Yasushi Sugano

Recent advances in bacterial cellulose production

Bacterial cellulose (BC), which is produced by some bacteria, has unique structural, functional, physical and chemical properties. Thus, the mass production of BC for industrial application has recently attracted considerable attention. To enhance BC production, two aspects have been considered, namely, the engineering and genetic viewpoints. The former includes the reactor design, nutrient selection, process control and optimization; and the latter the cloning of the BC synthesis gene, and the genetic modification of the speculated genes for higher BC production. In this review, recent advances in BC production from the two viewpoints mentioned above are described, mainly using the bacteriumGluconacetobacter xylinus.

DyP-type peroxidases comprise a novel heme peroxidase family

Abstract.Dye-decolorizing peroxidase (DyP) is produced by a basidiomycete (Thanatephorus cucumeris Dec 1) and is a member of a novel heme peroxidase family (DyP-type peroxidase family) that appears to be distinct from general peroxidases. Thus far, 80 putative members of this family have been registered in the PeroxiBase database (http://peroxibase.isbsib.ch/) and more than 400 homologous proteins have been detected via PSI-BLAST search. Although few studies have characterized the function and structure of these proteins, they appear to be bifunctional enzymes with hydrolase or oxygenase, as well as typical peroxidase activities. DyP-type peroxidase family suggests an ancient root compared with other general peroxidases because of their widespread distribution in the living world. In this review, firstly, an outline of the characteristics of DyP from T. cucumeris is presented and then interesting characteristics of the DyP-type peroxidase family are discussed.

DyP, a Unique Dye-decolorizing Peroxidase, Represents a Novel Heme Peroxidase Family : ASP171 REPLACES THE DISTAL HISTIDINE OF CLASSICAL PEROXIDASES

DyP, a unique dye-decolorizing enzyme from the fungus Thanatephorus cucumeris Dec 1, has been classified as a peroxidase but lacks homology to almost all other known plant peroxidases. The primary structure of DyP shows moderate sequence homology to only two known proteins: the peroxide-dependent phenol oxidase, TAP, and the hypothetical peroxidase, cpop21. Here, we show the first crystal structure of DyP and reveal that this protein has a unique tertiary structure with a distal heme region that differs from that of most other peroxidases. DyP lacks an important histidine residue known to assist in the formation of a Fe4+ oxoferryl center and a porphyrin-based cation radical intermediate (compound I) during the action of ubiquitous peroxidases. Instead, our tertiary structural and spectrophotometric analyses of DyP suggest that an aspartic acid and an arginine are involved in the formation of compound I. Sequence analysis reveals that the important aspartic acid and arginine mentioned above and histidine of the heme ligand are conserved among DyP, TAP, and cpop21, and structural and phylogenetic analyses confirmed that these three enzymes do not belong to any other families of peroxidase. These findings, which strongly suggest that DyP is a representative heme peroxidase from a novel family, should facilitate the identification of additional new family members and accelerate the classification of this novel peroxidase family.

Improvement of Bacterial Cellulose Production by Addition of Agar in a Jar Fermentor

Bacterial cellulose (BC) was produced by Acetobacter xylinum BPR 2001 and its acetan nonproducing mutant EP1 in corn steep liquor-fructose medium in a 10-l jar fermentor supplemented with different agar concentrations ranging from 0% to 1.0% (w/v). The BC productivity of the two strains was increased by adding agar. The maximum BC production of BPR 2001 at an agar concentration of 0.4% was 12.8 g/l compared with 8 g/l without agar. The mutant EP1 produced 11.6 g/l of BC at an agar concentration of 0.6%, while only 5.5 g/l was produced in the control. Enhanced productivity is associated with an increase in viscosity of the culture, dispersion of BC pellets, and number of free cells due to agar addition, suggesting that acetan produced by BPR 2001 has a critical role in enhanced BC production.

Efficient Heterologous Expression in Aspergillus oryzae of a Unique Dye-Decolorizing Peroxidase, DyP, of Geotrichum candidum Dec 1

ABSTRACT Efficient expression of the dye-decolorizing peroxidase, DyP, fromGeotrichum candidum Dec 1 in Aspergillus oryzaeM-2-3 was achieved by fusing mature cDNA encoding dyp with the A. oryzae α-amylase promoter (amyB). The activity yield of the purified recombinant DyP (rDyP) was 42-fold compared with that of the purified native DyP from Dec 1. No exogenous heme was necessary for the expression of rDyP in A. oryzae. From the N-terminal amino acid sequence analyses of native DyP and rDyP, the absence of a histidine residue in both DyPs, which was considered to be important for heme binding of DyP, was confirmed. These results suggest that rDyP without a typical heme-binding region produced by A. oryzae exhibits a function similar to that of native DyP.

Bacterial cellulose production by Acetobacter xylinum in a 50‐L internal‐loop airlift reactor

Bacterial cellulose (BC) production was realized in a batch cultivation of Acetobacter xylinum subsp. sucrofermentans BPR2001 in a 50-L internal-loop airlift reactor. When the bacterium was cultivated with air supply, 3.8 g/L of BC was produced after 67 hours. When oxygen-enriched gas was supplied, the concentration of BC was doubled and the production rate of BC was 0.116 g/L. h, which was two times higher than that of air-supplied culture and comparable to that in a mechanically agitated stirred-tank fermentor. Bacterial cellulose produced by the airlift reactor formed a unique ellipse pellet (BC pellet), different from the fibrous form which was produced in an agitated stirred-tank fermentor. The BC-pellet suspension was demonstrated to have a higher volumetric oxygen transfer coefficient than the fibrous BC suspension in a 50-L internal-loop airlift reactor. The mixing time of BC-pellet suspension in the airlift reactor was also shorter than that in water.

Degradation pathway of an anthraquinone dye catalyzed by a unique peroxidase DyP from Thanatephorus cucumeris Dec 1

The reactants produced by action of a purified unique dye-decolorizing peroxidase, DyP, on a commercial anthraquinone dye, Reactive Blue 5, were investigated using electrospray ionization mass spectrometry (ESI-MS), thin-layer chromatography (TLC), and 1H- and 13C- nuclear magnetic resonance (NMR). The results of ESI-MS analysis showed that phthalic acid, a Product 2 (molecular weight 472.5), and a Product 3 (molecular weight 301.5), were produced. Product 2 and Product 3 were generated by usual peroxidase reaction, whereas phthalic acid was generated by hydrolase- or oxygenase-catalyzed reaction. One potential associated product, o-aminobenzene sulfonic acid, was found to be converted to 2,2′-disulfonyl azobenzene by ESI-MS and NMR analyses. From these results, we propose, for the first time, the degradation pathway of an anthraquinone dye by the enzyme DyP.

cDNA cloning and genetic analysis of a novel decolorizing enzyme, peroxidase gene dyp from Geotrichum candidum Dec 1

A novel decolorizing peroxidase gene (dyp) was cloned from a cDNA library of a newly isolated strain of fungus Geotrichum candidum Dec 1. The open reading frame of 1494 nucleotides which corresponds to dyp predicts a primary translation product of 498 amino acids, M(r) 53,306. The deduced amino acid sequence of DyP does not contain the typical conserved motif which is characteristic of heme-containing peroxidases in the plant peroxidase superfamily. Comparison of the deduced amino acid sequence of DyP with that of a peroxidase from Polyporaceae sp. suggests that these proteins share highly homologous regions.

Effects of Acetan on Production of Bacterial Cellulose by Acetobacter xylinum

Acetan is a water-soluble polysaccharide produced by a bacterial cellulose (BC) producer, Acetobacter xylinum. An acetan-nonproducing mutant, EP1, was generated from wild-type A. xylinum BPR2001 by the disruption of aceA, which may act to catalyze the first step of the acetan biosynthetic pathway in this bacterium. EP1 produced less BC than the wild-type strain. However, when EP1 was cultured in a medium containing acetan, BC production was stimulated and the final yield of BC was equivalent to that of BPR2001. The culture broth containing acetan was more viscous and the free cell number was higher than that of the broth without the polysaccharide, so acetan may hinder the coagulation of BC in the broth. The addition of 1.5 g/l agar also increased BC production; we concluded that acetan and BC syntheses were not directly related on the genetic level.

Formation of Highly Twisted Ribbons in a Carboxymethylcellulase Gene-Disrupted Strain of a Cellulose-Producing Bacterium

Cellulases are enzymes that normally digest cellulose; however, some are known to play essential roles in cellulose biosynthesis. Although some endogenous cellulases of plants and cellulose-producing bacteria are reportedly involved in cellulose production, their functions in cellulose production are unknown. In this study, we demonstrated that disruption of the cellulase (carboxymethylcellulase) gene causes irregular packing of de novo-synthesized fibrils in Gluconacetobacter xylinus, a cellulose-producing bacterium. Cellulose production was remarkably reduced and small amounts of particulate material were accumulated in the culture of a cmcax-disrupted G. xylinus strain (F2-2). The particulate material was shown to contain cellulose by both solid-state (13)C nuclear magnetic resonance analysis and Fourier transform infrared spectroscopy analysis. Electron microscopy revealed that the cellulose fibrils produced by the F2-2 cells were highly twisted compared with those produced by control cells. This hypertwisting of the fibrils may reduce cellulose synthesis in the F2-2 strains.