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1.
Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified
enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima
at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from
40 to 70°C, with optimum at 55°C. It was stable for 2 h at temperatures below 60°C and stable over a broad pH range of 4.0–9.0
for 24 h. The apparent K
m and V
max of Chi72 for colloidal chitin were 0.23 mg ml−1 and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the
recycling of chitin waste. 相似文献
2.
Mishra A 《Applied biochemistry and biotechnology》2006,135(1):33-42
This article reports the production of high levels of l-asparaginase from a new isolate of Aspergillus niger in solid state fermentation (SSF) using agrowastes from three leguminous crops (bran of Cajanus cajan, Phaseolus mungo, and Glycine max). When used as the sole source for growth in SSF, bran of G. max showed maximum enzyme production followed by that of P. mungo and C. cajan. A 96-h fermentation time under aerobic condition with moisture content of 70%, 30 min of cooking time and 1205–1405 μ range
of particle size in SSF appeared optimal for enzyme production. Enzyme yield was maximum (40.9±3.35 U/g of dry substrate)
at pH 6.5 and temperature 30±2°C. The optimum temperature and pH for enzyme activity were 40°C and 6.5, respectively. The
study suggests that choosing an appropriate substrate when coupled with process level optimization improves enzyme production
markedly. Developing an asparaginase production process based on bran of G. max as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based
countries. 相似文献
3.
Ethanol production was studied in simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce at 42°C,
using a thermotolerant yeast. Three yeast strains of Kluyveromyces marxianus were compared in test fermentations. SSF experiments were performed with the best of these on 5% (w/w) of substrate at a
cellulase loading of 37 filter paper units/g of cellulose, and a β-glucosidase loading of 38 IU/gof cellulose. The detoxification
of the substrate and the lack of pH control in the experiments increased the final ethanol concentration. The final ethanol
yield was 15% lower compared to SSF with Saccharomyces cerevisiae at 37°C, owing to the cessation of ethanol fermentation after the first 10 h. 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.
Tsao George T. Xia Liming Cao Ningjun Gong Cheng S. 《Applied biochemistry and biotechnology》2000,84(1-9):743-749
Aspergillus niger NRRL3 was cultivated in a moist wheat bran and ground corncob solid medium supplemented with inorganic minerals for the production
of cellobiase (β-1,4-glucosidase, EC 3.2.1.21). With this method, A. niger NRRL3 was able to produce a high concentration of cellobiase (215 IU/gofsolid substrate) after 96 h of incubation. Temperature
and moisture content affected final cellobiase titers. The best conditions for cell obiase production from solid substrate
by A. niger NRRL3 were determined to be 70% moisture and 35°C. 相似文献
7.
Bacillus cellulyticus K-12 Avicelase (Avicelase I; EC 3.2.1.4) gene (ace A) has been cloned in Escherichia coli by using the vector pT7T3U19 and HindIII-HindIII libraries of the chromosomal inserts. The libraries were screened for the expression of avicelase by monitoring the immunoreaction
of the antiavicelase (immunoscreening). Positive clones (Ac-3, Ac-5, and Ac-7) contained the identical 3.5-kb HindIII fragment as determined by restriction mapping and Southern hybridization, and expressed avicelase efficiently and constitutively
using its own promoter in the heterologous host. From the immunoblotting analysis, a polypeptide that showed a carboxymethylcellulase
(CMCase) activity with an M
r
, of 64,000 was detected. The recombinant endo 1,4-β-
d
-glucanase I was purified to homogeneity from an intracellular fraction of E. coli by DEAE-Toyopearl M650, Phenyl Toyoperal M650, and TSK gel HW50S chromatography. The enzyme had a monomeric structure, its
relative molecular mass being 65 kDa by gel filtration and 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The pI was 5.3 and the optimal pH was 4.6, and the enzyme was stable at pH 4.0–10.5. The enzyme had a temperature optimum of 50°C
and was stable at 55°C for 48 h, and retained approx 20% of its activity after 30 min at 70°C. It showed high activity toward
carboxymethylcellulose (CMC) as well as p-nitrophenyl-β-d-cellobioside, 4-methylumbelliferyl cellobioside, Avicel, filter paper, and some cellooligosaccharides. K
m
values for CMC and Avicel were 7.6 and 85.2 mg/mL, respectively, whereas V
max values were 201 and 9.2 μmol · min−1 · mg−1, respectively. Cellotetraose (G4) was preferentially cleaved into cellobiose (G2) and cellopentaose (G5) was cleaved into
G2 + cellotriose (G3), whereas cellohexaose (G6) was cleaved into G4 + G2 and, to a lesser extent, into G3 + G3. G3 was not
cleaved at all. G2 was the main product of Avicel hydrolysis. G2 inhibited whereas Mg++ stimulated the activity of CMCase and Avicelase. Hydrolysis of CMC took place with a rapid decrease in viscosity but a slow
liberation of reducing sugars. Based on these results, it appeared that the cellulase should be regarded as endo type, although
it hydrolyzed Avicel. 相似文献
8.
Yu. A. Skorik E. V. Osintseva N. V. Podberezskaya A. V. Virovets L. K. Neudachina A. A. Vshivkov 《Russian Chemical Bulletin》2005,54(7):1563-1568
Protolytic equilibria and complexation of N-(2-carboxyethyl)anthranilic acid (H2CEAnt) with copper(II) ions in aqueous solutions were studied by UV spectroscopy and pH potentiometry. The H2CEAnt compound has no zwitterionic structure, and the protons are localized on the carboxy groups. The acid ionization constants
of H3CEAnt+ (T = 25 °C, I = 0.1 M KNO3) are pK
0 = 1.3±0.2 (=NH2
+), pK
1 = 3.88±0.02 (Alk-COOH), and pK
2 = 5.28±0.02 (Ar-COOH). The model of complexation of H2CEAnt with copper(II) ions involves two deprotonated complexes [Cu(CEAnt)] and [Cu(CEAnt)2]2− (logβ = 6.31±0.04 and 8.0±0.2, respectively). The [Cu(CEAnt)(H2O)]⋅H2O complex was synthesized by the reaction of H2CEAnt with (CuOH)2CO3, and its structure was established by X-ray diffraction. The coordination polyhedron of Cu is intermediate between the tetragonal
pyramid and trigonal bipyramid. The CEAnt2− ligand serves as a tetradentate chelating bridging ligand (Cu-O, 1.944(3) and 1.950(3) Å; Cu-O', 2.195(4) Å; Cu-N, 2.016(5)
Å), and the fifth position of the polyhedron is occupied by a water molecule (Cu-Ow, 1.976(4) Å).
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1518–1523, July, 2005. 相似文献
9.
To express high-active soluble d-amino acid oxidase (DAAO), a constitutive plasmid that is regulated by a native hydantoinase promoter (PHase), was constructed. A d-amino acid oxidase gene (dao) was ligated with the PHase and cloned into pGEMKT to constitutively express protein of DAAO without the use of any inducer such as isopropyl β-d-1-thiogalactopyranoside which is poisonous to the cells and environment. The ribosome binding site region, host strain, and
fermentation conditions were optimized to increase the expression level. When cultivated in a 5-m3 fermenter, the enzyme activity of JM105/pGEMKT-R-DAAO grown at 37 °C was found to be 32 U/mL and increase 16-fold over cells
of BL21(DE3)/pET-DAAO grown at 28 °C. These results indicate the success of our approaches to overproducing DAAO in soluble
form in Escherichia coli. 相似文献
10.
Yukiko Enomoto Hiroshi Kamitakahara Toshiyuki Takano Fumiaki Nakatsubo 《Cellulose (London, England)》2006,13(4):437-448
Novel cellobiose and cellulose (DP
n
=ca. 30) derivatives, N-(1-pyrenebutyloyl)-4-O-(β-d-glucopyranosyl)-β-d-glucopyranosylamine (6), N-(15-(1-pyrenebutyloylamino)-pentadecanoyl)-4-O-(β-d-glucopyranosyl)-β-d-glucopyranosylamine (7), N-(1-pyrenebutyloyl)-β-cellulosylamine (13), N-(15-(1-pyrenebutyloylamino)-pentadecanoyl)-β-cellulosylamine (14) carrying a pyrene group as a single fluorescent probe at the reducing end, were prepared in order to investigate their self-assembly systems in solutions. The relative intensity of the excimer emission at ca. 480 nm due to dimerized pyrenes (intensity I
E) to the monomer emission at ca. 380 nm due to isolated pyrene (intensity I
M), i.e., I
E/I
M, was monitored in various solutions. In water/dimethyl sulfoxide (DMSO) mixed solvent (0–98%, v/v), the ratio I
E/I
M remained low (0.04) for compound 6 over the range of water concentrations, indicating that pyrenes at C-1 position of compound 6 were diffused. On the other hand, the ratio I
E/I
M increased (0.04–4.96) for compound 7 with the increase in water concentration, indicating that pyrenes at C-1 position were associated. In aqueous NaOH solutions (4.4–17.5%, w/w), compound 14 showed a large increase in the ratio I
E/I
M (0.84–8.14) with the increase in NaOH concentration, compared to compound 13 (0.06–0.41). It was found that the association of hydrophobic groups at the reducing-end of cellulose could be controlled by the hydrophilic–hydrophobic balance of compounds and the solvent polarity. 相似文献
11.
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
12.
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
13.
Qing Li Liang Xu Ting-Ting Wang Ying Jia Zhi-Wei Wang Kai-Shun Bi 《Chromatographia》2008,67(7-8):627-631
Aidi injection is a clinical medicine used in China for the treatment of cancer. Calycosin-7-O-β-d-glucoside is the main effective components of the formulas. In this study, a high performance liquid chromatographic (LC)
method was developed to quantify calycosin-7-O-β-d-glucoside in rat plasma using a liquid–liquid extraction and ultraviolet (UV) absorbance detection. LC analysis was performed
on a Diamonsil C18 column (200 × 4.6 mm i.d., 5 μm particle size) with isocratic mobile phase consisting of acetonitrile–0.05% phosphoric acid
(19.5:80.5, v/v) of a flow rate of 1.0 mL min−1. The linear range was 0.11–17.6 μg mL−1 and the low quantification limit was 0.11 μg mL−1 (S/N = 10). The intra- and inter-day relative standard deviations (RSD) in the measurement of quality control (QC) samples
0.11, 0.22, 1.32 and 8.80 μg mL−1 ranged from 4.1 to 6.3 and 4.3 to 6.2%, respectively. The accuracy was from −6.7 to 4.3% in terms of relative error (RE).
Calycosin-7-O-β-d-glucoside was stable in storage at −20 °C for 2 weeks and stable after three freeze–thaw cycles in rat plasma. This method
was validated for specificity, accuracy, precision and was successfully applied to pharmacokinetic study of calycosin-7-O-β-d-glucoside in rat plasma after intravenous administration of Aidi lyophilizer. 相似文献
14.
The synthesis ofN-acetyl-β-l-fucosamine-1-phosphate and uridine 5′-diphospho-N-acetyl-β-l-fucosamine
P. A. Illarionov V. I. Torgov I. Hancock V. N. Shibaev 《Russian Chemical Bulletin》2000,49(11):1891-1894
Uridine 5′-(2-acetamido-2,6-dideoxy-β-l-galactopyranosyl) diphosphate (uridine 5′-diphospho-N-acetyl-β-l-fucosamine) was synthesized. The key intermediate, 3,4-di-O-acetyl-2-azido-2,6-dideoxy-β-l-galactopyranosyl dibenzyl phosphate, was prepared by a previously unknown reaction of cesium dibenzyl phosphate with the
corresponding α-glycosyl nitrate and was then converted into theN-acetylated glycosyl phosphate and nucleoside diphosphate sugarsvia 3,4-di-O-acetyl-2-amino-2,6-dideoxy-β-l-galactopyranosyl phosphate using mildN-acetylation andO-deacetylation as the last synthetic steps.
Published inIzvestiya Akademii Nauk, Seriya Khimicheskaya, No. 11, pp. 1919–1923, November, 2000. 相似文献
15.
Glycosyl hydrolase family 16 (GHF16) truncated Fibrobacter succinogenes (TFs) and GHF17 barley 1,3-1,4-β-d-glucanases (β-glucanases) possess different structural folds, β-jellyroll and (β/α)8, although they both catalyze the specific hydrolysis of β-1,4 glycosidic bonds adjacent to β-1,3 linkages in mixed β-1,3
and β-1,4 β-d-glucans or lichenan. Differences in the active site region residues of TFs β-glucanase and barley β-glucanase create binding
site topographies that require different substrate conformations. In contrast to barley β-glucanase, TFs β-glucanase possesses
a unique and compact active site. The structural analysis results suggest that the tyrosine residue, which is conserved in
all known 1,3-1,4-β-d-glucanases, is involved in the recognition of mixed β-1,3 and β-1,4 linked polysaccharide. 相似文献
16.
Barbagallo RN Spagna G Abbate C Azzaro G Palmeri R 《Applied biochemistry and biotechnology》2002,101(1):1-13
β-d-Glucopyranosidase (βG, EC 3.2.1.21) has been isolated from some collateral activities, α-l-arabinofuranosidase (Ara, EC3.2.1.55), α-l-rhamnopyranosidase (Rha, EC 3.2.1.40), and o-acetylesterase (Est, EC 3.1.1.53), using a commercial enzyme preparation and a simple method economically sustainable for
the food industry. The procedure comprises precipitation of extraneous substances by adding ethanol and CaCl2, ultrafiltration, and adsorption, first on bentonite and then on chitosan. The results obtained were the complete isolation
of βG from the above-mentioned activities, a drastic reduction in extraneous compounds, such as brown substances and polysaccharides,
and a slight increase in purification. 相似文献
17.
Liang Wang Xiaohua Jia Xiaohang Liu Zhi Yuan Jiaxian Huang 《Colloid and polymer science》2006,285(3):273-281
A class of novel amphiphilic diblock copolymer of MePEG-b-poly(DL-lactide-co-RS-β-malic acid) has been synthesized via the hydrogenation over palladium on charcoal of MePEG-b-poly(DL-lactide-co-RS-β-benzyl malolactonate), which was prepared by ring-opening copolymerization of DL-lactide and RS-β-benzyl malolactonate (MABz) using methyl-polyethylene glycol (MePEG) as the initiator and stannous octoate as the catalyst. The influence of copolymerization temperature, reaction time, macro-initiator (MePEG-5000) proportion and monomer ratio was studied. Gel permeation chromatography measurements revealed that the molecular weight decreased with increasing MABz feeding dose. The configurational structures of the protected and de-protected copolymers were determined by 13C nuclear magnetic resonance (NMR), 1H NMR and Fourier transform infrared. A water-swollen core of the nanospheres formed from the de-protected copolymer was discovered by transmission electron microscopy measurement. Additionally, the degradation experiments indicated that more hydrophilic malic acid content led to higher degradation rate. 相似文献
18.
The gene encoding a glycoside hydrolase family 39 xylosidase (BH1068) from the alkaliphile Bacillus halodurans strain C-125 was cloned with a C-terminal His-tag, and the recombinant gene product termed BH1068(His)6 was expressed in Escherichia coli. Of the artificial substrates tested, BH1068(His)6 hydrolyzed nitrophenyl derivatives of β-d-xylopyranose, α-l-arabinofuranose, and α-l-arabinopyranose. Deviation from Michaelis−Menten kinetics at higher substrate concentrations indicative of transglycosylation
was observed, and k
cat and K
m values were measured at both low and high substrate concentrations to illuminate the relative propensities to proceed along
this alternate reaction pathway. The pH maximum was 6.5, and under the conditions tested, maximal activity was at 47°C, and
thermal instability occurred above 45°C. BH1068(His)6 was inactive on arabinan, hydrolyzed xylooligosaccharides, and released only xylose from oat, wheat, rye, beech, and birch
arabinoxylan, and thus, can be classified as a xylosidase with respect to natural substrate specificity. The enzyme was not
inhibited by up to 200 mM xylose. The oligomerization state was tetrameric under the size-exclusion chromatography conditions
employed. 相似文献
19.
L. A. Baltina O. B. Flekhter E. V. Vasil'eva G. A. Tolstikov 《Russian Chemical Bulletin》1997,46(3):577-581
2-Deoxy-α-d-lyxo-hexopyranosides of 18β-glycyrrhetic acid, its 11-deoxo derivative and allobetulin were synthesized by glycosylation of oleonane-type
triterpene alcohols withd-galactal acetate in the presence ofN-iodosuccinimide followed by deiodination and deprotection.
Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 596–600. March, 1997. 相似文献
20.
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