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1.
Calsavara Luiza P. V. De Moraes Flávio F. Zanin Gisella M. 《Applied biochemistry and biotechnology》2001,91(1-9):615-626
The enzyme cellobiase from Novo was immobilized in controlled pore silica particles by covalent binding with the silane-glutaraldehyde
method with protein and activity yields of 67 and 13.7%, respectively. The activity of the free enzyme (FE) and immobilized
enzyme (IE) was determined with 2 g/L of cellobiose, from 40 to 75°C at pH 3.0–7.0 for FE and from 40 to 70°C at pH 2.2–7.0
for IE. At pH 4.8 the maximum specific activity for the FE and IE occurred at 65°C: 17.8 and 2.2 micromol of glucose/(min·mg
of protein), respectively. For all temperatures the optimum pH observed for FE was 4.5 whereas for IE it was shifted to 3.5.
The energy of activation was 11 kcal/mol for FE and 5 kcal/mol for IE at pH 4.5–5, showing apparent diffusional limitation
for the latter. Thermal stability of the FE and IE was determined with 2 g/L of cellobiose (pH 4.8) at temperatures from 40
to 70°C for FE and 40 to 75°C for IE. Free cellobiase maintained its activity practically constant for 240 min at temperatures
up to 55°C. The IE has shown higher stability, retaining its activity in the sametest up to 60°C. Half-life experimental results
for FE were 14.1, 2.1, and 0.17 h at 60, 65, and 70°C, respectively, whereas IE at the same temperatures had half-lives of
245, 21.3, and 2.9 h. The energy of thermal deactivation was 80.6 k cal/mol for the free enzyme and 85.2 k cal/mol for the
IE, suggesting stabilization by immobilization. 相似文献
2.
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
3.
Matioli Graciette Zanin Gisella M. De Moraes Flávio F. 《Applied biochemistry and biotechnology》2001,91(1-9):643-654
The enzyme cyclod extringly cosyltransferase (CGTase), EC2.4.1.19, which produces cyclodextrins (CDs) from starch, was obtained
from Bacillus firmus strain no. 37 isolated from Brazilian soil and characterized in the soluble form using as substrate 100 g/L of maltodextrin
in 0.05 M Tris-HCl buffer, 5 mM CaCl2, and appropriate buffers. Enzymatic activity and its activation energy were determined as a function of temperature and pH.
The activation energy for the production of β- and γ-CD was 7.5 and 9.9 kcal/mol, respectively. The energy of deactivation
was 39 kcal/mol. The enzyme showed little thermal deactivation in the temperature range of 35–60°C, and Arrhenius-type equations
were obtained for calculating the activity, deactivation, and half-life as a function of temperature. The molecular weight
of the enzyme was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, giving 77.6k Da. Results for CGTase
activity as a function of temperature gave maximal activity for the production of β-CD at 65°C, pH 6.0, and 7 1.5 mmol of
β-CD/(min·mg of protein), whereas for γ-CD it was 9.1 m mol of γ-CD/(min·mg of protein) at 70°C and pH 8.0. For long contact
times, the bestuse of the enzymatic activity occurs at 60°C oratalower temperature, and the reaction pH may be selected to
increase the vield of a desired CD. 相似文献
4.
Four myrosinase (β-thioglucosidase EC. 3.2.3.1) and seven disaccharase (β-fructofuranosidase, EC. 3.2.1.26) isoenzymes were isolated from turnip leaves. The most active enzymes were isolated in pure
form. Myrosinase and disaccharase mol wt was 62.0 × 103 and 69.5 × 103 dalton, respectively, on the basis of gel filtration on Sephadex G-200.
Myrosinase pH profile showed high activity between pH 5 and 7 with the optimum at pH 5.5. The purified enzyme was heat-stable
for 60 min at 30°C with only loss of 24% of activity. Its activity is strongly inhibited (100%) by Pb2+, Ba2+, Cu2+ and Ca2+ ions, and activated (70%) by EDTA at 0.04M. The pure enzyme failed to hydrolyze amylose, glycogen, lactose, maltose, and
sucrose. TheK
m andV
max values of myrosinase using sinigrin as specific substrate was 0.045 mM and 2.5 U, respectively.
The maximal activity of disaccharase enzyme was obtained at pH 4–5 and 35–37°C. The enzyme was heat-stable at 30°C for 30
min with only 10% loss of its activity. Its activity is strongly activated (70–240%) by Ca2+, Ba2+, Cu2+, and EDTA at 0.01M. The enzyme activity is specific to the disaccharide sucrose and failed to hydrolyze other disaccharides (maltose and lactose).
TheK
m andV
max of disaccharase were 0.123 mM and 3.33 U, respectively. 相似文献
5.
Preparation of γ-aminobutyric acid using E. coli cells with high activity of glutamate decarboxylase
A. Yu. Plokhov M. M. Gusyatiner T. A. Yampolskaya V. E. Kaluzhsky B. S. Sukhareva A. A. Schulga 《Applied biochemistry and biotechnology》2000,88(1-3):257-265
γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter synthesized in the central nervous system from glutamate
by glutamate decarboxylase (GAD). It has applications in the production of many drugs. The technology of GABA synthesis by
treating L-glutamic acid with the cells of the gene-engineered GAD superproducer strain of Escherichia coli GAD K10 was developed. Cell growing in the presence of 0.02 mM pyridoxal phosphate (PLP) causes the 2- to 2.5-fold increase of total productivity of the cells. The best way to prepare
the cells for the reaction was their thermal activation by pretreatment for 1 h at 53°C. The optimal conditions for this reaction
were 37°C and pH 4.6. The rate of the enzymatic reaction is the function of acetate concentration with the maximum at 0.5
M acetate. The total amount of GABA synthesized using 1 g of wet cells reached 23–25 g. The final concentration of GABA in
the reaction medium was 280–300 g/L. The yield of the product was about 99%. 相似文献
6.
Thereza Christina Vessoni Penna Marina Ishii Adalberto Pessoa Junior Olivia Cholewa 《Applied biochemistry and biotechnology》2004,114(1-3):469-483
The thermal stability of the recombinant green fluorescent protein (GFPuv) expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction chromatography was studied. The GFPuv
(3.5–9.0 μg of GFPuv/mL) was exposed to various pH conditions (4.91–9.03) and temperatures (75–95°C) in the 10 mM buffers: acetate (pH 5.0–7.0), phosphate (pH 5.5–8.0), and Tris-HCl (pH 7.0–9.0). The extent of protein denaturation (loss
of fluorescence intensity) was expressed in decimal reduction time (D-value), the time exposure required to reduce 90% of the initial fluorescence intensity of GFPuv. For pH 7.0 to 8.0, the thermostability
of GFPuv was slightly greater in phosphate buffer than in Tris-HCl. At 85°C, the D-values (pH 7.1–7.5) ranged from 7.24 (Tris-HCl) to 13.88 min (phosphate) The stability of GFPuv in Tris-HCl (pH>8.0) was
constant at 90 and 95°C, and the D-values were 7.93 (pH 8.38–8.92) and 6.0 min (pH 8.05–8.97), respectively. The thermostability of GFPuv provides the basis
for its potential utility as a fluorescent biologic indicator to assay the efficacy of moist-heat treatments at temperatures
lower than 100°C. 相似文献
7.
Cleide M. F. Soares Heizir F. de Castro Juliana E. Itako Flavio F. De Moraes Gisella M. Zanin 《Applied biochemistry and biotechnology》2005,123(1-3):845-859
Candida rugosa lipase was entrapped in silica sol-gel particles prepared by hydrolysis of methyltrimethoxysilane and assayed by p-nitrophenyl palmitate hydrolysis, as a function of pH and temperature, giving pH optima of 7.8 (free enzyme) and 5.0–8.0
(immobilized enzyme). The optimum temperature for the immobilized enzyme (50–55°C) was 19°C higher than for the free enzyme.
Thermal, operational, and storage stability were determined with n-butanol and bytyric acid, giving at 45°C a half-life 2.7 times greater for the immobilized enzyme; storage time was 21 d
at room temperature. For ester synthesis, the optimum temperature was 47°C, and high esterification conversions were obtained
under repeated batch cycles (half-life of 138 h). 相似文献
8.
A crude preparation of Aspergillus niger β-glucosidase (27.5 cello-biase U/mg protein at 40°C, pH 5.0) was immobilized on
concanavalin A-Sepharose (CAS). The cellobiase activity of the immobilized enzyme was 1334 U/mg dried CAS or 108 U/mL CAS
gel. The β-glucosidase-CAS complex was entrapped within crosslinked propylene glycol alginate/bone-geletin gel spheres that
possessed between 0.67 and 2.35 cellobiase U/mL spheres, depending on their size. The effect of cellobiose concentration (10–300
mM) on the activity of native, immobilized, and gel-entrapped enzyme was determined. It was shown that concentrations of cellobiose
between 10 and 180 mM were not inhibitory to the entrapped enzyme, although inhibition was found to occur with the native
and immobilized enzyme. Exogenous ion addition was not necessary to maintain the structural integrity of the spheres, which
were stable for 4 d at 40°C. 相似文献
9.
Mohamed A. Abdel-Naby Mona Y. Osman Ahmed F. Abdel-Fattah 《Applied biochemistry and biotechnology》1999,76(1):33-44
Three cellobiases, here called cellobiase A, B, and C, from the culture filtrate of Aspergillus niger A20, were purified by precipitation with ammonium sulphate, gel filtration through Sephadex G-75, and column chromatography
of DEAE-cellulose. The purified enzymes were homogeneous on polyacrylamide disk electrophoresis. The mol wt of the purified
enzymes were estimated by SDS-gelelectrophoresis to be 88,000, 80,000, and 71,000 for cellobiases A, B, and C, respectively.
The enzymes were active at pH 4.5 and 55–60°C. The pattern of their aminoacid compositions showed high contents of aspartic
acid, glutamic acid, threonine, serine, and glycine. The apparent Km values for cellobiose were 0.9, 1.63, and 1.0 mM for cellobiases A, B, and C, respectively. Calcium ions stimulated cellobiases B and C, and Co2+ and Mg2+ ions stimulated cell obiase A. The purified enzymes hydrolyzed cellobiose and aryl-β-d-glucosides, but they had no action on sucrose, maltose, and cellulose. The three cellobiases catalyzed transglycosylate reaction,
and the major product formed from cellobiose was tetramer of glucose. 相似文献
10.
Moradian F Khajeh K Naderi-Manesh H Ahmadvand R Sajedi RH Sadeghizadeh M 《Applied biochemistry and biotechnology》2006,134(1):77-87
Two Bacillus sp. strains, HR-08 and KR-8102, isolated from soil of the west and north parts of Iran were screened on gelatin agar medium
for their ability to produce alkaline protease. The enzymes were active in a wide pH range (6.0–11.0) and stable in the alkaline
range (7.0–12.0). The optimum temperatures for the protease from HR-08 and KR-8102 were 65 and 50°C, respectively. The irreversible
thermoinactivation of HR-08 and KR-8102 proteases showed that the stability of HR-08 enzyme was higher than that of KR-8102
and the half-lives of these enzymes were 95 and 32 min at 50°C, respectively. In the presence of 10 mM Ca2+, HR-08 retained 100, 90, and 20% of its initial activity after heating for 30 min at 50, 60, and 70°C, respectively. Enzymes
were inhibited by phenylmethylsulfonyl fluoride and iodoacetate. After inhibition by iodoacetate, both enzymes were reactivated
by dithiothreitol. These data show that the enzymes seem to be thiol-dependent serine alkaline proteases. The enzymes especially
from HR-08 were stable in the presence of H2O2, surfactants, and local detergents; their activities were enhanced in the presence of 5 mM Fe2+; and the presence of 5mM metal ions such as Mg2+, Cu2+, and Mn2+ produced almost no effect. 相似文献
11.
An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin
A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the
purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and
native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of
about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa,
thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing,
with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature
for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively.
The inulin hydrolysis activity was completely abolished with 1 mM Hg++, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower
K
m
(0.25 mM) and higher V
max (333.3 IU/mg) values for inulin. 相似文献
12.
Sari MM 《Applied biochemistry and biotechnology》2011,163(8):1020-1037
Cupric ion-chelated poly(hydroxyethyl methacrylate-n-vinyl imidazole) (poly(HEMA-VIM)) microspheres prepared by suspension polymerization were investigated as a specific adsorbent
for immobilization of yeast invertase in a batch system. They were characterized by scanning electron microscopy, surface
area, and pore size measurements. They have spherical shape and porous structure. The specific surface area of the p(HEMA-VIM)
spheres was found to be 81.2 m2/g with a size range of 70–120 μm in diameter, and the swelling ratio was 86.9%. Then, Cu(II) ion chelated on the microspheres
(546 μmol Cu(II)/g), and they were used in the invertase adsorption. Maximum invertase adsorption was 51.2 mg/g at pH 4.5.
Cu(II) chelation increases the tendency from Freundlich-type to Langmuir-type adsorption model. The optimum activity for both
free and adsorbed invertase was observed at pH 4.5. The optimum temperature for the poly(HEMA-VIM)/Cu(II)-invertase system
was found to be at 55 °C, 10 °C higher than that of the free enzyme at 45 °C. V
max values were determined as 342 and 304 U/mg enzyme, for free and adsorbed invertase, respectively. K
m values were found to be same for free and adsorbed invertase (20 mM). Thermal and pH stability and reusability of invertase
increased with immobilization. 相似文献
13.
Cyclodextrin glucanotransferase, produced by Bacillus megaterium, was characterized, and the biochemical properties of the purified enzyme were determined. The substrate specificity of the
enzyme was tested with different α-1,4-glucans. Cyclodextrin glucanotransferase displayed maximum activity in the case of
soluble starch, with a K
m value of 3.4 g/L. The optimal pH and temperature values for the cyclization reaction were 7.2 and 60 °C, respectively. The
enzyme was stable at pH 6.0–10.5 and 30 °C. The enzyme activity was activated by Sr2+, Mg2+, Co2+, Mn2+, and Cu2+, and it was inhibited by Zn2+and Ag+. The molecular mass of cyclodextrin glucanotransferase was established to be 73,400 Da by sodium dodecyl sulfate–polyacrylamide
gel electrophoresis, 68,200 Da by gel chromatography, and 75,000 Da by mass spectrometry. The monomer form of the enzyme was
confirmed by the analysis of the N-terminal amino acid sequence. Cyclodextrin glucanotransferase formed all three types of
cyclodextrins, but the predominant product was β-cyclodextrin. 相似文献
14.
Sene Luciane Felipe Maria G. A. Silva Silvio S. Vitolo Michele 《Applied biochemistry and biotechnology》2001,91(1-9):671-680
Candida guilliermondii FTI 20037 was cultured in sugarcane bagasse hydrolysate supplemented with 2.0 g/L of (NH4)2SO4, 0.1 g/L of CaCl2·2H2O, and 20.0 g/L of rice bran at 35°C; pH 4.0; agitation of 300 rpm; and aeration of 0.4, 0.6, or 0.8 vvm. The high xylitol
production (20.0 g/L) and xylose reductase (XR) activity (658.8 U/mg of protein) occurred at an aeration of 0.4 vvm. Under
this condition, the xylitol dehydrogenase (XD) activity was low. The apparent K
M for XR and XD against substrates and cofactors were as follows: for XR, 6.4×10−2
M (xylose) and 9.5×10−3 mM (NADPH); for XD, 1.6×10−1
M (xylitol) and 9.9×10−2 mM (NAD+). Because XR requires about 10-fold less xylose and cofactor than XD for the condition in which the reaction rate is
half of the V
max, some interference on the overall xylitol production by the yeast could be expected. 相似文献
15.
B. Szajáni Aranka Molnár Gabriella Klámar M. Kálmán 《Applied biochemistry and biotechnology》1987,14(1):37-47
A simple, one-step process, using 0.25Mp-benzoquinone dissolved in 20% dioxane at 50°C for 24 h was applied to the activation of polyacrylamide beads. The activated
beads were reacted with glucose oxidase isolated fromAspergillus niger. The coupling reaction was performed in 0.1M potassium phosphate at pH 8.5 and 0–4°C for 24 h. The protein concentration was
50 mg/mL. In such conditions, the highest activity achieved was about 100 U/g solid. The optimum pH for the catalytic activity
was shifted by about 1 pH unit in the acidic direction to pH 5.5. Between 35 and 50°C, the activity of the immobilized form
depends on the temperature to a smaller extent than that of the soluble form. Above 50°C, the activity of immobilized glucose
oxidase shows a sharper heat dependence. The enzyme-substrate interaction was not profoundly altered by the immobilization
of the enzyme. The heat resistance of the immobilized enzyme was enhanced. The immobilized glucose oxidase is most stable
at pH 5.5. The practical use of the immobilized glucose oxidase was tested in preliminary experiments for determination of
the glucose concentration in blood sera. 相似文献
16.
S. Coulon P. Chemardin Y. Gueguen A. Arnaud P. Galzy 《Applied biochemistry and biotechnology》1998,74(2):105-114
The lactic acid bacterium,Lactobacillus casei, produces an intracellular β-glucosidase when grown on Man-Rogosa-Sharpe (MRS) medium with cellobiose as carbon source. The
β-glucosidase activity is produced intracellulary, and no extracellulary activity was detected. The enzyme was purified by
ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase as estimated
by gel filtration was 480 kDa, consisting of six probably identical subunits. The enzyme exhibited optimum activity at 35°C
and pH 6.3 with citrate-phosphate buffer. The enzyme was active against soluble glycosides with (1→4)-β configuration and
from Lineweaver Burk plots, Km value of 16 mmol/L was found for β-pNPG. The β-glucosidase was competitively inhibited by glucose, and no glycosyl transferase
activity was observed in the presence of ethanol. 相似文献
17.
Nidetzky Bernd Griessler Richard Weinhausel Andreas Haltrich Dietmar Kulbe Klaus D. 《Applied biochemistry and biotechnology》1997,(1):159-172
Some important process properties of α-l,4-D-ghican phosphorylases isolated from the bacteriumCorynebacterium callunae and potato tubers (Solatium tuberosum) were compared. Apart from minor differences in their stability and specificity (represented by the maximum degree of maltodextrin
conversion) and a 10-fold higher affinity of the plant phosphorylase for maltodextrin (K
M of 1.3 g/L at 300 mM of orthophosphate), the performances of both enzymes in a continuous ultrafiltration membrane reactor
were almost identical. Product synthesis was carried out over a time course of 300–400 h in the presence or absence of auxiliary
pullulanase (increasing the accessibility of the glucan substrate for phosphorolytic attack up to 15–20%). The effect of varied
dilution rate and reaction temperature on the resulting productivities was quantitated, and a maximum operational temperature
of 40°C was identified. 相似文献
18.
Urease from pigeonpea (Cajanus cajan L.) was covalently linked to crab shell chitosan beads using glutaraldehyde. The optimum immobilization (64% activity) was
observed at 4°C, with a protein concentration of 0.24 mg/bead and 3% glutaraldehyde. The immobilized enzyme stored in 0.05
M Trisacetate buffer, pH 7.3, at 4°C had a t
1/2 of 110 d. There was practically no leaching of enzyme (<3%) from the immobilized beads in 30 d. The immobilized urease was
used 10 times at an interval of 24 h between each use with 80% residual activity at the end of the period. The chitosan-immobilized
urease showed a significantly higher Michaelis constant (8.3 mM) compared to that of the soluble urease (3.0 mM). Its apparent optimum pH also shifted from 7.3 to 8.5. Immobilized urease showed an optimal temperature of 77°C, compared
with 47°C for the soluble urease. Time-dependent kinetics of the thermal denaturation of immobilized urease was studied and
found to be monophasic in nature compared to biphasic in nature for soluble enzyme. This immobilized urease was used to analyze
blood urea of some of the clinical samples from the clinical pathology laboratories. The results compared favorably with those
obtained by the various chemical/biochemical methods employed in the clinical pathology laboratories. A column packed with
immobilized urease beads was also prepared in a syringe for the regular and continuous monitoring of serum urea concentrations. 相似文献
19.
Ping Xu Toshihiro Yano Kenji Yamamoto Hideyuki Suzuki Hidehiko Kumagai 《Applied biochemistry and biotechnology》1996,56(3):277-288
A lactate oxidase was purified about 36-fold from a newly screened strain KY6 of gram negative bacterium from soil to yield a homogeneous protein. The native enzyme had a molecular mass of 204 kDa measured by Sephadex G-200 and that of subunit on the SDS-PAGE was found to be 45 kDa. The enzyme was optimally active at pH 7.7 and showed stability at pH range of 5.7 to 9.5 for 24 h at 4?C. The optimum temperature was 70?C and the enzyme activity was stable for 10 min up to 45?C. The half-life of the enzyme activity was about 10 min at 55?C. The best substrate of the enzyme was D-lactate and Km value for D-lactate was 0.14 mM. The Km value for DL-lactate was 0.20 mM. Substrate inhibition of the enzyme was observed at higher concentrations than 20 mM of DL-lactate and 10 mM of D-lactate. 相似文献
20.
Asha Chaubey Manju Gerard V. S. Singh B. D. Malhotra 《Applied biochemistry and biotechnology》2001,96(1-3):303-311
Tetraethylorthosilicate (TEOS)-derived sol-gel films were utilized for the immobilization of lactate dehydrogenase (LDH) by
physical adsorption and sol-gel/LDH/sol-gel sandwich configuration. An attempt was made to ascertain the optimum pH and temperature
for the immobilized LDH. It was shown that TEOS-derived sol-gel films containing physically adsorbed LDH exhibited linearity
from 0.5 to 4 mM, whereas those containing LDH in sandwich configuration showed linearity from 0.5 to 3 mM
l-lactate. These sol-gel films, immobilized with LDH, were found to be stable for about 4 weeks at 4–10°C. 相似文献