaR1 control mycelial growth in response to low oxygen levels.     

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.     

Carboxymethylcellulase and avicelase activities from a cellulolytic Clostridium strain A11

The extracellular cellulase enzyme system of Clostridium A11 was fractionated by affinity chromatography on Avicel: 80% of the initial carboxymethylcellulase (CMCase) activity was adhered. This cellulase system was a multicomponent aggregate. Several CMCase activities were detected, but the major protein P1 had no detectable activity. Adhered and unadhered cellulases showed CMCase activity with the highest specific activity in Avicel-adhered fraction. However, only afhered fractions could degrade Avicel. Thus, efficiency of the enzymatic hydrolysis of Avicel was related to the cellulase-adhesion capacity. Carboxymethylcellulase and Avicelase activities were studied with the extracellular enzyme system and cloned cellulases. Genomic libraries from Clostridium A11 were constructed with DNA from this Clostridium, and a new gene cel1 was isolated. The gene(s) product(s) from cel1 exhibited CMCase and p-nitrophenylcellobiosidase (pNPCbase) activities. This cloned cellulase adhered to cellulose. Synergism between adhered enzyme system and cloned endoglucanases was observed on Avicel degradation. Conversely, no synergism was observed on CMC hydrolysis. Addition of cloned endoglucanase to cellulase complex led to increase of the V_max without significant K _m variation. Cloned endoglucanases can be added to cellulase complexes to efficiently hydrolyze cellulose.     

Bioconversion of lignocellulosic biomass: biochemical and molecular perspectives

In view of rising prices of crude oil due to increasing fuel demands, the need for alternative sources of bioenergy is expected to increase sharply in the coming years. Among potential alternative bioenergy resources, lignocellulosics have been identified as the prime source of biofuels and other value-added products. Lignocelluloses as agricultural, industrial and forest residuals account for the majority of the total biomass present in the world. To initiate the production of industrially important products from cellulosic biomass, bioconversion of the cellulosic components into fermentable sugars is necessary. A variety of microorganisms including bacteria and fungi may have the ability to degrade the cellulosic biomass to glucose monomers. Bacterial cellulases exist as discrete multi-enzyme complexes, called cellulosomes that consist of multiple subunits. Cellulolytic enzyme systems from the filamentous fungi, especially Trichoderma reesei, contain two exoglucanases or cellobiohydrolases (CBH1 and CBH2), at least four endoglucanases (EG1, EG2, EG3, EG5), and one β-glucosidase. These enzymes act synergistically to catalyse the hydrolysis of cellulose. Different physical parameters such as pH, temperature, adsorption, chemical factors like nitrogen, phosphorus, presence of phenolic compounds and other inhibitors can critically influence the bioconversion of lignocellulose. The production of cellulases by microbial cells is governed by genetic and biochemical controls including induction, catabolite repression, or end product inhibition. Several efforts have been made to increase the production of cellulases through strain improvement by mutagenesis. Various physical and chemical methods have been used to develop bacterial and fungal strains producing higher amounts of cellulase, all with limited success. Cellulosic bioconversion is a complex process and requires the synergistic action of the three enzymatic components consisting of endoglucanases, exoglucanases and β-glucosidases. The co-cultivation of microbes in fermentation can increase the quantity of the desirable components of the cellulase complex. An understanding of the molecular mechanism leading to biodegradation of lignocelluloses and the development of the bioprocessing potential of cellulolytic microorganisms might effectively be accomplished with recombinant DNA technology. For instance, cloning and sequencing of the various cellulolytic genes could economize the cellulase production process. Apart from that, metabolic engineering and genomics approaches have great potential for enhancing our understanding of the molecular mechanism of bioconversion of lignocelluloses to value added economically significant products in the future. JIMB 2008: BioEnergy - Special issue.