Enzymatic Characterization of Unused Biomass Degradation Using the Clostridium cellulovorans Cellulosome.

The cellulolytic system of Clostridium cellulovorans mainly consisting of a cellulosome that synergistically collaborates with non-complexed enzymes was investigated using cellulosic biomass. The cellulosomes were isolated from the culture supernatants with shredded paper, rice straw and sugarcane bagasse using crystalline cellulose. Enzyme solutions, including the cellulosome fractions, were analyzed by SDS-PAGE and Western blot using an anti-CbpA antibody. As a result, C. cellulovorans was able to completely degrade shredded paper for 9 days and to be continuously cultivated by the addition of new culture medium containing shredded paper, indicating, through TLC analysis, that its degradative products were glucose and cellobiose. Regarding the rice straw and sugarcane bagasse, while the degradative activity of rice straw was most active using the cellulosome in the culture supernatant of rice straw medium, that of sugarcane bagasse was most active using the cellulosome from the supernatant of cellobiose medium. Based on these results, no alcohols were found when C. acetobutylicum was cultivated in the absence of C. cellulovorans as it cannot degrade the cellulose. While 1.5 mM of ethanol was produced with C. cellulovorans cultivation, both n-butanol (1.67 mM) and ethanol (1.89 mM) were detected with the cocultivation of C. cellulovorans and C. acetobutylicum. Regarding the enzymatic activity evaluation against rice straw and sugarcane bagasse, the rice straw cellulosome fraction was the most active when compared against rice straw. Furthermore, since we attempted to choose reaction conditions more efficiently for the degradation of sugarcane bagasse, a wet jet milling device together with L-cysteine as a reducing agent was used. As a result, we found that the degradation activity was almost twice as high with 10 mM L-cysteine compared with without it. These results will provide new insights for biomass utilization.

Glutathione production from mannan-based bioresource by mannanase/mannosidase expressing Saccharomyces cerevisiae.

This work aims to produce glutathione directly from mannan-based bioresources using engineered Saccharomyces cerevisiae. Mannan proved to be a valuable carbon source for glutathione production by this organism. Mannan-hydrolyzing S. cerevisiae was developed by heterologous expression of mannanase/mannosidase on its cell surface. This strain efficiently produced glutathione from mannose polysaccharide, ß-1,4-mannan. Furthermore, it produced glutathione from locust bean gum (LBG), a highly dense and inexpensive mannan-based bioresource, as sole carbon source. Glutathione productivity from LBG was enhanced by engineering the glutathione metabolism of mannan-hydrolyzing S. cerevisiae. Expression of extracellular mannanase/mannosidase protein combined with intracellular metabolic engineering is potentially applicable to the efficient, environmentally friendly bioproduction of targeted products from mannan-based bioresources.

Complex carriers of affibody-displaying bio-nanocapsules and composition-varied liposomes for HER2-expressing breast cancer cell-specific protein delivery.

A bio-nanocapsule (BNC), a hollow particle composed of hepatitis B virus (HBV) surface antigen (HBsAg), and liposome (LP) conjugation method (BNC/LP) has been recently developed by Jung et al. (2008) . The BNC/LP complex carrier could successfully deliver fluorescence-labeled beads (100 nm) into liver cells. In this study, we report the promising delivery of proteins incorporated in the complex carriers, which were prepared by the BNC/LP conjugation method with specificity-altered BNC and composition-varied LPs. The specificity-altered BNC, Z(HER2)-BNC was developed by replacing the hepatocyte recognition site of BNC with Z(HER2) binding to HER2 receptor specifically. Using green fluorescent protein (GFP; 27 kDa) and cellular cytotoxic protein (exotoxin A; 66 kDa) for the delivery, we herein present the impact of different charges attributed to the composition of the LP on specific cell targeting and cellular uptake of the complex carriers. In addition, we demonstrate that the mixture prepared by mixing LPs with helper lipid possessing endosomal escaping ability boosts the functional expression of the cellular cytotoxic exotoxin A activity specifically. Finally, we further show the blending ratio of the LP mixture and Z(HER2)-BNC is a critical factor in determining the highly-efficient expression of the cytotoxic activity of exotoxin A.

Efficient heterologous expression and secretion in Aspergillus oryzae of a llama variable heavy-chain antibody fragment V(HH) against EGFR.

We have constructed a filamentous fungus Aspergillus oryzae that secretes a llama variable heavy-chain antibody fragment (V(HH)) that binds specifically to epidermal growth factor receptor (EGFR) in a culture medium. A major improvement in yield was achieved by fusing the V(HH) with a Taka-amylase A signal sequence (sTAA) and a segment of 28 amino acids from the N-terminal region of Rhizopus oryzae lipase (N28). The yields of secreted, immunologically active anti-EGFR V(HH) reached 73.8 mg/1 in a Sakaguchi flask. The V(HH) fragments were released from the sTAA or N28 proteins by an indigenous A. oryzae protease during cultivation. The purified recombinant V(HH) fragment was specifically recognized and could bind to the EGFR with a high affinity.

Enzymatic glutathione production using metabolically engineered Saccharomyces cerevisiae as a whole-cell biocatalyst.

We developed a novel enzymatic glutathione (GSH) production system using Saccharomyces cerevisiae as a whole-cell biocatalyst, and improved its GSH productivity by metabolic engineering. We demonstrated that the metabolic engineering of GSH pathway and ATP regeneration can significantly improve GSH productivity by up to 1.7-fold higher compared with the parental strain, respectively. Furthermore, the combination of both improvements in GSH pathway and ATP regeneration is more effective (2.6-fold) than either improvement individually for GSH enzymatic production using yeast. The improved whole-cell biocatalyst indicates its great potential for applications to other kinds of ATP-dependent bioproduction.