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1.
Kadokura K Sakamoto Y Saito K Ikegami T Hirano T Hakamata W Oku T Nishio T 《Biotechnology letters》2007,29(8):1209-1215
An open reading frame (ORF) encoding chitin oligosaccharide deacetylase (Pa-COD) gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome and its sequence was analyzed. The ORF encoded a 427 amino acid protein, including the 22 amino acid signal
sequence. The deduced amino acid sequence was highly similar to several bacterial chitin oligosaccharide deacetylases in carbohydrate
esterase family 4. An expression plasmid containing the gene was constructed and inserted into Escherichia coli cells and the recombinant enzyme was secreted into the culture medium with the aid of the signal peptide. The concentration
of the recombinant enzyme in the E. coli culture medium was 150 times larger than that of wild-type enzyme produced in the culture medium by V. parahaemolyticus KN1699. The recombinant enzyme was purified to homogeneity from culture supernatant in an overall yield of 16%. Substrate
specificities of the wild-type and the recombinant enzymes were comparable. 相似文献
2.
Kadokura K Sakamoto Y Saito K Ikegami T Hirano T Hakamata W Oku T Nishio T 《Bioscience, biotechnology, and biochemistry》2007,71(11):2848-2851
An open reading frame encoding the chitinase gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome. An expression plasmid containing the gene was introduced into Escherichia coli cells, and recombinant chitinase (Pa-rChi) was produced and secreted into the culture medium with the aid of the signal peptide. Pa-rChi was purified and its substrate specificity was determined. 相似文献
3.
Duriya Chantasingh Supattra Kitikhun Lily Eurwilaichitr Tanaporn Uengwetwanit 《Biocontrol Science and Technology》2011,21(6):677-686
A gene encoding chitinase was cloned from Ophiocordyceps unilateralis, a Formamidae-specific fungus, collected from Sirindhorn Peat Swamp Forest, Thailand. The O. unilateralis chitinase (OuChi) full-length gene (1311 bp) encodes 436 amino acids with the first 20 amino acids as a putative signal peptide. The gene showed highest identity (78%) to Isaria farinose endochitinase. To investigate if cross-species chitinase expression also enhances fungal toxicity, the mature OuChi gene was subcloned into an Agrobacterium binary vector pPZP-bar and then transformed into Beauveria bassiana strain BCC2659. Chitinase activity was detected using 4-methylumbelliferyl-β-D-N,N′-diacetylchitobioside. The fungal transformant expressing O. unilateralis chitinase showed higher toxicity against Spodoptera exigua. These results support the hypothesis that chitinolytic enzymes are one of several ‘virulence’ factors produced by entomopathogenic fungi during host encounter. 相似文献
4.
S. B. Hu P. Liu X. Z. Ding L. Yan Y. J. Sun Y. M. Zhang W. P. Li L. Q. Xia 《Applied microbiology and biotechnology》2009,82(6):1157-1167
Previous studies revealed that chitinase could enhance the insecticidal activity of Bacillus thuringiensis and it has been used in combination with B. thuringiensis widely. However, the expression of B. thuringiensis chitinase is rather low and needs induction by chitin, which limits its field application. It would make sense to constitutively
express the chitinase at a sufficiently high level to offer advantages in biological control of pests. In this study, a signal
peptide-encoding sequence-deleted chitinase gene from B. thuringiensis strain 4.0718 under the control of dual overlapping promoters plus Shine–Dalgarno sequence and terminator sequence of cry1Ac3 gene was cloned into shuttle vector pHT315 and introduced into an acrystalliferous B. thuringiensis strain Cry−B. The recombinant plasmid was stably maintained over 240 generations in Cry−B. Chitinase was overexpressed within the sporangial mother cells in the form of spherical crystal-like inclusion bodies.
The chitinase inclusions could be solubilized and exhibit chitinolytic activity in 30 mmol l−1 Na2CO3–0.2% β-mercaptoethanol buffer at a wide range of alkaline pH values, and what’s more, the chitinase inclusions potentiated
the insecticidal effect of Cry1Ac protoxin when used against larvae of Spodoptera exigua and Helicoverpa armigera. 相似文献
5.
《Bioscience, biotechnology, and biochemistry》2013,77(7):1518-1524
Genomic DNA encoding a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves in previous research (Biosci. Biotechnol. Biochem., 68, 1508–1517 (2004)). But this chitinase had an additional sequence composed of eight amino acids (a C-terminal extension) at the C-terminal, compared with class IV chitianses from other plants. In order to clarify the role of this C-terminal extension in cellular localization, plants and suspension-cultured cells of Nicotiana tabacum were transformed with either the cloned yam class IV chitinase gene carrying the C-terminal extension or its truncated gene by the Agrobacterium-mediated method, and then their localization was investigated. The results suggest that the C-terminal extension of yam class IV chitinase plays a role as a targeting signal for plant vacuoles. This is the first report presenting the existence of vacuolar type class IV chitinase. 相似文献
6.
Directed evolution for increased chitinase activity 总被引:3,自引:0,他引:3
Fan Y Fang W Xiao Y Yang X Zhang Y Bidochka MJ Pei Y 《Applied microbiology and biotechnology》2007,76(1):135-139
Directed evolution through DNA shuffling and screening was used to enhance the catalytic ability of a fungal, Beauveria bassiana, chitinase, Bbchit1. The Bbchit gene was first linked to various prokaryotic signal sequences and expressed in Escherichia coli. The signal peptide, PelB, from Erwinia carotovora resulted in greatest chitinase secretion into broth. The nucleotide sequence expressing PelB signal peptide was then incorporated
into an E. coli vector to express Bbchit1 variants generated by three rounds of DNA shuffling. A Bbchit1 library with 150,000 variants was constructed with a nucleotide point mutation frequency of 0.6% and screened for chitinolytic
activity. Two Bbchit1 variants (SHU-1 and SHU-2) were selected that showed increased chitinolytic activity compared to the
wild type. Sequence analysis of these variants revealed mutations in amino acid residues that would not normally be considered
for rational design of improved chitinase activity. The amino acid substitutions occurred outside of the two putative substrate-binding
sites and the catalytic region. 相似文献
7.
Yuka Sameshima-Yamashita Takashi Watanabe Takumi Tanaka Shun Tsuboi Tohru Yarimizu Tomotake Morita 《Bioscience, biotechnology, and biochemistry》2019,83(8):1547-1556
ABSTRACTThe basidiomycetous yeast Pseudozyma antarctica GB-4(0) esterase (PaE) is a promising candidate for accelerating degradation of used biodegradable plastics (BPs). To increase safety and reduce costs associated with the use of PaE, we constructed a self-cloning strain with high-PaE productivity. A Lys12 gene (PaLYS12)-deleted lysine auxotroph strain GB4-(0)-L1 was obtained from GB-4(0) by ultraviolet mutagenesis and nystatin enrichment. Subsequently, the PaE gene (PaCLE1) expression cassette consisting of GB-4(0)-derived PaCLE1, under the control of a xylose-inducible xylanase promoter with PaLYS12, was randomly introduced into the GB4-(0)-L1 genome. A PaE high-producing strain, PGB474, was selected from among the transformants by high throughput double-screening based on its ability to degrade emulsified polybutylene succinate-co-adipate. Quantitative PCR revealed that four copies of the PaE gene expression cassette were introduced into the PGB474 genome. PGB474 produced 2.0 g/L of PaE by xylose-fed-batch cultivation using a 3-L jar fermentor for 72 h. 相似文献
8.
A chitinase gene (pCHi58) encoding a 58 kDa chitinase was isolated from theSerratia marcescens KCTC 2172 cosmid library. The chitinase gene consisted of a 1686 bp open reading frame that encoded 562 amino acids.Escherichia coil harboring the pChi58 gene secreted a 58 kDa chitinase into the culture supernatant. The 58 kDa chitinase was purified using
a chitin affinity column and mono-S column. A nucleotide andN-terminal amino acid sequence analysis showed that the 58 kDa chitinase had a leader peptide consisting of 23 amino acids
which was cleaved prior to the 24th alanine. The 58 KDa chitinase exhibited a 98% similarity to that ofS. marcescens QMB 1466 in its nuclotide sequence. The chitinolytic patterns of the 58 kDa chitinase released N,N′-diacetyl chitobiose (NAG2)
as the major hydrolysis end-product with a trace amount ofN-acetylglucosamine. When a 4-methylumbellyferyl-N-acetylglucosamin monomer, dimmer, and tetramer were used as substrates, the 58 kDa chitinase did not digest the 4-Mu-NAG
monomer (analogue of NAG2), thereby indicating that the 58 kDa chitinase was likely an endochitinase. The optimum reaction temperature and pH of the
enzyme were 50°C and 5.0, respectively. 相似文献
9.
Yoon Gyo Lee Ki-Chul Chung Seung Gon Wi Jae Chang Lee Hyeun-Jong Bae 《Protein expression and purification》2009,65(2):244-250
The chitinase producing Penicillium sp. LYG 0704 was procured from soil of the Chonnam National University crop field. The chitinase activity was detected after the first day which increased gradually and reached its maximum after 3 days of cultivation. The chitinase was purified from a culture medium by precipitation with isopropanol and column chromatography with Mono Q and Butyl-Sepharose. The molecular mass of chitinase was estimated to be 47 kDa by SDS–PAGE. Optimal pH and temperature were 5.0 and 40 °C, respectively. The N-terminal amino acid sequence of the enzyme was determined to be 1AGSYRSVAYFVDWAI15. The fully cloned gene, 1287 bp in size, encoded a single peptide of 429 amino acids. BLAST search of the chitinase gene sequence showed similarity with chitinase of Aspergillus fumigatus Af293 chitinase gene (58%) and A. fumigatus class V chitinase ChiB1 gene (56%). 相似文献
10.
《Bioscience, biotechnology, and biochemistry》2013,77(10):2076-2078
An antimicrobial peptide, designated Pa-AMP, was purified by gel filtration on Sephadex G-75 followed by S-Sepharose, Cosmosil-SP, and reverse-phase HPLC from the seeds of pokeweed (Phytolacca americana). Pa-AMP is a basic peptide having an isoelectric point of over 10 and its extinction coefficient at 280 nm of 1% aqueous solution was 7.7. Pa-AMP has a molecular mass of 4 kDa and 3.4 kDa on tricine SDS-PAGE under nonreducing and reducing conditions, respectively. The N-terminal amino acid of Pa-AMP was blocked. The concentrations of peptide required for 50% inhibition (IC50) of the growth of plant pathogenic fungi, Gram-positive, and Gram-negative bacteria were 3 to 41 μg/ml. Differing from other peptides, Pa-AMP inhibited the growth of some Gram-negative bacteria. 相似文献
11.
Various chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences
and domains. We have isolated a tobacco gene that encodes a predicted polypeptide consisting of a 20-amino acid N-terminal
signal peptide, followed by a 245-amino acid chitinolytic domain. Although the predicted mature protein is basic and shows
greater sequence identity to basic class I chitinases (75%) than to acidic class II chitinases (67%), it lacks the N-terminal
cysteine-rich domain and the C-terminal vacuolar targeting signal that is diagnostic for class I chitinases. Therefore, this
gene appears to encode a novel, basic, class II chitinase, which we have designated NtChia2;B1. Accumulation of Chia2;B1 mRNA was induced in leaves in association with the local-lesion response to tobacco mosaic virus (TMV) infection, and in
response to treatment with salicylic acid, but was only slightly induced by treatment with ethephon. Little or no Chia2;B1 mRNA was detected in roots, flowers, and cell-suspension cultures, in which class I chitinase mRNAs accumulate to high concentrations.
Sequence comparisons of Chia2;B1 with known tobacco class I and class II chitinase genes suggest that Chia2;B1 might encode an ancestral prototype of the present-day class I and class II isoforms. Possible mechanisms for chitinase gene
evolution are discussed.
Received: 25 May 1998 / Accepted: 29 June 1998 相似文献
12.
Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter
was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression
of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R0 and R1 progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by
Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic
plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed
the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase
activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R1 lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation.
Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study
reporting the introduction of rice chitinase gene into finger millet for leaf blast resistance. 相似文献
13.
Distribution and evolution of chitinase genes in Streptomyces species: involvement of gene-duplication and domain-deletion 总被引:1,自引:0,他引:1
Streptomyces coelicolor A3(2) possesses nine genes for family 18 chitinases and two for family 19, showing high multiplicity. By hybridization analyses,
distribution of those chitinase genes was investigated in six other Streptomyces species covering the whole phylogenetic range based on 16S rDNA sequences. All strains showed high-multiplicity of chitinase
genes, like S. coelicolor A3(2). The phylogeny and gene organization of the family 18 chitinase genes cloned from Streptomyces species so far were then analyzed to investigate the gene evolution. It was concluded that Streptomyces already possessed a variety of chitinase genes prior to branching into many species, and that the ancestral genes of chiA and chiB have been generated by gene-duplication. In the course of the analyses, evidence that the chi30 and chi40 genes of S. thermoviolaceus were derived from their corresponding original chitinase genes by losing gene parts for substrate-binding domains and fibronectin
type III-like domains was obtained. It was thus shown that gene-duplication and domain-deletion were implicated in generating
the high diversity and multiplicity of chitinase genes in Streptomyces species.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
14.
A chitinase-producing bacterium, designated WS7b, was isolated from a soil sample obtained from a black-pepper plantation
on Bangka Island, Indonesia. Fatty-acid methyl-ester analysis indicated that the isolate was Aeromonas caviae. A chitinase gene from WS7b was cloned in a pUC19-based plasmid vector, but without its natural promoter. The complete nucleotide
sequence of the gene was determined, and the structural gene consisted of a 2748-bp region encoding 864 amino acids. DNA sequence
analysis indicated that the gene had been cloned without its promoter, and this was confirmed by chitinase-plate assay of
the truncated version of the gene in Escherichia coli. The chitinase gene product showed amino-acid sequence similarity to chiA from A. caviae. Chitinase enzyme activity was determined spectrophotometrically, using colloidal chitin azure as substrate for extracellular
and intracellular fractions. The ability of the chitinase cloned in E. coli to hydrolyze chitin was less than that of the enzyme in its indigenous host. 相似文献
15.
Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens 总被引:32,自引:0,他引:32 下载免费PDF全文
Analysis of clones isolated from a cosmid DNA library indicates that the Serratia marcescens chromosome contains at least two genes, chiA and chiB, which encode distinct secreted forms of the enzyme chitinase. These genes have been characterized by inspection of chitinase activity and secreted proteins in Escherichia coli strains containing subclones of these cosmids. The two chitinase genes show no detectable homology to each other. DNA sequence analysis of one of the genes predicts an amino acid sequence with an N-terminal signal peptide typical of genes encoding secreted bacterial proteins. This gene was mutagenized by cloning a neomycin phosphotransferase gene within its coding region, and the insertion mutation was recombined into the parental S. marcescens strain. The resulting chiA mutant transconjugant showed reduced chitinase production, reduced inhibition of fungal spore germination and reduced biological control of a fungal plant pathogen. 相似文献
16.
The chitinase gene of Manduca sexta was cloned into the expression vector, pET-28a, and expressed in Escherichia coli BL21 (DE3) host cells. The protein product was expressed in inclusion bodies. After denaturation and renaturation procedures using a Ni2+-NTA affinity chromatography column, soluble chitinase was obtained. The authenticity of the renatured protein was confirmed by Western blotting. Polyclonal antibodies to the purified protein were raised in rabbits. The antibody reacted specifically with the expressed chitinase and was used to quantify its presence in transgenic cotton being developed to resist attack by various insects.Revisions requested 24 September 2004; Revisions received 18 November 2004 相似文献
17.
Thirty-two strains of actinomycetes obtained from soil samples of Thailand were selected. Actinomycete strain SU-1 is the most effective in terms of antagonism of Fusarium moniliforme. It produces antifungal substances on agar medium against F. moniliforme. On the basis of microscopical observations of its morphology and biochemical tests as well as analysis of cell wall and fatty acid pattern, this strain was identified as Streptomyces fradiae. The chitinase gene B (chiB337) from Nocardiopsis prasina OPC-131 was inserted into an integrating plasmid pFIS318, an Escherichia coli–Streptomyces shuttle vector. The new plasmid pFIS319-1 carrying the chitinase gene was used to transform protoplasts of S. fradiae strain SU-1. The obtained recombinant strain SU-1 pFIS319-1 exhibited higher chitinase activity than the wild-type in chitinase induction medium. Chitinase activity after renaturing protein from SDS-PAGE was detected rapidly by using 4-methylumbelliferyl β-D-N,N′’-diacetylchitobioside as the substrate. S. fradiae SU-1 secreted two chitinases with estimated molecular masses of 26 kDa and 43 kDa whereas the recombinant strain secreted three chitinases of about 26 kDa, 31.5 kDa (ChiB), and 43 kDa. The supernatant of the recombinant strain grown in chitinase induction medium inhibited the hyphal extension of F. moniliforme. 相似文献
18.
Bacterial antagonism, responsible for biological control, may operate by antiobiosis, competition or parasitism. Parasitism relies on lytic enzymes for the degradation of cell walls of pathogenic fungi. Serratia marcescens was found to be an efficient biocontrol agent of Sclerotium rolfsii and Rhizoctonia solani under greenhouse conditions. Populations of 105 or 106 colony forming units g-1 soil were the most effective. Drench and drip application of S. marcescens suspension were more effective in controlling S. rolfsii than spraying, mixing in soil or seed coating. The highest population density of the bacteria in the rhizosphere was found on the proximal portion of the root, decreasing significantly until the tips, where it increased again. The isolated Serratia, found to possess chitinolytic activity, was able to release N-acetyl D-glucosamine from cell walls of S. rolfsii. The gene coding for chitinase was cloned into Escherichia coli and the enzyme was uniquely excreted from the bacterium into its growth medium. When S. rolfsii was sprayed by partially purified chitinase produced by the cloned gene, rapid and extensive bursting of the hyphal tips was observed. This chitinase preparation was effective in reducing disease incidence caused by S. rolfsii in beans and R. solani in cotton, under greenhouse conditions. A similar effect was obtained when a viable E. coli cell, containing the plasmid with the chitinase gene (pLCHIA), was applied. It appears that genetic engineering of the lytic enzymes, such as chitinase which play an important role in plant disease control, may improve the efficacy of biocontrol agents. 相似文献
19.
Guadalupe Medina-de la Rosa Moisés Graciano Carcaño-Montiel Jesús Francisco López-Olguín Miguel Ángel Hernández-Espinosa José Antonio Rivera-Tapia 《Archives Of Phytopathology And Plant Protection》2016,49(11-12):310-321
The chitinase enzyme was identified in isolated bacteria of maize rhizosphere as well as its potential for the biological control of fungi associated at seeds of the same plant. The production of chitinase enzyme was found in the genera identified as Acinetobacter, Bacterium, Burkholderia, Paenibacillus, Pseudomonas, Rhizobium, Shewanella, Sphingomonas and Stenotrophomonas. Bacterial isolates with ability to degrade fungal mycelium from maize fungi as Fusarium and Alternaria among others, were detected. Bacterial chitinase activity and the presence of the chiA gene were determined. The inoculation of chitinolytic bacteria showed a positive effect in the control of fungi in maize seeds. The results support the potential use of chitinase enzyme producing bacteria on the control of phytopathogenic fungi. 相似文献
20.
Fathi Hassan Jochen Meens Hans-Jrg Jacobsen Heiko Kiesecker 《Journal of biotechnology》2009,143(4):302-308
Embryo axes excised from mature seeds of pea (Pisum sativum L.) cv. ‘Sponsor’ were used as explants for Agrobacterium-mediated transformation using pGreenII 0229 binary vectors. The vectors harbored a chimeric chitinase gene (chit30), driven by the constitutive 35S promoter or the elicitor inducible stilbene synthase (vst) promoter from grape (Vitis vinifera L.). The secretion signal of the bacterial chitinase gene from Streptomyces olivaceoviridis ATCC 11238 (DSM 41433) was replaced by the A. thaliana basic chitinase leader sequence. Functional properties of the recombinant gene were tested in tobacco as a model system before the long process of pea transformation was undertaken. Several transgenic pea clones were obtained and the transgenic nature confirmed by different molecular methods. The accumulation and activity of chitinase in stably transformed plants were examined by Western blot analysis and in-gel assays, which showed the presence of an additional 3 isoform bands. Using in vitro bioassays with Trichoderma harzanium as a model, we found an inhibition or delay of hyphal extension, which might indicate enhanced antifungal activity compared with non-transformed pea plants. Up to the 4th generation, the transgenic plants did not show any phenotypic alterations compared with non-transgenic control plants. 相似文献