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Kentaro Isotani Junji Kurokawa Fumiko Suzuki Syunsuke Nomoto Takashi Negishi Michiko Matsuda Nobuya Itoh 《Applied and environmental microbiology》2013,79(4):1378-1384
We used the resting-cell reaction to screen approximately 200 microorganisms for biocatalysts which reduce 3-quinuclidinone to optically pure (R)-(−)-3-quinuclidinol. Microbacterium luteolum JCM 9174 was selected as the most suitable organism. The genes encoding the protein products that reduced 3-quinuclidinone were isolated from M. luteolum JCM 9174. The bacC gene, which consists of 768 nucleotides corresponding to 255 amino acid residues and is a constituent of the bacilysin synthetic gene cluster, was amplified by PCR based on homology to known genes. The qnr gene consisted of 759 nucleotides corresponding to 252 amino acid residues. Both enzymes belong to the short-chain alcohol dehydrogenase/reductase (SDR) family. The genes were expressed in Escherichia coli as proteins which were His tagged at the N terminus, and the recombinant enzymes were purified and characterized. Both enzymes showed narrow substrate specificity and high stereoselectivity for the reduction of 3-quinuclidinone to (R)-(−)-3-quinuclidinol. 相似文献
3.
Esmeralda Parra-Peralbo Manuel Pineda Miguel Aguilar 《Molecular biology reports》2009,36(8):2249-2258
A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris). A 2.4 kb cDNA clone of this gene (PVAS3) encodes a protein of 570 amino acids with a predicted molecular mass of 64,678 Da, an isoelectric point of 6.45, and a net
charge of −5.9 at pH 7.0. The PVAS3 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent
AS, and PVAS3 complemented an E. coli asparagine auxotroph, that demonstrates that it encodes a glutamine-dependent AS. PVAS3 displayed significant similarity
to other AS. It showed the highest similarity to soybean SAS3 (92.9% identity), rice AS (73.7% identity), Arabidopsis ASN2 (73.2%) and sunflower HAS2 (72.9%). A phylogenetic analysis revealed that PVAS3 belongs to class-II asparagine synthetases. Expression analysis by real-time RT-PCR revealed that PVAS3 is expressed ubiquitously and is not repressed by light. 相似文献
4.
Summary. The paper describes two methods of the synthesis of ethyl (3R,4S)- and (3S,4S)-4-[(benzyloxycarbonyl)amino]-5-[(tert-butyloxycarbonyl)amino]-3-hydroxypentanoates, useful for the syntheses of edeine analogs. Differently N-protected (S)-2,3-diaminopropanoic acid was used as a substrate in both procedures. The absolute configuration of newly generated asymmetric
carbon atoms C-3 in β-hydroxy-γ,δ-diamino products was assigned by means of 1H NMR spectroscopy after their transformation into corresponding piperidin-2-ones.
Received May 24, 2002 Accepted October 10, 2002 Published online December 18, 2002
Acknowledgment The authors are indebted to the Faculty of Chemistry, Technical University of Gdańsk for financial support.
Authors' address: Zbigniew Czajgucki, M. Sc., Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Technical University
of Gdańsk, 11/12 Narutowicza St., 80-952 Gdańsk, Poland, Fax +48 58 347 11 44, E-mail: zmczaj@wp.pl 相似文献
5.
A general in vitro cloning system was established for four Helleborus species: H. argutifolius, H. foetidus, H. niger and H. orientalis. The plant material was introduced in vitro from axillary buds. A Murashige and Skoog (MS)—based medium (Murashige and Skoog
1962) was used supplemented with 2% (w/v) sucrose, 2-isopentenyladenine (2-iP) and 6-benzylaminopurine (BA). Multiplication
rates depended on the genotype and varied from 1.3 for H. foetidus till 3.8 for H. niger. The first results showed that the rooting phase could be done ex vitro. Rooting was induced by a drench for one week in
a solution of indole-3-butyric acid (IBA -3 mg l−1) and 1-naphthaleneacetic acid (NAA-1 mg l−1) at 5°C. 相似文献
6.
Tarun Kumar Verinder Wahla Piyush Pandey R. C. Dubey D. K. Maheshwari 《World journal of microbiology & biotechnology》2009,25(2):277-285
Biological control of the cyst forming nematode Heterodera cajani was studied on sesame using plant growth promoting rhizobacteria (PGPR) Pseudomonas aeruginosa LPT3 and LPT5. Based on plant growth promoting attributes, two fluorescent pseudomonads, LPT3 and LPT5 were evaluated for
their efficacy against cyst forming nematode Heterodera cajani that parasitize Sesamum indicum. Pseudomonas aeruginosa LPT5 produced IAA, HCN, chitinase, glucanase and siderophore, and also solubilized inorganic phosphate in vitro. Moreover,
LPT5 resulted in mortality of second stage juveniles of H. cajani, which was 13% higher as compared to P. aeruginosa LPT3. Interestingly, when both strains were inoculated together for the management of H. cajani on Sesamum indicum the population of H. cajani was reduced significantly, in field trial. Approximately 60% reduction in cyst and juveniles population was recorded with
LPT5 coated seeds, while LPT3 resulted in 49% reduction in cyst and juvenile population as compared to control. Plants grown
with seeds bacterized with LPT5 and reduced doses of urea, diammonium phosphate (DAP), muriate of potash (K) and gypsum gave
maximum increase in yield, in comparison to that of plants raised under the influence of recommended or full doses of the
chemical fertilizers. Pseudomonas aeruginosa LPT5 also showed excellent root colonization. 相似文献
7.
Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences.
Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal
plots revealed K
m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N
3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate. 相似文献
8.
The xylitol dehydrogenase-encoding Arxula adeninivorans AXDH gene was isolated and characterized. The gene includes a coding sequence of 1107 bp encoding a putative 368 amino acid protein of 40.3 kDa. The identity of the gene was confirmed by a high degree of homology of the derived amino acid sequence to that of xylitol dehydrogenases from different sources. The gene activity was regulated by carbon source. In media supplemented with xylitol, D-sorbitol and D-xylose induction of the AXDH gene and intracellular accumulation of the encoded xylitol dehydrogenase was observed. This activation pattern was confirmed by analysis of AXDH promoter – GFP gene fusions. The enzyme characteristics were analysed from isolates of native strains as well as from those of recombinant strains expressing the AXDH gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins, a molecular mass of ca. 80 kDa was determined corresponding to a dimeric structure, an optimum pH at 7.5 and a temperature optimum at 35 °C. The enzyme oxidizes polyols like xylitol and D-sorbitol whereas the reduction reaction is preferred when providing D-xylulose, D-ribulose and L-sorbose as substrates. Enzyme activity exclusively depends on NAD+ or NADH as coenzymes. 相似文献
9.
The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMARTTM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the
gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide
of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus
sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases,
the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a
(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular
mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of
0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase
had the highest hydrolytic activity towards peanut oil. 相似文献
10.
The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose
in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO3), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO3 also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed
that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture
of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture
was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose. 相似文献
11.
Iwasaki A Matsumoto K Hasegawa J Yasohara Y 《Applied microbiology and biotechnology》2012,93(4):1563-1573
A novel (R)-amine transaminase, which catalyzed (R)-enantioselective transamination of chiral amine, was purified to homogeneity from Arthrobacter sp. KNK168 (FERM BP-5228). The molecular mass of the enzyme was estimated to be 148 kDa by gel filtration and 37 kDa by sodium
dodecyl sulfate polyacrylamide gel electrophoresis, suggesting a homotetrameric structure. The enzyme catalyzed transamination
between amines and pyruvate stereo-specifically. The reaction on 1-methylbenzylamine was (R)-enantioselective. Pyruvate was the best amino acceptor, but the enzyme showed broad amino acceptor specificity for various
ketone and aldehyde compounds. The apparent K
ms for (R)-1-methylbenzylamine and pyruvate were 2.62 and 2.29 mM, respectively. The cloned gene of the enzyme consists of an open
reading frame (ORF) of 993 bp encoding a protein of 330 amino acids, with a calculated molecular weight of 36,288. The deduced
amino acid sequence was found to be homologous to those of the aminotransferases belonging to fold class IV of pyridoxal-5′-phosphate-dependent
enzymes, such as branched-chain amino acid aminotransferases. 相似文献
12.
13.
Yu YJ Wu SC Chan HH Chen YC Chen ZY Yang MT 《Applied microbiology and biotechnology》2008,81(3):523-532
A novel microbial transglutaminase (TGase) from the cultural filtrate of Streptomyces netropsis BCRC 12429 (Sn) was purified. The specific activity of the purified TGase was 18.2 U/mg protein with an estimated molecular
mass of 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The TGase gene of S. netropsis was cloned and an open reading frame of 1,242 bp encoding a protein of 413 amino acids was identified. The Sn TGase was synthesized
as a precursor protein with a preproregion of 82 amino acid residues. The deduced amino acid sequence of the mature S. netropsis TGase shares 78.9–89.6% identities with TGases from Streptomyces spp. A high level of soluble Sn TGase with its N-terminal propeptide fused with thioredoxin was expressed in E. coli. A simple and efficient process was applied to convert the purified recombinant protein into an active enzyme and showed
activity equivalent to the authentic mature TGase.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
14.
To utilize Pichia pastoris to produce glutathione, an intracellular expression vector harboring two genes (gsh1 and gsh2) from Saccharomyces cerevisiae encoding enzymes involved in glutathione synthesis and regulated by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter
was transformed into P. pastoris GS115. Through Zeocin resistance and expression screening, a transformant that had higher glutathione yield (217 mg/L) in
flask culture than the host strain was obtained. In fed-batch culture process, this recombinant strain displayed high activity
for converting precursor amino acids into glutathione. The glutathione yield and biomass achieved 4.15 g/L and 98.15 g (dry
cell weight, DCW)/L, respectively, after 50 h fermentation combined with addition of three amino acids (15 mmol/L glutamic
acid, 15 mmol/L cysteine, and 15 mmol/L glycine). 相似文献
15.
Chitwadee Phithakrotchanakoon Ratama Daduang Arinthip Thamchaipenet Thidarat Wangkam Toemsak Srikhirin Lily Eurwilaichitr Verawat Champreda 《Applied microbiology and biotechnology》2009,82(1):131-140
A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His6-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase
containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights
of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V
max and K
m of 3.63 ± 0.16 μmol min−1 mg−1 and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that
PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm−2 h−1 for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films
was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique
will be of utility for further study of bioplastic degradation. 相似文献
16.
Nádia Skorupa Parachin Magnus Carlquist Marie F. Gorwa-Grauslund 《Applied microbiology and biotechnology》2009,84(3):487-497
In this study, the production of enantiomerically pure (1R,4S,6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one ((−)-2) through stereoselective bioreduction was used as a model reaction for the comparison of engineered Saccharomyces cerevisiae and engineered Escherichia coli as biocatalysts. For both microorganisms, over-expression of the gene encoding the NADPH-dependent aldo-keto reductase YPR1
resulted in high purity of the keto alcohol (−)-2 (>99% ee, 97–98% de). E. coli had three times higher initial reduction rate but S. cerevisiae continued the reduction reaction for a longer time period, thus reaching a higher conversion of the substrate (95%). S. cerevisiae was also more robust than E. coli, as demonstrated by higher viability during bioreduction. It was also investigated whether the NADPH regeneration rate was
sufficient to supply the over-expressed reductase with NADPH. Five strains of each microorganism with varied carbon flux through
the NADPH regenerating pentose phosphate pathway were genetically constructed and compared. S. cerevisiae required an increased NADPH regeneration rate to supply YPR1 with co-enzyme while the native NADPH regeneration rate was
sufficient for E. coli.
Nádia Skorupa Parachin and Magnus Carlquist have contributed equally to the paper. 相似文献
17.
An efficient succinic acid production process in a metabolically engineered <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> strain 总被引:1,自引:0,他引:1
Okino S Noburyu R Suda M Jojima T Inui M Yukawa H 《Applied microbiology and biotechnology》2008,81(3):459-464
A Corynebacterium glutamicum strain (ΔldhA-pCRA717) that overexpresses the pyc gene encoding pyruvate carboxylase while simultaneously exhibiting a disrupted ldhA gene encoding l-lactate dehydrogenase was investigated in detail for succinic acid production. Succinic acid was shown to be efficiently
produced at high-cell density under oxygen deprivation with intermittent addition of sodium bicarbonate and glucose. Succinic
acid concentration reached 1.24 M (146 g l−1) within 46 h. The yields of succinic acid and acetic acid from glucose were 1.40 mol mol−1 (0.92 g g−1) and 0.29 mol mol−1 (0.10 g g−1), respectively. The succinic acid production rate and yield depended on medium bicarbonate concentration rather than glucose
concentration. Consumption of bicarbonate accompanied with succinic acid production implied that added bicarbonate was used
for succinic acid synthesis. 相似文献
18.
We previously reported that the activities of two 3-hydroxybutyrate dehydrogenases (BDH1 and BDH2) were greatly influenced
by culture conditions when Ralstonia pickettii T1, a strain growing on extracellular poly-3-hydroxybutyrate (PHB), was grown on different carbon sources such as 3HB and
succinate. In this study, knockout mutants of bdh1 or bdh2 were constructed and characterized under different culture conditions. In addition, a novel BDH (BDH3) was found in bdh2 mutants, and bdh3 was cloned. Apparent kinetic parameters for the substrates of BDH3 indicated that the enzyme is suitable for the oxidation
reaction of 3-hydroxybutyrate (3HB) to acetoacetate. In Western blotting, it was clear that BDH3 is produced only in cells
grown on 3HB or PHB as a carbon source, while BDH1 and BDH2 are produced in cells grown on various carbon sources such as
sugars, amino acids, organic acids, 3HB, and PHB. Both the bdh1 and bdh2 mutants lagged behind the wild type in growth rates when the cells were cultured with 3HB, citrate, succinate, or nutrient
broth. A test of sensitivity to diamide as an oxidative stress revealed that the lack of BDH1 or BDH2 caused a decline in
the capacity to neutralize the stress. These results suggested that BDH1 and BDH2 are needed to regulate the cytoplasmic redox
state as well as to utilize 3HB, while BDH3 is specialized to utilize 3HB. The expression of bdh3 may be coordinately regulated with a gene encoding putative 3HB permease. 相似文献
19.
Mario C. N. Saparrat Geraldine E. Fermoselle Sebastián A. Stenglein Mónica B. Aulicino Pedro A. Balatti 《Mycopathologia》2009,168(1):41-47
Pseudocercospora griseola is the causal agent of angular leaf spot of common bean (ALS). It has undergone parallel coevolution with its host and two
major groups have been defined, “Andean” (P. griseola f. griseola) and “Mesoamerican” (P. griseola f. mesoamericana). The aim of this study was to analyze the nature and the level of the dark pigment synthesized by the representatives of
each group. After 21 days of incubation on potato dextrose agar medium, P. griseola f. griseola isolate S3b developed colonies with diameters of 17.5 ± 1.3 mm and concentric rings of pigmentation. Isolate T4 of P. griseola f. mesoamericana presented smaller colonies (9.9 ± 0.3 mm) with a uniform dark-gray color. Both isolates, S3b and T4, produced the same pigment,
a 1,8-dihydroxynaphthalene-melanin, although different in quantity and structural features as suggested by the IR spectrum.
The P. griseola f. griseola isolate S3b had a higher growth rate and melanin content as well as smaller sensitivity to melanin synthesis inhibitors compared
to the isolate T4 of P. griseola f. mesoamericana. These results suggest a possible link between melanin and growth in P. griseola. 相似文献