Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 68 , Issue 10
Showing 1-30 articles out of 30 articles from the selected issue
Review
  • Kunio YAMANE, Keigo BUNAI, Hiroshi KAKESHITA
    2004 Volume 68 Issue 10 Pages 2007-2023
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    Gram-positive sporulating Bacillus subtilis secretes high levels of protein. Its complete genome sequence, published in 1997, encodes 4,106 proteins. Bioinformatic searches have predicted that about half of all B. subtilis proteins are related to the cell membrane through export to the extracellular medium, insertion, and attachment. Key features of the B. subtilis protein secretion machinery are the absence of an Escherichia coli SecB homolog and the presence of an SRP (signal recognition particle) that is structurally rather similar to human SRP. In addition, B. subtilis contains five type I signal peptidases (SipS, T, U, V, and W). Our in vitro assay system indicated that co-operation between the SRP–protein targeting system to the cell membrane and the Sec protein translocation machinery across the cytoplasmic membrane constitutes the major protein secretion pathway in B. subtilis. Furthermore, the function of the SRP–Sec pathway in protein localization to the cell membrane and spore was analyzed.
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Organic Chemistry Regular Papers
  • Hideki KANHO, Sayaka YAOYA, Tomio ITANI, Takahisa NAKANE, Nobuo KAWAHA ...
    2004 Volume 68 Issue 10 Pages 2032-2039
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    Hairy roots of medicinal morning glory (Pharbitis nil) showed potent glucosylation activity against umbelliferone and aesculetin, so the glucosylation activity against several phenolic compounds was tested. Some coumarin derivatives and flavone derivatives having phenolic hydroxyl groups were incubated with the hairy roots. The coumarin derivatives and flavone derivatives almost disappeared from the culture medium in half a day. In the case of the coumarin derivatives, a 7-hydroxyl group was easily glucosylated. A methyl group at C-8 somewhat decreased the glucosylation to a hydroxyl group at C-7 of the coumarin skeleton. The 4-hydroxy coumarin derivatives were changed to acetophenone-type glucosides by incubation with the hairy roots through decarboxylation.
    Several flavonol derivatives were tested for glucosylation by the hairy roots. 3-Hydroxy flavone, 3.6-dihydroxyflavone and 3,7-dihydroxyflavone were glucosylated to give 3-glucosylated derivatives. Of these, 3,6-dihydroxyflavone was highly glucosylated, but not 3-hydroxyflavone or 3,7-dihydroxyflavone to the same degree. In the case of the flavones, a 3-hydroxy group could be predominantly glucosylated, and hydroxyl groups on the A and B ring of the flavones affected glucosylation by the hairy roots.
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  • Trevor M. KITSON
    2004 Volume 68 Issue 10 Pages 2165-2170
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    This work investigates the binding of the bioflavonoid, quercetin, to bovine serum albumin (BSA) by spectrophotometric techniques involving both the conventional and stopped-flow methods. Both the neutral and negatively-charged forms of quercetin bound to BSA with a red shift in the maximal absorption. At high pH values, quercetin was rapidly degraded in an oxygen-dependent process, but this decomposition was substantially slower when the flavonoid was bound to BSA. At pH 7.4, the difference spectrum of quercetin with and without BSA was maximal at 425 nm; this wavelength can be conveniently used to monitor the extent and speed of binding. Spectrophotometric studies with a range of equimolar mixtures of quercetin and BSA at pH 7.4 suggest the binding was maximal when the concentration was 10 μM. It is postulated that the binding site of BSA for quercetin was less available at higher protein concentrations, perhaps because of conformational change or self-association. The rate of spectrophotometric change when quercetin bound to BSA was fairly slow; the process was not quite complete within 45 seconds and was biphasic. When a pre-mixed equimolar mixture of BSA and quercetin was diluted with an equal volume of the buffer, there was a surprising further increase in absorbance at 425 nm (rather than the fall anticipated if the binary complex were to dissociate). It is concluded that, upon dilution, the effective concentration of BSA’s binding site increased, providing more scope for quercetin to bind.
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  • Hajime MOTOYOSHI, Masato HORIGOME, Ken ISHIGAMI, Tatsuhiko YOSHIDA, Su ...
    2004 Volume 68 Issue 10 Pages 2178-2182
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    The structure-activity relationship for FR901464, a potent cell-cycle inhibitor, was examined by synthesizing its analogs. A versatile method for converting FR901464 was devised. This method made it possible to synthesize biologically active FR901464-biotin conjugates which could be used to isolate the binding proteins.
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Organic Chemistry Notes
Organic Chemistry Communication
Biochemistry & Molecular Biology Regular Papers
  • Hajime YOSHISUE, Kazuhide HASEGAWA
    2004 Volume 68 Issue 10 Pages 2024-2031
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    While epidermal growth factor receptor (EGFR) plays a pivotal role in the repair process of epithelial cells, it is also involved in the overproduction of mucus and goblet cell hyperplasia (GCH), which occurs in chronic airway diseases such as asthma. Among the EGFR ligands, transforming growth factor (TGF)-α is thought to be the most important in the synthesis of mucus. Pro-TGF-α is cleaved to give an active form by members of the matrix metalloproteinases (MMP)/a disintegrin and metalloproteinases (ADAM) family. Thus MMP/ADAM inhibitors might prevent GCH by inhibiting transactivation of EGFR. Upon stimulation of differentiating normal human bronchial epithelial (NHBE) cells by IL-13, GCH was induced. The mucin genes MUC5AC, MUC5B, and MUC2 were upregulated whereas the expression of ciliated cell markers was greatly repressed. GM6001, a broad-spectrum inhibitor for MMP/ADAM, inhibited IL-13-induced mucin gene expression and mucus production as measured by periodic acid-Schiff staining. This was accompanied by an inhibition of TGF-α release. These results suggest that MMP/ADAMs play a pivotal role in the development of GCH in lung epithelial cells.
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  • Masahiko KAYA, Kengo MATSUMURA, Katsuya HIGASHIDA, Yoji HATA, Akitsugu ...
    2004 Volume 68 Issue 10 Pages 2040-2049
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    We cloned and characterized the gene and cDNA of Aspergillus oryzae cytochrome P450nor (Anor). The Anor gene (nicA; CYP55A5) has a different gene structure from other P450nor genes in that it has an extra intron. There were not only two kinds of mRNA but also two sets of TATA-box and CCAAT-box, and it appears that this gene has two expression patterns, like CYP55A1 of Fusarium oxysporum. A reporter analysis using the uidA gene indicated that gene expression of CYP55A5 was induced under anaerobic conditions, like CYP55A1. When the CYP55A5 gene was overexpressed in A. oryzae, a large amount of active Anor were accumulated as intracellular protein. Anor employed both NADH and NADPH as electron donors for reducing nitric oxide to nitrous oxide. Anor measured the amount of NO generated from 3-(2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)-1-propanamine (NOC5) with a spectrophotometer. The sensitivity was 10 nmol/ml.
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  • Masahiko NAGAKI, Yohei MIKI, Minori NAKADA, Jun KAWAKAMI, Haruo KITAHA ...
    2004 Volume 68 Issue 10 Pages 2070-2075
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    In order to develop synthetic methods for biologically active homoallylic terpene sulfates, we examined the applicability and substrate specificities of several prenyl chain elongating enzymes with respect to 4-methyl-4-pentenyl diphosphate (homoIPP).
    The reaction of dimethylallyl diphosphate with homoIPP by use of Bacillus stearothermophilus (all-trans)-farnesyl diphosphate synthase resulted in efficient yields of cis-(yield: 45.9%) and trans-4,8-dimethylnona-3,7-dien-1-ol (homoGOH, 25.5%), which has a carbon skeleton of 4,8-dimethylnona-3-en-1-sulfate, an antiproliferative compound from a marine organism (Aiello, A. et al., Tetrahedron, 53, 11489–11492 (1997)). The homoIPP was found to be also active as a homoallylic substrate in place of isopentenyl diphosphate for Sulfolobus acidocaldarius geranylgeranyl diphosphate synthase to give diphosphate of cis- and trans-4,8,12-trimethyltrideca-3,7,11-trien-1-ol, for Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase to give cis- and trans-4,8,12,16-tetramethylheptadeca-3,7,11,15-tetraen-1-ol (homoGGOH), and for Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase to give cis-homoGGOH exclusively.
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  • Mariko MIYASATO, Katsumi AJISAKA
    2004 Volume 68 Issue 10 Pages 2086-2090
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    The regioselectivity of β-galactosidase derived from Bacillus circulans ATCC 31382 (β-1,3-galactosidase) in transgalactosylation reactions using D-mannose as an acceptor was investigated. This D-mannose associated regioselectivity was found to be different from reactions using either GlcNAc or GalNAc as acceptors, not only for β-1,3-galactosidase but also for β-galactosidases of different origins. The relative hydrolysis rate of Galβ-pNP and D-galactosyl-D-mannoses, of various linkages, was also measured in the presence of β-1,3-galactosidase and was found to correlate well with the ratio of disaccharides formed by transglycosylation. The unexpected regioselectivity using D-mannose can therefore be explained by an anomalous specificity in the hydrolysis reaction. By utilizing the identified characteristics of both regioselectivity and hydrolysis specificity using D-mannose, an efficient method for enzymatic synthesis of β-1,3-, β-1,4- and β-1,6-linked D-galactosyl-D-mannose was subsequently established.
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  • Balan VENKATESH, Mohammad ARIFUZZAMAN, Hirotada MORI, Takahisa TAGUCHI ...
    2004 Volume 68 Issue 10 Pages 2096-2103
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    The folding of many proteins including luciferase in vivo requires the assistance of molecular chaperone proteins. To understand how a chaperone targets luciferase, we took three luciferases that give different bioluminescence with the same luciferin substrate and with differences in homology. The three luciferase genes, firefly luciferase (FF-Luc) (from Pyrocoelia miyako), and red (RE-Luc) and green (GR-Luc) bioluminescence-emitting luciferases (from Phrixothrix railroad-worms), were expressed in Escherichia coli to produce fusion proteins with predicted molecular masses. Subsequently, we observed that DnaK and GroEL were co-purified along with recombinant luciferase. Although the amount of co-purified DnaK was almost the same compared to FF-Luc, GroEL was 25 and 32 times higher in GR-Luc and RE-Luc respectively. Furthermore, co-expression of GroEL/GroES along with luciferase substantially refolded RE-Luc and GR-Luc compared to FF-Luc.
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  • Joel CORNISTA, Satoshi IKEUCHI, Mitsuru HARUKI, Atsuko KOHARA, Kazufum ...
    2004 Volume 68 Issue 10 Pages 2128-2137
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    Pitrilysin from Escherichia coli was overproduced, purified, and analyzed for enzymatic activity using 14 peptides as a substrate. Pitrilysin cleaved all the peptides, except for two of the smallest, at a limited number of sites, but showed little amino acid specificity. It cleaved β-endorphin (β-EP) most effectively, with a Km value of 0.36 μM and a kcat value of 750 min−1. β-EP consists of 31 residues and was predominantly cleaved by the enzyme at Lys19–Asn20. Kinetic analyses using a series of β-EP derivatives with N and/or C-terminal truncations and with amino acid substitutions revealed that three hydrophobic residues (Leu14, Val15, and Leu17) and the region 22–26 in β-EP are responsible for high-affinity recognition by the enzyme. These two regions are located on the N- and C-terminal sides of the cleavage site in β-EP, suggesting that the substrate binding pocket of pitrilysin spans its catalytic site.
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  • Hyongi CHON, Rikita NAKANO, Naoto OHTANI, Mitsuru HARUKI, Kazufumi TAK ...
    2004 Volume 68 Issue 10 Pages 2138-2147
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    The gene encoding RNase HIII from the thermophilic bacterium Bacillus stearothermophilus was cloned and overexpressed in Escherichia coli, and the recombinant protein (Bst–RNase HIII) was purified and biochemically characterized. Bst–RNase HIII is a monomeric protein with 310 amino acid residues, and shows an amino acid sequence identity of 47.1% with B. subtilis RNase HIII (Bsu–RNase HIII). The enzymatic properties of Bst–RNase HIII, such as pH optimum, metal ion requirement, and cleavage mode of the substrates, were similar to those of Bsu–RNase HIII. However, Bst–RNase HIII was more stable than Bsu–RNase HIII, and the temperature (T1⁄2) at which the enzyme loses half of its activity upon incubation for 10 min was 55 °C for Bst–RNase HIII and 35 °C for Bsu–RNase HIII. The optimum temperature for Bst–RNase HIII activity was also shifted upward by roughly 20 °C as compared to that of Bsu–RNase HIII. The availability of such a thermostable enzyme will facilitate structural studies of RNase HIII.
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  • Pingbo ZHANG, Kohji YAMAMOTO, Yongqiang WANG, Yutaka BANNO, Hiroshi FU ...
    2004 Volume 68 Issue 10 Pages 2148-2154
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    We compared the use of wet and dry two-dimensional electrophoresis (2-DE) gels for in-gel tryptic digestion and subsequent analysis by mass spectrometry, first using bovine serum albumin (BSA) as a model protein and then using unknown proteins from an extract of the silkworm midgut. The gel was either dried at 80 °C or left wet. Upon analysis of BSA, there was little difference in peptide recovery from 2-DE or in mass spectrum between the dry and the wet gels. The midgut extract was resolved into more than 1,100 protein spots by 2-DE, and 40 of these spots were sampled for further analysis. For all of the 40 proteins, the results obtained from dry and wet gels were quite similar in mass spectra and protein identification, although the relative amounts of peptides from tryptic digestion ranged from 45 to 146%. Based on these results, we confirmed the utility of dry electrophoretic gels for proteomics of insect extracts.
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Biochemistry & Molecular Biology Notes
Biochemistry & Molecular Biology Communication
Food & Nutrition Science Regular Papers
  • Aydan HATIPOGLU, Öznur KANBAGLI, Jale BALKAN, Mutlu KÜ&Ccedi ...
    2004 Volume 68 Issue 10 Pages 2050-2057
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    Hazelnut oil (HO) is rich in monounsaturated fatty acids and antioxidants. We wanted to investigate the effect of HO on lipid levels and prooxidant–antioxidant status in rabbits fed a high-cholesterol (HC) diet. An HC diet caused significant increases in lipids and lipid peroxide levels in the plasma, liver, and aorta together with histopathological atherosclerotic changes in the aorta. Glutathione levels, glutathione peroxidase, and glutathione transferase activities decreased significantly, but superoxide dismutase activity and vitamin E and C levels remained unchanged in the livers of rabbits following HC diet. HO supplementation reduced plasma, liver, and aorta lipid peroxide levels and aorta cholesterol levels together with amelioration in atherosclerotic lesions in the aortas of rabbits fed an HC diet, without any decreasing effect on cholesterol levels in the plasma or liver. HO did not alter the antioxidant system in the liver in the HC group. Our findings indicate that HO reduced oxidative stress and cholesterol accumulation in the aortas of rabbits fed an HC diet.
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  • Takumi YOSHIZAWA, Hiroaki KOHNO, Kazuyuki IKEDA, Tatsuya SHINODA, Hiro ...
    2004 Volume 68 Issue 10 Pages 2076-2085
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    We have developed and tested an enzyme-linked immunosorbent assay system for individual measurement of deoxynivalenol, nivalenol, and T-2 + HT-2 toxin using monoclonal antibodies for 3,4,15-triacetyl-nivalenol, for both 3,4,15-triacetyl-nivalenol and 3,15-diacetyl-deoxynivalenol, and for acetyl-T-2 toxin. The assay system comprised three kits (desinated the DON + NIV kit, the NIV kit, and the T-2 + HT-2 kit). The practical performance of the enzyme-linked immunosorbent assay system was assessed by assaying trichothecene mycotoxins in wheat kernels. The enzyme-linked immunosorbent assay system meets all the requirements for use in a routine assay in terms of sensitivity (detection limit: deoxynivalenol 80 ng/g, nivalenol 80 ng/g, T-2 toxin 30 ng/g), reproducibility (total coefficient of variation: 1.9–6.2%), accuracy (recovery: 93.8–112.0%), simplicity and rapidity (time required: <2 h), mass handling (>42 samples/assay), and a good correlation with gas chromatography–mass spectrometry (r=0.9146–0.9991). Components derived from the wheat extract did not interfere with the assay kits. The enzyme-linked immunosorbent assay system is a useful alternative method to gas chromatography–mass spectrometry, liquid chromatography–mass spectrometry, or liquid chromatography–ultraviolet absorption for screening cereals and foods for trichothecene mycotoxin contamination.
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  • Takeshi KAWAHARA, Daisuke KATAYAMA, Hajime OTANI
    2004 Volume 68 Issue 10 Pages 2091-2095
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    The effect of bovine β-casein (1-28) purified from commercial casein phosphopeptide preparations on human T, B, and monocyte cell lines was evaluated. Beta-casein (1-28) enhanced the proliferation of the following: T cell lines HUT-78, Jurkat Clone E6-1, and MOLT-4; B cell lines BALL, KHM-1B, and U266B1; and monocyte cell lines U937 and HL-60. Moreover, β-casein (1-28) stimulated IgA production by KHM-1B over 96 h of culture. Semiquantitative reverse transcriptional-polymerase chain reaction analysis indicated that β-casein (1-28) enhanced mRNA expression of interleukin (IL)-6 in U266B1 and KHM-1B. These results suggest that β-casein (1-28) exerts a mitogenic effect on human T, B, and monocyte cells, and an IgA-enhancing effect on B cells.
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  • Kaoru KOHYAMA, Tomoko SASAKI, Fumiyo HAYAKAWA, Eiko HATAKEYAMA
    2004 Volume 68 Issue 10 Pages 2104-2110
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    The effects of the cross-sectional area of food samples on bite force with molar teeth were investigated using raw carrots and surimi gels. We evaluated human bite force for food samples with different sizes between the upper and lower molars using a multiple-point sheet sensor and electromyography (EMG). The bite force curve and EMG clearly showed textural characteristics of the carrot and gel. In particular, the first peak in the bite curves corresponded to breaking point in the compression test. With increasing cross-sectional area of both foodstuffs, the bite force and contact area increased and the average stress to which the specimen was subjected (mean stress) tended to decrease, while the stress produced between the teeth and the specimen (active stress) did not change. Chewing rhythm and EMG activities were not greatly influenced by sample size. These findings suggest that higher bite force might cause difficulty in biting food with a larger cross-sectional area.
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  • Tatsuya MORITA, Hiroki TANABE, Kimio SUGIYAMA, Seiichi KASAOKA, Shuhac ...
    2004 Volume 68 Issue 10 Pages 2155-2164
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    The protective effect of a dietary high-amylose cornstarch (HAS) against trinitrobenzene sulfonic acid (TNBS)-induced colitis was examined in rats. Rats were fed a HAS-free basal diet or, a 15% or 30% HAS supplemented diet for 10 d, and then received intracolonic TNBS to induce colitis and fed the respective diets for a further 8 d. HAS ingestion significantly protected colonic injuries as evidenced by lower colonic myeloperoxidase activity. Rats fed the HAS diet showed greater cecal short-chain fatty acid (SCFA) production than those fed the basal diet. Further, just before TNBS administration, HAS ingestion dose-dependently increased fecal and cecal mucin contents, and protein and nucleic acid contents in the colonic mucosa. HAS ingestion also reduced colonic permeability. The protective effect of HAS ingestion on TNBS-induced colitis is perhaps exerted through alterations in colonic mucosa, possibly due to cecal SCFA production.
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Food & Nutrition Science Note
Microbiology & Fermentation Technology Regular Papers
  • Hiroya YURIMOTO, Maiko YAMANE, Yoshimi KIKUCHI, Hiroshi MATSUI, Nobuo ...
    2004 Volume 68 Issue 10 Pages 2058-2069
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    Transglutaminase (TGase) from the actinomycete Streptomyces mobaraensis is a useful enzyme in the food industry, and development of an efficient production system for it would be desirable. Herein we report secretion of TGase in an enzymatically active form by methylotrophic yeasts as expression hosts. Secretory production of active TGase required a pro-peptide from TGase. When an artificial Kex2-endopeptidase recognition site was placed between the pro-peptide and mature TGase, secretion and in vitro maturation of TGase depended on Kex2-dependent cleavage. Unexpectedly, coexpression of unlinked pro-peptide with mature TGase yielded efficient secretion of the active enzyme. These results indicate that the pro-peptide from TGase functions not only in an intramolecular but also in an intermolecular manner. Site-directed mutagenesis of putative N-glycosylation sites increased the productivity of the active TGase further. A recombinant Candida boidinii strain was found to secrete active TGase up to 1.83 U/ml (about 90 mg/l) after 119 h of cultivation.
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  • Rika NOBE, Yoichi SAKAKIBARA, Kihachiro OGAWA, Masahito SUIKO
    2004 Volume 68 Issue 10 Pages 2111-2119
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    The gene lamAI, which encodes a novel laminarinase AI of Trichoderma viride U-1, was cloned using RT-PCR in conjunction with the rapid amplification of cDNA ends (RACE) technique. The open reading frame consisted of 2,277 bp encoding a protein of 759 amino acid residues, including a 32-residue signal prepropeptide. The protein showed 91% sequence similarity to the putative Trichoderma virens β-1,3-glucanase BGN1, but no significant similarity to fungal β-1,6-glucanases or β-1,3-glucanases from other organisms. On 40 h incubation with a solo carbon source, northern analysis revealed that the gene was induced by 0.5% laminaran from Eisenia bicyclis but was not by the same concentration of glucose. The lamAI cDNA was functionally expressed in the methylotrophic yeast Pichia pastoris, resulting in a recombinant enzyme with as high activity against laminaran as native LAMAI. Based on these data, the probable existence of endo-β-1,3:1,6-glucan hydrolases as a subclass of endo-β-1,3-glucanases in some mycoparasitic fungi is suggested.
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  • Kohji ISHIHARA, Hitomi YAMAGUCHI, Taketo OMORI, Tadashi UEMURA, Nobuyo ...
    2004 Volume 68 Issue 10 Pages 2120-2127
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    We have achieved the purification of an α-keto ester reductase (SCKER) from S. coelicolor A3(2) whole cells. SCKER proved to be a homotetramer of 132 kDa containing one equivalent of zinc ion per subunit. The enzyme differed from other α-keto ester reductases from microorganisms with regard to subunit structure and metal ion dependency. From a computer search using the protein data banks, the N-terminal amino acid sequence of SCKER was consistent with that of a possible zinc containing alcohol dehydrogenase in S. coelicolor A3(2). None of three hypothetical proteins of S. coelocor A3(2) having a high homology sequence with those of already purified α-keto ester reductases from S. thermocyaneoviolaceus [Yamaguchi, H., et al., Biosci. Biotechnol. Biochem., 66, 588–597 (2002)] was identical with that of SCKER.
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  • Tadashi FUJII, Tatsuya NARIKAWA, Koji TAKEDA, Junichi KATO
    2004 Volume 68 Issue 10 Pages 2171-2177
    Published: 2004
    Released: October 23, 2004
    JOURNALS FREE ACCESS
    Biotransformation using alkane-oxidizing bacteria or their alkane hydroxylase (AH) systems have been little studied at the molecular level. We have cloned and sequenced genes from Gordonia sp. TF6 encoding an AH system, alkB2 (alkane 1-monooxygenase), rubA3 (rubredoxin), rubA4 (rubredoxin), and rubB (rubredoxin reductase). When expressed in Escherichia coli, these genes allowed the construction of biotransformation systems for various alkanes. Normal alkanes with 5 to 13 carbons were good substrates for this biotransformation, and oxidized to their corresponding 1-alkanols. Surprisingly, cycloalkanes with 5 to 8 carbons were oxidized to their corresponding cycloalkanols as well. This is the first study to achieve biotransformation of alkanes using the E. coli expressing the minimum component genes of the AH system. Our biotransformation system has facilitated assays and analysis leading to improvement of AH systems, and has indicated a cycloalkane oxidation pathway in microorganisms for the first time.
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