Yutaro Mori

Synergistic effect and application of xylanases as accessory enzymes to enhance the hydrolysis of pretreated bagasse

Recently, the new trend in the second-generation ethanol industry is to use mild pretreatments, in order to reduce costs and to keep higher content of hemicellulose in the biomass. Nevertheless, a high enzyme dosage is still required in the conversion of (hemi)cellulose. The interaction between cellulases and xylanases seems to be an effective alternative to reduce enzyme loading in the saccharification process. At first, to evaluate the synergism of xylanases on bagasse degradation, we have produced two xylanases from glycoside hydrolase family 10 (GH10) and three xylanases from glycoside hydrolase family 11 (GH11), from two thermophilic organisms, Thermobifida fusca and Clostridium thermocellum, and one mesophilic organism, Streptomyces lividans. Peracetic acid (PAA) pretreated bagasse was used as substrate. The combination of XynZ-C (GH10, from C. thermocellum), and XlnB (GH11, from S. lividans) presented the highest degree of synergy after 6h (3.62). However, the combination of XynZ-C and Xyn11A (GH11, from T. fusca) resulted in the highest total yield of reducing sugars. To evaluate the synergism between xylanases and cellulases, commercial cellulase preparation from Trichoderma reesei was combined with the selected xylanases, XynZ-C and Xyn11A. About 2-fold increase was observed in the concentration of reducing sugars, when both xylanases, XynZ-C and Xyn11A, were added together with T. reesei cellulases in the reaction mixture.

Aligning an endoglucanase Cel5A from Thermobifida fusca on a DNA scaffold: potent design of an artificial cellulosome

A novel multi-cellulase conjugate assembled on a double-stranded DNA scaffold, a DNA-(endoglucanase)n conjugate, exhibited unique hydrolytic activity toward crystalline cellulose (Avicel) depending on the cellulase/DNA ratio on the DNA-based artificial cellulosome.

Effect of pretreatment methods on the synergism of cellulase and xylanase during the hydrolysis of bagasse

The effect of pretreatment with peracetic acid (PAA) or an ionic liquid (1-ethyl-3-methylimidazolium acetate, [Emim][OAc]) on the synergism between endoglucanase and endoxylanase in the hydrolysis of bagasse was investigated. An endoglucanase, Cel6A, with a carbohydrate-binding module (CBM) and two endoxylanases, XynZ-C without a CBM and Xyn11A with an intrinsic xylan/cellulose binding module (XBM), were selected. The hemicellulose content, especially arabinan, and the cellulose crystallinity of bagasse were found to affect the cellulase-xylanase synergism. More specifically, higher synergism (above 3.4) was observed for glucan conversion, at low levels of arabinan (0.9%), during the hydrolysis of PAA pretreated bagasse. In contrast, [Emim][OAc] pretreated bagasse, showed lower cellulose crystallinity and achieved higher synergism (over 1.9) for xylan conversion. Ultimately, the combination of Cel6A and Xyn11A resulted in higher synergism for glucan conversion than the combination of Cel6A with XynZ-C, indicating the importance of the molecular architecture of enzymes for metabolic synergism.

Transglutaminase-mediated internal protein labeling with a designed peptide loop

Post-translational internal protein labeling was explored through the insertion of a 13-mer peptidyl loop specifically recognized by microbial transglutaminase (MTG). The peptidyl loop included one lysine residue (abbreviated as the K-loop), and was designed and inserted into two different regions of the protein bacterial alkaline phosphatase (BAP). MTG-mediated selective labeling of a lysine residue in the K-loop was achieved with a functional Gln-donor substrate. Internal protein labeling in the vicinity of the active site of BAP (residues 91-93) markedly decreased the activity of the enzyme. Conversely, insertion of the K-loop at a site distal from the active site (residues 219-221) afforded site-specific and covalent internal protein labeling without impairing the activity of the enzyme.

One-dimensional assembly of functional proteins: toward the design of an artificial cellulosome

In biological systems, proteins can form well-organized, higher-order structures with unique functions that would be difficult to achieve with a single protein. These proteinaceous supramolecular structures form by self-assembly, and the spatial arrangement of the protein building blocks in them is very important. In the present study, an artificial system was developed using recombinant proteins as building blocks, which were assembled in a one-dimensional manner. The assembly of these building blocks was based on the avidin–biotin interaction. A tetrameric biotin ligand unit was designed so that the 1 : 4 stoichiometry of the avidin–biotin interaction was altered to a 1 : 2 directional interaction between the streptavidin and tetrabiotinylated protein units. In a proof-of-concept study, site-specifically tetrabiotin-labeled endoglucanase and cellulose-binding module units were prepared, and then these components were self-assembled by mixing with streptavidin to mimic a natural cellulosome. The formation of one-dimensional assemblies of the protein units depended on the stoichiometry of the avidin–biotin interaction sites in the system. Interestingly, the saccharification efficiency improved when the component ratio of protein units in the assemblies was changed. The presence of the optimal ratio of the building blocks implies the modularity of the present protein assembly system.

Designing artificial metabolic pathways, construction of target enzymes, and analysis of their function

Artificial design of metabolic pathways is essential for the production of useful compounds using microbes. Based on this design, heterogeneous genes are introduced into the host, and then various analysis and evaluation methods are conducted to ensure that the target enzyme reactions are functionalized within the cell. In this chapter, we list successful examples of useful compounds produced by designing artificial metabolic pathways, and describe the methods involved in analyzing, evaluating, and optimizing the target enzyme reaction.

Substrate Engineering of Microbial Transglutaminase for Site-Specific Protein Modification and Bioconjugation

Microbial transglutaminase (MTG), a robust enzyme developed initially for the manipulation of edible proteins in the food industry, has now been widely recognized as a practical protein-modifying reagent in the range of biotechnological applications. In this chapter, we introduce the potential use of MTG through our basic studies on the design of novel glutamine (Gln) donor substrates for lysine (Lys)-specific protein modification. Based on the core structure of a conventional transglutaminase substrate, benzyloxycarbonyl-L-glutaminylglycine (Z-QG), new Gln-donor substrates have been developed for the conjugation of recombinant proteins with different functionalities. The first target site for the substrate engineering was the C-terminal carboxylic group of Z-QG, which is feasibly labeled with functional moieties. For the preparation of protein-nucleic acid conjugates with novel molecular architecture, a new nucleotidyl substrate, Z-QG-(d)UTP, was created. We have also explored substitution of the N-terminal protecting group (Z) with fluorophores and biotin, and found that MTG accepts diverse functional groups at the N-terminus by inserting a short linker, leading to an increase in the utility of MTG in site-specific modification of functional proteins. Our results demonstrated how the design of (small) Gln-donor substrates of MTG can expand the scope of enzymatic manipulation in biomolecular engineering.