Potential use of propolis-loaded quaternized chitosan/pectin hydrogel films as wound dressings: Preparation, characterization, antibacterial evaluation, and in vitro healing assay.

Quaternized chitosan (QCS) was blended with pectin (Pec) to improve water solubility and antibacterial activity of the hydrogel films. Propolis was also loaded into hydrogel films to improve wound healing ability. Therefore, the aim of this study was to fabricate and characterize the propolis-loaded QCS/Pec hydrogel films for use as wound dressing materials. The morphology, mechanical properties, adhesiveness, water swelling, weight loss, release profiles, and biological activities of the hydrogel films were investigated. Scanning Electron Microscope (SEM) investigation indicated a homogenous smooth surface of the hydrogel films. The blending of QCS and Pec increased tensile strength of the hydrogel films. Moreover, the blending of QCS and Pec improved the stability of the hydrogel films in the medium and controlled the release characteristics of propolis from the hydrogel films. The antioxidant activity of the released propolis from the propolis-loaded hydrogel films was ∼21-36 %. The propolis-loaded QCS/Pec hydrogel films showed the bacterial growth inhibition, especially against S. aureus and S. pyogenes. The propolis-loaded hydrogel films were non-toxicity to mouse fibroblast cell line (NCTC clone 929) and supported the wound closure. Therefore, the propolis-loaded QCS/Pec hydrogel films might be good candidates for use as wound dressing materials.

Characterization of 3 phylogenetically distinct membrane-bound d-gluconate dehydrogenases of Gluconobacter spp. and their biotechnological application for efficient 2-keto-d-gluconate production.

We identified a novel flavoprotein-cytochrome c complex d-gluconate dehydrogenase (GADH) encoded by gndXYZ of Gluconobacter oxydans NBRC 3293, which is phylogenetically distinct from previously reported GADHs encoded by gndFGH and gndSLC of Gluconobacter spp. To analyze the biochemical properties of respective GADHs, Gluconobacter japonicus NBRC 3271 mutant strain lacking membranous d-gluconate dehydrogenase activity was constructed. All GADHs (GndFGH, GndSLC, and GndXYZ) were successfully overexpressed in the constructed strain. The optimal pH and KM value at that pH of GndFGH, GndSLC, and GndXYZ were 5, 6, and 4, and 8.82 ± 1.15, 22.9 ± 5.0, and 11.3 ± 1.5 m m, respectively. When the mutants overexpressing respective GADHs were cultured in d-glucose-containing medium, all of them produced 2-keto-d-gluconate, revealing that GndXYZ converts d-gluconate to 2-keto-d-gluconate as well as other GADHs. Among the recombinants, the gndXYZ-overexpressing strain accumulated the highest level of 2-keto-d-gluconate, suggesting its potential for 2-keto-d-gluconate production.

Characterization of auxiliary iron-sulfur clusters in a radical S-adenosylmethionine enzyme PqqE from Methylobacterium extorquens AM1.

PqqE is a radical S-adenosyl-l-methionine (SAM) enzyme that catalyzes the initial reaction of pyrroloquinoline quinone (PQQ) biosynthesis. PqqE belongs to the SPASM (subtilosin/PQQ/anaerobic sulfatase/mycofactocin maturating enzymes) subfamily of the radical SAM superfamily and contains multiple Fe-S clusters. To characterize the Fe-S clusters in PqqE from Methylobacterium extorquens AM1, Cys residues conserved in the N-terminal signature motif (CX 3 CX 2C) and the C-terminal seven-cysteine motif (CX 9-15 GX 4 CX n CX 2 CX 5 CX 3 CX n C; n = an unspecified number) were individually or simultaneously mutated into Ser. Biochemical and Mössbauer spectral analyses of as-purified and reconstituted mutant enzymes confirmed the presence of three Fe-S clusters in PqqE: one [4Fe-4S]2+ cluster at the N-terminal region that is essential for the reductive homolytic cleavage of SAM into methionine and 5'-deoxyadenosyl radical, and one each [4Fe-4S]2+ and [2Fe-2S]2+ auxiliary clusters in the C-terminal SPASM domain, which are assumed to serve for electron transfer between the buried active site and the protein surface. The presence of [2Fe-2S]2+ cluster is a novel finding for radical SAM enzyme belonging to the SPASM subfamily. Moreover, we found uncommon ligation of the auxiliary [4Fe-4S]2+ cluster with sulfur atoms of three Cys residues and a carboxyl oxygen atom of a conserved Asp residue.

PqqE from Methylobacterium extorquens AM1: a radical S-adenosyl-l-methionine enzyme with an unusual tolerance to oxygen.

Methylobacterium extorquens AM1 is an aerobic facultative methylotroph known to secrete pyrroloquinoline quinone (PQQ), a cofactor of a number of bacterial dehydrogenases, into the culture medium. To elucidate the molecular mechanism of PQQ biosynthesis, we are focusing on PqqE which is believed to be the enzyme catalysing the first reaction of the pathway. PqqE belongs to the radical S-adenosyl-l-methionine (SAM) superfamily, in which most, if not all, enzymes are very sensitive to dissolved oxygen and rapidly inactivated under aerobic conditions. We here report that PqqE from M. extorquens AM1 is markedly oxygen-tolerant; it was efficiently expressed in Escherichia coli cells grown aerobically and affinity-purified to near homogeneity. The purified and reconstituted PqqE contained multiple (likely three) iron-sulphur clusters and showed the reductive SAM cleavage activity that was ascribed to the consensus [4Fe-4S](2+) cluster bound at the N-terminus region. Mössbauer spectrometric analyses of the as-purified and reconstituted enzymes revealed the presence of [4Fe-4S](2+) and [2Fe-2S](2+) clusters as the major forms with the former being predominant in the reconstituted enzyme. PqqE from M.extorquens AM1 may serve as a convenient tool for studying the molecular mechanism of PQQ biosynthesis, avoiding the necessity of establishing strictly anaerobic conditions.

Acetic acid bacteria: A group of bacteria with versatile biotechnological applications.

Acetic acid bacteria are gram-negative obligate aerobic bacteria assigned to the family Acetobacteraceae of Alphaproteobacteria. They are members of the genera Acetobacter, Gluconobacter, Gluconacetobacter, Acidomonas, Asaia, Kozakia, Swaminathania, Saccharibacter, Neoasaia, Granulibacter, Tanticharoenia, Ameyamaea, Neokomagataea, and Komagataeibacter. Many strains of Acetobacter and Komagataeibacter have been known to possess high acetic acid fermentation ability as well as the acetic acid and ethanol resistance, which are considered to be useful features for industrial production of acetic acid and vinegar, the commercial product. On the other hand, Gluconobacter strains have the ability to perform oxidative fermentation of various sugars, sugar alcohols, and sugar acids leading to the formation of several valuable products. Thermotolerant strains of acetic acid bacteria were isolated in order to serve as the new strains of choice for industrial fermentations, in which the cooling costs for maintaining optimum growth and production temperature in the fermentation vessels could be significantly reduced. Genetic modifications by adaptation and genetic engineering were also applied to improve their properties, such as productivity and heat resistance.

Adaptive mutation of Acetobacter pasteurianus SKU1108 enhances acetic acid fermentation ability at high temperature.

In vitro adaptation is one of the most challenging subjects in biology to understand adaptive evolution. Microbial adaptation to temperature is not only interesting in terms of understanding the adaptation mechanism, but also useful for industrial applications. In this study, we attempted the in vitro adaptation of Acetobacter pasteurianus SKU1108 by repeating its cultivation under high-temperature acetic acid fermentation conditions. As a result, thermo-adapted strains having the higher fermentation ability than the wild-type strain were obtained. Mutations and/or disruptions in several proteins of the adapted strains were detected with NGS sequencing technology. In particular, two different adapted strains had mutations or disruptions in three specific genes in common, suggesting that these genes are essential for thermotolerance or fermentation at higher temperature. In order to clarify their involvement in thermotolerance, two of the three genes were disrupted and their phenotype was examined. The results showed that mutations of the two proteins, MarR and an amino acid transporter, are partly responsible for higher fermentation ability and/or thermotolerance. Thus, it was suggested that these elevated abilities of the adapted strains are acquired by assembling several single gene mutations including the above two mutations.

Characterization of genes involved in D-sorbitol oxidation in thermotolerant Gluconobacter frateurii.

Further upstream of sldSLC, genes for FAD-dependent D-sorbitol dehydrogenase in Gluconobacter frateurii, three additional genes (sldR, xdhA, and perA) are found: for a transcriptional regulator, NAD(P)-dependent xylitol dehydrogenase, and a transporter protein, a member of major facilitator superfamily, respectively. xdhA and perA but not sldR were found to be in the same transcriptional unit. Disruption of sldR resulted in a dramatic decrease in sldSLC promoter activity, indicating that it is an activator for sldSLC expression. The recombinant protein of XdhA expressed in Escherichia coli showed NAD-dependent dehydrogenase activities with xylitol and D-sorbitol, but a mutant strain defective in this gene showed similar activities with both substrates as compared to the wild-type strain. Nonetheless, the growth of the xdhA mutant strain on D-sorbitol and xylitol was retarded, and so was that of a mutant strain defective in perA. These results indicate that xdhA and perA are involved in assimilation of D-sorbitol and xylitol.

Genome-wide phylogenetic analysis of differences in thermotolerance among closely related Acetobacter pasteurianus strains.

Acetobacter pasteurianus is a Gram-negative strictly aerobic bacterium that is widely used for the industrial production of vinegar. Three Acetobacter pasteurianus strains, SKU1108, NBRC 3283 and IFO 3191, have the same 16S rRNA sequence (100 % sequence identity) but show differences in thermotolerance. To clarify the relationships between phylogeny and thermotolerance of these strains, genome-wide analysis of these three strains was performed. Concatenated phylogenetic analysis of a dataset of 1864 orthologues has shown that the more thermotolerant strains, SKU1108 and NBRC 3283, are more closely related to each other than to the more thermosensitive strain, IFO 3191. In addition, we defined a dataset of 2010 unique orthologues among these three strains, and compared the frequency of amino acid mutations among them. Genes involved in translation, transcription and signal transduction are highly conserved among each unique orthologous dataset. The results also showed that there are several genes with increased mutation rates in IFO 3191 compared with the thermotolerant strains, SKU1108 and NBRC 3283. Analysis of the mutational directions of these genes suggested that some of them might be correlated with the thermosensitivity of IFO 3191. Concatenated phylogenetic analysis of these closely related strains revealed that there is a phylogenetic relationship associated with this phenotype among the thermotolerant and thermosensitive strains.

The crystal structure of l-sorbose reductase from Gluconobacter frateurii complexed with NADPH and l-sorbose.

l-Sorbose reductase from Gluconobacter frateurii (SR) is an NADPH-dependent oxidoreductase. SR preferentially catalyzes the reversible reaction between d-sorbitol and l-sorbose with high substrate specificity. To elucidate the structural basis of the catalytic mechanism and the substrate specificity of SR, we have determined the structures of apo-SR, SR in complex with NADPH, and the inactive mutant (His116Leu) of SR in complex with NADPH and l-sorbose at 2.83 Å, 1.90 Å, and 1.80 Å resolutions, respectively. Our results show that SR belongs to the short-chain dehydrogenase/reductase (SDR) family and forms a tetrameric structure. Although His116 is not conserved among SDR family enzymes, the structures of SR have revealed that His116 is important for the stabilization of the proton relay system and for active-site conformation as a fourth catalytic residue. In the ternary complex structure, l-sorbose is recognized by 11 hydrogen bonds. Site-directed mutagenesis of residues around the l-sorbose-binding site has shown that the loss of almost full enzymatic activity was caused by not only the substitution of putative catalytic residues but also the substitution of the residue used for the recognition of the C4 hydroxyl groups of l-sorbose (Glu154) and of the residues used for the construction of the substrate-binding pocket (Cys146 and Gly188). The recognition of the C4 hydroxyl group of l-sorbose would be indispensable for the substrate specificity of SR, which recognizes only l-sorbose and d-sorbitol but not other sugars. Our results indicated that these residues were crucial for the substrate recognition and specificity of SR.