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1.
An endoglycanase gene of Paenibacillus cookii SS-24 was cloned and sequenced. This Pgl8A gene had an open reading frame of 1,230 bp that encoded a putative signal sequence (31 amino acids) and mature enzyme (378
amino acids: 41,835 Da). The enzyme was most homologous to a β-1,3-1,4-glucanase of Bacillus circulans WL-12 with 84% identity. The recombinant enzyme hydrolyzed carboxymethyl cellulose, swollen celluloses, chitosan and lichenan
but not Avicel, chitin powder or xylan. With chitosan as the substrate, the optimum temperature and hydrolysis products of
the recombinant enzyme varied at pH 4.0 and 8.0. This is the first report that characterizes chitosanase activity under different
pH conditions. 相似文献
2.
Lee YS Yoo JS Chung SY Lee YC Cho YS Choi YL 《Applied microbiology and biotechnology》2006,73(1):113-121
A chitosanase-producing Bacillus sp. DAU101 was isolated from Korean traditional food. This strain was identified on the basis of phylogenetic analysis of the 16S rDNA sequence, gyrA gene, and phenotypic analysis. The gene encoding chitosanase (csn) was cloned and sequenced. The csn gene consisted of an open reading frame of 837 nucleotides and encodes 279 amino acids with a deduced molecular weight of 31,420 Da. The deduced amino acid sequence of the chitosanase from Bacillus sp. DAU101 exhibits 88 and 30 % similarity to those from Bacillus subtilis and Pseudomonas sp., respectively. The chitosanase was purified by glutathione S-transferase fusion purification system. The molecular weight of purified enzyme was about 27 kDa, which suggests the deletion of a signal peptide by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The pH and temperature optima of the enzyme were 7.5 and 50 °C, respectively. The enzyme activity was increased by about 1.6-fold by the addition of 5 or 10 mM Ca2+. However, Hg2+ and Ni+ ions strongly inhibited the enzyme. The enzyme produced, GlcN2–4, were the major products from a soluble chitosan. 相似文献
3.
Novel chitosanase from Streptomyces griseus HUT 6037 with transglycosylation activity 总被引:4,自引:0,他引:4
Tanabe T Morinaga K Fukamizo T Mitsutomi M 《Bioscience, biotechnology, and biochemistry》2003,67(2):354-364
Streptomyces griseus HUT 6037 inducibly produced two chitosanases when grown on chitosan. To elucidate the mechanism of degradation of chitinous compound by this strain, chitosanases I and II of S. griseus HUT 6037 were purified and characterized. The purified enzymes had a molecular mass of 34 kDa. Their optimum pH was 5.7, and their optimum temperature was 60 degrees C. They hydrolyzed not only partially deacetylated chitosan, but also carboxymethylcellulose. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the chitosanases were obtained for identification of the anomeric form of the reaction products. Both chitosanases produced the beta-form specifically, indicating that they were retaining enzymes. These enzymes catalyzed a glycosyltransfer reaction in the hydrolysis of chitooligosaccharides. The N-terminal and internal amino acid sequences of chitosanase II were identified. A PCR fragment corresponding to these amino acid sequences was used to screen a genomic library for the entire gene encoding chitosanase II. Sequencing of the choII gene showed an open reading frame encoding a protein with 359 amino acid residues. The deduced primary structure was similar to endoglucanase E-5 of Thermomonospora fusca, which enzyme belongs to family 5 of the glycosyl hydrolases. This is the first report of a family 5 chitosanase with transglycosylation activity. 相似文献
4.
Purification, Characterization, and Gene Analysis of a Chitosanase (ChoA) from Matsuebacter chitosanotabidus 3001 总被引:3,自引:0,他引:3 下载免费PDF全文
Jae Kweon Park Kumiko Shimono Nobuhisa Ochiai Kazutaka Shigeru Masako Kurita Yukari Ohta Katsunori Tanaka Hideyuki Matsuda Makoto Kawamukai 《Journal of bacteriology》1999,181(21):6642-6649
The extracellular chitosanase (34,000 M(r)) produced by a novel gram-negative bacterium Matsuebacter chitosanotabidus 3001 was purified. The optimal pH of this chitosanase was 4.0, and the optimal temperature was between 30 and 40 degrees C. The purified chitosanase was most active on 90% deacetylated colloidal chitosan and glycol chitosan, both of which were hydrolyzed in an endosplitting manner, but this did not hydrolyze chitin, cellulose, or their derivatives. Among potential inhibitors, the purified chitosanase was only inhibited by Ag(+). Internal amino acid sequences of the purified chitosanase were obtained. A PCR fragment corresponding to one of these amino acid sequences was then used to screen a genomic library for the entire choA gene encoding chitosanase. Sequencing of the choA gene revealed an open reading frame encoding a 391-amino-acid protein. The N-terminal amino acid sequence had an excretion signal, but the sequence did not show any significant homology to other proteins, including known chitosanases. The 80-amino-acid excretion signal of ChoA fused to green fluorescent protein was functional in Escherichia coli. Taken together, these results suggest that we have identified a novel, previously unreported chitosanase. 相似文献
5.
The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene
was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino
acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained
the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass
of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml
was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after
the hydrolysis of inulin with the crude recombinant inulinase. 相似文献
6.
Pradeep MA Jagadeesh J De AK Kaushik JK Malakar D Kumar S Dang AK Das SK Mohanty AK 《Theriogenology》2011,75(6):1005-1015
Oviduct-specific glycoprotein (oviductin) plays an important role during fertilization and early embryonic development. The oviductin cDNA was successfully cloned and sequenced in goat, which possessed an open reading frame of 1620 nucleotides representing 539 amino acids. Predicted amino acid sequence showed very high identity with sheep (97%) followed by cow (94%), porcine (77%), hamster (69%), human (66%), rabbit (65%), mouse (64%) and baboon (62%). The bioinformatics analysis of the sequences revealed the presence of a signal sequence of 21 amino acids, one potential N-linked glycosylation site at position 402, 21 potential O-linked glycosylation sites and 36 potential phosphorylation sites. The native oviductin was purified from the oviductal tissue, which showed three distinct bands on SDS-PAGE and western blot (MW ∼60-95 kDa). The predicted molecular weight of goat oviductin was 57.5 kDa, calculated from the amino acid sequences. The observed higher molecular weight has been attributed to the presence of large number of potential O-linked glycosylation sites. The lower concentration (10 μg/mL) of oviductin increased the cleavage rate, morula and blastocyst yield significantly (P < 0.05) as compared to higher concentration (100 μg/mL). Goat oviductin retarded the activity of pronase (0.1%) on zona solubility of oocytes significantly (P < 0.01). 相似文献
7.
By cDNA sequencing we have achieved the first, and complete, hemocyanin sequence of a bivalve (Nucula nucleus). This extracellular oxygen-binding protein consists of two immunologically distinguishable isoforms, here termed NnH1 and
NnH2. They share a mean sequence identity of 61%, both contain a linear arrangement of eight paralogous, ca.50-kDa functional units (FUs a-h), and in both isoforms the C-terminal FU-h possesses an extension of ca. 100 amino acids. The cDNA of NnH1 comprises 11,090 bp, subdivided into a 5′utr of 75 bp, a 3′utr of 791 bp, and an open
reading frame for a signal peptide of 19 amino acids plus a polypeptide of 3389 amino acids (M
r
= 385 kDa). The cDNA of NnH2 comprises 10,849 bp, subdivided into a 5′utr of 47 bp, a 3′utr of 647 bp, and an open reading
frame for a signal peptide of 16 amino acids plus a polypeptide of 3369 amino acids (M
r
= 387 kDa). In contrast to other molluscan hemocyanins, which are highly glycosylated, the bivalve hemocyanin sequence exhibits
only four potential N-glycosylation sites, and within both isoforms a peculiar indel is present, surrounding the highly conserved
copper-binding site CuA. Phylogenetic analyses of NnH1 and NnH2, compared to the known hemocyanin sequences of gastropods
and cephalopods, reveal a statistically sound closer relationship between gastropod and protobranch hemocyanin than to cephalopod
hemocyanin. Assuming a molecular clock, the last common ancestor of protobranch and gastropods lived 494 million ± 50 million
years ago, in conformity with fossil records from the late Cambrian.
[Reviewing Editor: Dr. Rüdiger Cerff]
The sequence reported in this paper has been deposited in the EMBL/GenBank database under accession number AJ786639 for NnH1
and AJ786640 for NnH2. 相似文献
8.
A novel phenylacetic acid (PAA)-induced CoA-ligase-encoding gene, designated as phlC, has been cloned from penicillin-producing fungus Penicillium chrysogenum. The open reading frame of phlC cDNA was 1671 bp and encoded a 556 amino acid residues protein with the consensus AMP binding site and a peroxisomal targeting
signal 1 on its C terminus. The deduced amino acid sequence showed 37% and 38% identity with characterized P. chrysogenum Phl and PhlB protein, respectively. Functional recombinant PhlC protein was overexpressed in Escherichia coli. The purified recombinant enzyme was capable to convert PAA into its corresponding CoA ester with a specific activity of
129.5 ± 3.026 pmol/min per mg protein. Similar to Phl and PhlB, PhlC displayed broad substrate spectrum and showed higher
activities to medium- and long-chain fatty acids. The catalytic properties of PhlC have been determined and compared to those
of Phl and PhlB. 相似文献
9.
Phytophthora capsici inflicts damage on numerous crop plants by secreting a series of pectinase including pectate lyase (PEL). Here, we report a pectate lyase gene (Pcpel1) from a genomic library of a highly virulent P. capsici strain SD33. Pcpel1 was identified as an open reading frame of 1233 bp encoding a protein of 410 amino acids with a predicted amino‐terminal signal sequence of 21 amino acids. The predicted protein of Pcpel1 has a calculated molecular mass of 43.8 kDa and a pI value of 6.8. Analysis of the amino acid sequence suggested that it was a member of the polysaccharide lyase family 1 that shows pectate lyase activity. Moreover, heterologous expression of Pcpel1 in Pichia pastoris produced proteins with molecular mass 66 kDa, very likely due to differential glycosylation by the yeast. By western blotting and northern blotting analysis, Pcpel1 was strongly expressed during interaction of P. capsici with the host plant, suggesting its involvement in the process of host infection. The role of Pcpel1 in cell wall disassembly and host/parasite interaction is discussed. 相似文献
10.
Parrinello N Vizzini A Arizza V Salerno G Parrinello D Cammarata M Giaramita FT Vazzana M 《Cell and tissue research》2008,334(2):305-317
A tumor necrosis factor-alpha (TNFα)-like gene from Ciona intestinalis (CiTNFα-like) body wall challenged with bacterial lipopolysaccharide (LPS) was cloned and sequenced 4 h after LPS inoculation.
An open reading frame of 936 bp encoding a propeptide of 312 amino acids (35.4 kDa) displaying a transmembrane domain from
positions 7 to 29, a TACE cleavage site, and a mature peptide domain of 185 amino acids (20.9 kDa), was determined with a
predicted isoelectric point of 9.4. The phylogenetic tree based on deduced amino acid sequences of invertebrate TNF-like protein
and vertebrate TNFs supported the divergence between the ascidian and vertebrate TNF families, whereas D. melanogaster Eiger A and B TNF-like sequences were distinctly separated from the chordate TNFs. Thus, the ascidian TNFα-like cytokine
was upregulated by in vivo LPS challenge supporting its pro-inflammatory role. In the pharynx, increased expression levels
were found following analysis by real-time polymerase chain reaction, whereas in situ hybridization assay showed positive
hemocytes both in the tissue and in circulating hemocytes. Finally, Western blot with monoclonal antibodies disclosed human
TNFα epitopes in a 15-kDa protein component of the hemolymph serum and in a 43-kDa protein contained in the hemocyte lysate
supernatant prepared in the presence of detergents. Both soluble and hemocyte-bound CiTNFα-like protein therefore appeared
to be modulated by the LPS challenge.
This work was supported by a research grant from the Italian Ministry of University and Scientific Research (PRIN 2006 to
N. Parrinello), co-funded by the University of Palermo. 相似文献
11.
Purification of a constitutive chitosanase produced by Bacillus sp. MET 1299 with cloning and expression of the gene 总被引:1,自引:0,他引:1
A chitosanase produced constitutively by Bacillus sp. MET 1299 was purified by SP-Sephadex column chromatography. The molecular weight was estimated to be 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimal enzyme activity was observed at a pH of 5.5 and temperature of 60 degrees C. The purified chitosanase showed high activity on 90% deacetylated colloidal chitosan and beta-glucan, but not on hydrolyzed colloidal chitin, CMC, or their derivatives. The N-terminal amino acid sequence of the enzyme was determined. The cloned full length gene, 1362 bp in size, encoded a single peptide of 453 amino acids and had a conserved amino acid sequence of glycosyl hydrolase family 8. A search of the cDNA sequence with NCBI BLAST showed homology with chitosanase of Bacillus sp. KTCC 0377BP and Bacillus sp. No. 7-M. The recombinant protein was expressed in Escherichia coli, purified using affinity chromatography and characterized. 相似文献
12.
Lingying Kong Daosen Guo Shiyi Zhou Xinlei Yu Guixue Hou Ronggui Li Boguang Zhao 《Archives of microbiology》2010,192(7):585-593
Pseudomonas fluorescens GcM5-1A was isolated from the pine wood nematode (PWN), Bursaphelenchus xylophilus, obtained from wilted Japanese black pine, Pinus thumbergii, in China. In this paper, a genomic library of the GcM5-1A strain was constructed and a toxin–producing clone was isolated
by bioassay. Nucleotide sequence analysis revealed an open reading frame of 1,290 bp encoding a protein of 429 amino acids
with N-terminal putative signal peptide of 36 amino acids, which shared a similarity of 83, 82 and 80% identity with hypothetical
protein PFLU2919 from P. fluorescens SBW25, Dyp-type peroxidase family protein from P. fluorescens Pf-5 and Tat-translocated enzyme from P. fluorescens Pf0-1, respectively. The gene encoding a full-length protein or without the putative signal peptide was cloned and expressed
as a soluble protein in E. coli. The recombinant protein was purified to electrophoretic homogeneity by affinity chromatography using a Ni2+ matrix column. Its relative molecular weight was estimated to be 48.5 kDa by SDS-PAGE for full-length protein, and 45.0 kDa
for the recombinant protein without putative signal peptide. Bioassay results showed that the recombinant protein with or
without the putative signal peptide was toxic to both suspension cells and P. thunbergii seedlings. HPLC analysis demonstrated that components in branch extracts of P. thunbergii were significantly changed after addition of the recombinant full-length protein and hydrogen peroxide, which indicated that
it is probably a peroxidase. This study offers information that can be used to determine the mechanism of pine wilt disease
caused by the PWN. 相似文献
13.
Cloning and characterization of the 14-3-3 protein gene from the halotolerant alga Dunaliella salina
Previous studies have demonstrated that 14-3-3 proteins exist in all the eukaryotic organisms studied; however, studies on
the 14-3-3 proteins have not been involved in the halotolerant, unicellular green alga Dunaliella salina so far. In the present study, a cDNA encoding 14-3-3 protein of D. salina was cloned and sequenced by PCR and rapid amplification of cDNA end (RACE) technique based on homologous sequences of the
14-3-3 proteins found in other organisms. The cloned cDNA of 1485 bp in length had a 29.2 kDa of molecular weight and contained
a 774 bp of open reading frame encoding a polypeptide of 258 amino acids. Like the other 14-3-3 proteins, the deduced amino
acid sequences of the D. salina 14-3-3 protein also contained two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore,
an EF hand motif characteristic for Ca2+-binding sites was located within the C-terminal part of this polypeptide (positions 208–219). Analysis of bioinformatics
revealed that the 14-3-3 protein of D. salina shared homology with that of other organisms. Real-time quantitative PCR demonstrated that expression of the 14-3-3 protein
gene is cell cycle-dependent. 相似文献
14.
Hemolysin of Vibrio fluvialis (VFH) was purified from culture supernatants by ammonium sulfate precipitation and successive column chromatographies on DEAE-cellulose and Mono-Q. N-terminal amino acid sequences of the purified VFH were determined. The purified protein exhibited hemolytic activity on many mammalian erythrocytes with rabbit erythrocytes being the most sensitive to VFH. Activity of the native VFH was inhibited by the addition of Zn2+, Ni2+, Cd2+ and Cu2+ ions at low concentrations. Pores formed on rabbit erythrocytes were approximately 2.8-3.7 nm in diameter, as demonstrated by osmotic protection assay. Nucleotide sequence analysis of the vfh gene revealed an open reading frame (ORF) consisting of 2200 bp which encodes a protein of 740 amino acids with a molecular weight of 82 kDa. Molecular weight of the purified VFH was estimated to be 79 kDa by SDS-PAGE and N-terminal amino acid sequence revealed that the 82 kDa prehemolysin is synthesized in the cytoplasm and is then secreted into the extracellular environment as the 79 kDa mature hemolysin after cleavage of 25 N-terminal amino acids. Deletion of 70 amino acids from the C-terminus exhibited a smaller hemolytic activity, while deletion of 148 C-terminal amino acids prevented hemolytic activity. 相似文献
15.
M.N. Heneghan L. McLoughlin P.G. Murray M.G. Tuohy 《Enzyme and microbial technology》2007,41(6-7):677-682
The aguA gene encoding α-glucuronidase was isolated from the thermophilic fungus Talaromyces emersonii by degenerate PCR. AguA has no introns and consists of an open reading frame of 2511 bp, encoding a putative protein of 837 amino acids. The N-terminus of the protein contains a putative signal peptide of 17 amino acids yielding a mature protein of 820 amino acids with a predicted molecular mass of 91.6 kDa. Twenty putative N-glycosylation sites and four O-glycosylation were identified. The T. emersonii α-glucuronidase falls into glycosyl hydrolase family 67, showing approximately 63% identity to similar enzymes from other fungi. Analysis of the aguA promoter revealed several possible regulatory motifs including two XlnR and a CreA binding site. Enzyme activity was optimal at 50 °C and pH 5. Enzyme production was investigated on a range of carbon sources and showed induction on beechwood, oat spelt and birchwood xylan, and repression by glucose or glucuronic acid. 相似文献
16.
17.
Qinggang Xu Hongwu Bian Ning Han Rong Hou Zhihe Zhang Muyuan Zhu 《Molecular biology reports》2010,37(6):2903-2907
Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor. It plays an important role in stimulating growth
hormone secretion, food intake, body weight gain and gastric motility. cDNA sequences coding for ghrelin precursor protein
(prepro-ghrelin) were isolated from the stomach of a giant panda. Two different mRNA sequences of ghrelin were obtained. The
long open reading frame of ghrelin (354 bp) encodes a precursor protein of 117 amino acids with a 23 amino acid signal peptide.
The short one (351 bp) encodes a precursor protein of 116 amino acids with the same 23 amino acid signal peptide. The presumed
giant panda mature ghrelin proteins also had two forms. Comparative analysis showed that the first and the fourth amino acids
(Gly and Phe) were completely conserved and the third amino acid (Ser) was also highly conserved in the mature ghrelin. RT-PCR
analysis of giant panda ghrelin mRNA in various tissues revealed high level of expression in stomach, relative lower levels
of expression in small intestine, liver and kidney, and no expression in thymus, spleen and heart. 相似文献
18.
Emi Miyamoto Yuri Tanioka Ayako Nishizawa-Yokoi Yukinori Yabuta Kouhei Ohnishi Haruo Misono Shigeru Shigeoka Yoshihisa Nakano Fumio Watanabe 《Archives of microbiology》2010,192(6):437-446
Significant accumulation of the methylmalonyl-CoA mutase apoenzyme was observed in the photosynthetic flagellate Euglena gracilis Z at the end of the logarithmic growth phase. The apoenzyme was converted to a holoenzyme by incubation for 4 h at 4°C with
10 μM 5′-deoxyadenosylcobalamin, and then, the holoenzyme was purified to homogeneity and characterized. The apparent molecular
mass of the enzyme was calculated to be 149.0 kDa ± 5.0 kDa using Superdex 200 gel filtration. SDS–polyacrylamide gel electrophoresis
of the purified enzyme yielded a single protein band with an apparent molecular mass of 75.0 kDa ± 3.0 kDa, indicating that
the Euglena enzyme is composed of two identical subunits. The purified enzyme contained one mole of prosthetic 5′-deoxyadenosylcobalamin
per mole of the enzyme subunit. Moreover, we cloned the full-length cDNA of the Euglena enzyme. The cDNA clone contained an open reading frame encoding a protein of 717 amino acids with a calculated molecular
mass of 78.3 kDa, preceded by a putative mitochondrial targeting signal consisting of nine amino acid residues. Furthermore,
we studied some properties and physiological function of the Euglena enzyme. 相似文献
19.
Rieske protein gene in the Pacific oyster Crassostrea gigas was obtained by in silico cloning for the first time, and its expression profiles and subcellular localization were determined,
respectively. The full-length cDNA of Cgisp is 985 bp in length and contains a 5′- and 3′-untranslated regions of 35 and 161 bp, respectively, with an open reading frame
of 786 bp encoding a protein of 262 amino acids. The predicted molecular weight of 30 kDa of Cgisp protein was verified by prokaryotic expression. Conserved Rieske [2Fe–2S] cluster binding sites and highly matched-pair tertiary
structure with 3CWB_E (Gallus gallus) were revealed by homologous analysis and molecular modeling. Eleven putative SNP sites and two conserved hexapeptide sequences,
box I (THLGC) and II (PCHGS), were detected by multiple alignments. Real-time PCR analysis showed that Cgisp is expressed in a wide range of tissues, with adductor muscle exhibiting the top expression level, suggesting its biological
function of energy transduction. The GFP tagging Cgisp indicated a mitochondrial localization, further confirming its physiological
function. 相似文献
20.
Jae-Sam Hwang Hyun-Jeong Go Tae-Won Goo Eun-Young Yun Kwang-Ho Choi Su-Il Seong Jong-Su Chang Sang-Mong Lee Bong-Hee Lee Ik-Soo Kim Nam Soon Kim Taehoon Chun Seok-Woo Kang 《Biotechnology letters》2005,26(19):1469-1473
LIM protein cDNA, from Bombyx mori that contains an open reading frame of 622 bp encoding 94 amino acids, was identified and characterized. The B. mori LIM protein homologue is classified into group 2 LIM proteins that contain glycine-rich LIM domain. B. mori LIM protein mRNA is up-regulated at late embryogenesis and detected in the mid-gut of 5th instar larvae. 相似文献