Biomass-degrading Enzymes Research Articles

Deciphering the Regulatory Network between the SREBP Pathway and Protein Secretion in Neurospora crassa

ABSTRACTSterol regulatory element binding proteins (SREBPs) are conserved from yeast to mammalian cells and function in the regulation of sterol homeostasis. In fungi, the SREBP pathway has been implicated in the adaptation to hypoxia and in virulence. In Neurospora crassa and Trichoderma reesei, the SREBP pathway also negatively regulates protein secretion under lignocellulolytic conditions. Here we utilized global transcriptional profiling combined with genetic and physiological analyses to address the regulatory link between the SREBP pathway and protein secretion in N. crassa. Our results demonstrated that the function of the SREBP pathway in ergosterol biosynthesis and adaptation to hypoxia was conserved in N. crassa. Under lignocellulolytic conditions, the SREBP pathway was highly activated, resulting in the reduced expression of lytic polysaccharide monooxygenases, which require molecular oxygen for catalytic activity. Additionally, activation of the SREBP pathway under lignocellulolytic conditions repressed a set of genes predicted to be involved in the endoplasmic reticulum stress response. Here we show that the inability of a hac-1 mutant, which bears a deletion of the major regulator of the unfolded protein response (UPR), to efficiently produce cellulases and utilize cellulose was suppressed by mutations in the SREBP pathway. The analyses presented here demonstrated new SREBP pathway functions, including linkages to the UPR, and provide new clues for genetic engineering of filamentous fungi to improve their production of extracellular proteins.

Genome sequencing and analysis of Talaromyces pinophilus provide insights into biotechnological applications

Species from the genus Talaromyces produce useful biomass-degrading enzymes and secondary metabolites. However, these enzymes and secondary metabolites are still poorly understood and have not been explored in depth because of a lack of comprehensive genetic information. Here, we report a 36.51-megabase genome assembly of Talaromyces pinophilus strain 1–95, with coverage of nine scaffolds of eight chromosomes with telomeric repeats at their ends and circular mitochondrial DNA. In total, 13,472 protein-coding genes were predicted. Of these, 803 were annotated to encode enzymes that act on carbohydrates, including 39 cellulose-degrading and 24 starch-degrading enzymes. In addition, 68 secondary metabolism gene clusters were identified, mainly including T1 polyketide synthase genes and nonribosomal peptide synthase genes. Comparative genomic analyses revealed that T. pinophilus 1–95 harbors more biomass-degrading enzymes and secondary metabolites than other related filamentous fungi. The prediction of the T. pinophilus 1–95 secretome indicated that approximately 50% of the biomass-degrading enzymes are secreted into the extracellular environment. These results expanded our genetic knowledge of the biomass-degrading enzyme system of T. pinophilus and its biosynthesis of secondary metabolites, facilitating the cultivation of T. pinophilus for high production of useful products.

Selective Isolation of a Eucalyptus spp. Woodchip Bacterial Community and Its Taxonomic and Metabolic Profiling

Taxonomic and metabolic profiles of bacterial inhabitants of Eucalyptus spp. woodchips from storage piles were analysed using whole genome shotgun metagenomics sequencing. The majority of the bacterial species present in the metagenome from the Eucalyptus spp. woodchips are found within four bacterial phyla: Proteobacteria (77%), Bacteroidetes (15%), Acidobacteria (3%) and Actinobacteria (2%). The most abundant species in the metagenome were Burkholderia spp. (25%; Proteobacteria) and Chryseobacterium spp. (37%; Bacteroidetes). Other species prevalent in this bacterial metagenome are Rhodanobacter spp. (7%), Klebsiella spp. (6%) and Pseudomonas spp. (5%). Both PhyloSift and Metagenome Rapid Annotation Subsystem Technology tools to estimate taxonomic diversity showed that the phylum Proteobacteria was the predominant group among the bacteria in the bacterial metagenome from the Eucalyptus spp. woodchips, followed by Bacteroidetes, Acidobacteria and Actinobacteria. Functional analysis revealed that clustering-based subsystem and carbohydrate metabolism which are key to degradation of the lignocellulose were the most abundant SEED subsystems representing 14% and 11% of the bacterial metagenome, respectively. Approximately 8% of the sequences were grouped under “miscellaneous functions” and 3% under metabolism of aromatic compounds. One thousand two hundred forty-seven potential contigs encoding biomass-degrading enzymes including glycoside hydrolases (GH, 525 contigs), glycosyl transferases (GT, 452 contigs) carbohydrate binding modules (CBM, 149 contigs), carbohydrate esterases (CE, 70 contigs), polysaccharide lyases (PL, 16 contigs) and auxiliary activity (AA, 35 contigs) were identified. Both taxonomic and functional classifications suggest that the bacterial inhabitants of the Eucalyptus spp. woodchips present a broader metabolic potential than the degradation of plant biomass.

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP).

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion. Graphical Abstract ᅟ.

Proteomic insights into mannan degradation and protein secretion by the forest floor bacterium Chitinophaga pinensis

Together with fungi, saprophytic bacteria are central to the decomposition and recycling of biomass in forest environments. The Bacteroidetes phylum is abundant in diverse habitats, and several species have been shown to be able to deconstruct a wide variety of complex carbohydrates. The genus Chitinophaga is often enriched in hotspots of plant and microbial biomass degradation. We present a proteomic assessment of the ability of Chitinophaga pinensis to grow on and degrade mannan polysaccharides, using an agarose plate-based method of protein collection to minimise contamination with exopolysaccharides and proteins from lysed cells, and to reflect the realistic setting of growth on a solid surface. We show that select Polysaccharide Utilisation Loci (PULs) are expressed in different growth conditions, and identify enzymes that may be involved in mannan degradation. By comparing proteomic and enzymatic profiles, we show evidence for the induced expression of enzymes and PULs in cells grown on mannan polysaccharides compared with cells grown on glucose. In addition, we show that the secretion of putative biomass-degrading enzymes during growth on glucose comprises a system for nutrient scavenging, which employs constitutively produced enzymes. Significance of this studyChitinophaga pinensis belongs to a bacterial genus which is prominent in microbial communities in agricultural and forest environments, where plant and fungal biomass is intensively degraded. Such degradation is hugely significant in the recycling of carbon in the natural environment, and the enzymes responsible are of biotechnological relevance in emerging technologies involving the deconstruction of plant cell wall material. The bacterium has a comparatively large genome, which includes many uncharacterised carbohydrate-active enzymes. We present the first proteomic assessment of the biomass-degrading machinery of this species, focusing on mannan, an abundant plant cell wall hemicellulose. Our findings include the identification of several novel enzymes, which are promising targets for future biochemical characterisation. In addition, the data indicate the expression of specific Polysaccharide Utilisation Loci, induced in the presence of different growth substrates. We also highlight how a constitutive secretion of enzymes which deconstruct microbial biomass likely forms part of a nutrient scavenging process.

Characterization of a laccase from a wood-feeding termite, Coptotermes formosanus.

Coptotermes formosanus Shiraki is a wood-feeding termite which secretes a series of lignolytic and cellulolytic enzymes for woody biomass degradation. However, the lignin modification mechanism in the termite is largely elusive, and the characteristics of most lignolytic enzymes in termites remain unknown. In this study, a laccase gene lac1 from C. formosanus was heterogeneously expressed in insect Sf9 cells. The purified Lac1 showed strong activities toward hydroquinone (305 mU/mg) and 2,6-dimethoxyphenol (2.9 mU/mg) with low Km values, but not veratryl alcohol or 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid). Lac1 could function well from pH 4.5 to 7.5, and its activity was significantly inhibited by H2 O2 at above 4.85 mmol/L (P < 0.01). In addition, the lac1 gene was found to be mainly expressed in the salivary glands and foregut of C. formosanus, and seldom in the midgut or hindgut. These findings suggested that Lac1 is a phenol-oxidizing laccase like RflacA and RflacB from termite Reticulitermes flavipes, except that Lac1 was found to be more efficient in phenol oxidation, and it did not require H2 O2 for its function. It is suspected that this kind of termite laccase might only be able to directly oxidize low redox-potential substrates, and the high redox-potential groups in lignin might be oxidized by other enzymes in the termite or by using the Fenton reaction.

Citrobacter freundii as a test platform for recombinant cellulose degradation systems.

Biofuels have been shown to be the best sustainable and alternative source of fuel to replace fossil fuels. Of the different types of feedstocks used for producing biofuels, lignocellulosic biomass is the most abundant. Converting this biomass to useful products has met with little success. Different approaches are being used and microbial platforms are the most promising and sustainable method. This study shows that Citrobacter freundii is a better test platform than Escherichia coli for testing various combinations of cellulases for the development of microbial systems for biomass conversion.

The fungal cultivar of leaf‐cutter ants produces specific enzymes in response to different plant substrates

Herbivores use symbiotic microbes to help derive energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, cultivating their mutualistic fungus Leucoagaricus gongylophorus on plant biomass that workers forage from a diverse collection of plant species. Here, we investigate the metabolic flexibility of the ants' fungal cultivar for utilizing different plant biomass. Using feeding experiments and a novel approach in metaproteomics, we examine the enzymatic response of L.gongylophorus to leaves, flowers, oats or a mixture of all three. Across all treatments, our analysis identified and quantified 1766 different fungal proteins, including 161 putative biomass-degrading enzymes. We found significant differences in the protein profiles in the fungus gardens of subcolonies fed different plant substrates. When provided with leaves or flowers, which contain the majority of their energy as recalcitrant plant polymers, the fungus gardens produced more proteins predicted to break down cellulose: endoglucanase, exoglucanase and β-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, while the mixed substrate treatment closely resembled the treatment with oats alone. This indicates that when provided a mixture of plant substrates, fungus gardens preferentially break down the simpler, more digestible substrates. This flexible, substrate-specific enzymatic response of the fungal cultivar allows leaf-cutter ants to derive energy from a wide range of substrates, which likely contributes to their ability to be dominant generalist herbivores.

High Throughput Screening: Developed Techniques for Cellulolytic and Xylanolytic Activities Assay.

High throughput screening (HTS) is a powerful tool in biotechnology. The search for new or improved enzymes with suitable biochemical properties for industrial processes, has resulted in high efforts and research activities to develop new methodologies for activity screening. In this context, important advances have been achieved for the screening of cellulases and xylanases activities from wild and recombinant microorganisms, and from sequence databases. These enzymes have a wide range of industrial applications, including food, animal feed, textile, pulp and paper industries and detergents. Cellulases and xylanases along with pectinases, represent 20% of the world enzyme market. Recently, cellulases and xylanases have been used on fermentable sugars recovered from lignocellulosic biomass for second-generation biorefineries, aimed to produce chemical and biofuel platforms. As a result, HTS methods for biomass or biomass-degrading enzymes are gaining importance. This article presents evidence of the studies carried out for HTS of cellulase and xylanase activities.

Engineering a highly active thermophilic β-glucosidase to enhance its pH stability and saccharification performance.

Backgroundβ-Glucosidase is an important member of the biomass-degrading enzyme system, and plays vital roles in enzymatic saccharification for biofuels production. Candidates with high activity and great stability over high temperature and varied pHs are always preferred in industrial practice. To achieve cost-effective biomass conversion, exploring natural enzymes, developing high level expression systems and engineering superior mutants are effective approaches commonly used.ResultsA newly identified β-glucosidase of GH3, Bgl3A, from Talaromyces leycettanus JCM12802, was overexpressed in yeast strain Pichia pastoris GS115, yielding a crude enzyme activity of 6000 U/ml in a 3 L fermentation tank. The purified enzyme exhibited outstanding enzymatic properties, including favorable temperature and pH optima (75 °C and pH 4.5), good thermostability (maintaining stable at 60 °C), and high catalytic performance (with a specific activity and catalytic efficiency of 905 U/mg and 9096/s/mM on pNPG, respectively). However, the narrow stability of Bgl3A at pH 4.0–5.0 would limit its industrial applications. Further site-directed mutagenesis indicated the role of excessive O-glycosylation in pH liability. By removing the potential O-glycosylation sites, two mutants showed improved pH stability over a broader pH range (3.0–10.0). Besides, with better stability under pH 5.0 and 50 °C compared with wild type Bgl3A, saccharification efficiency of mutant M1 was improved substantially cooperating with cellulase Celluclast 1.5L. And mutant M1 reached approximately equivalent saccharification performance to commercial β-glucosidase Novozyme 188 with identical β-glucosidase activity, suggesting its great prospect in biofuels production.ConclusionsIn this study, we overexpressed a novel β-glucosidase Bgl3A with high specific activity and high catalytic efficiency in P. pastoris. We further proved the negative effect of excessive O-glycosylation on the pH stability of Bgl3A, and enhanced the pH stability by reducing the O-glycosylation. And the enhanced mutants showed much better application prospect with substantially improved saccharification efficiency on cellulosic materials.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0560-8) contains supplementary material, which is available to authorized users.

Fungal communities in pressmud composting harbour beneficial and detrimental fungi for human welfare

Pressmud is a substrate derived from sugarcane juice filtrate, and around 26-40 kg of this residue are produced per ton of sugarcane. It is mainly used as fertilizer in crops, and its application in the field is often made without any prior treatment, but, in this research, it was studied for the risk this practice poses for human health. This research was stimulated by previous results indicating the presence of opportunistic pathogens in residues used in various composting systems and the extensive use of fresh pressmud in agriculture. Here, It was assessed the fungal diversity present in both fresh and composting pressmud using 454 pyrosequencing. In addition, heat-tolerant fungi were isolated and surveyed for their enzymatic repertoire of biomass-degrading enzymes (cellulase, xylanase, laccase and polygalacturonase). A wide range of opportunistic pathogens was found among the most abundant taxa in the fresh pressmud, such as Lomentospora prolificans (43.13 %), Trichosporon sp. (10.07 %), Candida tropicalis (7.91 %), and Hormographiella aspergillata (8.19 %). This indicates that fresh pressmud might be a putative source of human pathogenic fungi, presenting a potential threat to human health if applied as fertilizer without any treatment. With regard to the heat-tolerant fungi found in this substrate, all the 110 isolates screened were able to produce at least one of the tested enzymes. The pressmud composting process not only effectively reduces the load of pathogenic fungi, but also creates an interesting environment for fungi able to produce thermostable hydrolytic and oxidative enzymes with biotechnological applications.

Early-branching gut fungi possess a large, comprehensive array of biomass-degrading enzymes.

The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. We developed a systems-level approach that integrates transcriptomic sequencing, proteomics, phenotype, and biochemical studies of relatively unexplored basal fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, untreated plant biomass and are competitive with optimized commercial preparations from Aspergillus and Trichoderma. Compared to these model platforms, gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. These enzymes are universally catabolite-repressed and are further regulated by a rich landscape of noncoding regulatory RNAs. Additionally, we identified several promising sequence-divergent enzyme candidates for lignocellulosic bioprocessing.

Fueling biomass-degrading oxidative enzymes by light-driven water oxidation

Light-driven oxidation of water catalyzed by a photocatalyst can fuel the activity of LPMOs, key enzymes in the processing of recalcitrant polysaccharides.

糸状菌における植物バイオマス分解酵素遺伝子の発現制御

糸状菌における植物バイオマス分解酵素遺伝子の発現制御

Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris.

Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users’ community.

From gene to biorefinery: microbial β-etherases as promising biocatalysts for lignin valorization.

The set-up of biorefineries for the valorization of lignocellulosic biomass will be core in the future to reach sustainability targets. In this area, biomass-degrading enzymes are attracting significant research interest for their potential in the production of chemicals and biofuels from renewable feedstock. Glutathione-dependent β-etherases are emerging enzymes for the biocatalytic depolymerization of lignin, a heterogeneous aromatic polymer abundant in nature. They selectively catalyze the reductive cleavage of β-O-4 aryl-ether bonds which account for 45–60% of linkages present in lignin. Hence, application of β-etherases in lignin depolymerization would enable a specific lignin breakdown, selectively yielding (valuable) low-molecular-mass aromatics. Albeit β-etherases have been biochemically known for decades, only very recently novel β-etherases have been identified and thoroughly characterized for lignin valorization, expanding the enzyme toolbox for efficient β-O-4 aryl-ether bond cleavage. Given their emerging importance and potential, this mini-review discusses recent developments in the field of β-etherase biocatalysis covering all aspects from enzyme identification to biocatalytic applications with real lignin samples.

Thermotolerant and mesophylic fungi from sugarcane bagasse and their prospection for biomass-degrading enzyme production

Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3), A. sydowii (SBCM7) and A. fumigatus (SBC4). The best β-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g). For β-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g) and 4.0 (573 U/g), respectively. All these crude enzymes presented stability around pH 3.0–8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors.

Enrichment and Broad Representation of Plant Biomass-Degrading Enzymes in the Specialized Hyphal Swellings of Leucoagaricus gongylophorus, the Fungal Symbiont of Leaf-Cutter Ants.

Leaf-cutter ants are prolific and conspicuous constituents of Neotropical ecosystems that derive energy from specialized fungus gardens they cultivate using prodigious amounts of foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain plant biomass-degrading enzymes that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as ants incorporate it into the fungus garden. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plant biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous plant biomass-degrading enzymes likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three quarters of all biomass-degrading enzymes identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 40 of these enzymes enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass.

Isolation of aerobic cultivable cellulolytic bacteria from different regions of the gastrointestinal tract of giant land snail Achatina fulica.

The enzymatic hydrolysis of cellulose by cellulases is one of the major limiting steps in the conversion of lignocellulosic biomass to yield bioethanol. To overcome this hindrance, significant efforts are underway to identify novel cellulases. The snail Achatina fulica is a gastropod with high cellulolytic activity, mainly due to the abundance of glycoside hydrolases produced by both the animal and its resident microbiota. In this study, we partially assessed the cellulolytic aerobic bacterial diversity inside the gastrointestinal tract of A. fulica by culture-dependent methods and evaluated the hydrolytic repertoire of the isolates. Forty bacterial isolates were recovered from distinct segments of the snail gut and identified to the genus level by 16S rRNA gene sequence analysis. Additional phenotypic characterization was performed using biochemical tests provided by the Vitek2 identification system. The overall enzymatic repertoire of the isolated strains was investigated by enzymatic plate assays, containing the following substrates: powdered sugarcane bagasse, carboxymethylcellulose (CMC), p-nitrophenyl-β-D-glucopyranoside (pNPG), p-nitrophenyl-β-D-cellobioside (pNPC), 4-methylumbelliferyl-β-D-glucopyranoside (MUG), 4-methylumbelliferyl-β-D-cellobioside (MUC), and 4-methylumbelliferyl-β-D-xylopyranoside (MUX). Our results indicate that the snail A. fulica is an attractive source of cultivable bacteria that showed to be valuable resources for the production of different types of biomass-degrading enzymes.

Extreme Thermophilic Enzyme CelB-m Efficiently Degrades the Cellulose in Transgenic Arabidopsis thaliana.

Agricultural and forestry wastes abundant in the plant biomass are an important resource of green energy. However, little is known about how to exploit efficiently the resource. In this study, we isolated the CelB gene that encodes the extremely thermophilic cellulose-degrading enzyme from Thermotoga maritime. The enzyme-encoding gene CelB was optimized and reconstructed in N' codes by the code adaptability in Arabidopsis thaliana. Then, the optimized gene (CelB-m) or the recombinant gene (CBD-CelB) was fused with the plant binary vector which harbors the β-glucuronidase (GUS) gene that was transferred into Arabidopsis, respectively. GUS assay results showed CelB gene ubiquitous expression in transgenic plants. The enzyme-activity assays exhibited that the cellulase activity in the leaves of CelB-m transgenic plants were significantly higher than that of wild-type plants. The highest amount of enzymatic activity obtained was 131.2 U for every gram of fresh leaves in CBD-CelB plants. In addition, the enzymatic activity was stable at the temperature of 90 °C. These results suggested that the ectopic expression of pertinent biomass-degrading enzymes in transgenic plants can degrade effectively the plant biomass and lay a foundation on the application for the transgenic technique to crops.