• Molecules and Cells
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• v.23 no.3
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• pp.370-378
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• 2007

A superoxide dismutase was purified 62-fold in seven steps to homogeneity from Methylobacillus sp. strain SK1, an obligate methanol-oxidizing bacterium, with a yield of 9.6%. The final specific activity was 4,831 units per milligram protein as determined by an assay based on a 50% decrease in the rate of cytochrome c reduction. The molecular weight of the native enzyme was estimated to be 44,000. Sodium dodecyl sulfate gel electrophoresis revealed two identical subunits of molecular weight 23,100. The isoelectric point of the purified enzyme was found to be 4.4. Maximum activity of the enzyme was measured at pH 8. The enzyme was stable at pH range from 6 to 8 and at high temperature. The enzyme showed an absorption peak at 280 nm with a shoulder at 292 nm. Hydrogen peroxide and sodium azide, but not sodium cyanide, was found to inhibit the purified enzyme. The enzyme activity in cell-free extracts prepared from cells grown in manganese-rich medium, however, was not inhibited by hydrogen peroxide but inhibited by sodium azide. The activity in cell extracts from cells grown in iron-rich medium was found to be highly sensitive to hydrogen peroxide and sodium azide. One mol of native enzyme was found to contain 1.1 g-atom of iron and 0.7 g-atom of manganese. The N-terminal amino acid sequence of the purified enzyme was Ala-Tyr-Thr-Leu-Pro-Pro-Leu-Asn-Tyr-Ala-Tyr. The superoxide dismutase of Methylobacillus sp. strain SK1 was found to have antigenic sites identical to those of Methylobacillus glycogenes enzyme. The enzyme, however, shared no antigenic sites with Mycobacterium sp. strain JC1, Methylovorus sp. strain SS1, Methylobacterium sp. strain SY1, and Methylosinus trichosproium enzymes.

• Journal of Microbiology and Biotechnology
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• v.12 no.5
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• pp.819-825
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• 2002

Methanol dehydrogenase (MDH) is a key enzyme in the process of methanol oxidation in methylotrophic bacteria. However, information on MDH genes from genus Methylobacillus is limited. In this study, a 6.5-kb HindIII DNA fragment of Methylobacillus sp. SK-5 chromosomal DNA was isolated from the genomic library of the strain by using a degenerate oligonucleotide probe that was designed based on JV-terminal amino acid sequence of the MDH $\alpha$ subunit purified from the strain. Molecular analysis of the fragment revealed four tightly clustered genes (mxaFJGI) involved in the methanol oxidation. The first and fourth genes were very similar to mxaF (77% identity for nucleotides an 78% identity for amino acids) and mxaF (67% Identity for nucleotides and 68% Identity for amino acids) genes, respectively, from Methylovorus sp. SSI. Genes mxaF and mxaI encode $\alpha$ and $\beta$ subunits of MDH, respectively. The two subunits were identified from purified MDH from Methylobacillus sp. SK-5. A dendrogram constructed by comparison of amino acid sequences of MDH u subunits suggests that MxaF from Methylobacillus sp. SK-5 belongs to a subfamily cluster of MDH u subunits from $\beta$-subgroup Proteobacteria. The subfamily cluster is separated from the other subfamily that consists of $\beta$- and $\gamma$-subgroup Proteobacteria. This study provided information on mn genes from a methylotrophic bacterium in $\beta$-subgroup Proteobacteria, which would aid to better develop a gene probe to detect one-carbon metabolizing bacteria.

• KSBB Journal
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• v.5 no.3
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• pp.269-277
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• 1990

Methylobacillus sp. SK1, an obligate methylotroph, was cultivated in a fed-batch culture using DO as a methanol feeding indicator with a micro computer-aided control system. While 2.07g/l of cell density was obtained after 13 hr in the batch culture (initial methanol concentration: 1.0%(v/v)),45.3g/l of cell density was obtained after 17 hr by feeding methanol and metal ions in the fed-batch culture with oxygen supply. The high-density biomass was obtained in short cultuivation time by fed-batch culture with feedback control, and consequently the biomass productivity was significantly increased. It was mainly due to extension of logarithmic growth period by methanol feeding without methanol inhibition and intensive aeration without DO limitation with microcomputer-aided control system.

• Korean Journal of Microbiology
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• v.29 no.6
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• pp.380-386
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• 1991

Three types of soluble cytochrome c were purified to homogeneity from Methylobacillus sp. strain SK1 which grows only on methanol. Cytochrome c-I was purified 58.5-fold in seven steps. Cytochrome c-II and c-III were purified 57.3- and 122.1-fold in eight steps, respectively. The molecular weights of the cytochrome c-I was determined to be 12,500, while those of the cytochrome c-II and c-III were 16,000. The isoelectric points of the c-I, c-II and c-III were found to be 8.8, 6.6, and 6.6 respectively. The spectrum of reduced cytochrome c-I showed .alpha.-, .betha.-, .gamma.-peaks at 551.4, 522.2, and 416.6nm. The peaks for c-II were found at 551.0, 521.6, and 416.5nm, while those for c-III were shown at 551.2, 521.8, and 416.0 nm. The spectra of oxidized cytochrome c-I, c-II, and c-III showed .gamma.-peak at 411.8, 409.0, and 410.2 nm, respectively. The absorption coefficients of .alpha.- and .gamma.-peak for c-I in the reduced state were determined as 47 and 197 $mM^{-1}$ $cm^{-1}$ , respectively. The coefficients of .alpha.- and .gamma.-peak for c-II were determined to be 43 and 137 $mM^{-1}$ $cm^{-1}$ , while those for c-III were 41 and 172 $mM^{-1}$ $cm^{-1}$ , respectively. The c-I and c-III were found to bind carbon monoxide.

• Proceedings of the Zoological Society Korea Conference
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• 1994.10a
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• pp.225.1-225
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• 1994

• Proceedings of the Zoological Society Korea Conference
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• 1997.10b
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• pp.252.1-252
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• 1997

• Proceedings of the Zoological Society Korea Conference
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• 1997.10b
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• pp.253.1-253
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• 1997

• Korean Journal of Microbiology
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• v.29 no.4
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• pp.250-257
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• 1991

An obligate methanol-oxidizing bacterium, Methylobacillus sp. strain SK1, which grows only on methanol was isolated from soil. The isolate was nonmotile Gram-negtive rod. It does not have internal membrane system. The colonies were small, whitish-yellow, and smooth. The guanine plus cytosine content of the DNA was 48 mol%. Cellular fatty acids consisted predominantly of large amounts of straight-chain saturated $C_{16:0}$ acid and unsaturated $C_{16:1}$ acid. The major ubiquinone was Q-8, and Q-10 was present as minor component. The cell was obligately aerobic and exhibited catalase, but no oxidase, activity. Poly-.betha.-hydroxybutyrate, endospores, or cysts were not observed. the isolate could grow only on methanol in mineral medium. Growth factors were not required. The isolate was unable to use methane, formaldehyde, formate, methylamine, and several other organic compounds tested as a sole source of carbon and energy. Growth was optimal at 35.deg.C and pH 7.5. It could not grow at 42.deg.C. The doubling time was 1.2h at 30.deg.C when grown with 1.0%(v/v) methanol. The growth was not affected by antibiotics inhibiting cell wall synthesis and carbon monoxide but was completely suppressed by those inhibiting protein synthesis. Methanol was found to be assimilated through the ribulose monophosphate pathway. Cytochromes of b-, c-, and o- types were found. Cell-free extracts contained a phenazine methosulfate-linked methanol dehydrogenase activity, which required ammonium ions as an activator. Cells harvested after the late exponential phase seemed to contain blue protein.ein.

• Journal of Microbiology
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• v.34 no.2
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• pp.172-178
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• 1996

Methylobacillus sp. strain SK1, which grows only on methanol, was found to grow in the absence of added copper. The doubling time (t$_{d}$ = 1.3 h) of the bacterium growing at the exponential growth phase at 30.deg.C in the absence of copper was the same as that of the cell growing in the presence of copper. The bacterium growing after the exponential phase in the absence of copper, however, grew faster than the cell growing in the presence of copper. Cells harvested after thee arly stationary phase in the presence of copper were found to exhibit no methanol dehydrogenase (MDH) activity, but the amount and subunit structure of the enzyme in the cells were almost the same as that in cells harboring active MDH. Pellets of the cells harvested after the early stationary phase in the presence of copper were pale green. Cell-free extracts prepared from cells harvested at the early stationary phase in the presence of copper were pink and exhibited MDH activity, but it turned dark-green rapidly from the surface under air. The green-colored portions of the extracts showed no MDH activity and contained c-type cytochromes that were oxidized completely. The inactive MDH activity and contained c-type cytochromes that were oxidized completely. The inactive MDH proteins in the green portions were found to have antigenic sites identical to those of the active one as the inactive MDHs in cells grown in the presence of copper. The bacterium was found to accumulate copper actively during the exponential growth phase. MDH prepared from cells grown in the presence or absence of copper was found to be more stable under nitrogen gas than under air. Methanol at 10 mM was found to enhance the stability of the MDH under air.r.

• Korean Journal of Microbiology
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• v.31 no.4
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• pp.261-266
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• 1993

A new methyltrophic bacterium which utilizes methanol as a sole source of carbon and energy was isolated from soil. It was Gram-negative, nonmotile, nonspore-forming rod, and strictly aerobic bacterium. Catalase and oxidase activities were present. Nitrate was reduced to nitrite. Vitamins and other growth factors were not required. Generation time was 1.6 hr under the optimal condition. The isolate assimilated methanol via the ribulose mono-phosphate pathway (Enter-Doudoroff varient) and did not have .alpha.-ketoglutarate dehydrogenase. It assimilated ammonia through glutamate dehydrogenase. The guanine plus cytosine content of the DNA was 61.0 mol%. The celular fatty acid composition was primarily straight-chain saturated $C^{16 : 0}$ acids (palmitic acids) and unsaturated $C_{16 :1}$ acid (palmitoleic acids), and the isolate also contained two unidentified $C_{17}$ branched fatty acids. The major ubiquinone was Q-8, and Q-6 and Q-7 were present as minor components. Phosphatidylethanolamine and phosphatidylglycerol were predominantly present, and diphosphatidyglycerol was also detected. Based on the physiological and biochemical properties, the isolate was assigned to a novel species of the genus Methylobacillus, Methylobacillus methanolovorus sp. nov.