Simultaneous vinegar fermentation from a pineapple by-product using the co-inoculation of yeast and thermotolerant acetic acid bacteria and their physiochemical properties.

In the present study, a potential newly isolated thermotolerant acetic acid bacteria (TH-AAB), Acetobacter pasteurianus FPB2-3, with ethanol and acetic acid-tolerant properties was found to be very effective in the production of vinegar from pineapple peels as an alternative, inexpensive raw material using simultaneous vinegar fermentation (SVF). The results showed that using whole pineapple peel with the addition of diammonium phosphate (DAP) and MgSO4 at an initial pH of 5.5 gave a slightly higher acetic acid content than that produced from the squeezed juice. Subsequently, the effects of sugar concentration and inoculation time of A. pasteurianus FPB2-3 on acetic acid production were examined. The results revealed that an increase in sucrose concentration led to the high production of ethanol, which resulted in the suppression of acetic acid production. Allowing for the inoculated yeast to ferment prior to inoculation of the AAB for 1 or 2 days resulted in a longer lag time for ethanol oxidation. However, acetic acid accumulation commenced after 5 days and gradually increased to the maximum concentration of 7.2% (w/v) within 16 days. Furthermore, scaled-up fermentation in 6 l vessels resulted in slower acetic acid accumulation but still achieved a maximum acetic acid concentration of up to 6.5% (w/v) after 25 days. Furthermore, the antioxidant capacity of the vinegar produced from pineapple peels (PPV) was slightly higher than that produced from the squeezed juice (PJV), which was consistent with the higher total phenolic compound content found in the PPV sample. In addition to acetic acid, a main volatile acid present in vinegars, other volatile compounds, such as alcohols (isobutyl alcohol, isoamyl alcohol, and 2-phenyl ethanol), acids (3-methyl-butanoic acid), and esters (ethyl acetate, 3-methyl butanol acetate, and 2-phenylethyl acetate), were also detected and might have contributed to the observed differences in the odour and aroma of the pineapple vinegars.

A novel Na(+)(K(+))/H(+) antiporter plays an important role in the growth of Acetobacter tropicalis SKU1100 at high temperatures via regulation of cation and pH homeostasis.

A gene encoding a putative Na(+)/H(+) antiporter was previously proposed to be involved in the thermotolerance mechanism of Acetobacter tropicalis SKU 1100. The results of this study show that disruption of this antiporter gene impaired growth at high temperatures with an external pH>6.5. The growth impairment at high temperatures was much more severe in the absence of Na(+) (with only the presence of K(+)); under these conditions, cells failed to grow even at 30°C and neutral to alkaline pH values, suggesting that this protein is also important for K(+) tolerance. Functional analysis with inside-out membrane vesicles from wild type and mutant strains indicated that the antiporter, At-NhaK2 operates as an alkali cation/proton antiporter for ions such as Na(+), K(+), Li(+), and Rb(+) at acidic to neutral pH values (6.5-7.5). The membrane vesicles were also shown to contain a distinct pH-dependent Na(+)(specific)/H(+) antiporter(s) that might function at alkaline pH values. In addition, phylogenetic analysis showed that At-NhaK2 is a novel type of Na(+)/H(+) antiporter belonging to a phylogenetically distinct new clade. These data demonstrate that At-NhaK2 functions as a Na(+)(K(+))/H(+) antiporter and is essential for K(+) and pH homeostasis during the growth of A. tropicalis SKU1100, especially at higher temperatures.

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.

Global analysis of the genes involved in the thermotolerance mechanism of thermotolerant Acetobacter tropicalis SKU1100.

Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that grows even at 42 °C, a much higher temperature than the limit for the growth of mesophilic strains. To elucidate the mechanism underlying the thermotolerance of this strain, we attempted to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis followed by gene or genome analysis. Among the 4,000 Tn10-inserted mutants obtained, 32 exhibited a growth phenotype comparable to that of the parent strain at 30 °C but not at higher temperatures. We identified the insertion site of Tn10 on the chromosomes of all the mutant strains by TAIL (Thermal Asymmetric Interlaced)-PCR, and found 24 genes responsible for thermotolerance. The results also revealed a partial overlap between the genes required for thermotolerance and those required for acetic acid resistance. In addition, the origin and role of these thermotolerant genes are discussed.

Increased number of Arginine-based salt bridges contributes to the thermotolerance of thermotolerant acetic acid bacteria, Acetobacter tropicalis SKU1100.

Thermotolerant acetic acid bacteria (AAB), Acetobacter tropicalis SKU1100, can grow above 40°C. To investigate the basis of its thermotolerance, we compared the genome of A. tropicalis SKU1100 with that of mesophilic AAB strain Acetobacter pasteurianus IFO3283-01. The comparative genomic study showed that amino acid substitutions from large to small residue and Lys to Arg occur in many orthologous genes. Furthermore, comparative modeling study was carried out with the orthologous proteins between SKU1100 and IFO3283-01 strains, indicating that the number of Arg-based salt bridges increased in protein models. Since it has been reported that Arg-based salt bridges are important factor for thermo-stability of protein structure, our results strongly suggest that the increased number of Arg-based salt bridges may contributes to the thermotolerance of A. tropicalis SKU1100 (the thermo-stability of proteins in A. tropicalis SKU1100).

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.

Distinct physiological roles of two membrane-bound dehydrogenases responsible for D-sorbitol oxidation in Gluconobacter frateurii.

Two different membrane-bound enzymes oxidizing D-sorbitol are found in Gluconobacter frateurii THD32: pyroloquinoline quinone-dependent glycerol dehydrogenase (PQQ-GLDH) and FAD-dependent D-sorbitol dehydrogenase (FAD-SLDH). In this study, FAD-SLDH appeared to be induced by L-sorbose. A mutant defective in both enzymes grew as well as the wild-type strain did, indicating that both enzymes are dispensable for growth on D-sorbitol. The strain defective in PQQ-GLDH exhibited delayed L-sorbose production, and lower accumulation of it, corresponding to decreased oxidase activity for D-sorbitol in spite of high D-sorbitol dehydrogenase activity, was observed. In the mutant strain defective in PQQ-GLDH, oxidase activity with D-sorbitol was much more resistant to cyanide, and the H(+)/O ratio was lower than in either the wild-type strain or the mutant strain defective in FAD-SLDH. These results suggest that PQQ-GLDH connects efficiently to cytochrome bo(3) terminal oxidase and that it plays a major role in L-sorbose production. On the other hand, FAD-SLDH linked preferably to the cyanide-insensitive terminal oxidase, CIO.

L-sorbose reductase and its transcriptional regulator involved in L-sorbose utilization of Gluconobacter frateurii.

Upstream of the gene for flavin adenine dinucleotide (FAD)-dependent D-sorbitol dehydrogenase (SLDH), sldSLC, a putative transcriptional regulator was found in Gluconobacter frateurii THD32 (NBRC 101656). In this study, the whole sboR gene and the adjacent gene, sboA, were cloned and analyzed. sboR mutation did not affect FAD-SLDH activity in the membrane fractions. The SboA enzyme expressed and purified from an Escherichia coli transformant showed NADPH-dependent L-sorbose reductase (NADPH-SR) activity, and the enzyme was different from the NADPH-SR previously reported for Gluconobacter suboxydans IFO 3291 in molecular size and amino acid sequence. A mutant defective in sboA showed significantly reduced growth on L-sorbose, indicating that the SboA enzyme is required for efficient growth on L-sorbose. The sboR mutant grew on L-sorbose even better than the wild-type strain did, and higher NADPH-SR activity was detected in cytoplasm fractions. Reverse transcription-PCR experiments indicated that sboRA comprises an operon. These data suggest that sboR is involved in the repression of sboA, but not in the induction of sldSLC, on D-sorbitol and that another activator is required for the induction of these genes by D-sorbitol or L-sorbose.

Molecular properties of membrane-bound FAD-containing D-sorbitol dehydrogenase from thermotolerant Gluconobacter frateurii isolated from Thailand.

There are two types of membrane-bound D-sorbitol dehydrogenase (SLDH) reported: PQQ-SLDH, having pyrroloquinoline quinone (PQQ), and FAD-SLDH, containing FAD and heme c as the prosthetic groups. FAD-SLDH was purified and characterized from the PQQ-SLDH mutant strain of a thermotolerant Gluconobacter frateurii, having molecular mass of 61.5 kDa, 52 kDa, and 22 kDa. The enzyme properties were quite similar to those of the enzyme from mesophilic G. oxydans IFO 3254. This enzyme was shown to be inducible by D-sorbitol, but not PQQ-SLDH. The oxidation product of FAD-SLDH from D-sorbitol was identified as L-sorbose. The cloned gene of FAD-SLDH had three open reading frames (sldSLC) corresponding to the small, the large, and cytochrome c subunits of FAD-SLDH respectively. The deduced amino acid sequences showed high identity to those from G. oxydans IFO 3254: SldL showed to other FAD-enzymes, and SldC having three heme c binding motives to cytochrome c subunits of other membrane-bound dehydrogenases.