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1.
A recombinant putative glycoside hydrolase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 12 U mg−1 by heat treatment and His-Trap affinity chromatography, and identified as a single 56 kDa band upon SDS-PAGE. The native
enzyme is a dimer with a molecular mass of 112 kDa as determined by gel filtration. The enzyme exhibited its highest activity
when debranched arabinan (1,5-α-l-arabinan) was used as the substrate, demonstrating that the enzyme was an endo-1,5-α-l-arabinanase. The K
m, k
cat, and k
cat/K
m values were 18 mg ml−1, 50 s−1, and a 2.8 mg ml−1 s−1, respectively. Maximum enzyme activity was at pH 6.5 and 75°C. The half-lives of the enzyme at 65, 70 and 75°C were 2440,
254 and 93 h, respectively, indicating that it is the most thermostable of the known endo-1,5-α-l-arabinanases. 相似文献
2.
Molecular characterization of phenylalanine ammonia lyase gene from <Emphasis Type="Italic">Cistanche deserticola</Emphasis> 总被引:1,自引:0,他引:1
Hu GS Jia JM Hur YJ Chung YS Lee JH Yun DJ Chung WS Yi GH Kim TH Kim DH 《Molecular biology reports》2011,38(6):3741-3750
We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes
from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein
exhibited Michaelis–Menten kinetics with a K
m of 0.1013 mM, V
max of 4.858 μmol min−1, K
cat of 3.36 S−1, and K
cat/K
m is 33,168 M−1 S−1. The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol−1 when l-Phenylalanine was used as a substrate; l-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results
showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min
resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg2+, Pb2+, and Zn2+) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl
aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid
(tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a K
i of 8 μM. Competitive inhibitor AIP exhibited potent inhibition with K
i = 0.056 μM. 相似文献
3.
A mediator-free amperometric hydrogen peroxide biosensor was prepared by immobilizing horseradish peroxidase (HRP) enzyme
on colloidal Au modified platinum (Pt) wire electrode, which was modified by poly 2,6-pyridinediamine (pPA). The modified
process was characterized by electrochemical impedance spectroscopy (EIS), and the electrochemical characteristics of the
biosensor were studied by cyclic voltammetry, linear sweep voltammetry and chronoamperometry. The biosensor displayed an excellent
electrocatalytical response to reduction of H2O2 without the aid of an electron mediator, the linear range was 4.2 × 10−7–1.5 × 10−3 mol/L (r = 0.9977), with a detection limit of 1.4 × 10−7 mol/L. Moreover, the performance and factors influencing the resulted biosensor were studied in detail. The studied biosensor
exhibited permselectivity, good stability and good fabrication reproducibility. 相似文献
4.
Hee-Jung Moon Manish Tiwari Marimuthu Jeya Jung-Kul Lee 《Applied microbiology and biotechnology》2010,87(1):205-214
Ribitol dehydrogenase (RDH) catalyzes the conversion of ribitol to d-ribulose. A novel RDH gene was cloned from Zymomonas mobilis subsp. mobilis ZM4 and overexpressed in Escherichia coli BL21(DE3). DNA sequence analysis revealed an open reading frame of 795 bp, capable of encoding a polypeptide of 266 amino
acid residues with a calculated molecular mass of 28,426 Da. The gene was overexpressed in E. coli BL21(DE3) and the protein was purified as an active soluble form using glutathione S-transferase affinity chromatography. The molecular mass of the purified enzyme was estimated to be ∼28 kDa by sodium dodecyl
sulfate-polyacrylamide gel and ∼58 KDa with gel filtration chromatography, suggesting that the enzyme is a homodimer. The
enzyme had an optimal pH and temperature of 9.5 and 65°C, respectively. Unlike previously characterized RDHs, Z. mobilis RDH (ZmRDH) showed an unusual dual coenzyme specificity, with a k
cat of 4.83 s−1 for NADH (k
cat/K
m = 27.3 s−1 mM−1) and k
cat of 2.79 s−1 for NADPH (k
cat/K
m = 10.8 s−1 mM−1). Homology modeling and docking studies of NAD+ and NADP+ into the active site of ZmRDH shed light on the dual coenzyme specificity of ZmRDH. 相似文献
5.
Xuejun Hu Sylvain Robin Shane O’Connell Gary Walsh J. Gerard Wall 《Applied microbiology and biotechnology》2010,87(5):1773-1782
β-galactosidase is an enzyme administered as a digestive supplement to treat lactose intolerance, a genetic condition prevalent
in most world regions. The gene encoding an acid-stable β-galactosidase potentially suited for use as a digestive supplement
was cloned from Aspergillus niger van Tiegh, sequenced and expressed in Pichia pastoris. The purified recombinant protein exhibited kinetic properties similar to those of the native enzyme and thus was also competitively
inhibited by its product, galactose, at application-relevant concentrations. In order to alleviate this product inhibition,
a model of the enzyme structure was generated based on a Penicillium sp. β-galactosidase crystal structure with bound β-galactose. This led to targeted mutagenesis of an Asp258-Ser-Tyr-Pro-Leu-Gly-Phe amino acid motif in the A. niger van Tiegh enzyme and isolation from the resultant library of a mutant β-galactosidase enzyme with reduced sensitivity to
inhibition by galactose (K
i of 6.46 mM galactose, compared with 0.76 mM for the wildtype recombinant enzyme). The mutated enzyme also exhibited an increased
K
m (3.76 mM compared to 2.21 mM) and reduced V
max (110.8 μmol min−1 mg−1 compared to 172.6 μmol min−1 mg−1) relative to the wild-type enzyme, however, and its stability under simulated fasting gastric conditions was significantly
reduced. The study nevertheless demonstrates the potential to rationally engineer the A. niger van Tiegh enzyme to relieve product inhibition and create mutants with improved, application-relevant kinetic properties
for treatment of lactose intolerance. 相似文献
6.
Kemel Jellouli Ali Bougatef Laila Manni Rym Agrebi Rayda Siala Islem Younes Moncef Nasri 《Journal of industrial microbiology & biotechnology》2009,36(7):939-948
A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases
when it was grown at 30°C in media containing casein as carbon source (14,000 U ml−1). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular
weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence
of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide
range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity
of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic
constants K
m and K
cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 105 min−1, respectively. The catalytic efficiency (K
cat
/K
m) was 7.23 × 108 min−1 M−1. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability
and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that
of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme. 相似文献
7.
A direct electrochemical immunoassay system based on the immobilization of α-1-fetoprotein antibody (anti-AFP), as a model system, on the surface of core-shell Fe2O3/Au magnetic nanoparticles (MNP) has been demonstrated. To fabricate such an assay system, anti-AFP was initially covalently
immobilized on to the surface of core-shell Fe2O3/Au MNP. Anti-AFP-modified MNP (bio-nanoparticles) were then attached to the surface of carbon paste electrode with the aid
of a permanent magnet. The performance and factors influencing the performance of the resulting immunosensor were studied.
α-1-Fetoprotein antigen was directly determined by the change in current or potential before and after the antigen–antibody
reaction versus saturated calomel electrode. The electrochemical immunoassay system reached 95% of steady-state potential
within 2 min and had a sensitivity of 25.8 mV. The linear range for AFP determination was from 1 to 80 ng AFP ml−1 with a detection limit of 0.5 ng AFP ml−1. Moreover, the direct electrochemical immunoassay system, based on a functional MNP, can be developed further for DNA sensor
and enzyme biosensor.
Revisions requested 2 November 2005; Revisions received 17 January 2006 相似文献
8.
ZnO hollow spheres were firstly prepared. A new type of amperometric hydrogen peroxide biosensor was fabricated by entrapping
Hemoglobin (Hb) through the ZnO hollow spheres (ZHS) nanoparticles. The composition morphology and size were studied by transmission
electron microscopy. The surface topography of the prepared films was imaged by atomic force microscope (AFM). Several techniques,
including UV–vis absorption spectroscopy, cyclic voltammetry, chronoamperometry were employed to characterize the performance
of the biosensor. The results indicated that the ZHS nanoparticles had enhanced the performance of the hydrogen peroxide sensors.
The electrochemical parameters of Hb in the ZHS were calculated by the results of the electron-transfer coefficient (α) and
the apparent heterogeneous electron-transfer rate constant K
s as 0.5 and 3.1 s−1, respectively. The resulting biosensors showed a wide linear range from 2.1 × 10−6 to 5.18 × 10−3 M, with a low detection limit of 7.0 × 10−7 M (S/N = 3) under optimized experimental conditions. The results demonstrated that the ZHS matrix may improve the protein
loading with the retention of bioactivity and greatly promote the direct electron transfer, which can be attributed to its
unique morphology, high specific surface area, and biocompatibility. The biosensor obtained from this study possesses high
sensitivity, good reproducibility, and long-term stability. 相似文献
9.
The report is on an electrochemical biosensor with remarkably improved sensitivity toward nitrite. In this strategy, positively
charged gold nanoparticle (PCNA) is used in combination with multiwall carbon nanotubes (MWCNT) by electrostatic adsorption
for fabricating PCNA/MWCNT films. Then hemoglobin (Hb) biocatalyst will easily be attached to the surface of the combination
films aforementioned. After that, the Hb/PCNA films are immobilized onto the Hb/PCNA/MWCNT films through layer-by-layer assembly
technique. The (Hb/PCNA)2/MWNT/GC electrode thus prepared exhibits enhanced electrocatalytic behavior to the reduction of nitrite at −0.10 V versus
SCE in 0.05 M H2SO4 solution. On condition of the low detecting potential and low pH, interference caused by direct electrochemical oxidation or oxidizable
substances can be prevented. Therefore, the modified electrode shows fast response time, very high sensitivity, good selectivity
and stability. The current response of the sensor increases linearly with nitrite concentration from a range of 3.6 × 10−6 to 3.0 × 10−3 M with a detection limit(S /N = 3) of 9.6 × 10−7 M. 相似文献
10.
Alkaliphilic and halophilic Bacillus sp. BG-CS10 was isolated from Zabuye Salt Lake, Tibet. The gene celB, encoding a halophilic cellulase was identified from the genomic library of BG-CS10. CelB belongs to the cellulase superfamily
and DUF291 superfamily, with an unknown function domain and less than 58% identity to other cellulases in GenBank. The purified
recombinant protein (molecular weight: 62 kDa) can hydrolyze soluble cellulose substrates containing beta-1,4-linkages, such
as carboxylmethyl cellulose and konjac glucomannan, but has no exoglucanase and β-glucosidase activities. Thus, CelB is a
cellulase with an endo mode of action and glucomannanase activity. Interestingly, the enzyme activity was increased approximately
tenfold with 2.5 M NaCl or 3 M KCl. Furthermore, the optimal temperatures were 55°C with 2.5 M NaCl and 35°C without NaCl,
respectively. This indicates that NaCl can improve enzyme thermostability. The K
m
and k
cat values of CelB for CMC with 2.5 M NaCl were 3.18 mg mL−1 and 26 s−1, while the K
m
and k
cat values of CelB without NaCl were 6.6 mg mL−1 and 2.1 s−1. Thus, this thermo-stable, salt and pH-tolerant cellulase is a promising candidate for industrial applications, and provides
a new model to study salt effects on the structure of protein. 相似文献
11.
Daniel F. Visser Fritha Hennessy Konanani Rashamuse Maureen E. Louw Dean Brady 《Extremophiles : life under extreme conditions》2010,14(2):185-192
A purine nucleoside phosphorylase from the alkaliphile Bacillus halodurans Alk36 was cloned and overexpressed in Escherichia coli. The enzyme was purified fivefold by membrane filtration and ion exchange. The purified enzyme had a V
max of 2.03 × 10−9 s −1 and a K
m of 206 μM on guanosine. The optimal pH range was between 5.7 and 8.4 with a maximum at pH 7.0. The optimal temperature for
activity was 70°C and the enzyme had a half life at 60°C of 20.8 h. 相似文献
12.
Manish Kumar Tiwari Hee-Jung Moon Marimuthu Jeya Jung-Kul Lee 《Applied microbiology and biotechnology》2010,87(2):571-581
An NAD+-dependent xylitol dehydrogenase from Rhizobium etli CFN42 (ReXDH) was cloned and overexpressed in Escherichia coli. The DNA sequence analysis revealed an open reading frame of 1,044 bp, capable of encoding a polypeptide of 347 amino acid
residues with a calculated molecular mass of 35,858 Da. The ReXDH protein was purified as an active soluble form using GST
affinity chromatography. The molecular mass of the purified enzyme was estimated to be ∼34 kDa by sodium dodecyl sulfate–polyacrylamide
gel and ∼135 kDa with gel filtration chromatography, suggesting that the enzyme is a homotetramer. Among various polyols,
xylitol was the preferred substrate of ReXDH with a K
m = 17.9 mM and kcat
/K
m = 0.5 mM−1 s−1 for xylitol. The enzyme had an optimal pH and temperature of 9.5 and 70 °C, respectively. Heat inactivation studies revealed
a half life of the ReXDH at 40 °C of 120 min and a half denaturation temperature (T
1/2) of 53.1 °C. ReXDH showed the highest optimum temperature and thermal stability among the known XDHs. Homology modeling and
sequence analysis of ReXDH shed light on the factors contributing to the high thermostability of ReXDH. Although XDHs have
been characterized from several other sources, ReXDH is distinguished from other XDHs by its high thermostability. 相似文献
13.
A convenient and effective way for fabricating amperometric hydrogen peroxide (H2O2) biosensor was designed in this paper. First, the polyaniline (PANI) nanofibers membrane with good conductance and high surface
area was electropolymerized on a gold electrode surface. Then, Pt nanoparticle (PtNP) was electrochemically deposited on the
PANI nanofibers membrane. Finally, the hybrid film of gold nanoparticle, chitosan, and horseradish peroxidase (HRP) was cast
onto the modified electrode to form a stable biofunctional film, which was also employed as a protective layer to PtNP. The
proposed biosensor exhibited a rapid response to H2O2 with the linear range from 7.0 × 10−6 to 1.4 × 10−2 M and a detection limit of 2.8 × 10−6 M (S/N = 3). The sensitivity of 558 μA mM−1 cm−2 was obtained. The Michaelis–Menten constant,
K\textM\textapp K_{\text{M}}^{\text{app}} value was 1.90 mM suggesting a high affinity. Moreover, it displayed a good reproducibility and long-term stability. 相似文献
14.
A spiral packed-bed bioreactor inoculated with microorganisms obtained from activated sludge was used to conduct a feasibility
study for phenol removal. The reactor was operated continuously at various phenol loadings ranging from 53 to 201.4 g m−3 h−1, and at different hydraulic retention times (HRT) in the range of 20–180 min to estimate the performance of the device. The
results indicated that phenol removal efficiency ranging from 82.9 to 100% can be reached when the reactor is operated at
an HRT of 1 h and a phenol loading of less than 111.9 g m−3 h−1. At an influent phenol concentration of 201.4 g m−3, the removal efficiency increased from 18.6 to 76.9% with an increase in the HRT (20–120 min). For treatment of phenol in
the reactor, the maximum biodegradation rate (V
m) was 1.82 mg l−1 min−1; the half-saturation constant (K
s), 34.95 mg l−1. 相似文献
15.
Syed Ali Imran Bokhari Farooq Latif Muhammad Ibrahim Rajoka 《World journal of microbiology & biotechnology》2009,25(3):493-502
Xylanases produced from a locally isolated strain of Thermomyces lanuginosus and its mutant derivative were purified to a yield of 39.1 and 42.83% with specific activities of 15,501 and 17,778 IU mg−1 protein, respectively. The purification consisted of two steps i.e., ammonium sulphate precipitation, and gel filtration
chromatography. The mutant enzyme showed high affinity for substrate, with a K
m of 0.098 mg ml−1 as compared to wild type enzyme showing K
m of not less than 0.112 mg ml−1. It was found that pH values of 8.1 and 7.3 were best for activity of the mutant and wild-type-derived enzymes, respectively.
The values of pK
a of the acidic limbs of both enzymes were the same (5.0 and 4.9, respectively) but the pK
a value of the basic limb was slightly increased, indicating the participation of a carboxyl group present in a non-polar environment.
Temperatures of 70 and 65°C were found optimal for mutant and wild-derived xylanase, respectively. Enzymes displayed a high
thermostability showing a half life of 31.79 and 6.0 min (5.3-fold improvement), enthalpy of denaturation (ΔH*) of 146.06 and 166.95 kJ mol−1, entropy of denaturation (ΔS*) of 101.44 and 174.67, and free energy of denaturation (ΔG*) of 110.25 and 105.29 kJ mol−1 for mutant- and wild-organism derived enzyme, respectively at 80°C. Studies on the folding and stability of cellulase-less
xylanases are important, since their biotechnological employments require them to function under extreme conditions of pH
and temperature. The kinetic and thermodynamic properties suggested that the xylanase from the mutant organism is better as
compared to xylanase produced from the wild type and previously reported strains of same species, and may have a potential
usage in various industrial fields. 相似文献
16.
The specific activity of a recombinant β-glucosidase from Sulfolobus solfataricus for isoflavones was: daidzin > glycitin > genistin > malonyl genistin > malonyl daidzin > malonyl glycitin. The hydrolytic
activity of this enzyme for daidzin was highest at pH 5.5 and 90°C with a half-life of 18 h, a K
m of 0.5 mM, and a k
cat of 2532 s−1. The enzyme converted 1 mM daidzin to 1 mM daidzein after 1 h with a molar yield of 100% and a productivity of 1 mM h−1. Among β-glucosidases, that from S. solfataricus
β had the highest thermostability, k
cat, k
cat/K
m, conversion yield, and productivity in the hydrolysis of daidzin. 相似文献
17.
B Lin G Lu Y Zheng W Xie S Li Z Hu 《World journal of microbiology & biotechnology》2012,28(4):1691-1697
A β-agarase gene hz2 with 2,868 bp was cloned from the marine agarolytic bacterium Agarivorans sp. HZ105. It encoded a mature agarase HZ2 of 102,393 Da (920 amino acids). Based on the amino acid sequence similarity,
agarase HZ2 was assigned to the glycoside hydrolase family 50. The β-agarase shared a gene sequence identity of 98.6% with
the reported but much less characterized β-agarase agaB from Vibrio sp. JT0107. Its recombinant agarase rHZ2 was produced in E. coli cells and purified to homogeneity. The agarase rHZ2 degraded agarose and neoagarooligosaccharides with degrees of polymerization
above four, to yield neoagarotetraose as the dominant product, which was different from β-agarase agaB of Vibrio sp. JT0107. The agarose hydrolysis pattern suggested that rHZ2 was an endo-type β-agarase. Beta-mercaptoethanol (90 mM) and
dithiothreitol (9 mM) increased the agarase activity of rHZ2 by 72.9% and 17.3% respectively, while SDS (9 mM) inhibited the
activity completely. The agarase activity was independent of Na+, K+, Mg2+ and Ca2+. The maximal enzyme activity was observed at 40°C and pH 7. The kinetic parameters K
m, V
max, K
cat, and K
cat/K
m values toward agarose of agarase rHZ2 were 5.9 mg ml−1, 235 U mg−1, 401 s−1 and 6.8 × 105 M−1 s−1, respectively. Agarase rHZ2 could have a potential application in the production of bioactive neoagarotetraose. 相似文献
18.
C. Sivapathasekaran Palashpriya Das Soumen Mukherjee J. Saravanakumar Mahitosh Mandal Ramkrishna Sen 《International journal of peptide research and therapeutics》2010,16(4):215-222
A marine Bacillus circulans DMS-2 was able to grow and produce biosurfactant on glucose mineral salts medium (GMSM) with a reduction in the surface tension
up to 27 mN m−1. The microorganism produced 1.64 ± 0.1 g l−1 of crude biosurfactant. The lipopeptide nature of the produced biosurfactant was confirmed by primulin and ninhydrin assays
using High Performance Thin Layer Chromatography (HPTLC). Preparative thin layer chromatography (TLC) was performed to purify
the lipopeptides from the crude biosurfactant. The critical micelle concentrations (CMC) of the crude and purified products
were found to be 90 and 40 mg l−1 respectively. Fourier transform infrared spectrophotometer (FTIR) and matrix assisted laser desorption/ionization time of
flight (MALDI-ToF) mass spectral analysis revealed the identity of the produced lipopeptides as surfactin (m/z 1,023 Da) and fengycin (m/z 1,495 Da) isoforms. The purified marine lipopeptides displayed a significant antiproliferative activity against the human
colon cancer cell lines HCT-15 (IC50 80 μg ml−1) and HT-29 (IC50 120 μg ml−1). 相似文献
19.
Jung-Soo Kim Manish Kumar Tiwari Hee-Jung Moon Marimuthu Jeya Thangadurai Ramu Deok-Kun Oh In-Won Kim Jung-Kul Lee 《Applied microbiology and biotechnology》2009,83(2):273-283
Nitrile groups are catabolized to the corresponding acid and ammonia through one-step reaction involving a nitrilase. Here,
we report the use of bioinformatic and biochemical tools to identify and characterize the nitrilase (NitPf5) from Pseudomonas fluorescens Pf-5. The nitPf5 gene was identified via sequence analysis of the whole genome of P. fluorescens Pf-5 and subsequently cloned and overexpressed in Escherichia coli. DNA sequence analysis revealed an open-reading frame of 921 bp, capable of encoding a polypeptide of 307 amino acids residues
with a calculated isoelectric point of pH 5.4. The enzyme had an optimal pH and temperature of 7.0°C and 45°C, respectively,
with a specific activity of 1.7 and 1.9 μmol min−1 mg protein−1 for succinonitrile and fumaronitrile, respectively. The molecular weight of the nitrilase as determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography was 33,000 and 138,000 Da, respectively, suggesting
that the enzyme is homotetrameric. Among various nitriles, dinitriles were the preferred substrate of NitPf5 with a K
m = 17.9 mM and k
cat/K
m = 0.5 mM−1 s−1 for succinonitrile. Homology modeling and docking studies of dinitrile and mononitrile substrate into the active site of
NitPf5 shed light on the substrate specificity of NitPf5. Although nitrilases have been characterized from several other sources,
P. fluorescens Pf-5 nitrilase NitPf5 is distinguished from other nitrilases by its high specific activity toward dinitriles, which make
P. fluorescens NitPf5 useful for industrial applications, including enzymatic synthesis of various cyanocarboxylic acids. 相似文献
20.
Purification, immobilization and characterization of linoleic acid isomerase on modified palygorskite 总被引:2,自引:0,他引:2
Linoleic acid isomerase from Lactobacillus delbrueckii subsp. bulgaricus 1.1480 was purified by DEAE ion-exchange chromatography and gel filtration chromatography. An overall 5.1% yield and purification
of 93-fold were obtained. The molecular weight of the purified protein was ~41 kDa which was analyzed by SDS-PAGE. The purified
enzyme was immobilized on palygorskite modified with 3-aminopropyltriethoxysilane. The immobilized enzyme showed an activity
of 82 U/g. The optimal temperature and pH for the activity of the free enzyme were 30 °C and pH 6.5, respectively; whereas
those for the immobilized enzyme were 35 °C and pH 7.0, respectively. The immobilized enzyme was more stable than the free
enzyme at 30–60 °C, and the operational stability result showed that more than 85% of its initial activity was retained after
incubation for 3 h. The K
m and V
max values of the immobilized enzyme were found to be 0.0619 mmol l−1 and 0.147 mmol h−1 mg−1, respectively. The immobilized enzyme had high operational stability and retained high enzymatic activity after seven cycles
of reuse at 37 °C. 相似文献