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1.
Takimura Y Saito K Okuda M Kageyama Y Saeki K Ozaki K Ito S Kobayashi T 《Applied microbiology and biotechnology》2007,76(2):395-405
The presence of 11 genes encoding subtilisin-like serine proteases was demonstrated by cloning from the genome of alkaliphilic
Bacillus sp. strain KSM-LD1. This strain exoproduces the oxidatively stable alkaline protease LD-1 (Saeki et al. Curr Microbiol, 47:337–340,
2003). Among the 11 genes, six genes encoding alkaline proteases (SA, SB, SC, SD, SE, and LD-1) were expressed in Bacillus hosts. However, the other five genes for subtilisin-like proteases (SF, SG, SH, SI, and SJ) were expressed in neither Bacillus hosts nor Escherichia coli. The deduced amino acid sequences of SA, SB, SC, SF, SG, SH, SI, and SJ showed similarity to those of other subtilisin-like
proteases from Bacillus strains with only 38 to 86% identity. The deduced amino acid sequence of SD was completely identical to that of an oxidatively
stable alkaline protease from Bacillus sp. strain SD521, and that of SE was almost identical to that of a high-molecular mass subtilisin from Bacillus sp. strain D-6 with 99.7% identity. There are four to nine subtilisin-like serine protease genes in the reported genomes
of Bacillus strains. At least 11 genes for the enzymes present in the genome of Bacillus sp. strain KSM-LD1, and this is the greatest number identified to date. 相似文献
2.
Okuda M Sumitomo N Takimura Y Ogawa A Saeki K Kawai S Kobayashi T Ito S 《Extremophiles : life under extreme conditions》2004,8(3):229-235
Six genes encoding high-molecular-mass subtilisins (HMSs) of alkaliphilic Bacillus spp. were cloned and sequenced. Their open reading frames of 2,394–2,424 bp encoded prosubtilisins of 798–808 amino acids (aa) consisting of the prepropeptides of 151–158 aa and the mature enzymes of 640–656 aa. The deduced aa sequences of the mature enzymes exhibited 60–95% identity to those of FT protease of Bacillus sp. strain KSM-KP43, a subtilisin-like serine protease, and a minor serine protease, Vpr, of Bacillus strains. Three of the six recombinant enzymes were susceptible to proteolysis, but the others were autodigestion resistant. All enzymes had optimal pH values of 10.5–11.0, optimal temperatures of 40–45°C for hydrolysis of a synthetic substrate, and were heat labile. These alkaline proteases seem to form a new subtilisin family, as judged by their aa sequences and phylogenetic analysis.Communicated by K. Horikoshi 相似文献
3.
Kobayashi T Lu J Li Z Hung VS Kurata A Hatada Y Takai K Ito S Horikoshi K 《Applied microbiology and biotechnology》2007,75(1):71-80
A new high-alkaline protease (ALTP) was purified to homogeneity from a culture of the strictly anaerobic and extremely alkaliphilic
Alkaliphilus transvaalensis. The molecular mass was 30 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme showed the maximal
caseinolytic activity higher than pH 12.6 in KCl–NaOH buffer at 40°C. Hydrolysis of the oxidized insulin B-chain followed
by mass spectrometric analysis of the cleaved products revealed that as many as 24 of the total 29 peptide bonds are hydrolyzed
in a block-cutting manner, suggesting that ALTP has a widespread proteolytic functions. Calcium ion had no effect on the activity
and stability of ALTP, unlike known subtilisins. The deduced amino acid sequence of the enzyme comprised 279 amino acids plus
97 prepropeptide amino acids. The amino acid sequence of mature ALTP was confirmed by capillary liquid chromatography coupled
to tandem mass spectrometry, which was the 93% coverage of the deduced amino acid sequence. The mature enzyme showed moderate
homology to subtilisin LD1 from the alkaliphilic Bacillus sp. strain KSM-LD1 with 64% identity, and both enzymes formed a new subcluster at an intermediate position among true subtilisins
and high-alkaline proteases in a phylogenetic tree of subtilase family A. ALTP is the first high-alkaline protease reported
from a strict anaerobe in this family. 相似文献
4.
Amit Gupta Babu Joseph Abin Mani George Thomas 《World journal of microbiology & biotechnology》2008,24(2):237-243
In this communication, we report the presence of a newly identified serine alkaline protease producing bacteria, Virgibacillus pantothenticus (MTCC 6729) in the fresh chicken meat samples and the factors affecting biosynthesis as well as characterization of protease.
The strain produced only 14.3 U ml−1 protease in the standard medium after 72 h of incubation, while in optimized culture conditions the production of protease
was increased up to 18.2 U ml−1. The strain was able to produce protease at 40°C at pH 9.0. The addition of dextrose and casein improved protease production.
The protease was partially purified and characterized in terms of pH and temperature stability, effect of metal ions and inhibitors.
The protease was found to be thermostable alkaline by retaining its 100% and 85% stability at pH 10.0 and at 50°C respectively.
The protease was compatible with some of the commercial detergents tested, and was effective in removing protein stains from
cotton fabrics. The V. pantothenticus, MTCC 6729 protease appears to be potentially useful as an additive in detergents as a stain remover and other bio-formulations. 相似文献
5.
Konstantin V. Kiselev Anna V. Turlenko Galina K. Tchernoded Yuri N. Zhuravlev 《Plant cell reports》2009,28(8):1273-1278
It has been shown previously that the rolC gene from Agrobacterium tumefaciens gene was stably and highly expressed in 15-year-old Panax ginseng transgenic cell cultures. In the present report, we analyze in detail the nucleotide composition of the rolC and nptII (neomycin phosphotransferase) genes, which is the selective marker used for transgenic cell cultures of P.
ginseng. It has been established that the nucleotide sequences of the rolC and nptII genes underwent mutagenesis during cultivation. Particularly, 1–4 nucleotide substitutions were found per sequence in the
540 and 798 bp segments of the complete rolC and nptII genes, respectively. Approximately half of these nucleotide substitutions caused changes in the structure of the predicted
gene product. In addition, we attempted to determine the rate of accumulation of these changes by comparison of DNA extracted
from P.
ginseng cell cultures from 1995 to 2007. It was observed that the frequency of nucleotide substitutions for the rolC and nptII genes in 1995 was 1.21 ± 0.02 per 1,000 nucleotides analyzed, while in 2007, the nucleotide substitutions significantly increased
(1.37 ± 0.07 per 1,000 nucleotides analyzed). Analyzing the nucleotide substitutions, we found that substitution to G or to
C nucleotides significantly increased (in 1.9 times) in the rolC and nptII genes compared with P.
ginseng
actin gene. Finally, the level of nucleotide substitutions in the rolC gene was 1.1-fold higher when compared with the nptII gene. Thus, for the first time, we have experimentally demonstrated the level of nucleotide substitutions in transferred
genes in transgenic plant cell cultures. 相似文献
6.
The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae. 相似文献
7.
Farrakh Mehboob Howard Junca Gosse Schraa Alfons J. M. Stams 《Applied microbiology and biotechnology》2009,83(4):739-747
Microbial (per)chlorate reduction is a unique process in which molecular oxygen is formed during the dismutation of chlorite.
The oxygen thus formed may be used to degrade hydrocarbons by means of oxygenases under seemingly anoxic conditions. Up to
now, no bacterium has been described that grows on aliphatic hydrocarbons with chlorate. Here, we report that Pseudomonas chloritidismutans AW-1T grows on n-alkanes (ranging from C7 until C12) with chlorate as electron acceptor. Strain AW-1T also grows on the intermediates of the presumed n-alkane degradation pathway. The specific growth rates on n-decane and chlorate and n-decane and oxygen were 0.5 ± 0.1 and 0.4 ± 0.02 day−1, respectively. The key enzymes chlorate reductase and chlorite dismutase were assayed and found to be present. The oxygen-dependent
alkane oxidation was demonstrated in whole-cell suspensions. The strain degrades n-alkanes with oxygen and chlorate but not with nitrate, thus suggesting that the strain employs oxygenase-dependent pathways
for the breakdown of n-alkanes. 相似文献
8.
Yoshimune K Yamashita R Masuo N Wakayama M Moriguchi M 《Extremophiles : life under extreme conditions》2004,8(6):441-446
Salt-tolerant glutaminase (Micrococcus glutaminase, with an apparent molecular mass of 48.3 kDa, intact glutaminase) from the marine bacterium Micrococcus luteus K-3 was digested using protease derived from M. luteus K-3. The digestion products were a large fragment (apparent molecular mass of 38.5 kDa, the glutaminase fragment) and small fragments (apparent molecular mass of 8 kDa). The digestion was inhibited by phenylmethanesulfonyl fluoride (PMSF). Digestion of intact glutaminase by serine proteases including trypsin, elastase, lysyl endopeptidase, and arginylendopeptidase also produced the glutaminase fragment. The N-terminus of the glutaminase fragment was the same as that of intact glutaminase. The N-termini of two small fragments were Ala394 and Ala396, respectively. The enzymological and kinetic properties of the glutaminase fragment were almost the same as those of intact glutaminase except for salt-tolerant behavior. The glutaminase fragment was a higher salt-tolerant enzyme than the intact glutaminase, suggesting that Micrococcus glutaminase is digested in the C-terminal region by serine protease from M. luteus K-3 to confer salt tolerance on glutaminase. 相似文献
9.
Karbalaei-Heidari HR Amoozegar MA Hajighasemi M Ziaee AA Ventosa A 《Journal of industrial microbiology & biotechnology》2009,36(1):21-27
The production of a protease was investigated under conditions of high salinity by the moderately halophilic bacterium Halobacillus karajensis strain MA-2 in a basal medium containing peptone, beef extract, maltose and NaCl when the culture reached the stationary
growth phase. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed
that the maximum secretion occurred at 34°C, pH 8.0–8.5, and in the presence of gelatin. Replacement of NaCl by various concentrations
of sodium nitrate in the basal medium also increased the protease production. The secreted protease was purified 24-fold with
68% recovery by a simple approach including a combination of acetone precipitation and Q-Sepharose ion exchange chromatography.
The enzyme revealed a monomeric structure with a relative molecular mass of 36 kDa by running on SDS-PAGE. Maximum caseinolytic
activity of the enzyme was observed at 50°C, pH 9.0 and 0.5 M NaCl, although at higher salinities (up to 3 M) activity still
remained. The maximum enzyme activity was obtained at a broad pH range of 8.0–10.0, with 55 and 50% activity remaining at
pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), Pefabloc
SC and EDTA; indicating that it probably belongs to the subclass of serine metalloproteases. These findings suggest that the
protease secreted by Halobacillus karajensis has a potential for biotechnological applications from its haloalkaline properties point of view. 相似文献
10.
The interaction between Shigella dysenteriae or Shigella sonnei and Acanthamoeba castellanii was studied by viable counts, gentamicin assay and electron microscopy. The result showed that Shigella dysenteriae or Shigella sonnei grew and survived in the presence of amoebae for more than 3 weeks. Gentamicin assay showed that the Shigella were viable inside the Acanthamoeba castellanii which was confirmed by electron microscopy that showed the Shigella localized in the cytoplasm of the Acanthamoeba castellanii. In conclusion, the relationship between Shigella dysenteriae and Shigella sonnei with Acanthamoeba castellanii is symbiotic, and accordingly free-living amoebae may serve as a transmission reservoir for Shigella in water. 相似文献
11.
A gene encoding a Na(+)/H(+) antiporter was obtained from the genome of Halobacillus aidingensis AD-6(T), which was sequenced and designated as nhaH. The deduced amino acid sequence of the gene was 91% identical to the NhaH of H. dabanensis, and shared 54% identity with the NhaG of Bacillus subtilis. The cloned gene enable the Escherichia coli KNabc cell, which lack all of the major Na(+)/H(+) antiporters, to grow in medium containing 0.2 M NaCl or 10 mM LiCl. The nhaH gene was predicted to encode a 43.5 kDa protein (403 amino acid residues) with 11 putative transmembrane regions. Everted membrane vesicles prepared from E. coli KNabc cells carrying NhaH exhibited Na(+)/H(+) as well as Li(+)/H(+) antiporter activity, which was pH-dependent with the highest activity at pH 8.0, and no K(+)/H(+) antiporter activity was detected. The deletion of hydrophilic C-terminal amino acid residues showed that the short C-terminal tail was vital for Na(+)/H(+) antiporter activity. 相似文献
12.
13.
Lauro FM Bertoloni G Obraztsova A Kato C Tebo BM Bartlett DH 《Extremophiles : life under extreme conditions》2004,8(2):169-173
Three Clostridium strains were isolated from deep-sea sediments collected at a depth of 6.3–7.3 km in the Japan Trench. Physiological characterization and 16S rDNA analysis revealed that the three isolates were all closely related to Clostridium bifermentans. The spores of all three isolates were resistant to inactivation at high pressure and low temperature. However, despite the fact that the vegetative cells were halotolerant and eurythermal they did not appear to be adapted for growth or viability under the conditions prevailing in the deep-sea sediments from which they were obtained. The results suggest that the isolates had survived as spores in the deep-sea sediments and that the marine benthos could be a source of clostridia originating in other environments.Communicated by K. Horikoshi 相似文献
14.
15.
Stefanie Kimbacher Ingrid Gerstl Branko Velimirov Sylvia Hagemann 《Molecular genetics and genomics : MGG》2009,282(2):165-172
P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding
region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
16.
17.
Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species. 相似文献
18.
We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan. 相似文献
19.
Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic
expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase
and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic
activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant
strain was induced by 0.7 mmol l−1 isopropyl-β-D- thiogalactopyranoside (IPTG) at 20°C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic
of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases. 相似文献
20.
Fuhong Xie Yapeng Chao Zhiquan Xue Xiuqing Yang Guoqing Zhang Jiaji Shi Shijun Qian 《Journal of industrial microbiology & biotechnology》2009,36(5):739-746
In industry, fosfomycin is mainly prepared via chemical epoxidation of cis-propenylphosphonic acid (cPPA). The conversion yield of fosfomycin is less than 50% in the whole process and a large quantity
of waste is produced. Biotransformation by microorganisms is an alternative method of preparation. This kind of conversion
is more delicate, environmentally friendly, and the conversion yield of fosfomycin would be higher. In this work, an aerobic
bacterium capable of transforming cPPA to fosfomycin was isolated. The organism, designated as strain S101, was identified
as Bacillus simplex by morphological and physiological characteristics as well as by analysis of the gene encoding the 16S rRNA. Fosfomycin was
assayed by two means, bioassay and gas chromatography (GC). Glycerol was a good carbon source for growth and cPPA conversion
of strain S101. When cPPA was used as the sole carbon source, neither growth nor conversion to fosfomycin occurred. The optimum
cPPA concentration in the conversion medium was 2,000 μg ml−1. After 6 days of incubation, the concentration of fosfomycin reached its maximum level (1,838.2 μg ml−1), with a conversion ratio of 81.3%. Air was indispensable for the growth but not for the conversion to fosfomycin. Furthermore,
vanadium ions were found to be essential for the conversion. High concentrations of cPPA had fewer inhibitory effects on the
growth of strain S101. 相似文献