共查询到20条相似文献,搜索用时 15 毫秒
1.
B. Veith V.V. Zverlov N.A. Lunina O.V. Berezina C. Raasch G.A> Velikodvorskaya 《Biocatalysis and Biotransformation》2013,31(4-5):147-158
AbstractThe hyperthermophilic bacterium Thermotoga maritima contains an amylolytic gene cluster with two adjacent α-glucosidase genes, aglB and aglA. We have now identified a similar pair of α-glucosidase genes on a 5,451 bp fragment of T. neapolitana genomic DNA. Like in T. maritima, aglA of T. neapolitana is located downstream of aglB. The deduced AglB primary structure allows its assignment to glycoside hydrolase family 13 (GHF13), whereas AglA belongs to GHF4. The aglB gene of T. neapolitana and the corresponding gene from T. maritima were expressed in E. coli, and the recombinant enzymes were characterized. Both enzymes hydrolyzed cyclomaltodextrins and linear maltooligosaccharides to yield glucose and maltose. Evidence from the hydrolysis of non-natural oligosaccharides and the pseudo-tetrasaccharide acarbose suggests that linear malto-oligosaccharides are progressively degraded by T. neapolitana and T. maritima AglB from the reducing end, which is highly uncommon for α-glucosidases. AglB, in contrast to the cofactor-dependent (NAD+, Mn2+) α-glucosidase AglA, does not cleave maltose. The recent elucidation of the crystal structure of T. miritima AglA indicates that AglA and AglB employ different catalytic mechanisms for glycosidic bond cleavage. Possible reasons for the presence of two α-glucosidase genes in the same amylolytic gene cluster of Thermotoga species are discussed. 相似文献
2.
Robert L. Mach Bernhard Seiboth rey Myasnikov Ramon Gonzalez † Josef Strauss Anu M. Harkki Christian P. Kubicek 《Molecular microbiology》1995,16(4):687-697
We have investigated the effect of disruption of the bgl1-(β-glucosidase l-encoding) gene of Trichoderma reesei on the formation of other β-glucosidase activities and on the induction of cellulases. To this end the bgl1 locus was disrupted by insertion of the Aspergillus nidulans amdS (acetamidase-encoding) gene. The bgl1-disrupted strain did not produce the 75kDa extracellular β-glucosidase on cellulose or lactose, but still formed β-glucosidase activity on glucose, cellobiose, xylan or β-1,3-glucan, suggesting that the enzyme(s) exhibiting this β-glucosidase activity is (are) not encoded by bgl1. The cellulose-inducer sophorose induced the bgl1-encoded β-glucosidase, whereas the remaining β-glucosidase activity was induced by methyl-β-D-glucoside. The bgl1-gene product was mainly secreted into the medium, whereas the other β-glucosidase activity was mainly associated with the cells. A bgl1-multicopy strain formed higher amounts of cellulases than the parent strain. Nonsaturating concentrations of sophorose efficiently induced cellobiohydrolase I formation in the bgl1-multicopy strain, but less efficiently in the bgl1-disrupted strain. The multicopy strain and the parent strain were comparably efficient at saturating sophorose concentrations. The β-glucosidase inhibitor nojirimycin strongly inhibited induction in all strains. These data suggest that the bgl1-encoded β-glucosidase is not identical to the plasma-membrane-bound, constitutive, methyl-β-glucoside inducible β-glucosidase, but represents an extracellular cellulose-induced enzyme. Both enzymes contribute to rapid induction of cellulases by modifying the inducer sophorose. 相似文献
3.
Background
The relative ease of targeted gene disruption in the social amoeba Dictyostelium has stimulated its widespread use as an experimental organism for cell and developmental biology. However, the field has been hamstrung by the lack of techniques to recombine disrupted genes. 相似文献4.
Three forms of α-glucosidase have been isolated from 5-day-old green gram (Phaseolus vidissimus Ten.) seedlings, by a procedure including fractionation with ammonium sulfate and polyethylene glycol 6000, DEAE-cellulose column chromatography, SP-Sephadex column chromatography, preparative gel electrofocusing and preparative disc gel electrophoresis. The α-glucosidases isolated were designated as α-glucosidase I, α-glucosidase II–1 and α-glucosidase II–2. They were homogeneous on polyacrylamide disc gel electrophoresis. Their molecular weights were 145,000, 105,000 and 65,000, respectively. The three enzymes hydrolyzed maltose, maltotriose, phenyl α-maltoside, amylose and soluble starch liberating glucose, but did not act on sucrose. Their enzymes hydrolyzed phenyl α-maltoside into glucose and phenyl α-glucoside. They hydrolyzed amylose liberating α-glucose. Maltotriose was the main α-glucosyltransfer product formed from maltose by the three α-glucosidases. 相似文献
5.
Background
Development of the post-genomic age in Dictyostelium will require the existence of rapid and reliable methods to disrupt genes that would allow the analysis of entire gene families and perhaps the possibility to undertake the complete knock-out analysis of all the protein-coding genes present in Dictyostelium genome.Results
Here we present an optimized protocol based on the previously described construction of gene disruption vectors by in vitro transposition. Our method allows a rapid selection of the construct by a simple PCR approach and subsequent sequencing. Disruption constructs were amplified by PCR and the products were directly transformed in Dictyostelium cells. The selection of homologous recombination events was also performed by PCR. We have constructed 41 disruption vectors to target genes of unknown function, highly conserved between Dictyostelium and human, but absent from the genomes of S. cerevisiae and S. pombe. 28 genes were successfully disrupted.Conclusion
This is the first step towards the understanding of the function of these conserved genes and exemplifies the easiness to undertake large-scale disruption analysis in Dictyostelium. 相似文献6.
Nobuhisa Shimba Mai Shinagawa Wataru Hoshino Hideyuki Yamaguchi Naoyuki Yamada Ei-ichiro Suzuki 《Analytical biochemistry》2009,393(1):23-28
α-Glucosidase from Aspergillus niger is an enzyme that catalyzes hydrolysis of α-1,4 linkages and transglucosylation to form α-1,6 linkages. In this study, an analytical method of oligosaccharides by nuclear magnetic resonance (NMR) was used to provide quantitative estimation of the fractions of each sugar unit and was applied to characterize the α-glucosidase reaction. Our data indicated that α-glucosidase reacts with the nonreducing end of oligosaccharides to form an α-1,6 linkage, and then a sugar unit with two α-1,6 linkages is gradually produced. Data from mass spectrometry suggested that the sugar unit with two α-1,6 linkages originates mainly from a 3mer and/or 4mer when oligosaccharides are used as substrates. 相似文献
7.
In this study, a new α-glucosidase gene from Thermoanaerobacter ethanolicus JW200 was cloned and expressed in Escherichia coli by a novel heat-shock vector pHsh. The recombinant α-glucosidase exhibited its maximum hydrolytic activity at 70°C and pH 5.0∼5.5. With p-nitrophenyl-α-D-glucoside as a substrate and under the optimal condition (70°C, pH 5.5), K
m and V
max of the enzyme was 1.72 mM and 39 U/mg, respectively. The purified α-glucosidase could hydrolyze oligosaccharides with both α-1,4 and α-1,6 linkages. The enzyme also had strong transglycosylation activity when maltose was used as sugar donor. The transglucosylation
products towards maltose are isomaltose, maltotriose, panose, isomaltotriose and tetrasaccharides. The enzyme could convert
400 g/L maltose to oligosaccharides with a conversion rate of 52%, and 83% of the oligosaccharides formed were prebiotic isomaltooligosaccharides
(containing isomaltose, panose and isomaltotriose). 相似文献
8.
《Bioscience, biotechnology, and biochemistry》2013,77(11):2346-2352
Three kinds of α-glucosidases, I, II, and III, were purified from European honeybees, Apis mellifera L. In addition, an α-glucosidase was also purified from honey. Some properties, including the substrate specificity of honey α-glucosidase, were almost the same as those of α-glucosidase III. Specific antisera against the α-glucosidases were prepared to examine the localization of α-glucosidases in the organs of honeybees. It was immunologically confirmed for the first time that α-glucosidase I was present in ventriculus, and α-glucosidase II, in ventriculus and haemolymph. α-Glucosidase III, which became apparent to be honey α-glucosidase, was present in the hypopharyngeal gland, from which the enzyme may be secreted into nectar gathered by honeybees. Honey may be finally made up through the process whereby sucrose in nectar, in which glucose and fructose also are naturally contained, is hydrolyzed by secreted α-glucosidase III. 相似文献
9.
The methanolic extract of Pongamia pinnata L. Pierre (locally called as karanja) seed materials, an underutilized food legume collected from India was analyzed for antioxidant and type II diabetes related enzyme inhibition properties. The methanolic extract of raw seeds contained total free phenolic content of 14.85 ± 0.32 g catechin equivalent/100 g extract DM. Encouraging levels of ferric reducing/antioxidant power (FRAP, 1179 mmol Fe[II]/mg extract), inhibition of β-carotene degradation (41.13%) and radical scavenging activity against DPPH (54.64%) and superoxide (54.53%) were exhibited by the raw sample. Further, it also recorded 77.92% of α-amylase and 86.50% of α-glucosidase enzyme inhibition characteristics under in vitro starch digestion bioassay. Sprouting + oil-frying caused a apparent increase on the total free phenolic content and also significant improvement on the antioxidant and free radical scavenging capacity of P. pinnata seeds, while soaking + cooking as well as open-pan roasting treatments showed diminishing effects. Moreover, inhibition of α-amylase and α-glucosidase enzyme activities was declined to 24.24 and 45.14%, respectively during sprouting + oil-frying treatment, which are more desirable for the dietary management of type II diabetic patients. 相似文献
10.
Strasser R Steinkellner H Borén M Altmann F Mach L Glössl J Mucha J 《Glycoconjugate journal》1999,16(12):787-791
N-acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi enzyme involved in the biosynthesis of glycoprotein-bound N-linked oligosaccharides, catalysing an essential step in the conversion of oligomannose-type to complex N-glycans. GnTII activity has been detected in both animals and plants. However, while cDNAs encoding the enzyme have already been cloned from several mammalian sources no GnTII homologue has been cloned from plants so far. Here we report the molecular cloning of an Arabidopsis thalianaGnTII cDNA with striking homology to its animal counterparts. The predicted domain structure of A. thalianaGnTII indicates a type II transmembrane protein topology as it has been established for the mammalian variants of the enzyme. Upon expression of A. thalianaGnTII cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited GnTII activity. 相似文献
11.
An α-glucosidase active at acid pH and presumably lysosomal in origin has been purified from human liver removed at autopsy. The enzyme has both α-1,4-glucosidase and α-1,6-glucosidase activities. The Km of maltose for the enzyme is 8.9 mm at the optimal pH of 4.0. The Km of glycogen at the optimal pH of 4.5 is 2.5% (9.62 mm outerchain end groups). Isomaltose has a Km of 33 mm when α-1,6-glucosidase activity is tested at pH 4.2. The enzyme exists in several active charge isomer forms which have pI values between 4.4 and 4.7. These forms do not differ in their specific activities. Electrophoresis in polyacrylamide gels under denaturing conditions indicates that the protein is composed of two subunits whose approximate molecular weights are 88,000 and 76,000. An estimated molecular weight of 110,000 was obtained by nondenaturing polyacrylamide gel electrophoresis. When the protein was chromatographed on Bio-Gel P-200 it was separated into two partially resolved active peaks which did not differ in their charge isomer constitution or in subunit molecular weights. One peak gave a strongly positive reaction for carbohydrate by the periodic acid-Schiff method and the other did not. Both had the same specific activity. The enzyme was antigenic in rabbits, and the antibodies so obtained could totally inhibit the hydrolytic action of the enzyme on glycogen but were markedly less effective in inhibiting activity toward isomaltose and especially toward maltose. Using these antibodies it was found that liver and skeletal muscle samples from patients with the “infantile” form or with the “adult” form of Type II glycogen storage disease, all of whom lack the lysosomal α-glucosidase, do not have altered, enzymatically inactive proteins which are immunologically cross-reactive with antibodies for the α-glucosidase of normal human liver. 相似文献
12.
Omid Motabar Zhen-Dan Shi Ke Liu Ellen Sidransky Gary L. Griffiths 《Analytical biochemistry》2009,390(1):79-2002
Mutations in α-glucosidase cause accumulation of glycogen in lysosomes, resulting in Pompe disease, a lysosomal storage disorder. Small molecule chaperones that bind to enzyme proteins and correct the misfolding and mistrafficking of mutant proteins have emerged as a new therapeutic approach for the lysosomal storage disorders. In addition, α-glucosidase is a therapeutic target for type II diabetes, and α-glucosidase inhibitors have been used in the clinic as alternative treatments for this disease. We have developed a new fluorogenic substrate for the α-glucosidase enzyme assay, resorufin α-d-glucopyranoside. The enzyme reaction product of this new substrate emits at a peak of 590 nm, reducing the interference from fluorescent compounds seen with the existing fluorogenic substrate, 4-methylumbelliferyl-α-d-glucopyranoside. Also, the enzyme kinetic assay can be carried out continuously without the addition of stop solution due to the lower pKa of the product of this substrate. Therefore, this new fluorogenic substrate is a useful tool for the α-glucosidase enzyme assay and will facilitate compound screening for the development of new therapies for Pompe disease. 相似文献
13.
《Bioscience, biotechnology, and biochemistry》2013,77(11):2415-2423
The fungal strain Mortierella alliacea YN-15 is an arachidonic acid producer that assimilates soluble starch despite having undetectable α-amylase activity. Here, a α-glucosidase responsible for the starch hydrolysis was purified from the culture broth through four-step column chromatography. Maltose and other oligosaccharides were less preferentially hydrolyzed and were used as a glucosyl donor for transglucosylation by the enzyme, demonstrating distinct substrate specificity as a fungal α-glucosidase. The purified enzyme consisted of two heterosubunits of 61 and 31 kDa that were not linked by a covalent bond but stably aggregated to each other even at a high salt concentration (0.5 M), and behaved like a single 92-kDa component in gel-filtration chromatography. The hydrolytic activity on maltose reached a maximum at 55°C and in a pH range of 5.0-6.0, and in the presence of ethanol, the transglucosylation reaction to form ethyl-α-D-glucoside was optimal at pH 5.0 and a temperature range of 45-50°C. 相似文献
14.
Nobuyuki Takahashi Tokuji Shimomura Seiya Chiba 《Bioscience, biotechnology, and biochemistry》2013,77(13):2015-2024
Two kinds of αglucosidase which were homogeneous in disc electrophoretic and ultra-centrifugal analysis were isolated from rice seeds by means of ammonium sulfate fractionation and CM-cellulose, Sephadex G–100 and DEAE-cellulose column chromatography and designated as α-glucosidase I and α-glucosidase II.Both α-glucosidases hydrolyzed maltose and soluble starch to glucose and showed same optimal pH (4.0) on the both substrates. In addition, both enzymes acted on various α-linked gluco-oligosaccharides and soluble starch but little or not on α-linked hetero-glucosides and α-l,6-glucan (dextran).Activity of the enzymes on maltose and soluble starch was inhibited by Tris and erythritol. α-Glucosidase II was more sensitive to the inhibitors than α-glucosidase I.Km value for maltose was 1.1 mM for α-glucosidase I and 2.0 mM for α-glucosidase II. 相似文献
15.
Samar Ramzi 《Archives Of Phytopathology And Plant Protection》2013,46(17):2131-2138
Apodiphus amygdali or stink bug of fruit trees is one of the polyphagous species from pentatomid bugs that attack many of fruit trees and ornamental trees. In the current study, activities of α- and β-glucosidases were measured in the midgut of A. amygdali adults. It was found the higher activity of β-glucosidase than α-glucosidase in addition to different enzymatic properties of the enzymes. Optimal pHs for enzymatic activities were found to be 5 and 7 for α- and β-glucosidases, respectively. Values regarding optimal temperatures were obtained at 30?°C for both α- and β-glucosidases. Among ions used on α-glucosidase activity, K+ and Ca2+ significantly increased enzymatic activity, Na+ had no effect, and Cu2+, Fe2+ and Mg2+ had the significant negative effects on the enzyme activity. Ca2+ and Fe2+ increased β-glucosidase activity in the midgut of A. amygdali, Na+ had no effect, and other ions significantly decreased the enzyme activity. Ethylene glycol-bis (β-aminoethylether) N,N,N?,N-tetraacetic acid (EGTA), citric acid, ethylenediamide tetraacetic acid (EDTA) and sodium dodecylsulfate (SDS) significantly decreased α-glucosidase activity but EGTA, triethylenetetramine hexaacetic acid (TTHA), EDTA and SDS decreased β-glucosidase activity in the midgut of A. amygdali. Characterisation of digestive enzymes, especially the effect of inhibitors on enzyme activity, could be useful for better understanding of enzyme roles in nutritional physiology of insects in addition to reach safe and useful controls of insect pests. 相似文献
16.
A major proportion of N-glycoproteins are transiently glucosylated in the endoplasmic reticulum. 总被引:5,自引:0,他引:5
N-linked, high-mannose-type oligosaccharides lacking glucose residues may be transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum of mammalian, plant, fungal, and protozoan cells. The products formed have been identified as N-linked Glc1Man5-9GlcNAc2 and glucosidase II is apparently the enzyme responsible for the in vivo deglucosylation of the compounds. As newly glucosylated glycoproteins are immediately deglucosylated, it is unknown whether transient glucosylation involves all or nearly all N-linked glycoproteins or if, on the contrary, it only affects a minor proportion of them. In order to evaluate the molar proportion of N-linked oligosaccharides that are glucosylated, cells of the trypanosomatid protozoan Trypanosoma cruzi (a parasite transferring Man9GlcNAc2 in protein N-glycosylation) were grown in the presence of [14C]glucose and concentrations of the glucosidase II inhibitors deoxynojirimycin and castanospermine that were more than 1000-fold higher than those required to produce a 50% inhibition of the T. cruzi enzyme. About 52-53% total N-linked oligosaccharides appeared to have glucose residues. The compounds were identified as Glc1Man7-9GlcNAc2. The same percentage was obtained when cells were pulsed-chased with [14C]glucose in the presence of deoxynojirimycin for 60 min. No evidence for the presence of an endomannosidase yielding GlcMan from the glycosylated compounds was obtained. As the average number of N-linked oligosaccharides per molecule in glycoproteins is higher than one, these results indicate that more than 52-53% of total glycoproteins are glucosylated and that transient glucosylation is a major event in the normal processing of glycoproteins. 相似文献
17.
《Bioscience, biotechnology, and biochemistry》2013,77(11):2146-2149
An α-glucosidase was purified in an electrophoretically pure state from an extract of koji culture of Aspergillus sp. KT-11. This enzyme was found to have a transferring activity when the reaction was done in a high concentration of leucrose at pH 4.5. Two kinds of transfer products, fractions I and II, were obtained from leucrose by the enzyme and they were identified as [(α-D-glucopyranosyl-(1 →6)-α-D-glucopyranosyl-(1 →6)- α -D-glucopyranosyl-(1→5)-D-fructopyranose] and [α-D-glucopyranosyl-(1 →6)-α-D-glucopyranosyl-(1→5)-D- fructopyranose], respectively. These are considered to be novel oligosaccharides 相似文献
18.
Human NDP kinase B, product of the nm23-H2 gene, binds DNA. It has been suggested that a helix hairpin on the protein surface, part of the nucleotide substrate binding site, could accommodate DNA binding by swinging away. The presence of flexible regions was therefore investigated by 1H NMR dynamic filtering. Although TOCSY peaks could be assigned to five residues at the N terminus of Dictyostelium NDP kinase, no flexible region was detected in the human enzyme. These data favor the idea that the protein offers different binding sites to mono- and polynucleotides. Proteins 28:150–152, 1997. © 1997 Wiley-Liss Inc. 相似文献
19.
We describe a rapid method for creating Dictyo stelium gene disruption constructs, whereby the target gene is interrupted by a drug resistance cassette using in vitro transposition. A fragment of genomic DNA containing the gene to be disrupted is amplified by PCR, cloned into a plasmid vector using topoisomerase and then employed as the substrate in an in vitro Tn5 transposition reaction. The transposing species is a fragment of DNA containing a Dictyostelium blasticidin S resistance (bsr) cassette linked to a bacterial tetracycline resistance (tetr) cassette. After transposition the plasmid DNA is transformed into Escherichia coli and clones in which the bsr-tetr cassette is inserted into the Dictyostelium target DNA are identified. To demonstrate its utility we have employed the method to disrupt the gene encoding QkgA, a novel protein kinase identified from the Dictyostelium genome sequencing project. QkgA is structurally homologous to two previously identified Dictyostelium kinases, GbpC and pats1. Like them it contains a leucine-rich repeat domain, a small GTP-binding (ras) domain and a MEKK domain. Disruption of the qkgA gene causes a marked increase in growth rate and, during development, aggregation occurs relatively slowly to form abnormally large multicellular structures. 相似文献
20.
Ichiro Masai Emiko Suzuki Chun-Sik Yoon Ayako Kohyama Yoshiki Hotta 《Developmental neurobiology》1997,32(7):695-706
The Drosophila retinal degeneration A (rdgA) mutant has photoreceptor cells that degenerate within a week after eclosion. The degeneration starts with the disruption of the subrhabdomeric cisternae (SRC), which are the organelles essential for the transport of phospholipids to the photoreceptive membranes. Our previous biochemical and molecular studies suggested that the rdgA gene encodes an eye-specific diacylglycerol kinase (DGK). In this study, we show that retinal degeneration is prevented by the introduction of the eye-DGK gene in the rdgA mutant genome, suggesting that the DGK activity is crucial for the maintenance of the photoreceptor. Furthermore, by immunohistochemical analysis, we have demonstrated that the rdgA protein is predominantly associated with the SRC, suggesting that the conversion from diacylglycerol (DG) to phosphatidic acid (PA) most actively occurs in SRC. The analysis of the eyes of mutants homozygous for rdgA and eye-protein kinase C mutations indicates that retinal degeneration is caused by the deficiency of PA rather than excessive accumulation of DG. From these data, we conclude that the production of PA in the SRC membranes is essential for the maintenance of the photoreceptor. © 1997 John Wiley & Sons, Inc. J Neurobiol 32 : 695–706, 1997 相似文献