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1.
An amylase gene from Drosophila pseudoobscura was isolated from a genomic library constructed in pBR322 and cloned in Escherichia coli by selecting for the ability of its product to hydrolyze starch, a carbon source not normally utilized by E. coli. Hybridization of pAMY17F to D. pseudoobscura polytene chromosomes shows a positive signal at the amylase pseudogene locus (bank 78, chromosome 3). The chimeric plasmid pAMY17F, has been altered in such a way as to increase amylase expression. Southern and Northern hybridizations to the cloned amylase DNA indicate that the source of the gene is from D. pseudoobscura. Biochemical properties such as pH optima, substrate specificities, electrophoretic analyses, inhibitor sensitivities, heat stabilities, temperature responsiveness and molecular weights indicate that the amylases produced by the fly and bacterial clone are similar and have similar properties. It appears that E. coli/pAMY17F is producing an amylase like that found in D. pseudoobscura. 相似文献
2.
Nataša Božić Juan-Miguel Puertas Nikola Lončar Cristina Sans Duran Josep López-Santín Zoran Vujčić 《Process Biochemistry》2013,48(3):438-442
In this study, a new approach for extracellular production of recombinant α-amylase in Escherichia coli was investigated. A gene encoding a highly efficient raw-starch-digesting α-amylase from Bacillus licheniformis ATCC 9945a was cloned and expressed in E. coli. The gene encoding mature α-amylase was cloned into the pDAss expression vector, and secretion of the gene product was regulated by fusion to the signal peptide of DsbA, a well-characterized E. coli periplasmic protein. E. coli BL21 (DE3) carrying pDAss vector containing amylase gene had approximately 2.5-fold higher volumetric enzyme productivity than the natural system. The recombinant enzyme showed higher efficiency for digesting diverse raw starches when compared with the native enzyme and was similar to commercial α-amylase in its ability to hydrolyze raw starches. The properties of the recombinant enzyme demonstrate the potential of the DsbA signal peptide approach for the secretory production of the fully active, industrially important recombinant enzyme. 相似文献
3.
An alpha-amylase gene from Bacillus coagulans has previously been cloned in Escherichia coli and shown to direct the synthesis of an enzymically active protein of 60,000 Dal (Cornelis et al., 1982). In one particular E. coli host, strain HB101, amylase was found to accumulate in the periplasmic space. To study the processing and the location of the amylase, plasmid pAMY2 was introduced into E. coli 188 which is a strain constitutive for alkaline phosphatase, a periplasmic marker, and for beta-galactosidase, a cytoplasmic marker. Abnormally large amounts of both alpha-amylase and beta-galactosidase were found in the culture fluid of cells grown in rich medium. Furthermore a severe growth defect was found when cells containing pAMY2 were grown in maltose and glycerol media, while the ability to grow on glucose remained normal. This defect could be reversed by two types of spontaneous mutations. Mutations in the first class are located on the plasmid and correspond to the insertional inactivation of the amylase gene by IS1. Mutations in the second class are located on the host chromosome. These results suggest that the synthesis and export of B. coagulans alpha-amylase is deleterious to E. coli, especially in media containing maltose or glycerol as sole carbon source. 相似文献
4.
Cloning of a Gene Encoding Raw-Starch-Digesting Amylase from a Cytophaga sp. and Its Expression in Escherichia coli
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Chii-Ling Jeang Li-Shien Chen Ming-Yu Chen Rong-Jen Shiau 《Applied microbiology》2002,68(7):3651-3654
A raw-starch-digesting amylase (RSDA) gene from a Cytophaga sp. was cloned and sequenced. The predicted protein product contained 519 amino acids and had high amino acid identity to α-amylases from three Bacillus species. Only one of the Bacillus α-amylases has raw-starch-digesting capability, however. The RSDA, expressed in Escherichia coli, had properties similar to those of the enzyme purified from the Cytophaga sp. 相似文献
5.
Expression of 3-Ketoacyl-Acyl Carrier Protein Reductase (fabG) Genes Enhances Production of Polyhydroxyalkanoate Copolymer from Glucose in Recombinant Escherichia coli JM109
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Christopher T. Nomura Kazunori Taguchi Zhihua Gan Kazuhiro Kuwabara Tomoyo Tanaka Kazuma Takase Yoshiharu Doi 《Applied microbiology》2005,71(8):4297-4306
Polyhydroxyalkanoates (PHAs) are biologically produced polyesters that have potential application as biodegradable plastics. Especially important are the short-chain-length-medium-chain-length (SCL-MCL) PHA copolymers, which have properties ranging from thermoplastic to elastomeric, depending on the ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of applications for SCL-MCL PHA copolymers, it is important to develop and characterize metabolic pathways for SCL-MCL PHA production. In previous studies, coexpression of PHA synthase genes and the 3-ketoacyl-acyl carrier protein reductase gene (fabG) in recombinant Escherichia coli has been shown to enhance PHA production from related carbon sources such as fatty acids. In this study, a new fabG gene from Pseudomonas sp. 61-3 was cloned and its gene product characterized. Results indicate that the Pseudomonas sp. 61-3 and E. coli FabG proteins have different substrate specificities in vitro. The current study also presents the first evidence that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes can enhance the production of SCL-MCL PHA copolymers from nonrelated carbon sources. Differences in the substrate specificities of the FabG proteins were reflected in the monomer composition of the polymers produced by recombinant E. coli. SCL-MCL PHA copolymer isolated from a recombinant E. coli strain had improved physical properties compared to the SCL homopolymer poly-3-hydroxybutyrate. This study defines a pathway to produce SCL-MCL PHA copolymer from the fatty acid biosynthesis that may impact on PHA production in recombinant organisms. 相似文献
6.
The gene (tfa), encoding a maltotriose-producing α-amylase from Thermobifida fusca NTU22, was cloned, sequenced and expressed in Escherichia coli. The gene consists of 1,815 base pairs and encodes a protein of 605 amino acids. The base composition of the tfa coding sequence is 69% G+C and the protein has a predicted pI value of 5.5. The deduced amino acid sequence of the tfa amylase exhibited a high degree of similarity with amylases from Thermomonospora curvata and Streptomyces amylases. The purified amylase could be detected as a single band of about 65 kDa by SDS-polyacrylamide gel electrophoresis
and this agrees with the predicted size based on the nucleotide sequence. The optimal pH and temperature of the purified amylase
were 7.0 and 60°C, respectively. The properties of purified amylase from the E. coli transformant are similar to that of an amylase purified from the original T. fusca NTU22. 相似文献
7.
Eiko Matsumura Satoshi Yomoda Eiko Yamamoto Tatsu Kawano Takashi Shin Sawao Murao 《Bioscience, biotechnology, and biochemistry》2013,77(10):2675-2677
The β-1,3-glucanase (1,3-β-d-glucan glucanohydrolase, EC 3.2.1.6) gene from Flavobacterium dormitator var. glucanolyticae was cloned into Escherichia coli C600 with a vector plasmid, pBR322. The E. coli cells carrying a recombinant plasmid, pKUβG1 (8.2 kb), showed a high β-1,3-glucanase activity and a lytic activity on viable yeast cells. These activities were found in the peripiasmic space of E. coli clone cells. Southern hybridization analysis showed that the cloned gene was derived from F. dormitator chromosomal DNA. The gene products were purified from the periplasmic fraction of E. coli by ammonium sulfate fractionation and ion-exchange chromatography. The purified enzymes were demonstrated to be identical with a lytic endo-β-1,3-glucanase II and a nonlytic endo-β-1,3-glucanase I from F. dormitator from their enzymological and immunological properties. In the E. coli cells, endo-β-1,3-glucanase I was also formed by a proteolytic digestion of endo-β-1,3-glucanase II during the cultivation as in F. dormitator. Thus, the only endo-β-1,3-glucanase II was coded for in the cloned gene. 相似文献
8.
The gaf fimbrial gene cluster of Escherichia coli expresses a full-size and a truncated soluble adhesin protein
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Tanskanen J Saarela S Tankka S Kalkkinen N Rhen M Korhonen TK Westerlund-Wikström B 《Journal of bacteriology》2001,183(2):512-519
The GafD lectin of the G (F17) fimbriae of diarrhea-associated Escherichia coli was overexpressed and purified from the periplasm of E. coli by affinity chromatography on GlcNAc-agarose. The predicted mature GafD peptide comprises 321 amino acids, but the predominant form of GafD recovered from the periplasm was 19,092 Da in size and corresponded to the 178 N-terminal amino acid residues, as judged by mass spectrometry and amino acid sequencing, and was named ΔGafD. Expression of gafD from the cloned gaf gene cluster in DegP-, Lon-, and OmpT-deficient recombinant strains did not significantly decrease the formation of ΔGafD. The peptide was also detected in the periplasm of the wild-type E. coli strain from which the gaf gene cluster originally was cloned. We expressed gafD fragments encoding C-terminally truncated peptides. Peptides GafD1-252, GafD1-224, GafD1-189, and the GafD1-178, isolated from the periplasm by affinity chromatography, had apparent sizes closely similar to that of ΔGafD. Only trace amounts of truncated forms with expected molecular sizes were detected in spheroplasts. In contrast, the shorter GafD1-157 peptide was detected in spheroplasts but not in the periplasm, indicating that it was poorly translocated or was degraded by periplasmic proteases. Pulse-chase assays using gafD indicated that ΔGafD was processed from GafD and is not a primary translation product. The ΔGafD peptide was soluble by biochemical criteria and exhibited specific binding to GlcNAc-agarose. Inhibition assays with mono- and oligosaccharides gave a similar inhibition pattern in the hemagglutination by the G-fimbria-expressing recombinant E. coli strain and in the binding of [14C]ΔGafD to GlcNAc-agarose. ΔGafD bound specifically to laminin, a previously described tissue target for the G fimbria. Our results show that a soluble, protease-resistant subdomain of GafD exhibits receptor-binding specificity similar to that for intact G fimbriae and that it is formed when gafD is expressed alone or from the gaf gene cluster. 相似文献
9.
Molecular Cloning and Characterization of the Gene Coding for the Aerobic Azoreductase from Xenophilus azovorans KF46F 总被引:1,自引:0,他引:1
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The gene coding for an aerobic azoreductase was cloned from Xenophilus azovorans KF46F (formerly Pseudomonas sp. strain KF46F), which was previously shown to grow with the carboxylated azo compound 1-(4′-carboxyphenylazo)-2-naphthol (carboxy-Orange II) as the sole source of carbon and energy. The deduced amino acid sequence encoded a protein with a molecular weight of 30,278 and showed no significant homology to amino acid sequences currently deposited at the relevant data bases. A presumed NAD(P)H-binding site was identified in the amino-terminal region of the azoreductase. The enzyme was heterologously expressed in Escherichia coli and the azoreductase activities of resting cells and cell extracts were compared. The results suggested that whole cells of the recombinant E. coli strains were unable to take up sulfonated azo dyes and therefore did not show in vivo azoreductase activity. The turnover of several industrially relevant azo dyes by cell extracts from the recombinant E. coli strain was demonstrated. 相似文献
10.
Dosage Compensation of Genes on the Left and Right Arms of the X Chromosome of DROSOPHILA PSEUDOOBSCURA and DROSOPHILA WILLISTONI 总被引:5,自引:1,他引:4
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We have investigated the occurrence of dosage compensation in D. willistoni and D. pseudoobscura, two species whose X chromosome is metacentric with one arm homologous to the X and the other homologous to the left arm of chromosome 3 of D. melanogaster. Crude extracts were assayed for isocitrate dehydrogenase (XR), glucose-6-phosphate dehydrogenase (XL?), 6-phosphogluconate dehydrogenase (XL?), and α-glycerophosphate dehydrogenase (chromosome 2) in D. willistoni, and for esterase-5 (XR), glucose-6-phosphate dehydrogenase (XL?), 6-phosphogluconate dehydrogenase (XL?) and amylase (chromosome 3) in D. pseudoobscura. Our results indicate that a mechanism for dosage compensation is operative in both arms of the X chromosome of these two species. 相似文献
11.
Genetic Factors Affecting Recovery of Nonpoint Mutations in the Region of a Gene Coding for Ornithine Transcarbamylase: Involvement of Both the F Factor in Its Chromosomal State and the recA Gene 总被引:1,自引:0,他引:1
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Mutants of E. coli K12 that overproduce ornithine transcarbamylase can be identified in Car- strains because they permit utilization of citrulline as a carbamyl phosphate source, due to reversal of the normal OTCase reaction; they are called Cut mutants (citrulline utilizers). Hfr strains that carry the F factor adjacent to argF (one of two duplicate genes that code for ornithine transcarbamylase in E. coli K12) yield more Cut mutants than do F+ or F- strains, or Hfr strains in which the F factor is not adjacent to argF. When Hfr strains in which the F factor is integrated adjacent to argF are made recA, they yield few Cut mutants. Many of the Cut mutants recovered from one of the Hfr strains used in the investigation (Hfr P4X) are unstable; the properties of these unstable mutations suggest that they carry aberrations in the region of the argF gene. Thus, the increased yields of Cut mutants probably result from aberrations that occur when the F factor is integrated adjacent to argF. The nature of these aberrations is not yet known. The unstable Cut mutants are to a large extent stabilized by recA; such stabilization is one of the properties of duplications. Other data indicate that the aberrations may be more complex than simple gene duplications; in particular properties of segregants and some recombinants derived from unstable Cut mutants are most easily interpreted by assuming that segregation from, and possibly formation of, the unstable mutants occurs in several stages. 相似文献
12.
Population genetics of Drosophila amylase. V. Genetic background and selection on different carbohydrates
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Frequency changes in amylase allozymes and patterns of tissue-specific expression of amylase have been monitored in laboratory populations of Drosophila pseudoobscura maintained on media in which the only carbohydrate source was maltose or starch. Nonrandom changes occurred in patterns of expression, whereas no patterns in allozyme frequency changes were discernible. The nature of the pattern changes was similar to an identical study done on populations derived from a natural population several hundred miles from the population used in the present experiments. However, in the previous study nonrandom changes in allozyme frequencies were also noted. Evidently, selection on the Drosophila amylase system differs depending upon the genetic background of the population. Furthermore, the evolutionary dynamics of structural gene variants and those regions controlling its expression may be independent, a result consistent with DNA sequence data. 相似文献
13.
Sarika Sharma Farrah Gul Khan Ghulam Nabi Qazi 《Applied microbiology and biotechnology》2010,86(6):1821-1828
The increasing demand for novel biocatalysts stimulates exploration of resources from soil. Metagenomics, a culture independent
approach, represent a sheer unlimited resource for discovery of novel biocatalysts from uncultured microorganisms. In this
study, a soil-derived metagenomic library containing 90,700 recombinants was constructed and screened for lipase, cellulase,
protease and amylase activity. A gene (pAMY) of 909 bp encoding for amylase was found after the screening of 35,000 Escherichia coli clones. Amino acid sequence comparison and phylogenetic analysis indicated that pAMY was closely related to uncultured bacteria. The molecular mass of pAMY was estimated about 38 kDa by sodium dodecyl sulphate
polyacrylamide gel electrophoresis. Amylase activity was determined using soluble starch, amylose, glycogen and maltose as
substrates. The maximal activity (2.46 U/mg) was observed at 40 °C under nearly neutral pH conditions with amylose; whereas
it retains 90% of its activity at low temperature with all the substrates used in this study. The ability of pAMY to work
at low temperature is unique for amylases reported so far from microbes of cultured and uncultured division. 相似文献
14.
The ftsZ gene was cloned from the chromosomal DNA of Brevibacterium lactofermentum by the polymerase chain reaction (PCR) using two oligonucleotides designed from two conserved regions found in most of the previously cloned and sequenced ftsZ genes from other microorganisms. ftsZ is a single-copy gene in corynebacteria and is located downstream from ftsQ and murC, indicating linkage between genes involved in peptidoglycan synthesis (mur genes) and genes involved in cell division (fts genes). The organisation of the cluster is similar to that in Streptomyces and different from those of Escherichia coli or Bacillus subtilis because ftsA is not located upstream of ftsZ. The gene was expressed in E. coli using the T7 expression system; the calculated molecular weight of the expressed protein was 50?kDa. Expression of the B. lactofermentum ftsZ gene in E. coli inhibited cell division and led to filamentation. The ftsZ gene of this organism does not complement ftsZ mutations or deletions in E. coli, when cloned on low or high-copy-number vectors. 相似文献
15.
Nguyen Hoang Loc Le My Tieu Ngoc Tran Thuy Lan Le Quoc Viet Le Duc Thao Hoang Tan Quang Dinh Thi Bich Lan Phung Thang Long 《Indian journal of microbiology》2013,53(4):488-491
We cloned two genes coding F107-C and K88-1NT fimbrial subunits from strains E. coli C and 1NT isolated from Thua Thien Hue province, Vietnam. The mature peptide of faeG gene from strain E. coli 1NT (called faeG-1NT) is 100 % similarity with faeG gene, while the CDS of fedA gene from strain C (called fedA-C) has a similarity of 97 % with the fedA gene. Expression of the faeG-1NT and fedA-C genes in E. coli BL21 Star™ (DE3) produced proteins of ~31 and 22 kDa, respectively. The effect of IPTG concentration on the K88-1NT and F107-C fimbriae production was investigated. The results showed that 0.5 mM IPTG is suitable for higher expression of K88-1NT subunit, while 0.75 mM IPTG strongly stimulated expression of F107-C subunit. The optimal induction time for expression was also examined. Generally, highest expression of K88-1NT subunit occurred after 6 h of induction, while that of F107-C subunit is after 14 h. 相似文献
16.
In the present study, we identified, cloned and expressed a 40-kDa heat shock protein, DnaJ, from Bacillus halodurans. The open reading frame of the cloned gene contained 1116 bp and encoded 371 amino acid residues. The purified recombinant DnaJ contained a His-tag at the C-terminus and showed a single band at approximately 41-kDa on SDS-PAGE gel. The 3D structures of DnaJ obtained by I-TASSER showed that the overall structures of DnaJ from B. halodurans Guj1 and E. coli are very similar, with 45% sequence similarity. The present study revealed that the DnaJ protein from B. halodurans inhibits the heat-induced aggregation of insulin in a concentration-dependent manner as aggregation of the insulin B-chain was reduced by approximately 50% at 40 °C in the presence of 0.1 mg/ml of purified recombinant DnaJ. The overexpression of DnaJ improved thermotolerance properties in E. coli transformed with pET-28a + DnaJ. Salt resistance experiments indicated that the survival of E. coli transformed with DnaJ was enhanced 1.85-fold compared to that of the control cells in the presence of 0.5 M NaCl for 72 h. According to the results obtained, DnaJ from B. halodurans can potentially be used for improving the functional properties of enzymes and proteins in various applications. 相似文献
17.
The gene specifying a sequence-specific modification methylase of Bacillus centrosporus has been cloned in Escherichia coli using the restriction endonuclease HindIII and the plasmid pBR322. The selection was based on detection of new methylation properties rendering recombinant plasmids carrying the methylase gene nonsusceptible to BcnI endonuclease cleavage. The presence of a 3.2-kb HindIII fragment in either orientation conferred BcnI resistance on the recombinant plasmids. These results suggest that the BcnI methylase gene is expressed in E. coli under the control of a promoter located on the cloned fragment. The relative level of BcnI methylase enzyme in E. coli was similar to that in B. centrosporus. The recombinant clones do not exhibit any BcnI restriction-endonuclease activity. 相似文献
18.
《Journal of Fermentation and Bioengineering》1990,69(4):195-198
The genes coding for the GGPyPuCC-specific (BanI) and ATCGAT-specific (BanIII) restriction-modification systems of Bacillus aneurinolyticus IAM1077 were cloned and expressed in Escherichia coli using pBR322 as a vector. The plasmids carrying the BanI and BanIII restriction-modification genes were designated pBanIRM8 and pBanIIIRM12, respectively. The restriction maps of these recombinant plasmids were constructed. These two plasmids were stably maintained in E. coli HB101. However, when E. coli JM109 was used as a host, pBanIIIRM12 was efficiently propagated but pBanIRM8 was not. The HB101 cells carrying only the restriction gene of BanIII were viable, but the BanI restriction gene carrier could not form colonies on agar plates. The growth of bacteriophage λ was strongly restricted only in the F. coli HB101 cells harboring pBanIRM8. These facts indicate that the BanI restriction enzyme is expressed and functions more efficiently than BanI modification enzyme in E. coli. 相似文献
19.
Simon Vroemen Jacco Heldens Carol Boyd Bernard Henrissat Noel T. Keen 《Molecular genetics and genomics : MGG》1995,246(4):465-477
A β-glucosidase/xylosidase gene from Erwinia chrysanthemi strain D1 was cloned and sequenced. This gene, named bgxA, encodes a ca. 71 kDa protein product which, following removal of the leader peptide, resulted in a ca. 69 kDa mature protein that accumulated in the periplasmic space of E. chrysanthemi strain D1 and Escherichia coli cells expressing the cloned gene. The protein exhibited both β-glucosidase and β-xylosidase activities but gave no detectable activity on xylan or carboxymethyl cellulose. The enzyme was classified as a type 3 glycosyl hydrolase, but was unusual in having a truncated B region at the carboxyl-terminus. Several E. chrysanthemi strains isolated from corn produced the glucosidase/xylosidase activity but not those isolated from dicot plants. However, bgxA marker exchange mutants of strain D1 were not detectably altered in virulence on corn leaves. 相似文献
20.
Cloning and characterization of a putative UDP-rhamnose synthase 1 from Populus euramericana Guinier
L-Rhamnose is a constituent of plant primary cell wall polysaccharides including rhamnogalacturonan-I, rhamnogalacturonan-II, and other natural plant-based compounds. UDP-rhamnose serves as a rhamnose donor whose synthesis is catalyzed by UDP-rhamnose synthase (RHM). A RHM gene, PRHM was cloned from Populus euramericana Guinier. PRHM contains two domains: the NAD dependent epimerase/dehydratase family domain and the RmlD (dTDP-keto-rhamnose-4-keto-reductase) substrate-binding domain. Because the recombinant PRHM did not demonstrate any activity during an in vitro assay, complementation with an Escherichia coli mutant was carried out. The rfbD (dTDP-4-dehydrorhamnose reductase), which encodes an enzyme catalyzing the conversion of dTDP-4-keto-rhamnose to TDP-rhamnose, was mutated in E. coli. The mutant strain B-rfbD was transformed with PRHM gene and a flavonoid rhanmosyltransferase gene, AtUGT78D1. The resulting transformant was able to convert quercetin into quercetin 3-O-rhamnoside in a manner similar to that by the wild type E. coli strain harboring AtUGT78D1. This result indicated that PRHM catalyzed the conversion of UDP-glucose into UDP-rhamnose. 相似文献