Whole Genome Sequencing and Tn5-Insertion Mutagenesis of Pseudomonas taiwanensis CMS to Probe Its Antagonistic Activity Against Rice Bacterial Blight Disease.

The Gram-negative bacterium Pseudomonas taiwanensis is a novel bacterium that uses shrimp shell waste as its sole sources of carbon and nitrogen. It is a versatile bacterium with potential for use in biological control, with activities including toxicity toward insects, fungi, and the rice pathogen Xanthomonas oryzae pv.oryzae (Xoo). In this study, the complete 5.08-Mb genome sequence of P. taiwanensis CMS was determined by a combination of NGS/Sanger sequencing and optical mapping. Comparison of optical maps of seven Pseudomonas species showed that P. taiwanensis is most closely related to P. putida KT 2400. We screened a total of 11,646 individual Tn5-transponson tagged strains to identify genes that are involved in the production and regulation of the iron-chelator pyoverdine in P. taiwanensis, which is a key anti-Xoo factor. Our results indicated that the two-component system (TCS) EnvZ/OmpR plays a positive regulatory role in the production of pyoverdine, whereas the sigma factor RpoS functions as a repressor. The knowledge of the molecular basis of the regulation of pyoverdine by P. taiwanensis provided herein will be useful for its development for use in biological control, including as an anti-Xoo agent.

Construction of a Lactobacillus plantarum Strain Expressing the Capsid Protein of Porcine Circovirus Type 2d (PCV2d) as an Oral Vaccine.

Porcine circovirus type 2 (PCV2) is a pathogenic virus that causes high rates of porcine death, resulting in severe economic losses to the swine industry. In recent years, the prevalence of PCV2d genotype infection in pigs has increased, but most commercially available vaccines were developed against the PCV2a strain and do not ensure complete protection from PCV2d. Here, we first constructed an expression vector for the antigenic ORF2-encoded capsid protein of PCV2d (pLp3050-His6-tag-capsid). We then utilized Lactobacillus plantarum to express the protein at mucosal sites in orally vaccinated mice. After transducing L. plantarum with pLp3050-His6-tag-capsid, the expressed protein could be found in cell wall and cell-free supernatant fractions by Western blotting. Using flow cytometry, we found that L. plantarum cells with surface-displayed capsid protein increased with time after SppIP induction. Finally, mice that were orally immunized 18 times with capsid-expressing L. plantarum showed increased levels of capsid-specific sIgA and virus neutralizing activity at mucosal sites, suggesting mucosal immunity had been stimulated by the vaccine. Overall, our findings demonstrate the feasibility and utility of a PCV2d-based vaccine, which may be of great value in porcine agriculture.

Expression of the Clonostachys rosea lactonohydrolase gene by Lactobacillus reuteri to increase its zearalenone-removing ability.

BACKGROUND: Mycotoxins are secondary metabolites produced by filamentous fungi that can contaminate agricultural crops in the field as well as during harvest, transportation, processing, or storage. Zearalenone (ZEN), a non-steroidal estrogenic mycotoxin, produced by Fusarium species, has been shown to be associated with reproductive disorders in farm animals and to a lesser extent in hyperoestrogenic syndromes in humans. Thus, the decontamination of ZEN in foods and feeds is an important issue. RESULTS: In this study, the gene encoding ZHD101, a ZEN-degrading enzyme produced by Clonostachys rosea IFO 7063, was cloned into an Escherichia coli-Lactobacillus shuttle vector, pNZ3004, and the resultant plasmid pNZ-zhd101 was then introduced via electroporation into Lactobacillus reuteri Pg4, a probiotic strain isolated from the gastrointestinal tract of broilers. The transformed strain L. reuteri pNZ-zhd101 acquired the capacity to degrade ZEN. In addition, the production of recombinant ZHD101 did not affect cell growth, acid and bile salt tolerance, and had only a minor effect on the adhesion ability of L. reuteri pNZ-zhd101. CONCLUSIONS: To the best of our knowledge, this is the first report of successful expression of a ZEN-degrading enzyme by intestinal lactobacilli.

Characterization of an insecticidal toxin and pathogenicity of Pseudomonas taiwanensis against insects.

Pseudomonas taiwanensis is a broad-host-range entomopathogenic bacterium that exhibits insecticidal activity toward agricultural pests Plutella xylostella, Spodoptera exigua, Spodoptera litura, Trichoplusia ni and Drosophila melanogaster. Oral infection with different concentrations (OD = 0.5 to 2) of wild-type P. taiwanensis resulted in insect mortality rates that were not significantly different (92.7%, 96.4% and 94.5%). The TccC protein, a component of the toxin complex (Tc), plays an essential role in the insecticidal activity of P. taiwanensis. The ΔtccC mutant strain of P. taiwanensis, which has a knockout mutation in the tccC gene, only induced 42.2% mortality in P. xylostella, even at a high bacterial dose (OD = 2.0). TccC protein was cleaved into two fragments, an N-terminal fragment containing an Rhs-like domain and a C-terminal fragment containing a Glt symporter domain and a TraT domain, which might contribute to antioxidative stress activity and defense against macrophagosis, respectively. Interestingly, the primary structure of the C-terminal region of TccC in P. taiwanensis is unique among pathogens. Membrane localization of the C-terminal fragment of TccC was proven by flow cytometry. Sonicated pellets of P. taiwanensis ΔtccC strain had lower toxicity against the Sf9 insect cell line and P. xylostella larvae than the wild type. We also found that infection of Sf9 and LD652Y-5d cell lines with P. taiwanensis induced apoptotic cell death. Further, natural oral infection by P. taiwanensis triggered expression of host programmed cell death-related genes JNK-2 and caspase-3.

Structural analysis of a glycoside hydrolase family 11 xylanase from Neocallimastix patriciarum: insights into the molecular basis of a thermophilic enzyme.

The catalytic domain of XynCDBFV, a glycoside hydrolase family 11 (GH11) xylanase from ruminal fungus Neocallimastix patriciarum previously engineered to exhibit higher specific activity and broader pH adaptability, holds great potential in commercial applications. Here, the crystal structures of XynCDBFV and its complex with substrate were determined to 1.27-1.43 Å resolution. These structures revealed a typical GH11 ß-jelly-roll fold and detailed interaction networks between the enzyme and ligands. Notably, an extended N-terminal region (NTR) consisting of 11 amino acids was identified in the XynCDBFV structure, which is found unique among GH11 xylanases. The NTR is attached to the catalytic core by hydrogen bonds and stacking forces along with a disulfide bond between Cys-4 and Cys-172. Interestingly, the NTR deletion mutant retained 61.5% and 19.5% enzymatic activity at 55 °C and 75 °C, respectively, compared with the wild-type enzyme, whereas the C4A/C172A mutant showed 86.8% and 23.3% activity. These results suggest that NTR plays a role in XynCDBFV thermostability, and the Cys-4/Cys-172 disulfide bond is critical to the NTR-mediated interactions. Furthermore, we also demonstrated that Pichia pastoris produces XynCDBFV with higher catalytic activity at higher temperature than Escherichia coli, in which incorrect NTR folding and inefficient disulfide bond formation might have occurred. In conclusion, these structural and functional analyses of the industrially favored XynCDBFV provide a molecular basis of NTR contribution to its thermostability.

Improving the specific activity of ß-mannanase from Aspergillus niger BK01 by structure-based rational design.

ß-Mannanase has found various biotechnological applications because it is capable of degrading mannans into smaller sugar components. A highly potent example is the thermophilic ß-mannanase from Aspergillus niger BK01 (ManBK), which can be efficiently expressed in industrial yeast strains and is thus an attractive candidate for commercial utilizations. In order to understand the molecular mechanism, which helps in strategies to improve the enzyme's performance that would meet industrial demands, 3D-structural information is a great asset. Here, we present the 1.57Å crystal structure of ManBK. The protein adopts a typical (ß/α)8 fold that resembles the other GH5 family members. Polysaccharides were subsequently modeled into the substrate binding groove to identify the residues and structural features that may be involved in the catalytic reaction. Based on the structure, rational design was conducted to engineer ManBK in an attempt to enhance its enzymatic activity. Among the 23 mutants that we constructed, the most promising Y216W showed an 18±2.7% increase in specific activity by comparison with the wild type enzyme. The optimal temperature and heat tolerance profiles of Y216W were similar to those of the wild type, manifesting a preserved thermostability. Kinetic studies showed that Y216W has higher kcat values than the wild type enzyme, suggesting a faster turnover rate of catalysis. In this study we applied rational design to ManBK by using its crystal structure as a basis and identified the Y216W mutant that shows great potentials in industrial applications.

Structural and mutagenetic analyses of a 1,3-1,4-ß-glucanase from Paecilomyces thermophila.

The thermostable 1,3-1,4-ß-glucanase PtLic16A from the fungus Paecilomyces thermophila catalyzes stringent hydrolysis of barley ß-glucan and lichenan with an outstanding efficiency and has great potential for broad industrial applications. Here, we report the crystal structures of PtLic16A and an inactive mutant E113A in ligand-free form and in complex with the ligands cellobiose, cellotetraose and glucotriose at 1.80Å to 2.25Å resolution. PtLic16A adopts a typical ß-jellyroll fold with a curved surface and the concave face forms an extended ligand binding cleft. These structures suggest that PtLic16A might carry out the hydrolysis via retaining mechanism with E113 and E118 serving as the nucleophile and general acid/base, respectively. Interestingly, in the structure of E113A/1,3-1,4-ß-glucotriose complex, the sugar bound to the -1 subsite adopts an intermediate-like (α-anomeric) configuration. By combining all crystal structures solved here, a comprehensive binding mode for a substrate is proposed. These findings not only help understand the 1,3-1,4-ß-glucanase catalytic mechanism but also provide a basis for further enzymatic engineering.

Immobilization of Piromyces rhizinflata ß-glucanase on poly(dimethylsiloxane) and Si wafer and prediction of optimum reaction for enzyme activity.

EglA, a ß-1,4-glucanase isolated from the ruminal fungus Piromyces rhizinflata, shows promise in a wide range of industrial applications because of its broad substrate specificity. In this study, EglA was immobilized on different supporting materials including poly(dimethylsiloxane) (PDMS), Si wafer, textured Si wafer, and indium tin oxide-coated (ITO-coated) glass. The binding abilities of PDMS and Si wafer toward EglA were significantly higher than those of the other supporting materials. The optimized temperature and pH conditions for EglA immobilized on PDMS and on Si wafer were further determined by a response surface methodology (RSM) combined with a central composite design (CCD). The results indicated that the optimum pH and temperature values as well as the specific ß-glucanase activity of EglA on PDMS were higher than those of free-form EglA. In addition, EglA immobilized on PDMS could be reused up to six times with detectable enzyme activity, while the enzyme activity of Eg1A on Si wafer was undetectable after three cycles of enzyme reaction. The results demonstrate that PDMS is an attractive supporting material for EglA immobilization and could be developed into an enzyme chip or enzyme tube for potential industrial applications.

Increase of the adhesion ability and display of a rumen fungal xylanase on the cell surface of Lactobacillus casei by using a listerial cell-wall-anchoring protein.

BACKGROUND: Lactobacillus, which has great adhesion ability to intestinal mucosa and is able to hydrolyse plant cell walls, can be used more efficiently as a feed additive. To increase the adhesion ability and display a fungal xylanase on the cell surface of Lactobacillus casei, the Listeria monocytogenes cell-wall-anchoring protein gene, mub, was introduced into L. casei ATCC 393 cells and used as a fusion partner to display the rumen fungal xylanase XynCDBFV on the cell surface of the transformed strains. RESULTS: The transformed strain L. casei pNZ-mub, which harboured mub gene, displayed recombinant Mub on its cell surface and showed greater adhesion ability to Caco-2 cells than the parental strain. The transformed strain L. casei pNZ-mub/xyn, which harboured mub-xynCDBFV fusion gene, acquired the capacity to break down oat spelt xylan and exhibited greater competition ability against the adhesion of L. monocytogenes to Caco-2 cells, in comparison with the parental strain. CONCLUSION: Mub has a potential to be used as a fusion partner to display heterologous proteins on the cell surface of Lactobacillus. Moreover, this is the first report of the successful display of xylanase on the cell surface of Lactobacillus.

Screening and Characterization of a Chryseobacterium timonianum Strain with Aflatoxin B1 Removal Ability.

INTRODUCTION: Aflatoxin B1 (AFB1) is a potent hepatocarcinogenic mycotoxin found in animal feed and human food components. AFB1 contamination poses severe food safety and economic consequences. METHODS: In this study, we used a coumarin-selective medium to isolate bacterial strains that can remove AFB1. Among the isolated bacterial strains, strain c4a exhibited the highest AFB1 removal activity. This strain was subjected to biochemical and phylogenetic characterization. The AFB1 removal activity of the extracellular supernatant of this strain was optimized for growth medium, reaction temperature, pH, and metal ions. The degradation products were analyzed using UPLC-ESI MS/MS. RESULTS: Strain c4a was found to be most closely related to Chryseobacterium timonianum. The extracellular supernatant of C. timonianum c4a grown in a modified nutrient broth (with gelatin peptone and beef extract in a 4:1 ratio) demonstrated the highest AFB1 removal activity when incubated with 1 ppm AFB1 at 60°C, pH 8, and Mn2+ or Mg2+ supplementation for 72 h. Surprisingly, the autoclaved extracellular supernatant also retained AFB1 removal activity. UPLC-ESI MS/MS analysis suggested that AFB1 was transformed into a metabolite (m/z value 285.08) by water molecule addition on furan ring double bond. CONCLUSION: The AFB1 removal activity of C. timonianum c4a was extracellular, constitutive, and highly thermostable, structurally transforming AFB1 into a much less toxic product. Herein, we present the first evidence of thermostable AFB1 removal activity of a strain belonging to C. timonianum.

Substrate binding to a GH131 ß-glucanase catalytic domain from Podospora anserina.

ß-Glucanases have been utilized widely in industry to treat various carbohydrate-containing materials. Recently, the Podospora anserina ß-glucanase 131A (PaGluc131A) was identified and classified to a new glycoside hydrolases GH131 family. It shows exo-ß-1,3/exo-ß-1,6 and endo-ß-1,4 glucanase activities with a broad substrate specificity for laminarin, curdlan, pachyman, lichenan, pustulan, and cellulosic derivatives. Here we report the crystal structures of the PaGluc131A catalytic domain with or without ligand (cellotriose) at 1.8Å resolution. The cellotriose was clearly observed to occupy the +1 to +3 subsites in substrate binding cleft. The broadened substrate binding groove may explain the diverse substrate specificity. Based on our crystal structures, the GH131 family enzyme is likely to carry out the hydrolysis through an inverting catalytic mechanism, in which E99 and E139 are supposed to serve as the general base and general acid.

Preliminary X-ray diffraction analysis of thermostable ß-1,4-mannanase from Aspergillus niger BK01.

ß-1,4-Mannanase (ß-mannanase) is a key enzyme in decomposing mannans, which are abundant components of hemicelluloses in the plant cell wall. Therefore, mannan hydrolysis is highly valuable in a wide array of industrial applications. ß-Mannanase isolated from Aspergillus niger BK01 (ManBK) was classified into glycoside hydrolase family GH5. ManBK holds great potential in biotechnological applications owing to its high thermostability. Here, ManBK was expressed and purified in Pichia pastoris and the recombinant protein was crystallized. Crystals belonging to the orthorhombic space group C2221, with unit-cell parameters a=93.58, b=97.05, c=147.84 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 1.57 Šresolution. Structure determination using molecular-replacement methods is in progress.

Potential probiotics for regulation of the gut-lung axis to prevent or alleviate influenza in vulnerable populations.

Influenza, also known as "flu", is an infectious disease caused by influenza viruses. Three types of influenza virus, A, B, and C, are able to infect humans. In most people, influenza causes mild symptoms, but it can also induce severe complications and death. Annual influenza vaccines are currently the main intervention used to minimize mortality and morbidity. However, vaccination frequently fails to provide adequate protection, especially in the elderly. Traditional flu vaccine targets hemagglutinin to prevent virus infection, but the constant mutation of hemagglutinin means that it is a challenge to develop vaccines quickly enough to keep up with mutations. Thus, other methods of curbing influenza incidence would be welcomed, especially for vulnerable populations. Although influenza viruses primarily infect the respiratory tract, influenza virus infection also induces intestinal dysbiosis. Through gut microbiota-derived secreted products and the circulating immune cells, gut microbiota can affect pulmonary immunity. The crosstalk between the respiratory tract and gut microbiota, termed the "gut-lung axis", is observed in the regulation of immune responses against influenza virus infection or inflammation-induced lung damage, indicating the possibility of using probiotics to prevent influenza virus infection or alleviate respiratory symptoms. In this review, we summarize the current findings on the antiviral functions of particular probiotics and/or combinations and discuss the antiviral mechanisms and immunomodulatory activities of probiotics in vitro, in mice, and in humans. Clinical studies show probiotic supplements can provide health benefits, not only to the elderly or children with compromised immune systems, but also to young- and middle-aged adults.

Diverse substrate recognition mechanism revealed by Thermotoga maritima Cel5A structures in complex with cellotetraose, cellobiose and mannotriose.

The hyperthermophilic endoglucanase Cel5A from Thermotoga maritima can find applications in lignocellulosic biofuel production, because it catalyzes the hydrolysis of glucan- and mannan-based polysaccharides. Here, we report the crystal structures in apo-form and in complex with three ligands, cellotetraose, cellobiose and mannotriose, at 1.29Å to 2.40Å resolution. The open carbohydrate-binding cavity which can accommodate oligosaccharide substrates with extensively branched chains explained the dual specificity of the enzyme. Combining our structural information and the previous kinetic data, it is suggested that this enzyme prefers ß-glucosyl and ß-mannosyl moieties at the reducing end and uses two conserved catalytic residues, E253 (nucleophile) and E136 (general acid/base), to hydrolyze the glycosidic bonds. Moreover, our results also suggest that the wide spectrum of Tm_Cel5A substrates might be due to the lack of steric hindrance around the C2-hydroxyl group of the glucose or mannose unit from active-site residues.

Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop.

Cellulase 12A from Thermotoga maritima (TmCel12A) is a hyperthermostable ß-1,4-endoglucanase. We recently determined the crystal structures of TmCel12A and its complexes with oligosaccharides. Here, by using site-directed mutagenesis, the role played by Arg60 and Tyr61 in a unique surface loop of TmCel12A was investigated. The results are consistent with the previously observed hydrogen bonding and stacking interactions between these two residues and the substrate. Interestingly, the mutant Y61G had the highest activity when compared with the wild-type enzyme and the other mutants. It also shows a wider range of working temperatures than does the wild type, along with retention of the hyperthermostability. The k (cat) and K (m) values of Y61G are both higher than those of the wild type. In conjunction with the crystal structure of Y61G-substrate complex, the kinetic data suggest that the higher endoglucanase activity is probably due to facile dissociation of the cleaved sugar moiety at the reducing end. Additional crystallographic analyses indicate that the insertion and deletion mutations at the Tyr61 site did not affect the overall protein structure, but local perturbations might diminish the substrate-binding strength. It is likely that the catalytic efficiency of TmCel12A is a subtle balance between substrate binding and product release. The activity enhancement by the single mutation of Y61G provides a good example of engineered enzyme for industrial application.

Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-ß-D-glucanase.

1,3-1,4-ß-D-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-ß-D-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ∼30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity.

Investigation of microorganisms involved in biosynthesis of the kefir grain.

The purpose of this study was to understand the significance of each microorganism in grain formation by evaluating their microbial aggregation and cell surface properties during co-aggregation of LAB and yeasts together with an investigation of biofilm formation. Non-grain forming strains from viili were also evaluated as a comparison. Results indicated that the kefir grain strains, Lactobacillus kefiranofaciens and Saccharomyces turicensis possess strong auto-aggregation ability and that Lactobacillus kefiri shows significant biofilm formation properties. Significant co-aggregation was noted when S. turicensis and kefir LAB strains (Lb. kefiranofaciens and Lb. kefiri) were co-cultured. Most of the tested LAB strains are hydrophilic and had a negative charge on their cell surface. Only the kefir LAB strains, Lb. kefiranofaciens HL1 and Lb. kefiri HL2, possessed very high hydrophobicity and had a positive cell surface charge at pH 4.2. In contrast, the LAB and yeasts in viili did not show any significant self-aggregation or biofilm formation. Based on the above results, we propose that grain formation begins with the self-aggregation of Lb. kefiranofaciens and S. turicensis to form small granules. At this point, the biofilm producer, Lb. kefiri, then begins to attach to the surface of granules and co-aggregates with other organisms and components in the milk to form the grains. On sub-culturing, more organisms attach to the grains resulting in grain growth. When investigated by scanning electron microscopy, it was found that short-chain lactobacilli such as Lb. kefiri occupy the surface, while long-chain lactobacilli such as Lb. kefiranofaciens have aggregated towards the center of the kefir grains. These findings agree with the above hypothesis on the formation of grains. Taken together, this study demonstrates the importance of cell surface properties together with fermentation conditions to the formation of grains in kefir.

Screening of Lactic Acid Bacterial Strains with Antiviral Activity Against Porcine Epidemic Diarrhea.

Newly emerging and re-emerging viral infectious diseases cause significant economic losses in swine production. Efficacious vaccines have not yet been developed for several major swine infectious diseases, including porcine epidemic diarrhea virus (PEDV). We used the PEDV-infected Vero cell model to screen lactic acid bacteria (LAB) strains with antiviral activity. Sixty LAB strains were isolated from the feces of nursing piglets. After the elimination of LAB strains with high cytotoxicity to Vero cells, the protective effects of the remaining 6 strains against PEDV infection were determined. Vero cells pretreated with the intracellular extracts or cell wall fractions of YM22 and YM33 strains for 24 h before infection with PEDV showed significantly higher cell viabilities and lower mRNA expression of PEDV nucleocapsid (PEDV-N) than the unpretreated cells, indicating that the intracellular extracts and cell wall fractions of YM22 and YM33 possessed prophylactic effects on Vero cells against PEDV infection. PEDV-infection significantly increased the mRNA expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) in Vero cells. However, pretreatment of Vero cells with the cell wall fractions of YM22 and YM33 decreased the mRNA expression of TNF-α and IL-8, which could be a mechanism associated with the protective effects of YM22 and YM33 against PEDV. Based on the biochemical characteristics and phylogenetic analyses, YM22 and YM33 were identified as Ligilactobacillus agilis (basonym: Lactobacillus agilis) and Ligilactobacillus salivarius (basonym: Lactobacillus salivarius), respectively. These findings suggest that L. agilis YM22 and L. salivarius YM33 could provide some levels of protective effects against PEDV infections.

Crystal structure and substrate-binding mode of cellulase 12A from Thermotoga maritima.

Cellulases have been used in many applications to treat various carbohydrate-containing materials. Thermotoga maritima cellulase 12A (TmCel12A) belongs to the GH12 family of glycoside hydrolases. It is a ß-1,4-endoglucanase that degrades cellulose molecules into smaller fragments, facilitating further utilization of the carbohydrate. Because of its hyperthermophilic nature, the enzyme is especially suitable for industrial applications. Here the crystal structure of TmCel12A was determined by using an active-site mutant E134C and its mercury-containing derivatives. It adopts a ß-jellyroll protein fold typical of the GH12-family enzymes, with two curved ß-sheets A and B and a central active-site cleft. Structural comparison with other GH12 enzymes shows significant differences, as found in two longer and highly twisted ß-strands B8 and B9 and several loops. A unique Loop A3-B3 that contains Arg60 and Tyr61 stabilizes the substrate by hydrogen bonding and stacking, as observed in the complex crystals with cellotetraose and cellobiose. The high-resolution structures allow clear elucidation of the network of interactions between the enzyme and its substrate. The sugar residues bound to the enzyme appear to be more ordered in the -2 and -1 subsites than in the +1, +2 and -3 subsites. In the E134C crystals the bound -1 sugar at the cleavage site consistently show the α-anomeric configuration, implicating an intermediate-like structure.