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1.
Elsa Pimienta Julio C Ayala Caridad Rodríguez Astrid Ramos Lieve Van Mellaert Carlos Vallín Jozef Anné 《Microbial cell factories》2007,6(1):20
Background
Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat) pathway. 相似文献2.
Noda S Miyazaki T Miyoshi T Miyake M Okai N Tanaka T Ogino C Kondo A 《Journal of industrial microbiology & biotechnology》2011,38(5):643-648
Cinnamic acid production was demonstrated using Streptomyces as a host. A gene encoding phenylalanine ammonia lyase (PAL) from Streptomyces maritimus was introduced into Streptomyces lividans, and its expression was confirmed by Western blot analysis. After 4 days cultivation using glucose as carbon source, the
maximal level of cinnamic acid reached 210 mg/L. When glycerol (30 g/L) was used as carbon source, the maximal level of produced
cinnamic acid reached 450 mg/L. In addition, using raw starch, xylose or xylan as carbon source, the maximal level of cinnamic
acid reached 460, 300, and 130 mg/L, respectively. We demonstrated that S. lividans has great potential to produce cinnamic acid as well as other aromatic compounds. 相似文献
3.
Díaz M Adham SA Ramón D Gil JA Santamaría RI 《Applied microbiology and biotechnology》2004,65(4):401-406
The Aspergillus nidulans gene xlnA coding for the fungal xylanase X22 has been cloned and expressed in two heterologous bacterial hosts: Streptomyces lividans and Brevibacterium lactofermentum. Streptomyces strains yielded 10 units/ml of xylanase when the protein was produced with its own signal peptide, and 19 units/ml when its signal peptide was replaced by the one for xylanase Xys1 from Streptomyces halstedii. B. lactofermentum was also able to produce xylanase X22, affording 6 units/ml upon using either the Aspergillus xlnA signal peptide or Streptomyces xysA. These production values are higher than those previously reported for the heterologous expression of the A. nidulans xlnA gene in Saccharomyces cerevisiae (1 unit/ml). Moreover, the X22 enzyme produced by Streptomyces lividans showed oenological properties, indicating that this Streptomyces recombinant strain is a good candidate for the production of this enzyme at the industrial scale. 相似文献
4.
Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product
formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached
to the benzene ring and an electron donor, e.g., OH or NH2, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring.
Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including
HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K
M
values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K
cat/K
M
) were determined in the range of 430–1,110 s−1·M−1 with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to
C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan
synthase 7-DMATS from Aspergillus fumigatus. 相似文献
5.
Hai-Nan Su Bin-Bin Xie Xiu-Lan Chen Jin-Xia Wang Xi-Ying Zhang Bai-Cheng Zhou Yu-Zhong Zhang 《Journal of applied phycology》2010,22(1):65-70
Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple
and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding
hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed
by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude
phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0).
In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A
650/A
280 of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g−1 wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from
5.6 to 4.0. The absorbance ratio A
650/A
280 of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g−1 wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE. 相似文献
6.
A genetic transformation system has been developed for callus cells of Crataegus
aronia using Agrobacterium
tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with
5 mg l−1 Indole-3-butyric acid (IBA) and 0.5 mg l−1 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different
types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red
colored callus was obtained on MS medium supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l−1 kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli
were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l−1 hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this
is the first time to report an Agrobacterium-mediated transformation system in Crataegus
aronia. 相似文献
7.
The thermal and alkaline pH stability of Streptomyces lividans xylanase B was improved greatly by random mutagenesis using DNA shuffling. Positive clones with improved thermal stability
in an alkaline buffer were screened on a solid agar plate containing RBB-xylan (blue). Three rounds of directed evolution
resulted in the best mutant enzyme 3SlxB6 with a significantly improved stability. The recombinant enzyme exhibited significant
thermostability at 70°C for 360 min, while the wild-type lost 50% of its activity after only 3 min. In addition, mutant enzyme
3SlxB6 shows increased stability to treatment with pH 9.0 alkaline buffer. The K
m value of 3SlxB6 was estimated to be similar to that of wild-type enzyme; however k
cat was slightly decreased, leading to a slightly reduced value of k
cat/K
m, compared with wild-type enzyme. DNA sequence analysis revealed that eight amino acid residues were changed in 3SlxB6 and
substitutions included V3A, T6S, S23A, Q24P, M31L, S33P, G65A, and N93S. The stabilizing effects of each amino acid residue
were investigated by incorporating mutations individually into wild-type enzyme. Our results suggest that DNA shuffling is
an effective approach for simultaneous improvement of thermal and alkaline pH stability of Streptomyces lividans xylanase B even without structural information. 相似文献
8.
Fawzeia H. Elmhalli Katinka Pålsson Jan Örberg Thomas G. T. Jaenson 《Experimental & applied acarology》2009,48(3):251-262
The toxicity of para-menthane-3,8-diol (PMD), the main arthropod-repellent compound in the oil of the lemon eucalyptus, Corymbia citriodora, was evaluated against nymphs of Ixodes ricinus using five methods (A–E) of a contact toxicity bioassay. Mortality rates were estimated by recording numbers of dead nymphs
at 30 min intervals during the first 5 h after the start of exposure and at longer intervals thereafter. The mortality rate
increased with increasing concentration of PMD and duration of exposure with a distinct effect after 3.5 h. From the results
obtained by methods A, C and E, the LC50 range was 0.035–0.037 mg PMD/cm2 and the LC95 range was 0.095–0.097 mg PMD/cm2 at 4 h of exposure; the LT50 range was 2.1–2.8 h and the LT95 range was 3.9–4.2 h at 0.1 mg PMD/cm2. To determine the duration of toxic activity of PMD, different concentrations (0.002, 0.01, 0.1 mg PMD/cm2) were tested and mortality was recorded at each concentration after 1 h; thereafter new ticks were tested. This test revealed
that the lethal activity of PMD remained for 24 h but appeared absent after 48 h. The overall results show that PMD is toxic
to nymphs of I. ricinus and may be useful for tick control. 相似文献
9.
Nguyen VN Oh IJ Kim YJ Kim KY Kim YC Park RD 《Journal of industrial microbiology & biotechnology》2009,36(2):195-203
Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography.
The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and
showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino
acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe
and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46
were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag+ and Hg2+ while Chi46 by Hg2+ and Pb2+ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co2+. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc)
n
, n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase. 相似文献
10.
Zhang L Zhang L Han X Du M Zhang Y Feng Z Yi H Zhang Y 《Applied microbiology and biotechnology》2012,93(6):2335-2343
In this study, we first tested the capacity for eight different salts as stress-mediated bioprocesses in the production of
transglutaminase (TGase). A significant effect on the cell growth and TGase production was obtained with the highest yield
of TGase being observed at 96 h of incubation (4.3 U/ml) when the basic medium was supplemented 0.10 M MgCl2, as opposed to that observed with the basic medium control (2.1 U/ml at 120 h). Data from Western blot assays showed that
transformation of pro-TGase to its mature enzyme occurred more rapidly in MgCl2 medium. Furthermore, total protease, metalloprotease, and serine protease were also synthesized at a faster rate in the medium
containing MgCl2. The results demonstrate that MgCl2 enhanced the production of key proteases involved in the activation of TGase biosynthesis. To explore the mechanism, viability
assay was performed. The results show that MgCl2 induced the mycelia differentiation, decreased cell growth rate, and stimulated cell death. We argue that TGase production
was promoted by the stimulation of mycelium differentiation induced by MgCl2 stress. 相似文献
11.
Talita A. Sampaio e Silva Adriana Knob Célia R. Tremacoldi Marcia R. Brochetto-Braga Eleonora Cano Carmona 《World journal of microbiology & biotechnology》2011,27(11):2491-2497
Acid proteases represent an important group of enzymes, widely used in food, beverage and pharmaceutical industries. For most
of these applications the enzymatic preparation must be at least partially purified and free of substances that could change
the characteristics of the product or the process. Fungal proteases have replaced other sources because they are easily obtained
mainly from Mucor, Rhizopus, Penicillium
and
Aspergillus species. A strain of Aspergillus
clavatus was selected by producing high level of acid protease activity. An extracellular aspartatic protease from this strain was
purified 37.2 times with 37% recovery using (NH4)2SO4 fractionation and ion-exchange chromatography. The enzyme was found to be monomeric having a molecular mass of 30.4 kDa.
The purified enzyme is an acid protease with optimum pH of 5.5 and temperature for optimum activity of 50 °C. Its high pH
stability was verified in the range of 3.5–6.5. The acid protease was strongly inhibited by Hg+2 and partially inhibited by Cu+2, Zn+2 and Mn+2. The enzyme was sensitive to denaturing agent SDS and activated by thiol-containing reducing agent dithiotreitol (DTT). The
protease activity was not influenced by iodoacetic acid, E-64 and PMSF, while it was lightly actived by EDTA and totally inhibited
by pepstatin, with a Ki of 7.8 μM, indicating that is an aspartic protease. A.
clavatus acid protease presents interesting characteristics for biotechnological process, such as cheese and flavor manufacture and
dietary supplements, in which activity and stability in acid pH are required. 相似文献
12.
MA Nakisah MY Ida Muryany H Fatimah R Nor Fadilah MR Zalilawati S Khamsah M Habsah 《World journal of microbiology & biotechnology》2012,28(3):1237-1244
Crude methanol extracts of a marine sponge, Aaptos
aaptos, collected from three different localities namely Kapas, Perhentian and Redang Islands, Terengganu, Malaysia, were tested
in vitro on a pathogenic Acanthamoeba castellanii (IMR isolate) to examine their anti-amoebic potential. The examination of anti-Acanthamoebic activity of the extracts was
conducted in 24 well plates for 72 h at 30 °C. All extracts possessed anti-amoebic activity with their IC50 values ranging from 0.615 to 0.876 mg/mL. The effect of the methanol extracts on the surface morphology of A. castellanii was analysed under scanning electron microscopy. The ability of the extracts to disrupt the amoeba cell membrane was indicated
by extensive cell’s blebbing, changes in the surface morphology, reduced in cell size and with cystic appearance of extract-treated
Acanthamoeba. Number of acanthapodia and food cup was also reduced in this Acanthamoeba. Morphological criteria of apoptosis in Acanthamoeba following treatment with the sponge’s extracts was determined by acridine orange-propidium iodide staining and observed by
fluorescence microscopy. By this technique, apoptotic and necrotic cells can be visualized and quantified. The genotoxic potential
of the methanol extracts was performed by the alkaline comet assay. All methanol extracts used were significantly induced
DNA damage compared to untreated Acanthamoeba by having high percentage of scores 1, 2, and 3 of the DNA damage. Results from cytotoxicity and genotoxicity studies carried
out in the present study suggest that all methanol extracts of A. aaptos have anti-amoebic properties against A. castellanii. 相似文献
13.
Cytochrome c
6
, (cyt c
6) a soluble monoheme electron transport protein, was isolated and characterized from the chlorophyll d-containing cyanobacterium Acaryochoris marina, the type strain MBIC11017. The protein was purified using ammonium sulfate precipitation, ion exchange and gel filtration
column chromatography, and fast performance liquid chromatography. Its molecular mass and pI have been determined to be 8.87 kDa
and less than 4.2, respectively, by mass spectrometry and isoelectrofocusing (IEF). The protein has an alpha helical structure
as indicated by CD (circular dichroism) spectroscopy and a reduction midpoint potential (E
m) of +327 mV versus the normal hydrogen electrode (NHE) as determined by redox potentiometry. Its potential role in electron
transfer processes is discussed. 相似文献
14.
15.
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
16.
Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure l-lactic acid from both hexose and pentose sugars including l-arabinose with high yield, titer and productivity under thermophilic conditions. The l-arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant
L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated
to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn2+ was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic
pH than at alkaline pH. The kinetic studies showed that the K
m, V
max and k
cat/K
m for the conversion of l-arabinose were 106 mM, 84 U/mg and 34.5 mM−1min−1, respectively. The equilibrium ratio of l-arabinose to l-ribulose was 78:22 under optimal conditions. l-ribulose (97 g/L) was obtained from 500 g/l of l-arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion
of 19.4% and a production rate of 65 g L−1 h−1. 相似文献
17.
18.
Seven lipolytic genes were isolated and sequenced from a metagenomic library that was constructed following biomass enrichment
in a fed-batch bioreactor submitted to high temperature (50–70°C) and alkaline pH (7–8.5). Among those sequences, lipIAF1-6 was chosen for further study and cloned in Streptomyces lividans 10–164. The G+C content within the sequence was 64.3%. The encoded protein, LipIAF1-6, was related to various putative lipases
previously identified in different genome sequences. Homology of LipIAF-6 with the different lipases did not exceed 31%. The
optimum pH (8.5) and temperature (60°C) of the purified enzyme were in agreement with the enrichment conditions. Furthermore,
the enzyme was thermostable for as long as 30 min at 70°C. The maximum activity of the purified lipase was 4,287 IU/mg towards
p-nitrophenyl (p-NP) butyrate (60°C; pH 8.5). LipIAF1-6 does not seem to need the presence of metal ions for its activity. The enzyme was
slightly inhibited by 10 mM CoCl2 (14%), HgCl2 (12%), and dithiothreitol (DTT) (15%). The serine protease inhibitor phenylmethylsulphonyl fluoride (PMSF) reduced activity
by 39% and 71% when incubated at concentrations of 1 and 10 mM, respectively. Finally, LipIAF1-6 was stable in different organic
solvents, and against several surfactants and oxidative agents commonly found in detergent formulations. These results are
quite encouraging for further use of this enzyme in different industrial processes. 相似文献
19.
Thuy ML Kharel MK Lamichhane R Lee HC Suh JW Liou K Sohng JK 《Biotechnology letters》2005,27(7):465-470
An actinomycetes expression vector (pIBR25) was constructed and applied to express a gene from the kanamycin biosynthetic gene cluster encoding 2-deoxy-scyllo-inosose synthase (kanA) in Streptomyces lividans TK24. The expression of kanA in pIBR25 transformants reached a maximum after 72 h of culture. The plasmid pIBR25 showed better expression than pSET152, and resulted in the formation of insoluble KanA when it was expressed in Escherichia coli. This strategy thus provides a valuable tool for expressing aminoglycoside-aminocyclitols (AmAcs) biosynthetic genes in Streptomyces spp. 相似文献
20.
An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The Km and Vmax values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg−1 protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity
was strongly inhibited by Cu2+ and Hg2+ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose. 相似文献