共查询到20条相似文献,搜索用时 31 毫秒
1.
Katalin A Wilkinson Sandra M Newton Graham R Stewart Adrian R Martineau Janisha Patel Susan M Sullivan Jean-Louis Herrmann Olivier Neyrolles Douglas B Young Robert J Wilkinson 《BMC microbiology》2009,9(1):93
Background
The 19 kDa lipoprotein of Mycobacterium tuberculosis (MTB) is an important target of the innate immune response. To investigate the effect of post-translation modification of this protein on innate recognition in the context of the whole bacillus, we derived a recombinant M. tuberculosis H37Rv that lacked the 19 kDa gene (Δ19) and complemented this strain by reintroduction of the 19 kDa gene into the chromosome as a single copy to produce Δ19::19. We also reintroduced the 19 kDa gene in two modified forms that lacked motifs for acylation (Δ19::19NA) and O-glycosylation (Δ19::19NOG). 相似文献2.
Junpei Zhou Pengjun Shi Rui Zhang Huoqing Huang Kun Meng Peilong Yang Bin Yao 《Journal of industrial microbiology & biotechnology》2011,38(4):523-530
A pH-stable and protease-resistant xylanase (XynB119) was identified from Streptomyces sp. TN119, a strain isolated from the gut luminal contents of longhorned beetle (Batocera horsfieldi) larvae. Using the GC TAIL-PCR method, the 1,026-bp coding gene (xynB119) with 67.3% GC content was successfully cloned and expressed in Escherichia coli. It encodes a 341-residue polypeptide with a calculated molecular mass of 35.9 kDa, including a putative 41-residue signal
peptide, a catalytic domain of glycosyl hydrolase (GH) family 11, a short Gly/Pro-rich linker, and a family 2 cellulose-binding
domain (CBM 2). The deduced amino acid sequence is most similar to (61.9% identity) an endo-1,4-β-xylanase from Streptomyces thermoviolaceus OPC-520. Purified recombinant XynB119 exhibited peak activity at 50°C and pH 7.0, remained stable over a broad pH range (retaining >70%
activity after incubation at pH 1.0–11.0 for 1 h at 37°C without substrate), had strong protease resistance (retaining >90%
activity after proteolytic treatment at 37°C for 1 h) and SDS resistance (at 100 mM). These properties make XynB119 promising
for application in the feed industry and valuable for basic research. Compared to r-XynB119, the r-XynB119 derivative without
CBM 2 and linker region (r-XynB119d) exhibited a decreased pH stability of >25% at extreme pHs (pH 1.0–3.0 and pH 11.0–12.0). 相似文献
3.
4.
Jiaping Song Chuanwen Zhou Rui Liu Xudong Wu Di Wu Xiaojian Hu Yu Ding 《Molecular biology reports》2010,37(4):1823-1829
The crystal structures of almost all the enzymes of arginine metabolism have been determined, but arginine decarboxylase’s
structure is not resolved yet. In order to characterize and crystallize arginine decarboxylase, we overexpressed biosynthetic
arginine decarboxylase (ADC; EC 4.1.1.19, encoded by the speA gene) from Escherichia coli in the T7 expression system as a cleavable poly-His-tagged fusion construct. The expressed recombinant His10-ADC (77.3 kDa) was first purified by Ni–NTA affinity chromatography, then proteolytically digested with Tobacco Etch Virus
(TEV) protease to remove the poly-His fusion tag, and finally purified by anion exchange chromatography. The His10 tag removed recombinant ADC (74.1 kDa)’s typical yield was 90 mg from 1 l of culture medium with purity above 98%. The recombinant
ADC was assayed for decarboxylase activity, showing decarboxylase activity of 2.8 U/mg, similar to the purified native E. coli ADC. The decarboxylase activity assay also showed that the purified recombinant ADC tolerated broad ranges of pH (pH 6–9)
and temperature (20–80°C). Our research may facilitate further studies of ADC structure and function, including the determination
of its crystal structure by X-ray diffraction. 相似文献
5.
Finnegan PM Brumbley SM O'Shea MG Nevalainen H Bergquist PL 《Archives of microbiology》2005,183(2):140-147
Genes encoding dextranolytic enzymes were isolated from Paenibacillus strains Dex40-8 and Dex50-2. Single, similar but non-identical dex1 genes were isolated from each strain, and a more divergent dex2 gene was isolated from strain Dex50-2. The protein deduced from the Dex40-8 dex1 gene sequence had 716 amino acids, with a predicted Mr of 80.8 kDa. The proteins deduced from the Dex50-2 dex1 and dex2 gene sequences had 905 and 596 amino acids, with predicted Mr of 100.1 kDa and 68.3 kDa, respectively. The deduced amino acid sequences of all three dextranolytic proteins had similarity to family 66 glycosyl hydrolases and were predicted to possess cleavable N-terminal signal peptides. Homology searches suggest that the Dex40-8 and Dex50-2 Dex1 proteins have one and two copies, respectively, of a carbohydrate-binding module similar to CBM_4_9 (pfam02018.11). The Dex50-2 Dex2 deduced amino acid sequence had highest sequence similarity to thermotolerant dextranases from thermophilic Paenibacillus strains, while the Dex40-8 and Dex50-2 Dex1 deduced protein sequences formed a distinct sequence clade among the family 66 proteins. Examination of seven Paenibacillus strains, using a polymerase chain reaction-based assay, indicated that multiple family 66 genes are common within this genus. The three recombinant proteins expressed in Escherichia coli possessed dextranolytic activity and were able to convert ethanol-insoluble blue dextran into an ethanol-soluble product, indicating they are endodextranases (EC 3.2.1.11). The reaction catalysed by each enzyme had a distinct temperature and pH dependence. 相似文献
6.
Yun-Tao Li Mao-Teng Li Chu-Hua Fu Peng-Peng Zhou Jian-Min Liu Long-Jiang Yu 《Biotechnology letters》2009,31(7):1011-1017
Genes encoding Δ6 desaturase, Δ6 fatty acid elongase, and Δ5 desaturase from the alga, Phaeodactylum tricornutum, were co-expressed in Pichia pastoris to produce arachidonic acid (ARA; 20:4 Δ5, 8, 11, 14) and eicosapentaenoic acid (EPA; 20:5 Δ5, 8, 11, 14, 17). A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro
multimerization approach. ARA and EPA accumulated up to 0.3 and 0.1% of total fatty acids, respectively, in the recombinant
P. pastoris carrying with double copies of these three heterologous genes, as compared to 0.1 and 0.05%, respectively, in the recombinant
P. pastoris with single copy.
Yun-Tao Li and Mao-Teng Li contributed equally to this work. 相似文献
7.
Rui-Yu Zhao Wei Xiao Hai-Li Cheng Ping Zhu Ke-Di Cheng 《Journal of industrial microbiology & biotechnology》2010,37(11):1171-1182
The gene encoding squalene synthase (GfSQS) was cloned from Fusarium fujikuroi (Gibberella fujikuroi MP-C) and characterized. The cloned genomic DNA is 3,267 bp in length, including the 5′-untranslated region (UTR), 3′-UTR,
four exons, and three introns. A noncanonical splice-site (CA-GG, or GC-AG) was found at the first intron. The open reading
frame of the gene is 1,389 bp in length, corresponding to a predicted polypeptide of 462 amino acid residues with a MW 53.4 kDa.
The predicted GfSQS shares at least four conserved regions involved in the enzymatic activity with the SQSs of varied species.
The recombinant protein was expressed in E. coli and detected by SDS–PAGE and western blot. GC–MS analysis showed that the wild-type GfSQS could catalyze the reaction from
farnesyl diphosphate (FPP) to squalene, while the mutant mGfSQS (D82G) lost total activity, supporting the prediction that
the aspartate-rich motif (DTXED) in the region I of SQS is essential for binding of the diphosphate substrate. 相似文献
8.
Zhong-peng Guo Liang Zhang Zhong-yang Ding Zheng-Xiang Wang Gui-Yang Shi 《Journal of industrial microbiology & biotechnology》2011,38(8):935-943
The GPD2 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol-producing strain of Saccharomyces cerevisiae, was deleted. And then, either the non-phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus, or the NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Kluyveromyces lactis, was expressed in the obtained mutant AG2 deletion of GPD2, respectively. The resultant recombinant strain AG2A (gpd2Δ P
PGK
-gapN) exhibited a 48.70 ± 0.34% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.60 ± 0.12%
(relative to the amount of substrate consumed) increase in ethanol yield, while recombinant AG2B (gpd2Δ P
PGK
-GAPDH) exhibited a 52.90 ± 0.45% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.34 ± 0.15%
(relative to the amount of substrate consumed) increase in ethanol yield compared with the wild-type strain. More importantly,
the maximum specific growth rates (μ
max) of the recombinant AG2A and AG2B were higher than that of the mutant gpd2Δ and were indistinguishable compared with the wild-type strain in anaerobic batch fermentations. The results indicated that
the redox imbalance of the mutant could be partially solved by expressing the heterologous genes. 相似文献
9.
Juliana Branco Novo Maria Leonor Sarno Oliveira Geraldo Santana Magalhães Ligia Morganti Isaías Raw Paulo Lee Ho 《Molecular biotechnology》2010,46(3):279-286
Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher’s disease, the most common inherited storage
disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster
ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires
successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies
against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c
mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum
was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human
cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected
molecular masses of 59–66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization
of new biosimilar recombinant GCRs coming in the near future. 相似文献
10.
The present study was undertaken to determine the role of glutathione peroxidase3 (gpx3) in phospholipid protection in cells.
Wild-type (WT) cells showed an overall increase in phospholipids upon 50 μM cadmium (Cd)-treatment, whereas an untreated gpx3Δ strain showed a drastic reduction in overall phospholipids which was further reduced with 50 μM Cd. In WT cells, Cd-exposure
increased the short chain fatty acids and decreased the unsaturated fatty acids and the magnitude was high in Cd-treated gpx3Δ cells. Purified recombinant gpx3p showed higher activity with phospholipid hydroperoxides than shorter hydroperoxides. An
increase in gpx activity was observed in Cd-treated WT cells and no such alteration was observed in gpx3Δ. WT cells treated with Cd showed an increase in MDA over untreated, while untreated gpx3Δ cells themselves showed a higher level of MDA which was further enhanced with Cd-treatment. Iron, zinc and calcium levels
were significantly altered in WT and gpx3Δ cells during Cd-treatment. 相似文献
11.
Two carotenoid 1,2-hydratase (CrtC) genes from the photosynthetic bacteria Rubrivivax gelatinosus and Thiocapsa roseopersicina were cloned and expressed in Escherichia coli in an active form and purified by affinity chromatography. The biochemical properties of the recombinant enzymes and their
substrate specificities were studied. The purified CrtCs catalyze cofactor independently the conversion of lycopene to 1-HO-
and 1,1′-(HO)2-lycopene. The optimal pH and temperature for hydratase activity was 8.0 and 30°C, respectively. The apparent K
m and V
max values obtained for the hydration of lycopene were 24 μM and 0.31 nmol h−1 mg−1 for RgCrtC and 9.5 μM and 0.15 nmol h−1 mg−1 for TrCrtC, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed two protein bands of 44 and
38 kDa for TrCrtC, which indicate protein processing. Both hydratases are also able to convert the unnatural substrate geranylgeraniol
(C20 substrate), which functionally resembles the natural substrate lycopene. 相似文献
12.
A gene encoding an intracellular glucoamylase was identified in the genome of the extreme thermoacidophilic Archaeon Thermoplasma acidophilum. The gene taGA, consisting of 1,911 bp, was cloned and successfully expressed in Escherichia coli. The recombinant protein was purified 22-fold to homogeneity using heat treatment, anion-exchange chromatography, and gel
filtration. Detailed analysis shows that the glucoamylase, with a molecular weight of 66 kDa per subunit, is a homodimer in
its active state. Amylolytic activity was measured over a wide range of temperature (40–90°C) and pH (pH 3.5–7) and was maximal
at 75°C and at acidic condition (pH 5). The recombinant archaeal glucoamylase uses a variety of polysaccharides as substrate,
including glycogen and amylose. Maximal activity was measured towards amylopectin with a specific activity of 4.2 U/mg and
increased almost threefold in the presence of manganese. Calcium ions have a pronounced effect on enzyme stability; in the
presence of 5 mM CaCl2, the half-life increased from 15 min to 2 h at 80°C. 相似文献
13.
Finnegan PM Brumbley SM O'Shea MG Nevalainen H Bergquist PL 《Current microbiology》2004,49(5):327-333
Thermotolerant Paenibacillus strain Dex70-1B and unidentified strain Dex70-34 produce thermoactive dextran-degrading enzymes. Plasmid-based genomic DNA libraries constructed from mixed bacterial cultures containing Dex70-1B or Dex70-34 were screened for the ability to confer dextranolytic activity at 70°C onto Escherichia coli. One gene, designated dex1, was isolated from each strain. The Dex70-1B and Dex70-34 dex1 gene sequences were non-identical, and encoded proteins containing 597 (Mr 68.6 kDa) and 600 amino acids (Mr 69.2 kDa), respectively. The Dex1 amino acid sequences were most similar to one another, and formed a new clade among the family 66 glycosyl hydrolase sequences. Expression of the Dex1 proteins in E. coli produced dextranolytic activity that converted ethanol-insoluble blue dextran into an ethanol-soluble form, suggestive of endodextranases (EC 3.2.1.11). Both enzymes were most active at about 60°C and pH 5.5, and retained more than 70% maximal activity after incubation at 57°C for 9.5 h in the absence of substrate. 相似文献
14.
Purification of recombinant laccase from Trametes versicolor in Pichia methanolica and its use for the decolorization of anthraquinone dye 总被引:1,自引:0,他引:1
A recombinant laccase from Trametes versicolor in Pichia methanolica was produced constitutively in a defined medium. The recombinant laccase was purified using ultrafiltration, anion-exchange
chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 64 kDa by SDS-PAGE. The
purified recombinant laccase decolorized more than 90% of Remazol Brilliant Blue R (RBBR) initially at 80 mg l−1 after 16 h at 45°C and pH 5 when 25 U laccase ml−1 was used. The purified recombinant laccase could efficiently decolorize RBBR without additional redox mediators. 相似文献
15.
A fragment coding for a putative extracellular α-amylase, from the genomic library of the yeast Saccharomycopsis fibuligera KZ, has been subcloned into yeast expression vector pVT100L and sequenced. The nucleotide sequence revealed an ORF of 1,485 bp
coding for a 494 amino acid residues long protein with 99% identity to the α-amylase Sfamy from S. fibuligera HUT 7212. The S. fibuligera KZ α-amylase (Sfamy KZ) belongs to typical extracellular fungal α-amylases classified in the glycoside hydrolase family 13,
subfamily 1, as supported also by clustering observed in the evolutionary tree. Sfamy KZ, in addition to the essential GH13
α-amylase three-domain arrangement (catalytic TIM barrel plus domains B and C), does not contain any distinct starch-binding
domain. Sfamy KZ was expressed as a recombinant protein in Saccharomyces cerevisiae and purified to electrophoretic homogeneity. The enzyme had a molecular mass 53 kDa and contained about 2.5% of carbohydrate.
The enzyme exhibited pH and temperature optima in the range of 5–6 and 40–50 °C, respectively. Stable adsorption of the enzyme
to starch granules was not detected but a low degradation of raw starch in a concentration-dependent manner was observed. 相似文献
16.
A novel alkylsulfatase gene, sdsAP, was cloned from a newly isolated bacterium Pseudomonas sp. S9. It encoded a protein of 675 amino acids with a calculated molecular mass of 74.9 kDa. The protein contained a typical
N-terminal signal peptide of 41 amino acid residues, followed by a metallo-β-lactamase like domain at the N-terminus and a
SCP-2-like domain at the C-terminus. This domain organization mode suggested that it belonged to the type III sulfatase. The
mature alkylsulfatase was overexpressed in Escherichia coli. The optimal temperature and pH of the recombinant SdsAP were 70°C and 9.0, respectively. Notably, at optimal conditions,
the purified recombinant SdsAP had a high specific activity of 23.25 μmol min−1 mg−1, a K
m (app) of 264.3 μmol, and a V
max (app) of 33.8 μmol min−1 mg−1 for SDS. Additionally, it still retained more than 90% activity after incubation at 65°C for 1 h, which was much different
from other alkylsulfatases reported. The recombinant enzyme hydrolyzed the primary alkyl sulfate such as sodium octyl sulfate
and sodium dodecyl sulfate (SDS). It was a Zn2+-containing and Ca2+ activated alkylsulfatase. This is the first report to explore the various characteristics of the heterologous recombinant
alkylsulfatase in details. These favorable properties could make SdsAP attractive to be useful in the degradation of SDS-containing
waste. 相似文献
17.
Two chitosanases (CHSA1 and CHSA2) were purified from the culture supernatant of Acinetobacter calcoaceticus TKU024 with squid pen as the sole carbon/nitrogen source. The molecular masses of CHSA1 and CHSA2 determined by SDS-PAGE
were approximately 27 and 66 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of
CHSA1 and CHSA2 were (pH 6, 50°C, pH 4–10, <90°C) and (pH 7, 60°C, pH 6–11, <70°C), respectively. CHSA1 and CHSA2 had broad
pH and thermal stability. CHSA1 and CHSA2 were both inhibited by EDTA and were inhibited completely by 5 mM Mn2+. CHSA1 and CHSA2 degraded chitosan with DD ranging from 60 to 98%, and also degraded some chitin. The most susceptible substrate
was 60% deacetylated chitosan. Furthermore, TKU024 culture supernatant (1.5% SPP) incubated for 5 days has the most reducing
sugars (0.63 mg/ml). With this method, we have shown that shellfish wastes may have a great potential for the production of
bioactive materials. 相似文献
18.
Satomura T Zhang XD Hara Y Doi K Sakuraba H Ohshima T 《Applied microbiology and biotechnology》2011,89(4):1075-1082
The activity of a dye-linked l-proline dehydrogenase (dye-l-proDH) was found in the crude extract of an aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis JCM 11548, and was purified 163-fold through four sequential chromatography steps. The enzyme has a molecular mass of about
108 kDa and is a homodimer with a subunit molecular mass of about 46 kDa. The enzyme retained more than 90% of its activity
after incubation at 100 °C for 120 min (pH 7.5) or after incubation at pHs 4.5–9.0 for 30 min at 50 °C. The enzyme catalyzed
l-proline dehydrogenation to Δ1-pyroline-5-carboxylate using 2,6-dichloroindophenol (DCIP) as the electron acceptor and the Michaelis constants for l-proline and DCIP were 1.67 and 0.026 mM, respectively. The prosthetic group on the enzyme was identified as flavin adenine
dinucleotide by high-performance liquid chromatography. The subunit N-terminal amino acid sequence was MYDYVVVGAG. Using that
sequence and previously reported genome information, the gene encoding the enzyme (Pcal_1655) was identified. The gene was
then cloned and expressed in Escherichia coli and found to encode a polypeptide of 415 amino acids with a calculated molecular weight of 46,259. The dye-l-proDH gene cluster in P. calidifontis inherently differs from those in the other hyperthermophiles reported so far. 相似文献
19.
In-Hye Park Jie Chang Yong-Seok Lee Shu-Jun Fang Yong-Lark Choi 《The protein journal》2012,31(3):238-245
The bacterial strain Paenibacillus xylanilyticus KJ-03 was isolated from a sample of soil used for cultivating Amorphophallus konjac. The cellulase gene, cel5A was cloned using fosmid library and expressed in Escherichia coli BL21 (trxB). The cel5A gene consists of a 1,743 bp open reading frame and encodes 581 amino acids of a protein. Cel5A contains N-terminal signal
peptide, a catalytic domain of glycosyl hydrolase family 5, and DUF291 domain with unknown function. The recombinant cellulase
was purified by Ni-affinity chromatography. The cellulase activity of Cel5A was detected in clear band with a molecular weight
of 64 kDa by zymogram active staining. The maximum activity of the purified enzyme was displayed at a temperature of 40 °C
and pH 6.0 when carboxymethyl cellulose was used as a substrate. It has 44% of its maximum activity at 70 °C and retained
66% of its original activity at 45 °C for 1 h. The purified cellulase hydrolyzed avicel, CMC, filter paper, xylan, and 4-methylumbelliferyl
β-d-cellobiose, but no activity was detected against p-nitrophenyl β-d-glucoside. The end products of the hydrolysis of cellotetraose and cellopentaose by Cel5A were detected by thin layer chromatography,
while enzyme did not hydrolyze cellobiose and cellotriose. 相似文献
20.
Withu Choosri Regina Paukner Petra Wührer Dietmar Haltrich Christian Leitner 《World journal of microbiology & biotechnology》2011,27(6):1349-1353
The gene gaoA encoding the copper-dependent enzyme galactose oxidase (GAO) from Fusarium graminearum PH-1 was cloned and successfully overexpressed in E. coli. Culture conditions for cultivations in shaken flasks were optimized, and optimal conditions were found to be double-strength
LB medium, 0.5% lactose as inducer, and induction at the reduced temperature of 25°C. When using these cultivation conditions ~24 mg
of active GAO could be produced in shaken flasks per litre medium. Addition of copper to the fermentation medium decreased
the enzyme production significantly. The His-tagged recombinant enzyme could be purified conveniently with a single affinity
chromatography step. The purified enzyme showed a single band on SDS–PAGE with an apparent molecular mass of 66 kDa and had
kinetic properties similar to those of the fungal wild-type enzyme. 相似文献