共查询到20条相似文献,搜索用时 31 毫秒
1.
M. V. Semenova M. I. Drachevskaya O. A. Sinitsyna A. V. Gusakov A. P. Sinitsyn 《Biochemistry. Biokhimii?a》2009,74(9):1002-1008
Homogeneous β-xylosidases with molecular mass values 120 and 80 kDa (as shown by SDS-PAGE), belonging to the third family
of glycosyl hydrolases, were isolated by anion-exchange, hydrophobic, and gel-penetrating chromatography from enzyme preparations
based on the fungi Aspergillus japonicus and Trichoderma reesei, respectively. The enzymes exhibit maximal activity in acidic media (pH 3.5–4.0), and temperature activity optimum was 70°C
for the β-xylosidase of A. japonicus and 60°C for the β-xylosidase of T. reesei. Kinetic parameters of p-nitrophenyl β-xylopyranoside and xylooligosaccharide hydrolysis by the purified enzymes were determined, which showed that
β-xylosidase of A. japonicus was more specific towards low molecular weight substrates, while β-xylosidase of T. reesei preferred high molecular weight substrates. The competitive type of inhibition by reaction product (xylose) was found for
both enzymes. The interaction of the enzymes of different specificity upon hydrolysis of glucurono- and arabinoxylans was
found. The β-xylosidases exhibit synergism with endoxylanase upon hydrolysis of glucuronoxylan as well as with α-L-arabinofuranosidase
and endoxylanase upon hydrolysis of arabinoxylan. Addition of β-xylosidases increased efficiency of hydrolysis of plant raw
materials with high hemicellulose content (maize cobs) by the enzymic preparation Celloviridine G20x depleted of its own β-xylosidase. 相似文献
2.
Se-Wook Jung Tae-Kwon Kim Kwang-Woo Lee Yong-Hyun Lee 《Biotechnology and Bioprocess Engineering》2007,12(3):207-212
The catalytic properties of β-cyclodextrin glucanotransferase (β-CGTase) from alkalophilicBacillus sp. BL-12 specific for the intermolecular transglycosylation of stevioside were investigated. The molecular mass of purified
β-CGTase by ultra-filtration and β-cyclodextrin polymer affinity chromatography was estimated to be 90 kDa, which is high
compared to other known bacterial CGTases. The optimal pH and temperature were 9.0 and 50°C, respectively, and thermal stability
at 40°C was elevated 10-fold in the presence of 1% maltodextrin. The kinetic parameters of the new β-CGTase from alkalophilicBacillus sp. BL-12 indicate that it is more suitable for transglycosylation than the cyclization reaction. Maltodextrin was the most
suitable glycosyl donor for transglycosylation of stevioside. The transglycosylation of stevioside was carried out using 60
units of CGTase per gram of maltodextrin, 20 g/L stevioside as the glycosyl acceptor, and 50 g/L maltodextrin as the gycosyl
donor at 40°C for 6 h, and a conversion yield of stevioside as high as 76% was obtained. 相似文献
3.
Thierry Dintinger Delphine Dutheil-Bouëdec Michel Bouchonneau Charles Tellier 《Biotechnology letters》1994,16(7):689-692
Summary The synthetic potential ofAlmond β-glucosidase andJack bean α-mannosidase in the presence of high amounts of β-mercaptoethanol as glycosyl acceptor for the synthesis of β-mercaptoethyl-glycosides
was studied. The regioselectivity, O-glycosylation and/or S-glycosylation, and the stereoselectivity were analyzed with the
reverse hydrolysis and the transglycosylation methods. With both enzymes, high yields of condensation are obtained without
the use of chemical protective groups. 相似文献
4.
Summary The transglycosylation activity of β-galactosidase fromAchatina achatina digestive juice was tested for glycosylating protected hydroxy amino acids. Attractive yields of β-galactosyl-(Z-Ser-OMe)
(35%) and β-galactosyl-(Z-Hyp-OMe) (28%) could be obtained using lactose as glycosyl donor and the corresponding amino acid
methyl esters N-protected by a benzyloxycarbonyl group (Z) as glycosyl acceptors. 相似文献
5.
Nakagawa T Fujimoto Y Ikehata R Miyaji T Tomizuka N 《Applied microbiology and biotechnology》2006,72(4):720-725
In this study, we purified and molecularly characterized a cold-active β-galactosidase from Arthrobacter psychrolactophilus strain F2. The purified β-galactosidase from strain F2 exhibited high activity at 0°C, and its optimum temperature and pH were 10°C and 8.0, respectively. It was possible to inactivate the β-galactosidase rapidly at 45°C in 5 min. The enzyme was able to hydrolyze lactose as a substrate, as well as o-nitrophenyl-β-d-galactopyranoside (ONPG), the K
m values with ONPG and lactose being calculated to be 2.8 mM and 50 mM, respectively, at 10°C. Moreover, the bglA gene encoding the β-galactosidase of strain F2 was cloned and analyzed. The bglA gene consists of a 3,084-bp open reading frame corresponding to a protein of 1,028 amino acid residues. BglAp, the gene product derived from bglA, had several conserved regions for glycosyl hydrolase family 2, e.g., the glycosyl hydrolase 2 (GH2) sugar binding domain, GH2 acid-base catalyst, GH2 triosephosphate isomerase barrel domain, GH2 signature 1, and several other GH2 conserved regions. From these facts, we conclude that the β-galactosidase from A. psychrolactophilus strain F2, which is a new member of glycosyl hydrolase family 2, is a cold-active enzyme that is extremely heat labile and could have advantageous applications in the food industry. 相似文献
6.
The β-glycosidase gene of Thermus thermophilus KNOUC202 was cloned, expressed in Escherichia coli JM109(DE3), and the enzyme was purified and characterized. The gene (KNOUC202β-gly) was composed of 1296 bp encoding a β-glycosidase (KNOUC202β-glycosidase) of 431 a.a., belonging to the family 1 of glycosyl
hydrolase. The gene was expressed as monomer of 430 a.a. with amino terminal methionine excised in E. col JM109(DE3). The enzyme hydrolyzed β-glycosides whose glycone are galactose, glucose and fucose well, however showed no or
very low activity on β-D-glycosides whose glycone are disaccharides and xylose. k
cat of the enzyme for the hydrolysis of p-Nph-β-D-Glcp was lower than those for p-Nph-β-D-Galp and ONPG, however K
m for p-Nph-β-D-Glcp was highly lower than those for p-Nph-β-D-Galp and ONPG resulting in the catalytic efficiency(k
cat/K
m) for the hydrolysis of p-Nph-β-D-Glcp much higher than those for p-Nph-β-D-Galp and ONPG. Optimum pH and optimum temperature of the enzyme were pH 5.4 and 90°C. The enzyme has high thermostability, not
losing its activity at 80°C for 2 h in 0.05 M Na-phosphate buffer of pH 6.8 with T
m of 100.0 ± 0.031°C in 0.02 M Tris-HCl buffer of pH 8.2. The b-glycosidase produced a disaccharide composed of galactose as transglycosylation by-product during hydrolysis of lactose. 相似文献
7.
Su-Il Do 《Glycoconjugate journal》2009,26(5):567-575
Novel chimeric lacdiNAc (GalNAc(β1-4)GlcNAc) synthase (c-LacdiNAcS) was generated by gene fusion of α-lactalbumin (α-LA) and
β1,4-galactosyltransferase 1 (β1,4-GalT1). c-LacdiNAcS was expressed in Lec8 Chinese hamster ovary (Lec8 CHO) cells and exhibited
N-acetylgalactosaminyltransferase (GalNAcT) activity in the absence of exogenous α-LA as well as other glycosyltransferase
activities including lactose synthase (LacS), and β1,4-GalT. These glycosyltransferase activities of c-LacdiNAcS were compared
to those activities induced in LacS system under the co-presence of bovine β1,4-GalT1 and α-LA, indicating that each domain
of α-LA and β1,4-GalT1 on c-LacdiNAcS is not only folding correctly, but also interacting together. Furthermore, c-LacdiNAcS
was found to be auto-lacdiNAcylated and can synthesize lacdiNAc structures on cellular glycoproteins, demonstrating that GalNAcT
activity of c-LacdiNAcS is functional in Lec8 CHO cells. 相似文献
8.
We have developed a new procedure based on the random glycosyl reaction of a partially benzoylated glycosyl acceptor with a glycosyl donor containing a 4,6-O-(4-methoxycarbonylbenzylidene) protecting group as a masked/caged ion-tag. Glycosylated products are ionically tagged by saponification of the methyl ester and the use of this anion-tag greatly simplifies the separation of the desired oligosaccharides from unreacted or excess glycosyl acceptors as well as from over-glycosylated oligosaccharides. In addition, the use of partially benzoylated acceptors greatly improves their solubility in dichloromethane increasing the yield of product formation and, also, of altering the distribution of positional isomers in favor of products derived by reaction of the donors at hydroxyl groups which otherwise would be considerably less reactive. Using this new approach in random glycosyl reactions, several oligosaccharide libraries were readily prepared in overall yields of 60–70% and the individual positional isomers present in the libraries were identified using the ‘reductive-cleavage’ method. 相似文献
9.
Tamarind xyloglucan was oxidised by reaction with sodium hypochlorite in the presence of 2,2,6,6-tetramethyl-1-piperidinyloxy
free radical (TEMPO). Galactose residues and non-xylosylated glucose residues were thus converted into galacturonic and glucuronic
acid residues, respectively, producing an anionic polysaccharide. Acid hydrolysis of oxidised xyloglucan yielded two aldobiouronic
acids, deduced to be β-d-GalpA-(1→2)-d-Xyl and β-d-GlcpA-(1→4)-d-Glc. Anionic xyloglucan had a decreased ability to hydrogen-bond to cellulose and to complex with iodine. It was almost totally
resistant to digestion by cellulase [endo-(1→4)-β-glucanase] and did not serve as a donor substrate for xyloglucan endotransglucosylase
(XET) activity. Like several other anionic polysaccharides, it promoted XET activity when unmodified (non-ionic) xyloglucan
was used as donor substrate. Anionic xyloglucan may mimic polyanions whose presence in the plant cell wall promotes the action
of endogenous XTH proteins. NaOCl with TEMPO oxidised the heptasaccharide, XXXG, to form XXX-glucarate, which did serve as
an acceptor substrate although at a rate approximately fourfold less than XXXG itself. Anionic derivatives of xyloglucan,
acting as acceptor but not donor substrates, may be valuable tools for exploring the biological roles of XTHs in the integration
versus the re-structuring of xyloglucan in the plant cell wall. 相似文献
10.
Park TH Choi KW Park CS Lee SB Kang HY Shon KJ Park JS Cha J 《Applied microbiology and biotechnology》2005,69(4):411-422
The gene encoding β-glucosidase of the marine hyperthermophilic eubacterium Thermotoga neapolitana (bglA) was subcloned and expressed in Escherichia coli. The recombinant BglA (rBglA) was efficiently purified by heat treatment at 75°C, and a Ni-NTA affinity chromatography and its molecular mass were determined to be 56.2 kDa by mass spectrometry (MS). At 100°C, the enzyme showed more than 94% of its optimal activity. The half-life of the enzyme was 3.6 h and 12 min at 100 and 105°C, respectively. rBglA was active toward artificial (p-nitrophenyl β-d-glucoside) and natural substrates (cellobiose and lactose). The enzyme also exhibited activity with positional isomers of cellobiose: sophorose, laminaribiose, and gentiobiose. Kinetic studies of the enzyme revealed that the enzyme showed biphasic behavior with p-nitrophenyl β-d-glucoside as the substrate. Whereas metal ions did not show any significant effect on its activity, dithiothreitol and β-mercaptoethanol markedly increased enzymatic activity. When arbutin and cellobiose were used as an acceptor and a donor, respectively, three distinct intermolecular transfer products were found by thin-layer chromatography and recycling preparative high-performance liquid chromatography. Structural analysis of three arbutin transfer products by MS and nuclear magnetic resonance indicated that glucose from cellobiose was transferred to the C-3, C-4, and C-6 in the glucose unit of acceptor, respectively. 相似文献
11.
A general method is described for the assay of glycosyltransferase activity, which makes use of synthetic glycoside acceptors attached to hydrophobic aglycones. The products formed by incubation of an enzyme with acceptor and radiolabelled sugarnucleotide can then be rapidly (one minute) separated from interfering radioactivity by adsorption on to reverse-phase C-18 cartridges. After aqueous washing, products are easily isolated by elution with methanol. The utility of the method for the assay of (1–4)galactosyltransferase, (1–2)fucosyltransferase andN-acetylglucosaminyltransferase I and V is demonstrated. 相似文献
12.
Lucien Patrice Kouamé Sébastien Niamké Jacques Diopoh Bernard Colas 《Biotechnology letters》2001,23(19):1575-1581
The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates. 相似文献
13.
The moderately thermophilic aerobic ascomycete Talaromyces emersonii secretes, under selected growth conditions, several β-glucan hydrolases including an exo-1,3-β-glucanase. This enzyme was
purified to apparent homogeneity in order to characterise its biochemical properties and investigate hydrolysis of different
β-glucans, including laminaran, a 1,3-β-glucan from brown algae. The native enzyme is monomeric with a molecular mass of ~40 kDa
and a pI value of 4.3, and is active over broad ranges of pH and temperature, with optimum activity observed at pH 5.4 and 65 °C.
At pH 5.0, the enzyme displays strict specificity for laminaran (apparent K
m 1.66 mg mL−1; V
max 7.69 IU mL−1) and laminari-oligosaccharides and did not yield activity against 1,4-β-glucans, 1,3;1,4-β-glucans or 4-nitrophenyl- and
methylumbelliferyl-β-d-glucopyranosides. Analysis of hydrolysis products formed during time-course hydrolysis of laminaran by high-performance anion
exchange chromatography with pulsed amperometric detection revealed a strict exo mode of action, with glucose being the sole
reaction product even at the initial stages of hydrolysis. The T. emersonii exo-1,3-β-glucanase was inhibited by glucono-δ-lactone (K
i 1.25 mM) but at significantly higher concentrations than typically inhibitory for exo-glycosidases such as β-glucosidase.
‘De novo’ sequence analysis of the purified enzyme suggests that it belongs to family GH5 of the glycosyl hydrolase superfamily.
The results clearly show that the exo-1,3-β-glucanase is yet another novel enzyme present in the β-glucanolytic enzyme system
of T. emersonii. 相似文献
14.
Wayllace NZ Valdez HA Merás A Ugalde RA Busi MV Gomez-Casati DF 《Molecular biology reports》2012,39(1):585-591
The metabolic pathways leading to the synthesis of bacterial glycogen involve the action of several enzymes, among which glycogen
synthase (GS) catalyzes the elongation of the α-1,4-glucan. GS from Agrobacterium tumefaciens uses preferentially ADPGlc, although UDPGlc can also be used as glycosyl donor with less efficiency. We present here a continuous
spectrophotometric assay for the determination of GS activity using ADP- or UDPGlc. When ADPGlc was used as the substrate,
the production of ADP is coupled to NADH oxidation via pyruvate kinase (PK) and lactate dehydrogenase (LDH). With UDPGlc as
substrate, UDP was converted to ADP via adenylate kinase and subsequent coupling to PK and LDH reactions. Using this assay,
we determined the kinetic parameters of GS and compared them with those obtained with the classical radiochemical method.
For this purpose, we improved the expression procedure of A. tumefaciens GS using Escherichia coli BL21(DE3)-RIL cells. This assay allows the continuous monitoring of glycosyltransferase activity using ADPGlc or UDPGlc as
sugar-nucleotide donors. 相似文献
15.
An enzyme-linked immunosorbent assay (ELISA)-based glycosyltransferase assay has been used to measure UDP-Gal:N-acetylglucosamine beta-1,4-galactosyl-transferase (EC 2.4.1.38) activity in detergent extracts of chinese hamster ovary (CHO) cells. LEC11 cells (a mutant of the CHO cell line, Pro -5), which are known to express a complex array of carbohydrate structures, were used to develop the assay for use with whole cell extracts. A detergent-solubilized preparation of the enzyme from whole cells was used to convert the substrate, lactotriglycosylceramide, to the product, neolactotetraglycosylceramide. The monoclonal antibody, 1B2, which specifically binds to the Gal beta 1-4GlcNAc epitope, was used in an ELISA to identify and quantify the product. The enzyme activity in the preparations was found to be similar to that obtained by conventional radioactive assay methods. The beta-galactosyltransferase found in LEC11 cell detergent extracts exhibited an absolute requirement for the nucleotide sugar and MnCl2. The activity of the enzyme was also strictly dependent on the presence of exogenous glycolipid acceptor. When Triton X-114 was used to solubilize the LEC11 beta-galactosyltransferase, activity was found in both the hydrophilic and the hydrophobic phases, suggesting the presence of two forms of the enzyme. The ELISA-based assay was used to compare beta-1,4-galactosyltransferase activity in detergent extracts of four CHO cell lines: Pro-5, Lec1, LEC11, and LEC12 and in detergent-solubilized microsomes from human leukemia cells. The results from this study demonstrate the utility of the ELISA-based assay for measuring glycosyltransferase activity in detergent-solubilized whole cells and microsome preparations. 相似文献
16.
Birichevskaya LL Kvach SV Sivets GG Kalinichenko EN Zinchenko AI Mikhailopulo IA 《Biotechnology letters》2007,29(4):585-591
Enzymatic 5′-monophosphorylation and 5′-phosphatidylation of a number of β-l- and β-d-nucleosides was investigated. The first reaction, catalyzed by nucleoside phosphotransferase (NPT) from Erwinia herbicola, consisted of the transfer of the phosphate residue from p-nitrophenylphosphate (p-NPP) to the 5′-hydroxyl group of nucleoside; the second was the phospholipase d (PLD)-catalyzed transphosphatidylation of l-α-lecithin with a series of β-l- and β-d-nucleosides as the phosphatidyl acceptor resulted in the formation of the respective phospholipid-nucleoside conjugates.
Some β-l-nucleosides displayed similar or even higher substrate activity compared to the β-d-enantiomers. 相似文献
17.
Raju M Lakshminarayana R Krishnakantha TP Baskaran V 《Molecular and cellular biochemistry》2006,288(1-2):7-15
Improving the bioavailability of β-carotene is vital to manage vitamin A deficiency. The influence of micellar oleic (OA), linoleic (LA) and eicosapentaenoic (EPA) acids on plasma β-carotene response and its conversion to retinol has been studied in rats employing single (9 h time course) and repeated (10 days) dose administrations. After a single dose, the levels (area under the curve) of plasma β-carotene and retinyl palmitate in OA and EPA groups were higher (p < 0.05) by 13, 7 and 11, 6 folds than LA group. The liver β-carotene level in OA and EPA groups were higher (p < 0.05) by 3 and 1.2 folds than LA group. After repeated dose, the plasma β-carotene and retinyl palmitate levels in OA (6.2%, 51.7%) and EPA (25.4%, 17.23%) groups were higher (p < 0.05) than LA group. The liver β-carotene level in OA (21.2%) and EPA (17.6%) groups were higher (p < 0.05) than LA group. In both the experiments, the activity of β-carotene 15,15′-dioxygenase in the intestinal mucosa and plasma triglyceride levels were also higher in OA and EPA groups than LA group. β-Carotene excreted through urine and feces of OA and EPA groups was lower than the LA group. These results demonstrate an improved absorption and metabolism of β-carotene when fed mixed micelles with OA or EPA compared with LA. Although the mechanism involved in selective absorption of fatty acids needs further studies, intestinal β-carotene uptake and its conversion to vitamin A can be modulated using specific fatty acids. 相似文献
18.
An operon, bglABC, that encodes two sugar permeases and a β-glucosidase was cloned from a cellulolytic actinomycete, Thermobifida fusca, into Escherichia coli and sequenced. The bglC gene encoding an intracellular β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) belonging to glycosyl hydrolase
family 1 was subcloned and expressed in E. coli. The purified enzyme (MW 53,407 Da; pI 4.69) hydrolyzed substrates containing both β 1 → 4 and β 1 → 2 glycosidic bonds,
and was most active against cellobiose (Vmax= 29, K
m
= 0.34 mm), cellotriose, cellotetraose, and sophorose. The enzyme also showed aryl-β-glucosidase activity on p-nitrophenyl-β-d-glucopyranoside and p-nitrophenyl-β-d-cellobioside. BglC had a pH optimum of 7.0 and a temperature optimum
of 50°C. The enzyme was stable at 60°C, but was rapidly inactivated at 65°C. BglC was inhibited by low concentrations of gluconolactone,
but was insensitive to end-product inhibition by glucose and was not affected by Ca or Mg ions or EDTA. Its properties are
well suited for use in a process to hydrolyze biomass cellulose to glucose.
Received: 21 August 2000 / Accepted: 4 October 2000 相似文献
19.
Moreland D. Gibbs Rosalind A. Reeves Anwar Sunna Peter L. Bergquist 《Current microbiology》1999,39(6):351-357
A β-mannanase gene (manA) was isolated from the extremely thermophilic bacterium Dictyoglomus thermophilum Rt46B.1. ManA is a single-domain enzyme related to one group of β-mannanases (glycosyl hydrolase family 26). The manA gene was expressed in the heat-inducible vector pJLA602 and the expression product, ManA, purified to homogeneity. The recombinant
ManA is a monomeric enzyme with a molecular mass of 40 kDa and an optimal temperature and pH for activity of 80°C and 5.0.
In the absence of substrate, the enzyme showed no loss of activity at 80°C over 16 h, while at 90°C the enzyme had a half-life
of 5.4 min. Hydrolysis of the galactomannan locust bean gum (LBG) by purified ManA released mainly mannose, mannobiose, and
mannotriose, confirming that ManA is an endo-acting β-mannanase. Sequence comparisons with related β-mannanases has allowed
the design of consensus PCR primers for the identification and isolation of related genes.
Received: 7 June 1999 / Accepted: 6 July 1999 相似文献
20.
Ha Young Chung Jeon Hwang-Bo Seong-Ki Kim Nam In Baek Youn Hyung Lee In Sik Chung Jong-Hwa Park 《Biotechnology and Bioprocess Engineering》2011,16(4):801-807
Arabidopsis thaliana sterol glycosyltransferase (SGT), UGT80A2, was expressed from stably transformed Drosophila melanogaster Schneider 2 (S2) cells. Recombinant SGT was detected in both intracellular and extracellular fractions with a molecular mass
of approximately 76 kDa. Secreted recombinant SGT accounted for approximately 60% of the total recombinant SGT production.
Recombinant SGT in the extracellular fractions was purified to homogeneity using a simple one-step Ni-NTA affinity fractionation.
Radiometrical assay using uridine diphospho-d-[U-14C]glucose (UDP-14C-glucose) as a sugar donor and sterols, β-sitosterol and stigmasterol, as sugar acceptors showed that the purified recombinant
SGT contained UDP-glycosyltransferase activity and could attach 14C-glucose to β-sitosterol and stigmasterol. Recombinant SGT contained higher catalytic activity with β-sitosterol, which was
similar to the recombinant SGT produced by a bacterial expression system. The transfer of 14C-glucose by recombinant SGT was further determined by gas chromatography-mass spectrometry (GC-MS) analysis of cellulase-treated
14C-glucosetransferred β-sitosterol and stigmasterol reactants. 相似文献