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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.
Okamoto Y Monjushiro H Fukumoto T Watarai H 《Analytical and bioanalytical chemistry》2006,385(8):1430-1438
A new analytical technique, spinning microtube fluorometry (SMF), was developed and applied to the study of interfacial hydrolysis
of 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG) by β-galactosidase (β-gal) in the toluene–water system. The nonfluorescent lactone form of C12FDG in the toluene phase was converted at the interface to 5-dodecanoylaminofluorescein (C12F), which was fluorescent in the aqueous phase as a dianion at pH 7.3, though some part of C12F was extracted into the toluene phase as its nonfluorescent lactone form. The distribution ratios of C12FDG and C12F at pH 7.3 were determined as 1.4×102 and 1.97, respectively. The interfacial adsorption constants from the toluene phase to the interface at pH 7.3 were 4.8×10−4 and 1.7×10−2 dm for C12FDG and C12F, respectively. The kinetic experiments with the SMF method concluded that the rate-determining step of the enzymatic hydrolysis
at the interface and in the aqueous phase was the 1:1 reaction of C12FDG and β-gal and that the hydrolysis reaction rate constant at the interface at pH 7.3 was 1.84×103 M−1s−1, almost equal to that in the aqueous solution, 1.76×103 M−1s−1. Finally, the SMF method revealed that the contribution of the interfacial reaction to the overall hydrolysis reaction rate
of the toluene–water system was as high as 97%. 相似文献
3.
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
4.
Smaali M. Issam Gargouri Mohamed Legoy Marie Dominique Maugard Thierry Limam Farid Marzouki Nejib 《Applied biochemistry and biotechnology》2004,112(2):63-77
The filamentous fungus Sclerotinia sclerotiorum, grown on a xylose medium, was found to excrete one β-glucosidase (β-glu x). The enzyme was purified to apparent homogeneity
by ammonium sulfate precipitation, gel filtration, anion-exchange chromatography, and high-performance liquid chromatography
(HPLC) gel filtration chromatography. Its molecular mass was estimated to be 130 kDa by HPLC gel filtration and 60 kDa by
sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that β-glu x may be a homodimer. For p-nitrophenyl β-d-glucopyranoside hydrolysis, apparent K
m and V
max values were found to be 0.09 mM and 193 U/mg, respectively, while optimum temperature and pH were 55–60°C and pH 5.0, respectively. β-Glu x was strongly
inhibited by Fe2+ and activated about 35% by Ca2+. β-Glu x possesses strong transglucosylation activity in comparison with commercially available β-glucosidases. The production
rate of total glucooligosaccharides (GOSs) from 30% cellobiose at 50°C and pH 5.0 for 6 h with 0.6 U/mL of enzyme preparation
was 80 g/L. It reached 105 g/L under the same conditions when using cellobiose at 350 g/L (1.023 M). Finally, GOS structure was determined by mass spectrometry and 13C nuclear magnetic resonance spectroscopy. 相似文献
5.
Christakopoulos P Katapodis P Hatzinikolaou DG Kekos D Macris BJ 《Applied biochemistry and biotechnology》2000,87(2):127-133
An α-l-arabinofuranosidase from Fusarium oxysporum F3 was purified to homogeneity by a two-step ion exchange intercalated by a gel filtration chromatography. The enzyme had
a molecular mass of 66 kDa and was optimally active at pH 6.0 and 60°C. It hydrolyzed aryl α-l-arabinofuranosides and cleaved arabinosyl side chains from arabinoxylan and arabinan. There was a marked synergistic effect
between the α-l-arabinofuranosidase and an endo-(1 →4)-β-d-xylanase produced by F. oxysporum in the extensive hydrolysis of arabinoxylan. 相似文献
6.
A. Najafi F. Golestani-Fard H. R. Rezaie N. Ehsani 《Journal of Sol-Gel Science and Technology》2011,59(2):205-214
Nano sized β-SiC particles were synthesized from sol–gel process. Mono dispersed β-SiC nano particles with semi spherical
morphology were obtained by employing APC as a dispersant agent and adjusting pH in the range of 2.5–4. Phenolic resin and
TEOS were employed as precursors and heat treatment was conducted up to 1500 °C. Different techniques such as XRD, DTA, FTIR,
PSA, SEM and TEM were used to characterize the formation of β-SiC. The (Si–O-C) bonds were formed by hydrolysis and condensation
reactions in the gel while the nucleation of crystalline β-SiC was found to be initiated at 1400 °C. The primary particles
in the sol were found to be (< 10 nm) while the size distribution in the final product was recorded in the range of 30–50 nm. 相似文献
7.
Shengtang Zhang Sufang Gao Guoqiang Gao 《Applied biochemistry and biotechnology》2010,160(5):1386-1393
A study of the cross-linking of β-galactosidase on magnetic beads is reported here. The magnetic beads were prepared from
artemisia seed gum, chitosan, and magnetic fluid in the presence of a cross-linking regent (i.e., glutaraldehyde). The reactive
aldehyde groups of the magnetic beads allowed the reaction of the amino groups of the enzymes. The animated magnetic beads
were used for the covalent immobilization of β-galactosidase. The effect of various preparation conditions on the activity
of the immobilized β-galactosidase, such as immobilizing time, amount of enzyme, and the concentration of glutaraldehyde,
were investigated. The influence of pH and temperature on the activity and the stability of the enzyme, both free and immobilized,
have been studied. And o-nitrophenyl-β-d-galactopyranoside (ONPG) was chosen as a substrate. The β-galactosidase immobilized on the magnetic beads resulted in an
increase in enzyme stability. Optimum operational temperature for immobilized enzyme was 10 °C higher than that of free enzyme
and was significantly broader. 相似文献
8.
Wojciech Zielenkiewicz Małgorzata Koźbiał Bożenna Golankiewicz Jarosław Poznański 《Journal of Thermal Analysis and Calorimetry》2010,101(2):555-560
Solubilities of tricyclic acyclovir derivatives in buffered aqueous solutions of hydroxypropyl-β-cyclodextrin (HP-β-CD) at
pH 5.5 and 7.0 were determined at 25 and 37 °C. Complexation of these compounds with HP-β-CD resulted in a noticeable increase
of their solubility; nevertheless it was limited to tricyclic derivatives of acyclovir carrying an aryl substituent. Combination
of 1H NMR and DSC techniques demonstrated the existence of inclusion complexes between acyclovir derivatives and HP-β-CD. The
stability constants, estimated using the Higuchi–Connors method, were found in the range of 10–100 M−1. Additionally, the pK
a values at 25 °C and molar extinction coefficients in aqueous buffered solutions were also determined for all studied compounds. 相似文献
9.
A crude cellulase preparation from Aspergillus niger was used to depolymerize chitosan. The depolymerization process was followed by measuring the apparent viscocity and the
intrinsic viscosity. The optimum conditions for enzymatic hydrolysis were investigated. On the selected optimum conditions
(pH 5.0, temperature 50 °C, and an enzyme to substrate ratio of 1:5), chitosan was hydrolyzed for 1, 4, 8, and 24 h, its viscosity-average
molecular weights were 3.49 × 104, 1.18 × 104, 5.83 × 103, and 1.13 × 103, respectively. Compared with chitosan having viscosity-average molecular weight of 5.18 × 105 before enzymatic hydrolysis, the crude cellulase preparation had rather apparent effect on depolymerization of chitosan.
Through the comparison of different origin of cellulases, the prepared cellulase has good ability of enzymatic hydrolysis.
The reproducibility and reversibility for enzymatic hydrolysis was appraised. The data are of value for the production of
low-molecular weight chitosans and chitooligomers of medical and biotechnological interest. 相似文献
10.
Laura L. G. Fuentes Sarita C. Rabelo Rubens Maciel Filho Aline C. Costa 《Applied biochemistry and biotechnology》2011,163(5):612-625
The objective of this work was to determine the optimum conditions of sugarcane bagasse pretreatment with lime to increase
the enzymatic hydrolysis of the polysaccharide component and to study the delignification kinetics. The first stage was an
evaluation of the influence of temperature, reaction time, and lime concentration in the pretreatment performance measured
as glucose release after hydrolysis using a 23 central composite design and response surface methodology. The maximum glucose yield was 228.45 mg/g raw biomass, corresponding
to 409.9 mg/g raw biomass of total reducing sugars, with the pretreatment performed at 90°C, for 90 h, and with a lime loading
of 0.4 g/g dry biomass. The enzymes loading was 5.0 FPU/dry pretreated biomass of cellulase and 1.0 CBU/dry pretreated biomass
of β-glucosidase. Kinetic data of the pretreatment were evaluated at different temperatures (60°C, 70°C, 80°C, and 90°C),
and a kinetic model for bagasse delignification with lime as a function of temperature was determined. Bagasse composition
(cellulose, hemicellulose, and lignin) was measured, and the study has shown that 50% of the original material was solubilized,
lignin and hemicellulose were selectively removed, but cellulose was not affected by lime pretreatment in mild temperatures
(60–90°C). The delignification was highly dependent on temperature and duration of pretreatment. 相似文献
11.
de Almeida MN Guimarães VM Bischoff KM Falkoski DL Pereira OL Gonçalves DS de Rezende ST 《Applied biochemistry and biotechnology》2011,165(2):594-610
The aim of this work was to have cellulase activity and hemicellulase activity screenings of endophyte Acremonium species (Acremonium zeae EA0802 and Acremonium sp. EA0810). Both fungi were cultivated in submerged culture (SC) containing l-arabinose, d-xylose, oat spelt xylan, sugarcane bagasse, or corn straw as carbon source. In solid-state fermentation, it was tested as
carbon source sugarcane bagasse or corn straw. The highest FPase, endoglucanase, and xylanase activities were produced by
Acremonium sp. EA0810 cultivated in SC containing sugarcane bagasse as a carbon source. The highest β-glucosidase activity was produced
by Acremonium sp. EA0810 cultivated in SC using d-xylose as carbon source. A. zeae EA0802 has highest α-arabinofuranosidase and α-galactosidase activities in SC using xylan as a carbon source. FPase, endoglucanase,
β-glucosidase, and xylanase from Acremonium sp. EA0810 has optimum pH and temperatures of 6.0, 55 °C; 5.0, 70 °C; 4.5, 60 °C; and 6.5, 50 °C, respectively. α-Arabinofuranosidase
and α-galactosidase from A. zeae EA0802 has optimum pH and temperatures of 5.0, 60 °C and 4.5, 45 °C, respectively. It was analyzed the application of Acremonium sp. EA0810 to hydrolyze sugarcane bagasse, and it was achieved 63% of conversion into reducing sugar and 42% of conversion
into glucose. 相似文献
12.
K. Tebbji H. Oudda B. Hammouti M. Benkaddour S. S. Al-Deyab A. Aouniti S. Radi A. Ramdani 《Research on Chemical Intermediates》2011,37(8):985-1007
The effect of some prepared compounds, namely 3,5-dimethyl-1H-pyrazole (P1), 3(5)-amino-5(3)-methylpyrazole (P2), and 1′,3,5,5′-tetramethyl-1′H-1,3′-bipyrazole (P3), on the corrosion behaviour of steel in 1.0 M hydrochloric acid solution as corrosive medium has been investigated at 308 K using weight-loss measurement, potentiodynamic
polarisation, linear polarisation, and impedance spectroscopy (EIS). Generally, inhibition efficiency of the investigated
compounds was found to depend on the concentration and nature of the inhibitor. P3 was a better inhibitor than P1 and P2,
and its inhibition efficiency increased with increasing concentration of inhibitor, attaining 94% above 10−3
M. Potentiodynamic polarisation studies clearly reveal that P3 acts essentially as a cathodic inhibitor. E (%) values obtained from different methods are in reasonably good agreement. EIS measurements show an increase of transfer
resistance with inhibitor concentration. Partial π-charge on atoms was calculated. Correlation between the highest occupied
molecular orbital energy E
HOMO and inhibition efficiencies was sought. The temperature effect on the corrosion behaviour of steel in 1.0 M HCl without and with different concentrations of inhibitor P3 was studied in the temperature range 308 to 343 K. Thermodynamic
data, for example heat of adsorption (
\Updelta H\textads° \Updelta H_{\text{ads}}^{^\circ } ), entropy of adsorption (
\Updelta S\textads° \Updelta S_{\text{ads}}^{^\circ } ) and free energy of adsorption (
\Updelta G\textads° \Updelta G_{\text{ads}}^{^\circ } ) were calculated by use of thermodynamic equations. Kinetic activation data, for example E
a, ΔH*, ΔS* and pre-exponential factor, were calculated, and are discussed. The inhibiting action of P3 on the corrosion of steel in
1–10 M hydrochloric acid was also studied by weight-loss measurement. The rate constant and reaction constant were calculated for
the corrosion reactions. Adsorption of P3 on the steel surface in 1.0 M HCl follows the Langmuir isotherm model. 相似文献
13.
Ashok Pandey Simon Joseph L. Ashakumary P. Selvakumar Carlos R. Soccol 《Applied biochemistry and biotechnology》1999,82(2):103-114
A newly isolated mesophilic bacterial strain from dahlia rhizosphere, identified as Staphylococcus sp. and designated as RRL-M-5, was evaluated for inulinase synthesis in submerged cultivation using different carbon sources
individually or in combination with inulin as substrate. Inulin appeared as the most favorable substrate at a 0.5–1.0% concentration.
Media pH influenced the enzyme synthesis by the bacterial strain, which showed an optimum pH at 7.0–7.5. Supplementation of
fermentation medium with external nitrogen (organic and inorganic) showed a mixed impact on bacterial activity of enzyme synthesis.
The addition of soybean meal and corn steep solid resulted in about an 11% increase in enzyme titers. Among inorganic nitrogen
sources, ammonium sulfate was found to be the most suitable. Maximum enzyme activities (446 U/L) were obtained when fermentation
was carried out at 30°C for 24 h with a medium containing 0.5% inulin as a sole carbon source and 0.5% soybean meal as the
nitrogen source. Bacterial inulinase could be a good source for the hydrolysis of inulin for the production of d-fructose. 相似文献
14.
Daniel Luciano Falkoski Valéria Monteze Guimarães Maíra Nicolau de Almeida Acelino Couto Alfenas Jorge Luiz Colodette Sebastião Tavares de Rezende 《Applied biochemistry and biotechnology》2012,166(6):1586-1603
The aim of this work was to evaluate the biochemical features of the white-rot fungi Pycnoporus sanguineus cellulolytic complex and its utilization to sugarcane bagasse hydrolysis. When cultivated under submerged fermentation using
corn cobs as carbon source, P. sanguineus produced high FPase, endoglucanase, β-glucosidase, xylanase, mannanase, α-galactosidase, α-arabinofuranosidase, and polygalacturonase
activities. Cellulase activities were characterized in relation to pH and temperature. β-Glucosidase and FPase activities
were higher at 55 °C, pH 4.5, and endoglucanase activity was higher at 60 °C, in a pH range of 3.5–4.0. All cellulase activities
were highly stable at 40 and 50 °C through 48 h of pre-incubation. Crude enzymatic extract from P. sanguineus was applied in a saccharification experiment using acid-treated and alkali-treated sugarcane bagasse as substrate, and the
hydrolysis yields were compared to that obtained by a commercial cellulase preparation. Reducing sugar yields of 60.4% and
64.0% were reached when alkali-treated bagasse was hydrolyzed by P. sanguineus extract and commercial cellulase, respectively. Considering the glucose production, it was observed that P. sanguineus extract and commercial cellulase ensured yields of 22.6% and 36.5%, respectively. The saccharification of acid-treated bagasse
was lower than that of alkali-treated bagasse regardless of the cellulolytic extract. The present work showed that P. sanguineus has a great potential as an enzyme producer for biomass saccharification. 相似文献
15.
Stefanie K. Nguyen Supaporn Sophonputtanaphoca Eugene Kim Michael H. Penner 《Applied biochemistry and biotechnology》2009,158(2):352-361
A low, but significant, fraction of the carbohydrate portion of herbaceous biomass may be composed of fructose/fructosyl-containing
components (“fructose equivalents”); such carbohydrates include sucrose, fructooligosaccharides, and fructans. Standard methods
used for the quantification of structural-carbohydrate-derived neutral monosaccharide equivalents in biomass are not particularly
well suited for the quantification of fructose equivalents due to the inherent instability of fructose in conditions commonly
used for hemicellulose/cellulose hydrolysis (>80% degradation of fructose standards treated at 4% sulfuric acid, 121°C, 1 h).
Alternative time, temperature, and acid concentration combinations for fructan hydrolysis were considered using model fructans
(inulin, β-2,1, and levan, β-2,6) and a grass seed straw (tall fescue, Festuca arundinacea) as representative feedstocks. The instability of fructose, relative to glucose and xylose, at higher acid/temperature combinations
is demonstrated, all rates of fructose degradation being acid and temperature dependent. Fructans are shown to be completely
hydrolyzed at acid concentrations well below that used for the structural carbohydrates, as low as 0.2%, at 121°C for 1 h.
Lower temperatures are also shown to be effective, with corresponding adjustments in acid concentration and time. Thus, fructans
can be effectively hydrolyzed under conditions where fructose degradation is maintained below 10%. Hydrolysis of the β-2,1
fructans at temperatures ≥50°C, at all conditions consistent with complete hydrolysis, appears to generate difructose dianhydrides.
These same compounds were not detected upon hydrolysis of levan, sucrose, or straw components. It is suggested that fructan
hydrolysis conditions be chosen such that hydrolysis goes to completion; fructose degradation is minimized, and difructose
dianhydride production is accounted for. 相似文献
16.
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
17.
Munish Puri Shivani Gupta Parveen Pahuja Aneet Kaur J. R. Kanwar J. F. Kennedy 《Applied biochemistry and biotechnology》2010,160(1):98-108
β-D-galactosidase (EC 3.2.1.23) from Kluyveromyces marxianus YW-1, an isolate from whey, has been studied in terms of cell disruption to liberate the useful enzyme. The enzyme produced
in a bioreactor on a wheat bran medium has been successfully immobilized with a view to developing a commercially usable technology
for lactose hydrolysis in the food industry. Three chemical and three physical methods of cell disruption were tested and
a method of grinding with river sand was found to give highest enzyme activity (720 U). The enzyme was covalently immobilized
on gelatin. Immobilized enzyme had optimum pH and temperature of 7.0 and 40 °C, respectively and was found to give 49% hydrolysis
of lactose in milk after 4 h of incubation. The immobilized enzyme was used for eight hydrolysis batches without appreciable
loss in activity. The retention of high catalytic activity compared with the losses experienced with several previously reported
immobilized versions of the enzyme is significant. The method of immobilization is simple, effective, and can be used for
the immobilization of other enzymes. 相似文献
18.
Characterization of Thermo-stable Endoinulinase from a New Strain <Emphasis Type="Italic">Bacillus Smithii</Emphasis> T7 总被引:1,自引:0,他引:1
A new thermophilic inulinase-producing strain, which grows optimally at 60 °C, was isolated from soil samples with medium
containing inulin as a sole carbon source. It was identified as a Bacillus smithii by analysis of 16s rDNA. Maximum inulinase yield of 135.2 IU/ml was achieved with medium pH7.0, containing inulin 2.0%, (NH4)H2PO4 0.5%, yeast extract 0.5%, at 50 °C 200 rpm shaker for 72-h incubation. The purified inulinase from the extracellular extract
of B. smithii T7 shows endoinulinolytic activity. The optimum pH for this endoinulinase is 4.5 and stable at pH range of 4.0–8.0. The optimum
temperature for enzyme activity was 70 °C, the half life of the endoinulinase is 9 h and 2.5 h at 70 °C and 80 °C respectively.
Comparatively lower Michaelis–Menten constant (4.17 mM) and higher maximum reaction velocity (833 IU/mg protein) demonstrate
the endoinulinase’s greater affinity for inulin substrate. These findings are significant for its potential industrial application. 相似文献
19.
Manchumas Hengsakul Prousoontorn Supranee Pantatan 《Journal of inclusion phenomena and macrocyclic chemistry》2007,57(1-4):39-46
Cyclodextrin glycosyltransferase (CGTase) isolated and purified from Paenibacillus sp. A11 was immobilized on various carriers by covalent linkage using bifunctional agent glutaraldehyde. Among tested carriers,
alumina proved to be the best carrier for immobilization. The effects of several parameters on the activation of the support
and on the immobilization of enzyme were optimized. The best preparation of immobilized CGTase retained 31.2% of its original
activity. After immobilization, the enzymatic properties were investigated and compared with those of the free enzyme. The
optimum pH of the immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60°C). Free
and immobilized CGTase showed similar pH stability profile but the thermal stability of the immobilized CGTase was 20% higher.
Kinetic data (K
M and V
max) for the free and immobilized enzymes were determined from the rate of β-CD formation and it was found that the immobilized
form had higher K
M and lower V
max. The immobilized CGTase also exhibited higher stability when stored at both 4°C and 25°C for 2 months. The enzyme immobilized
on alumina was further used in a batch production of 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin. The yield of AA-2G was 2.92% and the immobilized CGTase retained
its activity up to 74.4% of the initial catalytic activity after being used for 3 cycles. The immobilized CGTase would have
a promising application in the production of various transglycosylated compounds and in the production of cyclodextrin by
the hydrolysis of starch. 相似文献
20.
Pharmacological chaperones (PCs) represent a promising therapeutic strategy for treatment of lysosomal storage disorders based
on enhanced stabilization and trafficking of mutant protein upon orthosteric and/or allosteric binding. Herein, we introduce
a simple yet reliable enzyme assay using capillary electrophoresis (CE) for inhibitor screening of PCs that target the lysosomal
enzyme, β-glucocerebrosidase (GCase). The rate of GCase-catalyzed hydrolysis of the synthetic substrate, 4-methylumbelliferyl-β-d-glucopyranoside was performed using different classes of PCs by CE with UV detection under standardized conditions. The pH
and surfactant dependence of inhibitor binding on recombinant GCase activity was also examined. Enzyme inhibition studies
were investigated for five putative PCs including isofagomine (IFG), ambroxol, bromhexine, diltiazem, and fluphenazine. IFG
was confirmed as a potent competitive inhibitor of recombinant GCase with half-maximal inhibitory concentration (IC
50
) of 47.5 ± 0.1 and 4.6 ± 1.4 nM at pH 5.2 and pH 7.2, respectively. In contrast, the four other non-carbohydrate amines were
demonstrated to function as mixed-type inhibitors with high micromolar activity at neutral pH relative to acidic pH conditions
reflective of the lysosome. CE offers a convenient platform for characterization of PCs as a way to accelerate the clinical
translation of previously approved drugs for oral treatment of rare genetic disorders, such as Gaucher disease. 相似文献