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1.
A 66-kDa thermostable family 1 Glycosyl Hydrolase (GH1) enzyme with β-glucosidase and β-galactosidase activities was purified
to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family. N-terminal and partial internal amino acid sequences showed significant resemblance to plant GH1 enzymes. Kinetic
studies showed that enzyme hydrolyzed p-nitrophenyl β-d-glucopyranoside (pNP-Glc) with higher efficiency (K
cat/K
m = 2.27 × 104 M−1 s−1) as compared to p-nitrophenyl β-d-galactopyranoside (pNP-Gal; K
cat/K
m = 1.15 × 104 M−1 s−1). The optimum pH for β-galactosidase activity was 4.8 and 4.4 in citrate phosphate and acetate buffers respectively, while
for β-glucosidase it was 4.6 in both buffers. The activation energy was found to be 10.6 kcal/mol in the temperature range
30–65 °C. The enzyme showed maximum activity at 65 °C with half life of ~40 min and first-order rate constant of 0.0172 min−1. Far-UV CD spectra of enzyme exhibited α, β pattern at room temperature at pH 8.0. This thermostable enzyme with dual specificity
and higher catalytic efficiency can be utilized for different commercial applications. 相似文献
2.
Ah-Reum Park Hye-Jung Kim Jung-Kul Lee Deok-Kun Oh 《Applied biochemistry and biotechnology》2010,160(8):2236-2247
We expressed a putative β-galactosidase from Sulfolobus acidocaldarius in Escherichia coli and purified the recombinant enzyme using heat treatment and Hi-Trap ion-exchange chromatography. The resultant protein gave
a single 57-kDa band by SDS-PAGE and had a specific activity of 58 U/mg. The native enzyme existed as a dimer with a molecular
mass of 114 kDa by gel filtration. The maximum activity of this enzyme was observed at pH 5.5 and 90 oC. The half-lives of
the enzyme at 70, 80, and 90 oC were 494, 60, and 0.2 h, respectively. The hydrolytic activity with p-nitrophenyl(pNP) substrates followed the order p-nitrophenyl-β-d-fucopyranoside > pNP-β-d-glucopyranoside > pNP-β-d-galactopyranoside > pNP-β-d-mannopyranoside > pNP-β-d-xylopyranoside, but not toward aryl-α-glycosides or pNP-β-l-arabinofuranoside. Thus, the enzyme was actually a β-glycosidase. The β-glycosidase exhibited transglycosylation activity
with pNP-β-d-galactopyranoside, pNP-β-d-glucopyranoside, and pNP-β-d-fucopyranoside in decreasing order of activity, in the reverse order of its hydrolytic activity. The hydrolytic activity
was higher toward cellobiose than toward lactose, but the transglycosylation activity was lower with cellobiose than with
lactose. 相似文献
3.
A simple one-step synthesis of β-d-galactopyranosyl azide from 0-nitrophenyl-β-d-galactopyranoside and azide catalyzed by E461G-β-galactosidase is described. The synthesis is quantitative in the presence
of excess azide and only the β anomer is produced. The product was purified (71% yield) from the other reaction components
by extraction with ethyl acetate, silica gel chromatography, and crystallization. The purity was verified by GLC, TLC, and
NMR. Thus, E461G-β-galactosidase is able to specifically and quantitatively from β-d-galactopyranosyl-azide. The purified β-d-galactopyranosylazide inhibited the growth of Escherichia coli that express β-galactosidase but not of E. coli that do not. Growth is stopped because β-galactosidase catalyzes the hydrolysis of the β-galactopyranosyl-azide, and the
azide that is produced inhibits cell growth. This selective inhibition of growth has potential application in molecular biology
screening. 相似文献
4.
Triblock cooligomers consisting of tri-O-methyl-glucopyranosyl and unmodified glucopyranosyl residues, methyl 2,3,4,6-tetra-O-methyl-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-α-d-glucopyranoside (1: ABA triblock cooligomer; DS = 2.1) and β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1 → 4)-d-glucopyranose (2: BAB triblock cooligomer; DS = 1.8) were prepared. Compound 1 dissolved both in distilled water and chloroform but compound 2 dissolved in distilled water not in chloroform, though compounds 1 and 2 consist of 4 tri-O-methyl-glucopyranosyl and 2 unmodified anhydro glucopyranosyl units. 相似文献
5.
Agnese Jurkevica H. Brian Halsall Carl J. Seliskar William R. Heineman 《Analytical and bioanalytical chemistry》2010,397(7):3133-3136
Viable Escherichia coli can be detected by an immunoassay in which live bacteria captured on antibody-coated paramagnetic beads are induced to synthesize
the enzyme β-galactosidase, which catalyzes the hydrolysis of the slightly fluorescent substrate 4-methyl umbelliferyl-β-d-galactoside to the highly fluorescent product 7-hydroxy-4-methylcoumarin for detection. The effects of bacterial strain,
presence of dead bacteria, and some environmental stresses on assay performance were evaluated. 相似文献
6.
Xu Z Li S Fu F Li G Feng X Xu H Ouyang P 《Applied biochemistry and biotechnology》2012,166(4):961-973
d-tagatose is a ketohexose that can be used as a novel functional sweetener in foods, beverages, and dietary supplements. This
study was aimed at developing a high-yielding d-tagatose production process using alginate immobilized Lactobacillus fermentum CGMCC2921 cells. For the isomerization from d-galactose into d-tagatose, the immobilized cells showed optimum temperature and pH at 65 °C and 6.5, respectively. The alginate beads exhibited
a good stability after glutaraldehyde treatment and retained 90% of the enzyme activity after eight cycles (192 h at 65 °C)
of batch conversion. The addition of borate with a molar ratio of 1.0 to d-galactose led to a significant enhancement in the d-tagatose yield. Using commercial β-galactosidase and immobilized L. fermentum cells, d-tagatose was successfully obtained from lactose after a two-step biotransformation. The relatively high conversion rate and
productivity from d-galactose to d-tagatose of 60% and 11.1 g l−1 h−1 were achieved in a packed-bed bioreactor. Moreover, lactobacilli have been approved as generally recognized as safe organisms,
which makes this L. fermentum strain an attracting substitute for recombinant Escherichia coli cells among d-tagatose production progresses. 相似文献
7.
Jordan DB 《Applied biochemistry and biotechnology》2008,146(1-3):137-149
Catalytically efficient β-d-xylosidase from Selenomonas ruminantium (SXA) exhibits pK
as 5 and 7 (assigned to catalytic base, D14, and catalytic acid, E186) for k
cat/K
m with substrates 1,4-β-d-xylobiose (X2) and 1,4-β-d-xylotriose (X3). Catalytically inactive, dianionic SXA (D14−E186−) has threefold lower affinity than catalytically active, monoanionic SXA (D14−E186H) for X2 and X3, whereas D14−E186− has twofold higher affinity than D14−E186H for 4-nitrophenyl-β-d-xylopyranoside (4NPX), and D14−E186− has no affinity for 4-nitrophenyl-α-l-arabinofuranoside. Anomeric isomers, α-d-xylose and β-d-xylose, have similar affinity for SXA. 4-Nitrophenol competitively inhibits SXA-catalyzed hydrolysis of 4NPX. SXA steady-state
kinetic parameters account for complete progress curves of SXA-catalyzed hydrolysis reactions.
The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of
Agriculture over other firms or similar products not mentioned. 相似文献
8.
Hiroshi Kamitakahara Kaoru Murata-Hirai Yoshimasa Tanaka 《Cellulose (London, England)》2012,19(1):171-187
Bioimaging is a key to understanding immune responses, cell differentiation, and development. Quantum dots (QDs) conjugated
with monoclonal antibodies and other biomolecules are currently utilized for flow cytometry and immunohistochemistry, but
monoclonal antibody–QD complexes are of limited use when cell surface markers are not available. In this study, we synthesized
novel amphiphilic blockwise alkylated tetrasaccharides and developed a simple method for labeling a wide variety of live cells
with organic QDs encapsulated with these carbohydrates. The novel amphiphilic blockwise alkylated tetrasaccharides were as
follows: methyl β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-d-glucopyranoside (1), methyl β-d-galactopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-methyl-d-glucopyranoside (2), ethyl β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-ethyl-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-ethyl-d-glucopyranoside, (3), and ethyl β-d-galactopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-ethyl-β-d-glucopyranosyl-(1 → 4)-2,3,6-tri-O-ethyl-d-glucopyranoside (4). The newly synthesized blockwise alkylated tetrasaccharides spontaneously assembled into micelle-like particles, in which
the hydrophobic moiety of the blockwise alkylated tetrasaccharides played an important role. They were less toxic to human
cells than octyl β-d-glucopyranoside, a commonly used amphiphilic glucoside. Flow cytometry and confocal laser scanning microscopy revealed that
the blockwise alkylated tetrasaccharide–organic QD complexes were stably attached to live cells. The affinity of compounds
1 and 2 to the live cell surface was slightly higher than that of compounds 3 and 4. Because the preparation of these carbohydrate–QD
complexes is simple and does not require sophisticated equipment, and because the complexes can be autonomously attached to
a wide spectrum of cell lines, they can be used as cell labeling reagents in biomedical studies. 相似文献
9.
Liang Xiang Feng Qi DaZhang Dai Chun Li YuanDa Jiang 《Applied biochemistry and biotechnology》2010,162(3):654-661
Effects of simulated microgravity (SMG) on bacteria have been studied in various aspects. However, few reports are available
about production of recombinant protein expressed by bacteria in SMG. In this study growth of E. coli BL21 (DE3) cells transformed with pET-28a (+)-pgus in double-axis clinostat that could model low shear SMG environment and
the recombinant β-d-glucuronidase (PGUS) expression have been investigated. Results showed that the cell dry weights in SMG were 16.47%, 38.06%,
and 28.79% more than normal gravity (NG) control, and the efficiency of the recombinant PGUS expression in SMG were 18.33%,
19.36%, and 33.42% higher than that in NG at 19 °C, 28 °C, and 37 °C, respectively (P < 0.05). 相似文献
10.
Serra B Gamella M Reviejo AJ Pingarrón JM 《Analytical and bioanalytical chemistry》2008,391(5):1853-1860
The use of lectins for microorganism biosensors fabrication is proposed. Lectins are immobilised onto a gold-plated quartz
crystal for direct piezoelectric label-free transduction of the bacteria–lectin binding event using an electrochemical quartz
crystal microbalance (EQCM). Concanavalin A (Con A) and Escherichia coli were used for the evaluation of the lectin immobilisation method and the biosensor performance. Adsorption on nonpolarised
and polarised (−0.200 V) gold-coated quartz crystals and immobilisation through avidin–biotin binding were checked for Con
A surface attachment. Lectin–bacteria binding was evaluated in all cases. With a crystal modified with Con A via avidin–biotin
immobilisation we obtained a linear calibration plot between 5.0 × 106 and 2.0 × 107 cfu mL−1 by measuring frequency changes with E. coli concentration 1 h after bacteria addition. A remarkable increase in sensitivity was achieved when the analytical solution
contained free biotinylated Con A, as a consequence of multiple lectin adhesion to Escherichia coli cell wall, which produced an accumulation of Con A–E. coli conjugates in the form of multilayers at the electrode surface. A detection limit of approximately 1.0 × 104 cfu mL−1 was achieved. Moreover nonspecific adsorptions were minimised. Using Con A and lectin from Arachis hypogaea, different response profiles were achieved for Escherichia coli, Staphylococcus aureus and Mycobacterium phlei, thus demonstrating the feasibility of bacteria discrimination. An approach involving filtering of free and lectin-bound
bacteria and introduction of a filter in the measuring cell allowed a significant frequency change to be obtained for an E. coli concentration of 1.0 × 103 cfu mL−1 in order to further increase the sensitivity and discriminate between viable and nonviable cells; an approach using electrochemical
measurements of bacterial catalase activity was also checked. 相似文献
11.
Mixtures of diblock co-oligomers of tri-O-methylated and unmodified cello-oligosaccharides have been found to be amphiphilic, as reported before. In order to clarify
their accurate amphiphilic property, diblock co-oligomers of tri-O-methylated and unmodified cello-oligosaccharides with monodispersity, methyl β-d-glucopyranosyl-(1→4)-2,3,6–tri-O-methyl-β-d-glucopyranosyl-(1→4)-2,3,6–tri-O-methyl-β-d-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-d-glucopyranoside (1, pentamer), methyl β-d-glucopyranosyl-(1→4)- β-d-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-d-glucopyranoside (2, hexamer), and methyl β-d-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1→4)- 2,3,6-tri-O-methyl-d-glucopyranoside (3, trimer) were synthesized independently. These compounds had higher surface activities compared to the mixture of diblock
co-oligomers of tri-O-methylated and unmodified cello-oligosaccharides and commercially available methylcellulose (MC) SM-4. This paper describes
the methods of synthesis of these compounds, and the influence of amphiphilic character on their surface activity. A new class
of carbohydrate-based nonionic surfactant without long alkyl chain was discovered. 相似文献
12.
Piyasena ME Real LJ Diamond RA Xu HH Gomez FA 《Analytical and bioanalytical chemistry》2008,392(5):877-886
Teicoplanin (teic) from Actinoplanes teichomyceticus is a glycopeptide antibiotic used to treat many Gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial
growth by binding to carboxy-terminal d-Ala-d-Ala intermediates in the peptidoglycan of the cell wall of Gram-positive bacteria. In this paper we report the derivatization
of magnetic microspheres with teic (teic-microspheres). Fluorescence-based techniques have been developed to analyze the binding
properties of the microspheres to two d-Ala-d-Ala terminus peptides. The dissociation constant for the binding of carboxyfluorescein-labeled d-Ala-d-Ala-d-Ala to teic on microspheres was established via fluorimetry and flow cytometry and was determined to be 0.5 × 10−6 and 3.0 × 10−6 mol L−1, respectively. The feasibility of utilizing microparticles with fluorescence methods to detect low levels (the limit of bacterial
detection was determined to be 30 colon-forming units; cfu) of Gram-positive bacteria has been demonstrated. A simple microfluidic
experiment is reported to demonstrate the possibility of developing microsphere-based affinity assays to study peptide–antibiotic
interaction. 相似文献
13.
Amperometric determination of live <Emphasis Type="Italic">Escherichia coli</Emphasis> using antibody-coated paramagnetic beads 总被引:2,自引:0,他引:2
Boyaci IH Aguilar ZP Hossain M Halsall HB Seliskar CJ Heineman WR 《Analytical and bioanalytical chemistry》2005,382(5):1234-1241
Detecting and enumerating fecal coliforms, especially Escherichia coli, as indicators of fecal contamination, are essential for the quality control of supplied and recreational waters. We have developed a sensitive, inexpensive, and small-volume amperometric detection method for E. coli -galactosidase by bead-based immunoassay. The technique uses biotin-labeled capture antibodies (Ab) immobilized on paramagnetic microbeads that have been functionalized with streptavidin (bead–Ab). The bead–Ab conjugate captures E. coli from solution. The captured E. coli is incubated in Luria Bertani (LB) broth medium with the added inducer isopropyl -D-thiogalactopyranoside (IPTG). The induced -galactosidase converts p-aminophenyl -D-galactopyranoside (PAPG) into p-aminophenol (PAP), which is measured by amperometry using a gold rotating disc electrode. A good linear correlation (R2=0.989) was obtained between log cfu mL–1 E. coli and the time necessary to product a specific concentration of PAP. Amperometric detection enabled determination of 2×106 cfu mL–1 E. coli within a 30 min incubation period, and the total analysis time was less than 1 h. It was also possible to determine as few as 20 cfu mL–1 E. coli under optimized conditions within 6–7 h. This process may be easily adapted as an automated portable bioanalytical device for the rapid detection of live E. coli. 相似文献
14.
Omar M. Ali 《Monatshefte für Chemie / Chemical Monthly》2007,138(9):917-922
Summary. 2-(Arylamino)pyrimidin-4-ones were synthesized, silylated, and condensed with l,2,3,5-tetra-O-acetyl-β- d-ribofuranoside to afford the corresponding N
2-aryl protected isocytidines. Deprotection of the acetylated isocytidines using saturated NH3 in MeOH solution gave 1-(β-d-ribofuranosyl)-2-(arylamino)-4-pyrimidinones. Methyl 2-deoxy-3,5-di-O-toluyl-α/β-d-ribofuranoside was prepared and condensed with the previously silylated bases to afford the anomeric mixture of protected
nucleosides. The pure β-anomers were synthesized with better yield by treating the sodium salts of N
2-arylisocytosine derivatives with 2-deoxy-3,5-di-O-toluyl-α-d-ribofuranosyl chloride. Deprotection of the latter anomers afforded the corresponding free hydroxyl derivatives. The synthesized
free nucleosides are under antiviral and oligonucleotide investigations. 相似文献
15.
Abstract
tert-Butyldimethylsilyl (4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-β-d-glucopyranosyl)-(1 → 4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside (Kawada and Yoneda [MOCHEM-D-09-00120], 2009), designed as a repeating disaccharide unit in a β-glucan having two different faces, was converted into a glycosyl donor and an acceptor. The glycosyl acceptor was glycosylated with the donor to afford a chito-tetrasaccharide derivative in good yield. Phthalimido and azido groups in the tetrasaccharide were successively converted into acetamido and free amino groups, and all other protecting groups were cleaved to obtain the chito-tetrasaccharide (2-amino-2-deoxy-β-d-glucopyranosyl)-(1 → 4)-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1 → 4)-(2-amino-2-deoxy-β-d-glucopyranosyl)-(1 → 4)-2-acetamido-2-deoxy-d-glucopyranose. 相似文献16.
Rapid multiplexed analysis of microorganisms is important in water analysis to control bacterial contamination for health
and safety reasons. Direct quantification of bacteria by means of flow-through microarray immunoassays requires new analysis
strategies for optimising sensitivity and the analysis time. For bacteria and for particles, hydrodynamic forces and sedimentation
are the dominating effects for binding on surfaces in a flow-through system, whereas diffusion is insignificant. Therefore,
we have implemented a stop and flow technique for quantification of viable E. coli cells. The method, with alternation of resting volume elements and pumping the elements forward, was more effective than
continuous-flow approaches for analysis of bacteria. For quantification of viable E. coli cells, a chemiluminescence sandwich immunoassay test format was performed by means of antibody microarrays and flow-injection-based
microarray analysis. Antibodies, which served as selective capture molecules, were immobilised on polymer-modified glass surfaces
serving as microarray substrate. For the bacteria recognition step, a second detection antibody was used, forming a sandwich
immunoassay at each spot of the microarray. Detection was carried out with a horseradish peroxidase catalysed chemiluminescence
reaction. All assay steps were conducted with an automated flow-through chemiluminescence microarray readout system. Living
E. coli cells could be detected in 67 min with a detection limit of 4 × 105 cells mL−1. By introduction of the stopped-flow technique and optimisation of interaction time and interaction steps the achieved detection
of E. coli was faster and two orders of magnitude more sensitive than with a conventional ELISA technique in microplates. 相似文献
17.
Two new polyhydroxysteroids and five new glycosides were isolated from the starfishCeramaster patagonicus and their structures were elucidated: 5α-cholestane-3β,6α,15β,16β,26-pentol, (22E)-5α-cholest-22-ene-3β,6α,8,15α,24-pentol, (22E)-28-O-[O-(2-O-methyl-β-d-xylopyranosyl)-(1→2)-β-d-galactofuranosyl]-24-hydroxymethyl-5α-cholest-22-ene-3β,4β, 6α,8,15β,16β,28-heptol (ceramasteroside C1), (22E)-28-O-[O-(2,4-di-O-methyl-β-d-xylopyranosyl)-(1→2)-β-d-galactofuranosyl]-24-hydroxymethyl-5α-cholest-22-ene-3β, 6α,8,15β,16β,28-hexol (ceramasteroside C2), (22E)-28-O-[O-methyl-β-d-xylopyranosyl)-(1→2)-β-d-galactofuranosyl]-24-hydroxymethyl-5α-cholest-22-ene-3β,6α,8,15β,16β 28-hexol (eramasteroside C3), (22E)-28-O-[O-(2-O-methyl-β-d-xylopyranosyl)-(1→2)-β-d-galactofuranosyl]-24-methyl-5α-cholest-22-ene-3β,4β,6α,8, 15β, 26-hexol (ceramasteroside C4), and (22E)-28-O-[O-(2-O-methyl-β-d-xylopyranosyl)-(1→2)-β-d-xylopyranosyl]-5α-cholest-22-ene-3β,6α,8,15β,24-pentol (ceramasteroside C5)). Three known polyhydroxysteroids (24-methylene-5α-cholestane-3β,6α,8,15β,16β,26-hexol, 5α-cholestane-3β,6α,8,15β,16β,26-hexol,
and 5α-cholestane-3β,6β,15α,16β,26-pentol) were also isolated.
Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 190–195, January, 1997. 相似文献
18.
A. A. Kicha N. V. Ivanchina T. T. T. Huong A. I. Kalinovsky P. S. Dmitrenok P. Q. Long 《Russian Chemical Bulletin》2010,59(11):2133-2136
A new minor asterosaponin (20S)-6-O-{β-d-fucopyranosyl-(1→2)-[β-d-fucopyranosyl-(1→4)-β-d-quinovopyranosyl-(1→2)]-β-d-quinovopyranosyl-(1→3)-β-d-quinovopyranosyl}-3β,6α,20-trihydroxycholest-9(11)-en-23-one 3-sulfate (archasteroside C) was isolated from the starfish
Archaster typicus collected in shallow coastal waters of Vietnam. The structure of archasteroside C was determined by 2D NMR spectroscopy and
electrospray ionization (ESI) tandem mass spectrometry. 相似文献
19.
A novel β-galactosidase of 120 kDa (BgaBM) from Bacillus megaterium 2-37-4-1 was purified, and its gene (bgaBM) was analyzed and expressed. It displayed wide acceptor specificity for transglycosylation with a series of acceptors, including
pentose, hexose, hydroxyl, and alkyl alcohol using o-nitrophenyl-β-d-galactoside (ONPG) as a donor. BgaBM preferentially hydrolyzed ONPG in all tested substrates and showed maximum activity
at pH 7.5–8.0 and 55 °C. It was stable at pH 6.0–9.0 below 40 °C. The K
m and V
max values for ONPG and lactose were 9.5 mM, 16.6 mM/min and 12.6 mM, 54.4 mM/min, respectively. The nucleotide sequence of the
bgaBM gene consists of an ORF of 3,105 bp corresponding to 118 kDa protein, which indicates that BgaBM is a modular enzyme in the
glycosyl hydrolase family 2, including conserved sugar-binding domain, acid–base catalyst, and immunoglobulin-like beta-sandwich
domain. The possible acid/base and nucleophile sites of BgaBM were estimated to be E481 and E547, respectively. Furthermore,
expression of the bgaBM gene in Escherichia coli and purification of the recombinant enzyme were performed. The recombinant enzyme showed similar biochemical characteristics
to natural enzyme. 相似文献
20.
Functional expression of a β-d-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60°C, whereas the enzyme activity was optimal at 40°C when expressed
by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted
in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa.
The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up. 相似文献