Detecting cellulase penetration into corn stover cell walls by immuno‐electron microscopy

In general, pretreatments are designed to enhance the accessibility of cellulose to enzymes, allowing for more efficient conversion. In this study, we have detected the penetration of major cellulases present in a commercial enzyme preparation (Spezyme CP) into corn stem cell walls following mild‐, moderate‐ and high‐severity dilute sulfuric acid pretreatments. The Trichoderma reesei enzymes, Cel7A (CBH I) and Cel7B (EG I), as well as the cell wall matrix components xylan and lignin were visualized within digested corn stover cell walls by immuno transmission electron microscopy (TEM) using enzyme‐ and polymer‐specific antibodies. Low severity dilute‐acid pretreatment (20 min at 100°C) enabled <1% of the thickness of secondary cell walls to be penetrated by enzyme, moderate severity pretreatment at (20 min at 120°C) allowed the enzymes to penetrate ～20% of the cell wall, and the high severity (20 min pretreatment at 150°C) allowed 100% penetration of even the thickest cell walls. These data allow direct visualization of the dramatic effect dilute‐acid pretreatment has on altering the condensed ultrastructure of biomass cell walls. Loosening of plant cell wall structure due to pretreatment and the subsequently improved access by cellulases has been hypothesized by the biomass conversion community for over two decades, and for the first time, this study provides direct visual evidence to verify this hypothesis. Further, the high‐resolution enzyme penetration studies presented here provide insight into the mechanisms of cell wall deconstruction by cellulolytic enzymes. Biotechnol. Bioeng. 2009;103: 480–489. © 2009 Wiley Periodicals, Inc.     

Enhanced cellulose degradation using cellulase-nanosphere complexes

Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS) and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC); however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production.     

Modifying plants for biofuel and biomaterial production

The productivity of plants as biofuel or biomaterial crops is established by both the yield of plant biomass per unit area of land and the efficiency of conversion of the biomass to biofuel. Higher yielding biofuel crops with increased conversion efficiencies allow production on a smaller land footprint minimizing competition with agriculture for food production and biodiversity conservation. Plants have traditionally been domesticated for food, fibre and feed applications. However, utilization for biofuels may require the breeding of novel phenotypes, or new species entirely. Genomics approaches support genetic selection strategies to deliver significant genetic improvement of plants as sources of biomass for biofuel manufacture. Genetic modification of plants provides a further range of options for improving the composition of biomass and for plant modifications to assist the fabrication of biofuels. The relative carbohydrate and lignin content influences the deconstruction of plant cell walls to biofuels. Key options for facilitating the deconstruction leading to higher monomeric sugar release from plants include increasing cellulose content, reducing cellulose crystallinity, and/or altering the amount or composition of noncellulosic polysaccharides or lignin. Modification of chemical linkages within and between these biomass components may improve the ease of deconstruction. Expression of enzymes in the plant may provide a cost‐effective option for biochemical conversion to biofuel.     

Synergistic Activity of Plant Extracts with Microbial Cellulases for the Release of Free Sugars

Agricultural lignocellulosic waste such as corn stover is a potential source of inexpensive, abundant, and renewable biomass for the production of bioethanol. The enzymatic process for the economically viable breakdown of cellulose to ethanol relies on the availability of inexpensive microbial cellulases. Although the cost of cellulase has decreased in recent years, current costs still preclude the production of economically viable bioethanol from lignocellulose. Substantive efforts in this lab are being directed to transgenic production of cellulases in maize in order to boost efficiency both of production of enzymes and degradation of corn stover. We serendipitously observed that the addition of non-transgenic maize seed extracts to cellulose and microbial enzymes potentiated free sugar release by as much as 20-fold. Further, this synergistic effect between cellulase enzymes and extract was seen with a variety of plant species and tissue extracts, but varied in efficiency, and was optimal at low concentrations of cellulases. Although the nature of the synergistic molecule is not known, the use of extracts to potentiate cellulose breakdown provides opportunities for a clearer mechanistic understanding of the degradation process as well as an economical way to improve the efficiency of cellulases to produce more cost-effective bioethanol from agricultural waste.     

Application of Molecular Simulations Toward Understanding Cellulase Mechanisms

Computational approaches have provided new biological insights into the chemical mechanism of action of cellulases, which are used in the industrial production of bioethanol. Fine-grained methods, such as molecular dynamics and quantum mechanics, as well as coarse-grained methods, such as elastic network models, were used to investigate how the chemistry and structural dynamics of these enzymes contribute to their function. In this review, we highlight recent computational studies to understand this crucial biofuel enzyme class’s chemistry and structural dynamics, as well as their significance in revealing enzymatic mechanism of action. Computational methods can complement and amplify the findings of experimental methods, which can be used in tandem to create more efficient industrial enzymes.     

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.     

Plant biotechnology for lignocellulosic biofuel production

Lignocelluloses from plant cell walls are attractive resources for sustainable biofuel production. However, conversion of lignocellulose to biofuel is more expensive than other current technologies, due to the costs of chemical pretreatment and enzyme hydrolysis for cell wall deconstruction. Recalcitrance of cell walls to deconstruction has been reduced in many plant species by modifying plant cell walls through biotechnology. These results have been achieved by reducing lignin content and altering its composition and structure. Reduction of recalcitrance has also been achieved by manipulating hemicellulose biosynthesis and by overexpression of bacterial enzymes in plants to disrupt linkages in the lignin–carbohydrate complexes. These modified plants often have improved saccharification yield and higher ethanol production. Cell wall‐degrading (CWD) enzymes from bacteria and fungi have been expressed at high levels in plants to increase the efficiency of saccharification compared with exogenous addition of cellulolytic enzymes. In planta expression of heat‐stable CWD enzymes from bacterial thermophiles has made autohydrolysis possible. Transgenic plants can be engineered to reduce recalcitrance without any yield penalty, indicating that successful cell wall modification can be achieved without impacting cell wall integrity or plant development. A more complete understanding of cell wall formation and structure should greatly improve lignocellulosic feedstocks and reduce the cost of biofuel production.     

Crystal structure of CelM2, a bifunctional glucanase-xylanase protein from a metagenome library

Degradation of polysaccharides by cellulases and xylanases plays an important role in the carbon cycle, but only occurs in plant cell walls, a few bacteria and some animals. This process is also critical in processes such as biomass degradation and fuel production in the conversion cycles of cellulosic biomass. The enzyme CelM2 is bifunctional, because it is able to effectively hydrolyze barley glucan and xylan. Here, we show the crystal structure of the bifunctional enzyme CelM2, isolated from a metagenome library, and describe the sequence information and structure of its two domains. We believe that CelM2 is attractive as an industrial enzyme and that the structural results presented herein provide insights that are relevant to the genetic engineering of multifunctional enzymes.     

Microbial diversity of cellulose hydrolysis

Enzymatic hydrolysis of cellulose by microorganisms is a key step in the global carbon cycle. Despite its abundance only a small percentage of microorganisms can degrade cellulose, probably because it is present in recalcitrant cell walls. There are at least five distinct mechanisms used by different microorganisms to degrade cellulose all of which involve cellulases. Cellulolytic organisms and cellulases are extremely diverse possibly because their natural substrates, plant cell walls, are very diverse. At this time the microbial ecology of cellulose degradation in any environment is still not clearly understood even though there is a great deal of information available about the bovine rumen. Two major problems that limit our understanding of this area are the vast diversity of organisms present in most cellulose degrading environments and the inability to culture most of them.     

High-temperature enzymatic breakdown of cellulose

Cellulose is an abundant and renewable biopolymer that can be used for biofuel generation; however, structural entrapment with other cell wall components hinders enzyme-substrate interactions, a key bottleneck for ethanol production. Biomass is routinely subjected to treatments that facilitate cellulase-cellulose contacts. Cellulases and glucosidases act by hydrolyzing glycosidic bonds of linear glucose β-1,4-linked polymers, producing glucose. Here we describe eight high-temperature-operating cellulases (TCel enzymes) identified from a survey of thermobacterial and archaeal genomes. Three TCel enzymes preferentially hydrolyzed soluble cellulose, while two preferred insoluble cellulose such as cotton linters and filter paper. TCel enzymes had temperature optima ranging from 85°C to 102°C. TCel enzymes were stable, retaining 80% of initial activity after 120 h at 85°C. Two modes of cellulose breakdown, i.e., with endo- and exo-acting glucanases, were detected, and with two-enzyme combinations at 85°C, synergistic cellulase activity was observed for some enzyme combinations.     

Chimeric Cellulase Matrix for Investigating Intramolecular Synergism between Non-hydrolytic Disruptive Functions of Carbohydrate-binding Modules and Catalytic Hydrolysis

The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose.     

Plant genetic engineering to improve biomass characteristics for biofuels

Currently, most ethanol produced in the United States is derived from maize kernel, at levels in excess of four billion gallons per year. Plant lignocellulosic biomass is renewable, cheap and globally available at 10-50 billion tons per year. At present, plant biomass is converted to fermentable sugars for the production of biofuels using pretreatment processes that disrupt the lignocellulose and remove the lignin, thus allowing the access of microbial enzymes for cellulose deconstruction. Both the pretreatments and the production of enzymes in microbial tanks are expensive. Recent advances in plant genetic engineering could reduce biomass conversion costs by developing crop varieties with less lignin, crops that self-produce cellulase enzymes for cellulose degradation and ligninase enzymes for lignin degradation, or plants that have increased cellulose or an overall biomass yield.     

Genome analyses highlight the different biological roles of cellulases

Cellulolytic enzymes have been the subject of renewed interest owing to their potential role in the conversion of plant lignocellulose to sustainable biofuels. An analysis of ～1,500 complete bacterial genomes, presented here, reveals that ～40% of the genomes of sequenced bacteria encode at least one cellulase gene. Most of the bacteria that encode cellulases are soil and marine saprophytes, many of which encode a range of enzymes for cellulose hydrolysis and also for the breakdown of the other constituents of plant cell walls (hemicelluloses and pectins). Intriguingly, cellulases are present in organisms that are usually considered as non-saprophytic, such as Mycobacterium tuberculosis, Legionella pneumophila, Yersinia pestis and even Escherichia coli. We also discuss newly emerging roles of cellulases in such non-saprophytic organisms.     

Over-expression of the cucumber expansin gene (Cs-EXPA1) in transgenic maize seed for cellulose deconstruction

Plant cell wall degradation into fermentable sugars by cellulases is one of the greatest barriers to biofuel production. Expansin protein loosens the plant cell wall by opening up the complex of cellulose microfibrils and polysaccharide matrix components thereby increasing its accessibility to cellulases. We over-expressed cucumber expansin in maize kernels to produce enough protein to assess its potential to serve as an industrial enzyme for applications particularly in biomass conversion. We used the globulin-1 embryo-preferred promoter to express the cucumber expansin gene in maize seed. Expansin protein was targeted to one of three sub-cellular locations: the cell wall, the vacuole, or the endoplasmic reticulum (ER). To assess the level of expansin accumulation in seeds of transgenic kernels, a high throughput expansin assay was developed. The highest expressing plants were chosen and enriched crude expansin extract from those plants was tested for synergistic effects with cellulase on several lignocellulosic substrates. Activity of recombinant cucumber expansin from transgenic kernels was confirmed on these pretreated substrates. The best transgenic lines (ER-targeted) can now be used for breeding to increase expansin expression for use in the biomass conversion industry. Results of these experiments show the success of expansin over-expression and accumulation in transgenic maize seed without negative impact on growth and development and confirm its synergistic effect with cellulase on deconstruction of complex cell wall substrates.     

Outlook for cellulase improvement: screening and selection strategies

Cellulose is the most abundant renewable natural biological resource, and the production of biobased products and bioenergy from less costly renewable lignocellulosic materials is important for the sustainable development of human beings. A reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. Here, we review quantitative cellulase activity assays using soluble and insoluble substrates, and focus on their advantages and limitations. Because there are no clear relationships between cellulase activities on soluble substrates and those on insoluble substrates, soluble substrates should not be used to screen or select improved cellulases for processing relevant solid substrates, such as plant cell walls. Cellulase improvement strategies based on directed evolution using screening on soluble substrates have been only moderately successful, and have primarily targeted improvement in thermal tolerance. Heterogeneity of insoluble cellulose, unclear dynamic interactions between insoluble substrate and cellulase components, and the complex competitive and/or synergic relationship among cellulase components limit rational design and/or strategies, depending on activity screening approaches. Herein, we hypothesize that continuous culture using insoluble cellulosic substrates could be a powerful selection tool for enriching beneficial cellulase mutants from the large library displayed on the cell surface.     

Cellulases: ambiguous nonhomologous enzymes in a genomic perspective

The key material for bioethanol production is cellulose, which is one of the main components of the plant cell wall. Enzymatic depolymerization of cellulose is an essential step in bioethanol production, and can be accomplished by fungal and bacterial cellulases. Most of the biochemically characterized bacterial cellulases come from only a few cellulose-degrading bacteria, thus limiting our knowledge of a range of cellulolytic activities that exist in nature. The recent explosion of genomic data offers a unique opportunity to search for novel cellulolytic activities; however, the absence of clear understanding of structural and functional features that are important for reliable computational identification of cellulases precludes their exploration in the genomic datasets. Here, we explore the diversity of cellulases and propose a genomic approach to overcome this bottleneck.     

In situ label-free imaging of hemicellulose in plant cell walls using stimulated Raman scattering microscopy

Background

Plant hemicellulose (largely xylan) is an excellent feedstock for renewable energy production and second only to cellulose in abundance. Beyond a source of fermentable sugars, xylan constitutes a critical polymer in the plant cell wall, where its precise role in wall assembly, maturation, and deconstruction remains primarily hypothetical. Effective detection of xylan, particularly by in situ imaging of xylan in the presence of other biopolymers, would provide critical information for tackling the challenges of understanding the assembly and enhancing the liberation of xylan from plant materials.

Results

Raman-based imaging techniques, especially the highly sensitive stimulated Raman scattering (SRS) microscopy, have proven to be valuable tools for label-free imaging. However, due to the complex nature of plant materials, especially those same chemical groups shared between xylan and cellulose, the utility of specific Raman vibrational modes that are unique to xylan have been debated. Here, we report a novel approach based on combining spectroscopic analysis and chemical/enzymatic xylan removal from corn stover cell walls, to make progress in meeting this analytical challenge. We have identified several Raman peaks associated with xylan content in cell walls for label-free in situ imaging xylan in plant cell wall.

Conclusion

We demonstrated that xylan can be resolved from cellulose and lignin in situ using enzymatic digestion and label-free SRS microscopy in both 2D and 3D. We believe that this novel approach can be used to map xylan in plant cell walls and that this ability will enhance our understanding of the role played by xylan in cell wall biosynthesis and deconstruction.