Antioxidant Capacities and Total Phenolic Contents of Selected Actinomycetes sp.

Objective: To investigate antioxidant activity and the optimal condition for antioxidant production from actinomycetes isolate SR3.97. Method: Cultivation of actinomycetes isolate SR3.97 was done by inoculation into maltose yeast extract (MYEB), tryptone-yeast extract broth (ISP-1) and Bennett’s broth (BN). The cultures were incubated on rotary shaker at 200 rpm, 37 ºC, for 7 days. The culture supernatants were obtained and subsequently lyophilized, and further subject to the analysis of antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl radical-scavenging (DPPH) method. Actinomycetes isolate was cultured in select liquid medium and the lyophilized portion was taken for analysis of antioxidant activity by DPPH, 2,2’-azinobis (3-ethylbensothiazoline-6-sulfonic acid) assay (ABTS), Ferric-reducing Antioxidant Power (FRAP) assay and total phenolic content. Optimal conditions for antioxidant production on initial pH at 4, 5, 6, 7, 8 and 9, temperatures at 25, 30, 35, 37, 40 and 45 ºC and incubation time of 1, 3, 5, 7, 9, 11, 13, 15 days were investigated. Identification of actinomycetes isolate SR3.97 was carried out based on 16S rRNA sequence analysis. Results: Actinomycetes isolate SR3.97 was selected as the best isolate for producing antioxidant in MYEB medium. The MYEB medium exhibited antioxidant activity as measured by DPPH, ABTS and FRAP analysis with IC50 values of 71.52 ± 0.76 µg/ml, 35.88 ± 0.84 µg/ml and 156.91 ± 1.34 µM Fe(II)/mg, respectively and total phenolic content of 57.82 ± 0.1 µg GEA/mg. The optimal culture conditions for antioxidant production were initial culture pH at 7, incubation temperature of 37ºC, and incubation time for 7 days. Actinomycetes isolate SR3.97 was identified based on 16S rRNA gene sequence analysis. The isolate was 99.40% resembled with Streptomyces chrysomallus subsp. fumigatus NBRC 15393T. Conclusion: Difference antioxidant levels were produced from actinomycetes isolate SR3.97 when different media were used. The temperature, pH and incubation time affected antioxidant production. Keywords: Actinomycetes, Antioxidant, Phenolic compound, Streptomyces chrysomallu

Antioxidant Capacities and Total Phenolic Contents of Selected Actinomycetes sp.

Objective: To investigate antioxidant activity and the optimal condition for antioxidant production from actinomycetes isolate SR3.97. Method: Cultivation of actinomycetes isolate SR3.97 was done by inoculation into maltose yeast extract (MYEB), tryptone-yeast extract broth (ISP-1) and Bennett’s broth (BN). The cultures were incubated on rotary shaker at 200 rpm, 37 ºC, for 7 days. The culture supernatants were obtained and subsequently lyophilized, and further subject to the analysis of antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl radical-scavenging (DPPH) method. Actinomycetes isolate was cultured in select liquid medium and the lyophilized portion was taken for analysis of antioxidant activity by DPPH, 2,2’-azinobis (3-ethylbensothiazoline-6-sulfonic acid) assay (ABTS), Ferric-reducing Antioxidant Power (FRAP) assay and total phenolic content. Optimal conditions for antioxidant production on initial pH at 4, 5, 6, 7, 8 and 9, temperatures at 25, 30, 35, 37, 40 and 45 ºC and incubation time of 1, 3, 5, 7, 9, 11, 13, 15 days were investigated. Identification of actinomycetes isolate SR3.97 was carried out based on 16S rRNA sequence analysis. Results: Actinomycetes isolate SR3.97 was selected as the best isolate for producing antioxidant in MYEB medium. The MYEB medium exhibited antioxidant activity as measured by DPPH, ABTS and FRAP analysis with IC50 values of 71.52 ± 0.76 µg/ml, 35.88 ± 0.84 µg/ml and 156.91 ± 1.34 µM Fe(II)/mg, respectively and total phenolic content of 57.82 ± 0.1 µg GEA/mg. The optimal culture conditions for antioxidant production were initial culture pH at 7, incubation temperature of 37ºC, and incubation time for 7 days. Actinomycetes isolate SR3.97 was identified based on 16S rRNA gene sequence analysis. The isolate was 99.40% resembled with Streptomyces chrysomallus subsp. fumigatus NBRC 15393T. Conclusion: Difference antioxidant levels were produced from actinomycetes isolate SR3.97 when different media were used. The temperature, pH and incubation time affected antioxidant production. Keywords: Actinomycetes, Antioxidant, Phenolic compound, Streptomyces chrysomallu

Whole-genome analyses reveal genetic instability of Acetobacter pasteurianus

Acetobacter species have been used for brewing traditional vinegar and are known to have genetic instability. To clarify the mutability, Acetobacter pasteurianus NBRC 3283, which forms a multi-phenotype cell complex, was subjected to genome DNA sequencing. The genome analysis revealed that there are more than 280 transposons and five genes with hyper-mutable tandem repeats as common features in the genome consisting of a 2.9-Mb chromosome and six plasmids. There were three single nucleotide mutations and five transposon insertions in 32 isolates from the cell complex. The A. pasteurianus hyper-mutability was applied for breeding a temperature-resistant strain grown at an unviable high-temperature (42°C). The genomic DNA sequence of a heritable mutant showing temperature resistance was analyzed by mutation mapping, illustrating that a 92-kb deletion and three single nucleotide mutations occurred in the genome during the adaptation. Alpha-proteobacteria including A. pasteurianus consists of many intracellular symbionts and parasites, and their genomes show increased evolution rates and intensive genome reduction. However, A. pasteurianus is assumed to be a free-living bacterium, it may have the potentiality to evolve to fit in natural niches of seasonal fruits and flowers with other organisms, such as yeasts and lactic acid bacteria

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

Screening of Thermotolerant Gluconobacter Strains for Production of 5-Keto-d-Gluconic Acid and Disruption of Flavin Adenine Dinucleotide-Containing d-Gluconate Dehydrogenase▿

We isolated thermotolerant Gluconobacter strains that are able to produce 5-keto-d-gluconic acid (5KGA) at 37°C, a temperature at which regular mesophilic 5KGA-producing strains showed much less growth and 5KGA production. The thermotolerant strains produced 2KGA as the major product at both 30 and 37°C. The amount of ketogluconates produced at 37°C was slightly less than the amount produced at 30°C. To improve the yield of 5KGA in these strains, we disrupted flavin adenine dinucleotide-gluconate dehydrogenase (FAD-GADH), which is responsible for 2KGA production. Genes for FAD-GADH were cloned by using inverse PCR and an in vitro cloning strategy. The sequences obtained for three thermotolerant strains were identical and showed high levels of identity to the FAD-GADH sequence reported for the genome of Gluconobacter oxydans 621 H. A kanamycin resistance gene cassette was used to disrupt the FAD-GADH genes in the thermotolerant strains. The mutant strains produced 5KGA exclusively, and the final yields were over 90% at 30°C and 50% at 37°C. We found that the activity of pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase, which is responsible for 5KGA production, increased in response to addition of PQQ and CaCl2 in vitro when cells were grown at 37°C. Addition of 5 mM CaCl2 to the culture medium of the mutant strains increased 5KGA production to the point where over 90% of the initial substrate was converted. The thermotolerant Gluconobacter strains that we isolated in this study provide a promising new option for industrial 5KGA production