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1.
The nucleotide sequence of a 4.39-kb DNA fragment encoding the alpha-glucosidase gene of Candida tsukubaensis is reported. The cloned gene contains a major open reading frame (ORF 1) which encodes the alpha-glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa. N-terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the alpha-glucosidase precursor is proteolytically processed by removal of an N-terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63-65 kDa and 50-52 kDa, respectively. Both subunits are secreted by the heterologous host S. cerevisiae in a glycosylated form. Coincident with its efficient expression in the heterologous host, the C. tsukubaensis alpha-glucosidase gene contains many of the canonical features of highly expressed S. cerevisiae genes. There is considerable sequence similarity between C. tsukubaensis alpha-glucosidase, the rabbit sucrase-isomaltase complex (proSI) and human lysosomal acid alpha-glucosidase. The cloned DNA fragment from C. tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids. The function and identity of the polypeptide encoded by ORF 2 is not known. 相似文献
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Schweiger P Volland S Deppenmeier U 《Journal of molecular microbiology and biotechnology》2007,13(1-3):147-155
The Gluconobacter oxydans 621H genome contains two genes (gox1122 and gox0499) that encode putative cytosolic NAD(P)-dependent aldehyde dehydrogenases. Each gene was expressed in Escherichia coli, and the recombinant enzymes were purified and characterized. The native protein Gox1122 exhibited an apparent molecular mass of 50.1 kDa, and the subunit mass was 50.5 kDa, indicating a monomeric structure of the native enzyme. The preferred substrates were acetaldehyde and NADP. The enzyme also oxidized other short-chained aliphatic and aromatic aldehydes at lower rates. Recombinant protein Gox0499 was composed of a single subunit and had an apparent molecular mass of 49.5 kDa. The substrate spectrum of Gox0499 was broad with a preference for long-chained aliphatic and aromatic aldehydes. Highest activities were obtained using dodecanal and NAD as substrates. RT real-time PCR showed that genes gox0499 and gox1122 were expressed at an elevated level (about 3-fold) when the cells were exposed to ethanol and dodecanal in comparison to control cells. 相似文献
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Xu Liu Rong Chen Zhongwei Yang Jiale Wang Jinping Lin Dongzhi Wei 《Molecular biotechnology》2014,56(4):285-295
A gene encoding an NADH-dependent short-chain dehydrogenase/reductase (gox2036) from Gluconobacter oxydans 621H was cloned and heterogeneously expressed in Escherichia coli. The protein (Gox2036) was purified to homogeneity and biochemically characterized. Gox2036 was a homotetramer with a subunit size of approximately 28 kDa. Gox2036 had a strict requirement for NAD+/NADH as the cofactor. Gox2036 displayed preference for oxidation of secondary alcohols and 2,3-diols as well as for reduction of α-diketones, hydroxy ketones, α-ketoesters, and β-ketoesters. However, Gox2036 was poorly active on 1,2-diols and acetoin and showed no activity on primary alcohols, polyols, and aldehydes. The optimum pH values for the oxidation and reduction reactions were 9 and 6, respectively. Gox2036 was highly selective in the reduction of various β-ketones and β-ketoesters. Among the substrates tested, ethyl 4-chloro acetoacetate was reduced to ethyl (R)-4-chloro-3-hydroxybutanoate ester with an excellent conversion yield of 96.9 % and optical purity of >99 % e.e. using an efficient in situ NADH-recycling system involving glucose and a glucose dehydrogenase from Bacillus subtilis (BsGDH). 相似文献
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Gluconobacter oxydans is an industrially important bacterium that possesses many uncharacterized oxidoreductases, which might be exploited for novel biotechnological applications. In this study, gene gox1801 was homologously overexpressed in G. oxydans and it was found that the relative expression of gox1801 was 13-fold higher than that in the control strain. Gox1801 was predicted to belong to the 3-hydroxyisobutyrate dehydrogenase-type proteins. The purified enzyme had a native molecular mass of 134 kDa and forms a homotetramer. Analysis of the enzymatic activity revealed that Gox1801 is a succinic semialdehyde reductase that used NADH and NADPH as electron donors. Lower activities were observed with glyoxal, methylglyoxal, and phenylglyoxal. The enzyme was compared to the succinic semialdehyde reductase GsSSAR from Geobacter sulfurreducens and the γ-hydroxybutyrate dehydrogenase YihU from Escherichia coli K-12. The comparison revealed that Gox1801 is the first enzyme from an aerobic bacterium reducing succinic semialdehyde with high catalytic efficiency. As a novel succinic semialdehyde reductase, Gox1801 has the potential to be used in the biotechnological production of γ-hydroxybutyrate. 相似文献
6.
A D Barone J J Silva D D Ho R C Gallo F F Wong-Staal N T Chang 《Journal of immunology (Baltimore, Md. : 1950)》1986,137(2):669-673
Randomly sheared DNA fragments from HTLV-III proviral DNA were cloned into an E. coli open reading frame (ORF) expression vector. The inserted ORF DNA was expressed in E. coli transformants as a polypeptide fused to the lambda CI protein at the amino terminus and to beta-galactosidase at the carboxyl terminus. The reactivity of the recombinant peptides with antibodies from sera of AIDS patients was determined by the Western blot technique. The coordinates of the DNA inserts of the immunoreactive clones were then determined by DNA sequencing. A clone, ORF 628, was found to contain a short DNA segment located between the sor and env genes (nucleotide positions 5367 to 5597), a region previously thought to be noncoding. Inspection of the DNA sequences of this clone and of other HTLV-III isolates revealed the presence of a small ORF located between nucleotide position 5411 and 5625, capable of encoding a polypeptide of 72 amino acids. The biosynthesis of the polypeptide of ORF 628 initiates from an ATG codon within the HTLV-III insert. The fusion protein of ORF 628 was partially purified by affinity chromatography on CH Sepharose 4B coupled to a beta-galactosidase ligand, and tested against a panel of sera from AIDS patients by Western blot analysis. Approximately 35% of the sera from patients with AIDS or ARC contained antibodies reactive with the peptide. The DNA region spanned by ORF 628 is now thought to be the major functional element of the trans-activator gene, tat. 相似文献
7.
The iutA gene from marine Vibrio species SD004, which encoded a ferric aerobactin receptor for the uptake of iron(III), was cloned onto a multicopy plasmid, pUC 18, in Escherichia coli. Identification of the positive clone was achieved on the basis of its deferrization activity and was detected as a halo formation on the chrome azurol S (CAS)-containing selective plate. Nucleotide sequence analysis of the cloned DNA fragment revealed an open reading frame (ORF) which encoded a polypeptide of 706 amino acid residues, and the deduced molecular mass of this polypeptide was 77.906 kD. The amino acid sequence showed a 41% homology with that of the lutA protein from E. coli. The cloned gene was iutA, which encoded the ferric aerobactin receptor. Another incomplete ORF was found 100 bp upstream of the iutA gene, which was homologous (31 out of 49 amino acids) with the C-terminal region of the luc D protein of E. coli. It is suggested that aerobactin biosynthesis and the transport genes are located tandemly on the Vibrio chromosome and may form an aerobactin operon. 相似文献
8.
Cloning and characterization of the gene encoding glutamate 1-semialdehyde 2,1-aminomutase, which is involved in delta-aminolevulinic acid synthesis in Propionibacterium freudenreichii. 总被引:2,自引:0,他引:2
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The gene from Propionibacterium freudenreichii that encodes glutamate 1-semialdehyde 2,1-aminomutase (EC 5.4.3.8), which is involved in the C5 pathway for synthesis of delta-aminolevulinic acid (ALA), a precursor in heme and cobalamin biosynthesis, was cloned onto a multicopy plasmid, pUC18, via complementation of an ALA-deficient mutant (hemL) of Escherichia coli. Subcloning of fragments from the initial 3.3-kb chromosomal fragment allowed the isolation of a 1.9-kb fragment which could complement the hemL mutation. Nucleotide sequence analysis of the 1.9-kb DNA fragment revealed an open reading frame (ORF) that was located downstream from a potential ribosome-binding site. The ORF encoded a polypeptide of 441 amino acid residues, and the deduced molecular mass of this polypeptide is 45,932 Da. A high G+C content (70 mol%) of the codons of the ORF was found and was consistent with the taxonomic features of Propionibacterium species. The amino acid sequence showed a high degree of homology with those of the HemL proteins from other organisms, and a putative binding site for pyridoxal 5'-phosphate was conserved, with the exception of a single substitution of phenylalanine for leucine. These results suggest that ALA is synthesized via the C5 pathway in a producer of vitamin B12, P. freudenreichii. 相似文献
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Molecular cloning and structure of the gene for 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase from a Pseudomonas strain. 总被引:10,自引:6,他引:4
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A Pseudomonas strain produced an enzyme capable of deacylating 7 beta-(4-carboxybutanamido)cephalosporanic acid to 7-aminocephalosporanic acid in response to glutaric acid. The gene for the enzyme was cloned within the PstI site of pBR325 as a 7.35-kilobase-pair DNA segment from a mutant of this strain whose enzyme is produced constitutively. The gene expression in the primary clone appeared to be low in Escherichia coli but was significantly enhanced by reducing the size of the initial segment coupled with E. coli promoters. Subsequent subcloning resulted in localization of the gene to a 2.45-kilobase-pair fragment. Three clone-specific polypeptides with molecular weights of ca. 16,000, 54,000, and 70,000 were shown by maxicell analysis. The former two corresponded to the small and large subunits of the purified enzyme from the Pseudomonas strain, and the third polypeptide was suggested to be their precursor. This was supported by DNA sequence study together with amino acid sequencing of the amino terminus of both subunits: the sequences for the small and large subunits were localized contiguously in this order on the structural gene without termination codons between them. The nucleotide sequence also disclosed the presence of a signallike sequence preceding that for the small subunit, consistent with the previous observation that the enzyme might be periplasmic in the Pseudomonas strain. Those results suggest a process for the formation of an active enzyme complex from a precursor through two steps of processing. 相似文献
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The organization of genes encoding the blood group M-specific hemagglutinin (M-agglutinin) of Escherichia coli strain IH11165 was studied with a cloned 6.5-kb DNA segment. This DNA segment contains at least five genes which code for the polypeptides of 12.5, 30, 80, 18.5 and 21 kDa. The 30-, 80- and 21-kDa polypeptides are synthesized as precursors that are approximately 2 kDa larger. The 21-kDa polypeptide was identified as the M-agglutinin subunit by its reactivity with anti-M-agglutinin serum. Nucleotide sequence analysis of the corresponding gene showed that the M-agglutinin precursor had a 24-amino acid (aa) signal sequence, while the mature protein is 146 aa residues long. Although the organization of the M-agglutinin gene cluster resembles those of other E. coli adhesins, there is no significant sequence homology between the M-agglutinin subunit and the subunits of the other potentially related proteins in E. coli. 相似文献
11.
P Tavares M A Santos R Lurz G Morelli H de Lencastre T A Trautner 《Journal of molecular biology》1992,225(1):81-92
The virulent Bacillus subtilis bacteriophage SPP1 encapsidates its DNA by a headful mechanism. Analyzing phage missense mutants, which package less DNA than SPP1 wild-type but show no other affected properties, we have identified a gene whose product is involved in the sizing of phage DNA during maturation. Characterization of this gene and its product provides an experimental access to the poorly understood mechanism of DNA sizing in packaging. The gene (gene 6 or siz) was cloned and sequenced. An open reading frame (ORF) coding for a 57.3 kDa polypeptide was identified. All the single nucleotide substitutions present in different siz mutants affect the net charge of that protein. The gene was further characterized by assignment of several nonsense mutations (sus) to the ORF. Phages carrying the latter type of mutations could be complemented in trans when gene 6 is provided by a plasmid. 相似文献
12.
The peripheral (catalytic) sector of vacuolar H+-ATPases contains five different polypeptides denoted as subunits A-E in order of decreasing molecular masses from 72 to 33 kDa. The gene encoding subunit B (57 kDa) of yeast vacuolar H+-ATPase was cloned on a 5-kilobase pair genomic DNA fragment and sequenced. Four open reading frames were identified in the sequenced DNA. One of them encodes a protein of 504 amino acids with a calculated Mr of 56,557. Hydropathy plot revealed no apparent transmembrane segments. Southern analysis demonstrated that a single gene encodes this polypeptide in the yeast genome. The amino acid sequence exhibits extensive identity with the homologous protein from the plant Arabidopsis (77%). This polypeptide also contains regions of homology with the alpha subunits of H+-ATPases from mitochondria, chloroplasts, and bacteria. However, less similarity was detected when it was compared with the beta subunits of those enzymes. The implication of these phenomena on the evolution of proton pumps is discussed. 相似文献
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Valdivieso-Ugarte M Ronchel C Bañuelos O Velasco J Adrio JL 《Biotechnology progress》2006,22(4):1096-1101
Fructo-oligosaccharides (FOS) represent the most abundantly supplied and utilized group of nondigestible oligosaccharides as food ingredients. These prebiotics can be produced from sucrose using the transglycosylating activity of beta-fructofuranosidases (EC 3.2.1.26) at high concentrations of the starting material. The main problem during FOS synthesis is that the activity of the enzyme is inhibited by the glucose generated during the reaction, and therefore the maximum FOS content in commercial products reaches up to 60% on a dry substance basis. The glucose oxidase (gox) gene from Aspergillus niger BT18 was cloned and integrated, as part of an expression cassette, into the ribosomal DNA of a Saccharomyces cerevisiae host strain. One of the recombinant strains with a high copy number of the gox gene and showing a high GOX specific activity was used to produce the enzyme. Addition of the extracellular glucose oxidase to the FOS synthesis reaction helped to remove the glucose generated, avoiding the inhibition of the fungal beta-fructofuranosidase. As a result, a final syrup containing up to 90% of FOS was obtained. 相似文献
16.
Genetic and biochemical analysis of the flagellar hook of Treponema phagedenis. 总被引:8,自引:4,他引:4
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The periplasmic flagellum of Treponema phagedenis consists of the flagellar filament and hook-basal body. We report here a characterization of the hook gene and flagellar hook of T. phagedenis, and in the process of this analysis we found evidence that the hook polypeptide is likely cross-linked in situ. A T. phagedenis genomic library was screened with a Treponema pallidum antiserum, and the DNA segments from several positive plaques were subcloned and sequenced. DNA sequencing of two overlapping segments revealed a 1,389-nucleotide (nt) open reading frame (ORF) with a deduced amino acid sequence that was 36% identical to that of FlgE, the hook polypeptide of Salmonella typhimurium. This gene was designated T. phagedenis flgE. Beginning at 312 nt downstream from flgE was a partial ORF of 486 nt with a deduced amino acid sequence that was 33% identical to that of MotA of Bacillus subtilis, a polypeptide that enables flagellar rotation. Upstream of flgE, separated by 39 nt, was a partial (291-nt) ORF with a deduced amino acid sequence that was homologous to that of ORF8, a polypeptide of unknown function located in an operon encoding polypeptides involved in motility of B. subtilis. The T. phagedenis flgE gene was cloned into an Escherichia coli protein expression plasmid, and the purified recombinant protein was used to prepare a FlgE antiserum. Western blots (immunoblots) of whole-cell lysates probed with this antiserum revealed a 55-kDa polypeptide and a ladder of polypeptide bands with increasing molecular masses. T. phagedenis hooks were then isolated and purified, and electron microscopic analysis revealed that the morphology of the hooks resembled that in other bacteria. The hooks were slightly curved and had an average length of 69 +/- 8 nm and a diameter of 23 +/- 1 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots of purified hook preparations using the FlgE antiserum also revealed a polypeptide ladder, suggesting that the hooks are composed of a covalently cross-linked polypeptide. 相似文献
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Sequence of the phosphoprotein gene of pneumonia virus of mice: expression of multiple proteins from two overlapping reading frames. 总被引:3,自引:3,他引:0
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The gene encoding the phosphoprotein of the pneumovirus pneumonia virus of mice (PVM) has been cloned and sequenced. The gene is 903 nucleotides in length and contains a long open reading frame (ORF) capable of encoding a polypeptide of 295 amino acid residues. A smaller, second, overlapping ORF encoding a polypeptide 137 amino acids in length was also present. The large ORF directed the synthesis of a 39-kDa polypeptide and four additional polypeptides with masses of 37 kDa, 26 kDa, 23 kDa, and 16 kDa in vitro. The smaller polypeptides were generated by internal initiation on in-frame AUG initiation codons to generate carboxy co-terminal products. Western immunoblot analysis indicated that at least two of these proteins and several other related polypeptides are present in infected cells, and the possible origins of these are discussed. Western blot analysis using antiserum raised against a synthetic peptide and specific for the predicted second ORF product identified a polypeptide of 23 kDa in PVM-infected cells. The pattern of PVM P gene expression is unlike that of the closely related respiratory syncytial virus and is reminiscent of that of paramyxoviruses such as Sendai virus. This is the first example of a pneumovirus encoding multiple polypeptide products from a single mRNA in vivo. 相似文献
19.
Kato N Suyama S Shirokane M Kato M Kobayashi T Tsukagoshi N 《Applied and environmental microbiology》2002,68(3):1250-1256
Aspergillus nidulans possessed an alpha-glucosidase with strong transglycosylation activity. The enzyme, designated alpha-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising 74- and 55-kDa subunits and catalyzed hydrolysis of maltose along with formation of isomaltose and panose. Approximately 50% of maltose was converted to isomaltose, panose, and other minor transglycosylation products by AgdB, even at low maltose concentrations. The agdB gene was cloned and sequenced. The gene comprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. The deduced amino acid sequence contained the chemically determined N-terminal and internal amino acid sequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptide precursor. AgdB showed low but overall sequence homology to alpha-glucosidases of glycosyl hydrolase family 31. However, AgdB was phylogenetically distinct from any other alpha-glucosidases. We propose here that AgdB is a novel alpha-glucosidase with unusually strong transglycosylation activity. 相似文献
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Homology between the photoreactivation genes of Saccharomyces cerevisiae and Escherichia coli 总被引:11,自引:0,他引:11
A cloned fragment of Saccharomyces cerevisiae chromosomal DNA carrying the photoreactivation gene (PHR) has been sequenced. The fragment contains a 1695-bp intronless open reading frame (ORF) coding for a polypeptide of 564 amino acids (aa). The phr gene of Escherichia coli was also sequenced, and the sequence is in agreement with the published data. The yeast PHR gene has a G + C content of 36.2%, whereas 53.7% was found for the E. coli gene. Despite the difference in G + C content there is a 35% homology between the deduced aa sequences. This homology suggests that both genes have originated from a common ancestral gene. 相似文献