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1.
Jei Oh Yon Ji Seon Lee Bo Geum Kim Sang Dal Kim Doo Hyun Nam 《Biotechnology and Bioprocess Engineering》2008,13(1):102-107
The immobilization of phospholipase D produced by Streptomyces sp. YU100 was evaluated to see it would be practical for industrial applications. To accomplish this, the purified enzyme,
which contained 53 unit/mg of protein, was subjected to immobilization on various matrices. When immobilization supports including
calcium alginate gel, polyacrylamide gel, and macroporous resin were evaluated, the highest enzyme retention ratio (> 42%)
was observed on a Dowex MSA-2 macro-porous resin. This may have occurred as a result of the ability of the hydrophobic domain
of phospholipase D to interact with the polystyrene backbone of the resin, as well as the ability of the dimethylethanolamine
group of the MSA-2 resin to retain the enzyme by forming hydrogen bonds with the acidic residues of the enzyme. Upon the operation
of a reactor packed with enzyme that had been immobilized on a Dowex MSA-2 resin, greater than 80% of the initial enzyme activity
was retained for 16 days. During the reaction, phosphatidylcholine became bound to the immobilized resin and interfered with
the enzyme reaction, therefore, the resin was washed with ethyl ether every 2 h. A process for recovering excessive l-serine from phospholipids using the Dowex MR-3 resin was designed, and the separated l
-serine was employed again after replacing the amount that was used. 相似文献
2.
A cDNA encoding a phosphoinositide-specific phospholipase C (PI-PLC) has been isolated from Zea mays by screening a cDNA library. The cDNA, designated ZmPLC, encodes a polypeptide of 586 amino acids, containing the catalytic X, Y and C2 domains found in all PI-PLCs from plants. Northern blot analysis showed that the expression of the ZmPLC gene in roots is up-regulated under conditions of high salt, dehydration, cold or low osmotic stress conditions. Recombinant ZmPLC protein was expressed in Esch- erichia coli, purified and used to produce polyclonal antibody, this polyclonal antibody is important for further studies to assess the ultimate function of the ZmPLC gene in plants. 相似文献
3.
Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain. 相似文献
4.
Hong-Guan Xie Yong-Gang Bao Li-ping Bai Jun-Jie Shan Rong Jiang Yang Zhang Lian-Hong Guo Ren Zhang Yuan Li 《Journal of microbiology (Seoul, Korea)》2009,47(2):193-200
Streptomyces sp. 139 generates a novel exopolysaccharide (EPS) designated as Ebosin, which exerts an antagonistic effect on IL-1R in vitro and anti-rheumatic arthritis activity in vivo. A ste gene cluster for Ebosin biosynthesis consisting of 27 ORFs was previously identified in our laboratory. In this paper, ste16 was expressed in Escherichia coli BL21 and the recombinant protein was purified, which has the ability to catalyze the transfer of the methyl group from S-adenosylmethionine
(AdoMet) to dTDP-4-keto-6-deoxy-D-glucos, which was thus identified as a methyltransferase. In order to determine the function
of ste16 in Ebosin biosynthesis, the gene was disrupted with a double crossover via homologous recombination. The monosaccharide composition
of EPS-m generated by the mutant strain Streptomyces sp. 139 (ste16) was found to differ from that of Ebosin. The IL-1R antagonist activity of EPS-m was markedly lower than that of Ebosin.
These experimental results have shown that the ste16 gene codes for a methyltransferase which is involved in Ebosin biosynthesis.
These authors contributed equally to this work. 相似文献
5.
Anaerobic fungi belonging to the family Neocallimastigaceae are native inhabitants in the rumen of the most herbivores, such
as cattle, sheep and goats. A member of this unique group, Neocallimastix sp. GMLF2 was isolated from cattle feces and screened for its xylanase encoding gene using polymerase chain reaction. The
gene coding for a xylanase (xyn2A) was cloned in Escherichia coli and expression was monitored. To determine the enzyme activity, assays were conducted for both fungal xylanase and cloned
xylanase (Xyl2A) for supernatant and cell-associated activities. Optimum pH and temperature of the enzyme were found to be
6.5 and 50°C, respectively. The enzyme was stable at 40°C and 50°C for 20 min but lost most of its activity when temperature
reached 60°C for 5-min incubation time. Rumen fungal xylanase was mainly released to the supernatant of culture, while cloned
xylanase activity was found as cell-associated. Multiple alignment of the amino acid sequences of Xyl2A with published xylanases
from various organisms suggested that Xyl2A belongs to glycoside hydrolase family 11. 相似文献
6.
A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a
and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly
disulfide-bonded β-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for
production of refolded and active T. molitor antifreeze protein in bacteria was obtained. 相似文献
7.
The gene (choB
b
), encoding cholesterol oxidase from Brevibacterium sp. CCTCC M201008, was cloned and sequenced by PCR (GenBank accession number: DQ345780). The gene consists of 1653 base pairs
and encodes a protein of 551 amino acids. ChoB
b
exhibited a homology of 98% with cholesterol oxidase gene from Brevibacterium
sterolicum ATCC 21387. The cholesterol oxidase gene, cloned in the vector pET-28a, was over-expressed in Escherichia
coli BL21–CodonPlus (DE3)-RP grown at 23°C in Luria-Bertani medium containing 50 μM riboflavin, the precursor of the FAD coenzyme
of the enzyme. A maximum activity of 3.7 U/mg was obtained from cell free extract of E. coli BL21-CodonPlus (DE3)-RP harboring the pET-28a-choBb. 相似文献
8.
A full-length cDNA of a StCONSTANS-like (StCOL) gene was cloned from potato (Solanum tuberosum L.) by RTPCR and RACE. The predicted amino acid sequence of this cDNA has a high degree of identity with other homologous
members of the CO or COL family. Analysis of mRNA levels for StCOL shows that it is highly expressed in leaves and becomes weaker during tuberization; moreover, is independent of gibberellin
A3 and sucrose.
Published in Russian in Biokhimiya, 2007, Vol. 72, No. 11, pp. 1525–1531. 相似文献
9.
The endochitinase DNA and cDNA from Trichoderma sp. were cloned, sequenced and expressed. The cloned DNA and cDNA sequences were 1,476 and 1,275 bp in length, respectively.
There were three introns in DNA sequence in comparison with the cDNA sequence. The endochitinase protein contained three regions:
the signal peptide, the prepro-region and the mature protein region. The gene fragment encoding the mature endochitinase was
ligated into the expression vector pET-28a+, yielding pET-1. The plasmid pET-1 was transformed into the Escherichia coli BL21 (DE3). The clone bearing pET-1 was picked and cultured at 30°C for the expression of endochitinase. SDS-PAGE analysis
showed that the endochitinase was expressed in the periplasmic space and the purified protein showed a single band. The activity
of 70.2 U/mg was obtained from the cellular extract of the recombinant strain. The activity of endochitinase was 2.5-fold
higher at 24 h than at 16 h in the periplasmic space. The optimal pH and temperature of the recombinant endochitinase were
determined to be 7.0 and 35°C, respectively. It was relatively stable within the pH range of 5–8. Significant activity stimulation
by 1 mM Mg2+ and 5 mM Fe2+ and inhibition by 5 mM Co2+ and 5 mM Hg2+ were observed. The kinetic constants Km, Vmax and Kcat for the hydrolysis of the colloidal chitin were 1.5 mM, 1.37 μmol min−1 and 6.23 min−1, respectively. 相似文献
10.
Jing-Wei Lin Li-Xia Hao Gui-Xue Xu Fei Sun Feng Gao Ren Zhang Li-Xia Liu 《World journal of microbiology & biotechnology》2009,25(3):383-390
The fungal immunomodulatory proteins (FIPs) are a new protein family identified from several edible and medical mushrooms
and play an important role in anti-tumor, anti-allergy and immunomodulating activities. A gene encoding the FIP was cloned
from the mycelia of Changbai Lingzhi (Ganoderma lucidum) and recombinant expressed in the Pichia pastoris expression system. SDS-PAGE, amino acid composition and circular dichroism analyses of the recombinant FIP (reFIP) indicated
that the gene was correctly and successfully expressed. In vitro assays of biological activities revealed that the reFIP exhibited
similar immunomodulating capacities as native FIPs. The reFIP significantly stimulated the proliferation of mouse spleen lymphocytes
and apparently enhanced the expression level of interleukin-2 released from the mouse splenocytes. In addition, anti-tumor
activity assay showed that the reFIP could inhibit the proliferation of human leukemia-NB4 by inducing the cell apoptosis
to a degree of about 32.4%. Taken together, the FIP gene from Changbai G.
lucidum has been integrated into the yeast genome and expressed effectively at a high level (about 191.2 mg l−1). The reFIP possessed very similar biological activities to native FIPs, suggesting its potential application as a food supplement
or immunomodulating agent in pharmaceuticals and even medical studies.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
11.
F. L. Zhang Z. M. Chi K. L. Zhu J. Li M. J. Li L. K. Liang L. F. Wu 《World journal of microbiology & biotechnology》2007,23(3):331-337
The full length empA gene encoding Vibrio anguillarum metalloprotease was amplified by PCR and fused to the expression vector pBAD24. The carboxy-terminal 6xHis-tagged recombinant
metalloprotein (rEmpA) was expressed from plasmid pBAD-VAP6his in E. coli TOP10 and purified with affinity chromatography using a Ni-NTA column. SDS-PAGE analysis and Western blotting revealed a
molecular mass of the mature rEmpA predicted to be 36 kDa. The optimal temperature and pH for the purified rEmpA were 37°C
and 8.0, respectively. The enzyme was stable below 30°C and between pH 5.0 and 8.0, respectively. The results show that Ca2+, Na+ and Mg2+ had an activating effect on the enzyme while Zn2+ and Cu2+ acted as inhibitors of the enzyme. The purified rEmpA was characterized as a zinc metalloprotease as it was inhibited by
zinc- and metal-specific inhibitors, such as 1,10-phenanthroline, EDTA and EGTA. The results indicate that some characteristics
of EmpA from marine V. anguillarum had been modified after expression and processing in the engineered E. coli. The purified rEmpA showed degradation activity towards various kinds of proteins, indicating its potential role in pathogenesis. 相似文献
12.
Gram-positive bacteria, notably Bacillus and Streptomyces, have been used extensively in industry. However, these microorganisms have not yet been exploited for the production of the biodegradable polymers, polyhydroxyalkanoates (PHAs). Although PHAs have many potential applications, the cost of production means that medical applications are currently the main area of use. Gram-negative bacteria, currently the only commercial source of PHAs, have lipopolysaccharides (LPS) which co-purify with the PHAs and cause immunogenic reactions. On the other hand, Gram- positive bacteria lack LPS, a positive feature which justifies intensive investigation into their production of PHAs. This review summarizes currently available knowledge on PHA production by Gram- positive bacteria especially Bacillus and Streptomyces. We hope that this will form the basis of further research into developing either or both as a source of PHAs for medical applications. 相似文献
13.
During antioxidant screening using 1,1-diphenyl-picrylhydrazyl (DPPH) and a lipid peroxidation assay, a streptomycete strain
was found to produce herbimycin A and dihydroherbimycin A as antioxidants in the culture filtrate. These molecules were identified
by using spectral analyses, including infrared, ultraviolet, mass spectrum, and nuclear magnetic resonance assays. In the
DPPH radical-scavenging assay, dihydroherbimycin A exhibited more potent antioxidant activity (IC50, 1.3 μM) than α-tocopherol (IC50, 2.7 μM) that was used as a reference compound. In the lipid peroxidation assay, both herbimycin A and dihydroherbimycin
A demonstrated antioxidant activities of 61% and 72%, respectively, at 100 μg/ml, while α-tocopherol exhibited an activity
of 93% at the same concentration. Therefore, dihydroherbimycin A might have the potential to be developed into a new therapeutic
agent. 相似文献
14.
Sun QL Wang LY Shan JJ Jiang R Guo LH Zhang Y Zhang R Li Y 《Archives of microbiology》2007,188(4):333-340
Streptomyces sp. 139 produces a novel exopolysaccharide (EPS) designated Ebosin which has antagonistic activity for IL-1R in vitro and
remarkable anti-rheumatic arthritis activity in vivo. We previously identified a ste (Streptomyces eps) gene cluster consisting of 27 ORFs responsible for Ebosin biosynthesis. The gene product of ste15 shows high homology to known glycosyltransferases (GTFs). To elucidate its function in Ebosin biosynthesis, the ste15 gene was knocked out with a double crossover via homologous recombination. Our analysis of monosaccharide composition for
EPS-m produced by the mutant strain Streptomyces sp. 139 (ste15
−) showed that glucose was significantly diminished compared to its natural counterpart Ebosin. This derivative of Ebosin lost
the antagonistic activity for IL-1R in vitro and its molecular mass was smaller than Ebosin. These results have demonstrated
that the ste15 gene codes for a GTF for glucose, which is functionally involved in Ebosin biosynthesis. 相似文献
15.
Gholizadeh A Kohnehrouz BB Santha IM Lodha ML Kapoor HC 《Biochemistry. Biokhimii?a》2005,70(9):1005-1010
A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ~57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome-inactivating site. We nominate this short cDNA fragment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714. 相似文献
16.
Expression of <Emphasis Type="Italic">Escherichia coli</Emphasis> AppA2 phytase in four yeast systems 总被引:4,自引:0,他引:4
To develop an effective fermentation system for producing Escherichia coliphytase AppA2, we expressed the enzyme in three inducible yeast systems: Saccharomyces cerevisiae (pYES2), Schizosaccharomyces pombe (pDS472a), and Pichia pastoris (pPICZ A), and one constitutive system: P. pastoris (pGAPZA). All four systems produced an extracellular functional AppA2 phytase with apparent molecular masses ranging from 51.5 to 56 kDa. During 8-day batch fermentation in shaking flasks, the inducible Pichia system produced the highest activity (272 units ml–1 medium), whereas the Schizo. pombesystem produced the lowest activity (2.8 units ml–1). The AppA2 phytase expressed in Schizo. pombehad 60–75% lower Kmfor sodium phytate and 28% higher heat-stability at 65 °C than that expressed in other three systems. However, all four recombinant AppA2 phytases had pH optimum at 3.5 and temperature optimum at 55 °C and similar efficacy in hydrolyzing phytate–phosphate from soybean meal.Revisions requested 18 November 2004; Revisions received 7 January 2005 相似文献
17.
Lee JH Moon YH Kim N Kim YM Kang HK Jung JY Abada E Kang SS Kim D 《Biotechnology letters》2008,30(4):749-754
The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K
m of 3 mM with sucrose as a substrate; optimum activity was at 37°C and pH 6.7. The purified 742SPase transferred the glucosyl
moiety of sucrose to cytosine monophosphate (CMP).
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
18.
H. M. El Shafey S. Ghanem A. Guyonvarch 《World journal of microbiology & biotechnology》2009,25(3):367-373
Nucleotide and amino acid sequences of Corynebacterium glutamicum recA genes, from GenBank, were compared in silico. On the basis of the identity found between sequences, two degenerate primers were designed on the two sides of the deduced open reading frame (ORF) of the recA gene. PCR experiments, for amplifying the recA ORF region, were done. pGEM®-T Easy vector was selected to be used for cloning PCR products. Then recA ORF was placed under the control of Escherichia coli hybrid trc promoter, in pKK388-1 vector. pKK388-1 vector, containing recA ORF, was transformed to E. coli DH5α ΔrecA (recombinant deficient strain), in an attempt to phenotypically complement it. Ultraviolet (u.v.) exposure experiments of the transformed and non-transformed E. coli DH5α ΔrecA cells revealed tolerance of transformed cells up to dose 0.24 J/cm2, while non-transformed cells tolerated only up to dose 0.08 J/cm2. It is concluded that phenotypic complementation of E. coli DH5α ΔrecA with recA ORF of C. glutamicum, could be achieved and RecA activity could be restored. 相似文献
19.
20.
Yoshimune K Ninomiya Y Wakayama M Moriguchi M 《Journal of industrial microbiology & biotechnology》2004,31(9):421-426
The overproduction of d-aminoacylase (d-ANase, 233.8 U/mg), N-acyl-d-glutamate amidohydrolase (d-AGase, 38.1 U/mg) or N-acyl-d-aspartate amidohydrolase (d-AAase, 6.2 U/mg) in Escherichia coli is accompanied by aggregation of the overproduced protein. To facilitate the expression of active enzymes, the molecular chaperones GroEL-GroES (GroELS), DnaK-DnaJ-GrpE (DnaKJE), trigger factor (TF), GroELS and DnaKJE or GroELS and TF were coexpressed with the enzymes. d-ANase (313.3 U/mg) and d-AGase (95.8 U/mg) were overproduced in an active form at levels 1.3- and 1.8-fold higher, respectively, upon co-expression of GroELS and TF. An E. coli strain expressing the d-AAase gene simultaneously with the TF gene exhibited a 4.3-fold enhancement in d-AAase activity (32.0 U/mg) compared with control E. coli expressing the d-AAase gene alone. 相似文献