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1.
Kang HJ Uegaki K Fukada H Ishikawa K 《Extremophiles : life under extreme conditions》2007,11(2):251-256
A hyperthermophilic β-1,4 endoglucanase (EGPh) from the hyperthermophilic archaeon Pyrococcus horikoshii exhibits a strong hydrolyzing activity toward crystalline cellulose. The characteristic features of EGPh are: (1) it appears
to have disulfide bonds, which is rare among anaerobic hyperthermophilic archaeon proteins, and (2) it lacks a carbohydrate-binding
domain, which is necessary for effective hydrolysis of cellulose. We first examined the relationship between the disulfide
bonds and the catalytic activity by analyzing various cysteine mutations. The activities of the mutated enzymes toward carboxy
methyl cellulose (CMC) increased without any loss in thermostability. Second, we prepared a fusion enzyme so that the thermostable
chitin-binding domain of chitinase from P. furiosus was joined to the C-terminus of EGPh and its variants. These fusion enzymes showed stronger activities than did the wild-type
EGPh toward both CMC and crystalline cellulose (Avicel). 相似文献
2.
3.
A chitinase (VpChiA) and its C-terminal truncated G589 mutant (VpChiAG589) of Vibrio parahaemolyticus were cloned by polymerase chain reaction (PCR) techniques. To study the role of the C-terminal 30 amino acids of VpChiA in
the enzymatic hydrolysis of chitin, both the recombinant VpChiA and VpChiAG589 encoded in 1,881 and 1,791 bp DNA fragments,
respectively, were expressed in Escherichia coli using the pET-20b(+) expression system. The His–Tag affinity purified VpChiA and VpChiAG589 enzymes had a calculated molecular
mass of 65,713 and 62,723 Da, respectively. The results of biochemical characterization including kinetic parameters, spectroscopy
of fluorescence and circular dichroism, chitin-binding and hydrolysis, and thermostability, both VpChiA and VpChiAG589, had
very similar physicochemical properties such as the optimum pH (6), temperature (40°C), and kinetic parameters of Km and kcat against the 4MU–(GlcNAc)2 or 4MU–(GlcNAc)3 soluble substrates. The significant increase of thermostability and the drastic decrease of the hydrolyzing ability of VpChiAG589
toward the insoluble α-chitin substrate suggested that a new role could be played by the C-terminal 30 amino acids. 相似文献
4.
Qi-Kai Zeng Hong-Li Du Jing-Fang Wang Dong-Qing Wei Xiao-Ning Wang Yi-Xue Li Ying Lin 《Biotechnology letters》2009,31(7):1025-1029
A major problem when xylose is used for ethanol production is the intercellular redox imbalance arising from different coenzyme
specificities of xylose reductase (XR) and xylitol dehydrogenase. The residue Lys21 in XR from Pichia stipitis was subjected to site-directed mutagenesis to alter its coenzyme specificity. The N272D mutant exhibited improved catalytic
efficiency when NADH was the coenzyme. Both K21A and K21A/N272D preferred NADH to NADPH, their catalytic efficiencies for
NADPH were almost zero. The catalytic efficiency of K21A/N272D for NADH was almost 9-fold and 2-fold that of K21A and the
wild-type enzyme, respectively. Complete reversal of coenzyme specificity toward NADH and improved catalytic efficiency were
achieved.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Qi-Kai Zeng, Hong-Li Du, Jing-Fang Wang have contributed equally to this work. 相似文献
5.
Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain
showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and
chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources,
arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum. 相似文献
6.
Hui J Cao Y Xiao F Zhang J Li H Hu F 《Journal of industrial microbiology & biotechnology》2008,35(9):975-980
Staphylococcal enterotoxin C2 (SEC2) is one member of bacterial superantigens produced by Staphylococcus aureus. It can be attributed to its superantigenic activity to cross-link major histocompatibility complex class II molecules with T-cell receptors and activate a large number of resting T cells resulting in release of massive cytokines, which will produce significant tumor inhibition in vivo and in vitro. However, it could be not broadly applied to cure malignant tumors in clinic because of emetic activity of SEC2. The aim of this study was to inactivate emetic activity of SEC2 through site-directed mutagenesis. Cys93, Cys110 and His118 were selected as substitutional sites based on the functional sites responsible for emesis. The mutated proteins were used to determine Peripheral blood mononuclear cell proliferation activity and anti-tumor activity in vitro. Results showed that these mutated proteins efficiently stimulated T cell and exhibited the same tumor-inhibition effect as SEC2. It is possible to inactivate emetic activity of SEC2 through site-directed mutagenesis and provide satisfying agents for tumor treatment in clinic. 相似文献
7.
Zhi Hua Liu Yu Cheng Wang Xiao Tian Qi Chuan Ping Yang 《Molecular biology reports》2010,37(5):2447-2453
Chitinases are digestive enzymes that break down glycosidic bonds in chitin. In the current study, an endochitinase gene Lbchi31 was cloned from Limonium bicolor. The cDNA sequence of Lbchi31 was 1,107 bp in length, encoding 322 amino acid residues with a calculated molecular mass of 31.7 kDa. Clustal analysis showed
that there was a highly conserved chitin-binding domains in Lbchi31 protein, containing four sulfide bridges. The Lbchi31 gene was inserted into the pPIC9 vector and transferred into yeast Pichia pastoris GS115 and KM71 for heterologous expression. The transformant harboring the Lbchi31 gene showed a clearly visible protein band with a molecular mass of more than 31 kDa in the SDS-PAGE gel, indicating that
it had been translated in P. pastoris. Enzyme characterization showed that the optimal reaction condition for chitinase LbCHI31 activity was: 40°C, pH of 5.0 and
5 mmol l−1 of Mn2+. The maximum enzyme activity was 0.88 U ml−1 following exposure to the cell wall chitin of Valsa sordida. The LbCHI31 enzyme can efficiently degrade cell wall chitin of the phytopathogenic Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum, V. sordida, Septoria tritici and Phytophthora sojae, suggesting that it has the biocontrol function to fungal phytopathogen. 相似文献
8.
Morimoto K Yoshimoto M Karita S Kimura T Ohmiya K Sakka K 《Applied microbiology and biotechnology》2007,73(5):1106-1113
A novel chitinase gene chiC of Clostridium paraputrificum M-21, a chitinolytic and hydrogen-gas-producing bacterium, was characterized along with its translated product. The chi18C gene encodes 683 amino acids (signal peptide included) with a deduced molecular weight of 74,651. Chi18C is a modular enzyme
composed of a family-18 catalytic module of glycoside hydrolases, two reiterated modules of unknown function, and a family-12
carbohydrate-binding module. Recombinant Chi18C was active toward soluble and insoluble chitin preparations, and synthetic
substrates such as 4-methylumbelliferyl-β-d-N-N′-N″-triacetylchitotriose, but not active toward 4-MU-N-acetylglucosamine or 4-MU-β-d-N-N′-diacetylchitobioside. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunological analyses suggested that
the expression of chi18C was inducible with chitinous substrates and that Chi18C was secreted into the culture medium. A possible role of Chi18C in
the chitinolytic system of C. paraputrificum M-21 is discussed. 相似文献
9.
S. Bharali R. K. Purama A. Majumder C. M. G. A. Fontes A. Goyal 《Indian journal of microbiology》2007,47(2):109-118
The non-catalytic, family 11 carbohydrate binding module (CtCBM11) belonging to a bifunctional cellulosomal cellulase from Clostridium thermocellum was hyper-expressed in E. coli and functionally characterized. Affinity electrophoresis of CtCBM11 on nondenaturing PAGE containing cellulosic polysaccharides showed binding with β-glucan, lichenan, hydroxyethyl cellulose
and carboxymethyl cellulose. In order to elucidate the involvement of conserved aromatic residues Tyr 22, Trp 65 and Tyr 129
in the polysaccharide binding, site-directed mutagenesis was carried out and the residues were changed to alanine. The results
of affinity electrophoresis and binding adsorption isotherms showed that of the three mutants Y22A, W65A and Y129A of CtCBM11, two mutants Y22A and Y129A showed no or reduced binding affinity with polysaccharides. These results showed that tyrosine
residue 22 and 129 are involved in the polysaccharide binding. These residues are present in the putative binding cleft and
play a critical role in the recognition of all the ligands recognized by the protein. 相似文献
10.
Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes. 相似文献
11.
Bourgois TM Van Craeyveld V Van Campenhout S Courtin CM Delcour JA Robben J Volckaert G 《Applied microbiology and biotechnology》2007,76(6):1309-1317
The complete genome sequence of Bacillus subtilis reveals that sequences encoding several hemicellulases are co-localised with a gene (xynD) encoding a putative family 43 glycoside hydrolase that has not yet been characterised. In this work, xynD has been isolated from genomic DNA of B. subtilis subsp. subtilis ATCC 6051 and cloned for cytoplasmatic expression in Escherichia coli. Recombinant XynD (rXynD) was purified using ion-exchange chromatography and gel permeation chromatography. The enzyme had
a molecular mass of approximately 52 kDa, a pI above 9.0 and releases α-l-arabinose from arabinoxylo-oligosaccharides as well as arabinoxylan polymers with varying degree of substitution. Using para-nitrophenyl-α-l-arabinofuranoside as substrate, maximum activity was observed at pH 5.6 and 45°C. The enzyme retained its activity over a
large pH range, while activity was lost after pre-incubation above 50°C. Gas–liquid chromatography and proton nuclear magnetic
resonance spectrometry analysis indicated that rXynD specifically releases arabinofuranosyl groups from mono-substituted C-(O)-2 and C-(O)-3 xylopyranosyl residues on the xylan backbone. As rXynD did not display endoxylanase, xylosidase or arabinanase activity
and was inactive on arabinan, we conclude that this enzyme is best described as an arabinoxylan arabinofuranohydrolase. 相似文献
12.
The conserved histidine residues, His-191, His-227, His-345, and His-378, in Bacillus stearothermophilus leucine aminopeptidase II (LAPII) were replaced with leucine by site-directed mutagenesis. The overexpressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular masses were approximately 44.5 kDa. Under assay conditions, no LAP activity was detected in H345L and H378L. Although the Km value for H191L increased more than 30% with respect to the wild-type LAPII, alteration in this residue did not lead to a significant change on the catalytic efficiency. The 39% decrease in Kcat/Km for H227L was partly caused by a 3.9-fold increase in Km value. Based on these results, it is suggested that His-345 and His-378 play a crucial role in the catalytic reaction of B. stearothermophilus LAPII. 相似文献
13.
Ali Dehestani Kamal Kazemitabar Gholamreza Ahmadian Nadali Babaeian Jelodar Ali Hatef Salmanian Mehdi Seyedi Heshmat Rahimian Seyedhadi Ghasemi 《Biotechnology letters》2010,32(4):539-546
The Bacillus pumilus SG2 chitinase gene (ChiS) and its truncated form lacking chitin binding (ChBD) and fibronectin type III (FnIII) domains were transformed to Arabidopsis plants and the expression, functionality and antifungal activity of the recombinant proteins were investigated. Results showed
that while the two enzyme forms showed almost equal hydrolytic activity toward colloidal chitin, they exhibited a significant
difference in antifungal activity. Recombinant ChiS in plant protein extracts displayed a high inhibitory effect on spore
germination and radial growth of hyphae in Alternaria brassicicola, Fusarium graminearum and Botrytis cinerea, while the activity of the truncated enzyme was strongly abolished. These findings demonstrate that ChBD and FnIII domains
are not necessary for hydrolysis of colloidal chitin but play an important role in hydrolysis of chitin–glucan complex of
fungal cell walls. Twenty microgram aliquots of protein extracts from ChiS transgenic lines displayed strong antifungal activity
causing up to 80% decrease in fungal spore germination. This is the first report of a Bacillus pumilus chitinase expressed in plant system. 相似文献
14.
The stylar component to gametophytic self-incompatibility in Solanaceae is an S-RNase. Its primary structure has a characteristic pattern of two hypervariable regions, involved in pollen recognition, and five constant regions. Two of the latter (C2 and C3) constitute the active site, while the highly hydrophobic C1 and C5 are believed to be involved in protein stability. We analyzed the role of the C4 region by site-directed mutagenesis. A GGGG mutant, in which the four charged residues in the C4 region were replaced with glycine, did not accumulate the protein to detectable levels in styles, suggestive of a role in protein stability. A R115G mutant, in which a charged amino acid was eliminated to reduce the potential binding affinity, had no effect on the pollen rejection phenotype. This suggests the C4 does not interact with partners such as potential pollen tube receptors facilitating S-RNase uptake. Finally, a K113R mutant replaced a potential ubiquitination target with arginine. However, this RNase acted as the wild type in both incompatible and compatible crosses. The latter crosses rule out the role of the conserved C4 lysine in ubiquitination. 相似文献
15.
16.
The composition of membrane-bound electron-transferring proteins from denitrifying cells of Haloarcula marismortui was compared with that from the aerobic cells. Accompanying nitrate reductase catalytic NarGH subcomplex, cytochrome b-561, cytochrome b-552, and halocyanin-like blue copper protein were induced under denitrifying conditions. Cytochrome b-561 was purified to homogeneity and was shown to be composed of a polypeptide with a molecular mass of 40 kDa. The cytochrome
was autooxidizable and its redox potential was −27 mV. The N-terminal sequence of the cytochrome was identical to the deduced
amino acid sequence of the narC gene product encoded in the third ORF of the nitrate reductase operon with a unique arrangement of ORFs. The sequence of
the cytochrome was homologous with that of the cytochrome b subunit of respiratory cytochrome bc. A possibility that the cytochrome bc and the NarGH constructed a supercomplex was discussed. 相似文献
17.
A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan 相似文献
18.
Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner.
The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate
larvae Galleria
mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host. 相似文献
19.
Noel H. Holmgren 《Brittonia》2018,70(1):115-139
A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations. 相似文献
20.
A genetic transformation system has been developed for callus cells of Crataegus
aronia using Agrobacterium
tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with
5 mg l−1 Indole-3-butyric acid (IBA) and 0.5 mg l−1 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different
types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red
colored callus was obtained on MS medium supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l−1 kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli
were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l−1 hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this
is the first time to report an Agrobacterium-mediated transformation system in Crataegus
aronia. 相似文献