共查询到20条相似文献,搜索用时 62 毫秒
1.
Canakci S Belduz AO Saha BC Yasar A Ayaz FA Yayli N 《Applied microbiology and biotechnology》2007,75(4):813-820
The gene encoding an α-l-arabinofuranosidase from Geobacillus caldoxylolyticus TK4, AbfATK4, was isolated, cloned, and sequenced. The deduced protein had a molecular mass of about 58 kDa, and analysis
of its amino acid sequence revealed significant homology and conservation of different catalytic residues with α-l-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. A histidine tag was introduced at the N-terminal
end of AbfATK4, and the recombinant protein was expressed in Escherichia coli BL21, under control of isopropyl-β-D-thiogalactopyranoside-inducible T7 promoter. The enzyme was purified by nickel affinity
chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 236 kDa, suggesting a
homotetrameric structure. AbfATK4 was active at a broad pH range (pH 5.0–10.0) and at a broad temperature range (40–85°C),
and it had an optimum pH of 6.0 and an optimum temperature of 75–80°C. The enzyme was more thermostable than previously described
arabinofuranosidases and did not lose any activity after 48 h incubation at 70°C. The protein exhibited a high level of activity
with p-nitrophenyl-α-l-arabinofuranoside, with apparent K
m and V
max values of 0.17 mM and 588.2 U/mg, respectively. AbfATK4 also exhibited a low level of activity with p-nitrophenyl-β-d-xylopyranoside, with apparent K
m and V
max values of 1.57 mM and 151.5 U/mg, respectively. AbfATK4 released l-arabinose only from arabinan and arabinooligosaccharides. No endoarabinanase activity was detected. These findings suggest
that AbfATK4 is an exo-acting enzyme. 相似文献
2.
An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60–65°C.
The apparent K
m with citrus pectin was 1.46 mg/ml and the V
max was 2433.3 μmol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50°C for 1 h
and showed a half-life of 10 min at 60°C. Polygalacturonase was stable at pH 5.0–5.5 and maintained 33% of initial activity
at pH 9.0. Metal ions, such as Zn+2, Mn+2, and Hg+2, inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing
mono, di and tri-galacturonic acids within 10 min of hydrolysis. 相似文献
3.
Chitwadee Phithakrotchanakoon Ratama Daduang Arinthip Thamchaipenet Thidarat Wangkam Toemsak Srikhirin Lily Eurwilaichitr Verawat Champreda 《Applied microbiology and biotechnology》2009,82(1):131-140
A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His6-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase
containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights
of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V
max and K
m of 3.63 ± 0.16 μmol min−1 mg−1 and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that
PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm−2 h−1 for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films
was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique
will be of utility for further study of bioplastic degradation. 相似文献
4.
Laila Oukhattar Tarik Baibai Adnane Moutaouakkil Omar Assobhei Abdelaziz Soukri 《Reviews in Fish Biology and Fisheries》2008,18(3):263-271
The NAD+ dependent cytosolic Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from arms of Octopus vulgaris, Cuvier, 1787, (Octopoda, Cephalopoda) was purified to homogeneity and its kinetic properties investigated. The purification
method consisted of ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulting in a 26-fold
increase in specific activity and a final yield of approximately 16%. The apparent molecular weight of the purified native
enzyme was 153 kDa. The protein is an homotetramer, composed of identical subunits with an apparent molecular weight of approximately
36 kDa. The Michaelis constants Km for both NAD+ and d-G3P were 66 μM and 320 μM, respectively. The maximal velocity Vmax of the purified enzyme was estimated to be 21.8 U/mg. Only one GAPDH isoform (pI 6.6) was obtained by isoelectrofocusing in polyacrylamide slab gels holding ampholyte generated pH gradients. Under the conditions
of assay, the optimum activity occurs at pH 7.0 and at temperature of 35°C. Polyclonal antibodies raised in rabbits against
the purified GAPDH immunostained a single 36 kDa GAPDH band on crude extract protein preparations blotted onto nitrocellulose. 相似文献
5.
Purification and characterization of an ADP-dependent phosphofructokinase from Thermococcus zilligii 总被引:1,自引:0,他引:1
R. S. Ronimus Jurre Koning H. W. Morgan 《Extremophiles : life under extreme conditions》1999,3(2):121-129
The ADP-dependent phosphofructokinase (PFK) from Thermococcus zilligii has been purified 950 fold; it had a specific activity of 190 U mg−1. The enzyme required Mg2+ ions for optimal activity and was specific for ADP. The forward reaction kinetics were hyperbolic for both cosubstrates (pH
optimum of 6.4), and the apparent K
m values for ADP and fructose-6-phosphate were 0.6 mM (apparent V
max of 243 U mg−1) and 1.47 mM (apparent V
max of 197 U mg−1), respectively. Significantly, the enzyme is indicated to be nonallosteric but was slightly activated by some monovalent
cations including Na+ and K+. The protein had a subunit size of 42.2 kDa and an estimated native molecular weight of 66 kDa (gel filtration). Maximal
reaction rates for the reverse reaction were attained at pH 7.5–8.0, and the apparent K
m values for fructose-1,6-bisphosphate and AMP were 0.56 mM (apparent V
max of 2.9 U mg−1) and 12.5 mM, respectively. The biochemical characteristics of this unique ADP-dependent enzymatic activity are compared
to ATP and pyrophosphate-dependent phosphofructokinases.
Received: August 14, 1998 / Accepted: December 2, 1998 相似文献
6.
Jin Zhou Ju Chu Yong-Hong Wang Si-Liang Zhang Ying-Ping Zhuang Zhong-Yi Yuan 《World journal of microbiology & biotechnology》2008,24(6):789-796
An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH4)2SO4 fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed
the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular
weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric
point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5
and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature
stability was up to 45 °C. Metal ions, such as, Mn2+ and K+ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg2+ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu2+ and Ag2+) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K
m of 120 and 330 μM and V
max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively. 相似文献
7.
We have identified two types of invertases, one bound ionically and the other covalently to the particulate fraction in grains
of heat tolerant C 306 and heat susceptible WH 542 cultivars of wheat (Triticum aestivum L.). The cell walls contained a high level of invertase activity, of which 79.2–72.8% was extractable by 2 M NaCl and 14.9–21.1%
by 0.5% EDTA in C 306 and WH 542, respectively. The NaCl-released invertase constituted the predominant fraction. Using 5–100 mM
sucrose and pH range of 4.0–7.0, the apparent Michaelis constant (K
m, enzyme substrate affinity measure) of enzyme ranged from 5.73 to 16.06 mM for C 306 and from 6.08 to 19.86 mM for WH 542.
The V
max (maximum catalytic rate) values at these pH were higher in C 306 (0.63–11.04 μg sucrose hydrolysed min−1) than WH 542 (0.51–8.73 μg sucrose hydrolysed min−1). By employing photo-oxidation and by studying the effect of pH on K
m and V
max, the involvement of histidine and α-carboxyl groups at the active site of the enzyme was indicated. The two cultivars also
showed differential response in terms of thermodynamic properties of the enzyme i.e. energy of activation (E
a), enthalpy change (ΔH) and entropy change (ΔS). NaCl-released invertase showed differential response to metal ions in two cultivars suggesting their distinctive nature.
Mn2+, Cu2+, Hg2+, Mg2+, Zn2+ and Cd2+ were strong inhibitors in WH 542 as compared to C 306 while K+, Ca2+ were stimulators in both the cultivars. Overall the results suggest that genetic differences exist in wall bound invertase
properties of wheat grains as evident in its altered kinetic behaviour. 相似文献
8.
The first unique step in bacterial and plant methionine biosynthesis involves the acylation of the gamma-hydroxyl of homoserine. In Haemophilus influenzae, acylation is accomplished via an acetyl-CoA-dependent acetylation catalyzed by homoserine transacetylase. The activity of this enzyme regulates flux of homoserine into multiple biosynthetic pathways and, therefore, represents a critical control point for cell growth and viability. We have cloned homoserine transacetylase from H. influenzae and present the first detailed enzymatic study of this enzyme. Steady-state kinetic experiments demonstrate that the enzyme utilizes a ping-pong kinetic mechanism in which the acetyl group of acetyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine to form the final product, O-acetylhomoserine. The maximal velocity and V/K(homoserine) were independent of pH over the range of values tested, while V/K(acetyl)(-)(CoA) was dependent upon the ionization state of a single group exhibiting a pK value of 8.6, which was required to be protonated. Solvent kinetic isotope effect studies yielded inverse effects of 0.75 on V and 0.74 on V/K(CoA) on the reverse reaction and effects of 1.2 on V and 1.7 on V/K(homoserine) on the forward reaction. Direct evidence for the formation of an acetyl-enzyme intermediate was obtained using rapid-quench labeling studies. On the basis of these observations, we propose a chemical mechanism for this important member of the acyltransferase family and contrast its mechanism with that of homoserine transsuccinylase. 相似文献
9.
Jeyagowri Kiddinamoorthy Alfredo J. Anceno Gulelat D. Haki Sudip K. Rakshit 《World journal of microbiology & biotechnology》2008,24(5):605-612
Production of extracellular xylanase from Bacillus sp. GRE7 using a bench-top bioreactor and solid-state fermentation (SSF) was attempted. SSF using wheat bran as substrate
and submerged cultivation using oat-spelt xylan as substrate resulted in an enzyme productivity of 3,950 IU g−1 bran and 180 IU ml−1, respectively. The purified enzyme had an apparent molecular weight of 42 kDa and showed optimum activity at 70°C and pH
7. The enzyme was stable at 60–80°C at pH 7 and pH 5–11 at 37°C. Metal ions Mn2+ and Co2+ increased activity by twofold, while Cu2+ and Fe2+ reduced activity by fivefold as compared to the control. At 60°C and pH 6, the K
m for oat-spelt xylan was 2.23 mg ml−1 and V
max was 296.8 IU mg−1 protein. In the enzymatic prebleaching of eucalyptus Kraft pulp, the release of chromophores, formation of reducing sugars
and brightness was higher while the Kappa number was lower than the control with increased enzyme dosage at 30% reduction
of the original chlorine dioxide usage. The thermostability, alkali-tolerance, negligible presence of cellulolytic activity,
ability to improve brightness and capacity to reduce chlorine dioxide usage demonstrates the high potential of the enzyme
for application in the biobleaching of Kraft pulp. 相似文献
10.
Halvor Aarnes 《Planta》1978,140(2):185-192
Homoserine kinase was purified 700-fold by fractional ammonium sulfate precipitation, heat treatment, CM-Sephadex C-50 and DEAE-Sephadex A-50 ion exchange chromatography, and Sephadex G-100 gel filtration. The reaction products O-phosphohomoserine and ADP were the only compounds which caused considerable inhibition of homoserine kinase activity. Product inhibition studies showed non-competitive inhibition between ATP and O-phosphohomoserine and between homoserine and O-phosphohomoserine, and competitive inhibition between ATP and ADP. ADP showed non-competitive inhibition versus homoserine at suboptimal concentrations of ATP. At saturating concentrations of ATP no effect of ADP was observed. The homoserine kinase activity was negligible in the absence of K+ and the Km value for K+ was observed to be 4.3 mmol l–1. A non-competitive pattern was observed with respect to the substrates homoserine and ATP. Threonine synthase in the first green leaf of 6-day-old barley seedlings was partially purified 15-fold by ammonium sulfate fractionation and Sephadex G-100 gel chromatography. Threonine synthase was shown to require pyridoxal 5-phosphate as coenzyme for optimum activity and the enzyme was strongly activated by S-adenosyl-L-methionine. The optimum pH for threonine synthase activity was 7 to 8.Abbreviations PLP
Pyridoxal 5-phosphate
- SAM
S-adenosyl-L-methionine
- HSP
O-phosphohomoserine 相似文献
11.
Modrak-Wójcik A Kirilenko A Shugar D Kierdaszuk B 《European biophysics journal : EBJ》2008,37(2):153-164
Kinetics of the reactions of purine nucleoside phosphorylases (PNP) from E. coli (PNP-I, the product of the deoD gene) and human erythrocytes with their natural substrates guanosine (Guo), inosine (Ino), a substrate analogue N(7)-methylguanosine
(m7Guo), and orthophosphate (Pi, natural cosubstrate) and its thiophosphate analogue (SPi), found to be a weak cosubstrate, have been studied in the pH range 5–8. In this pH range Guo and Ino exist predominantly
in the neutral forms (pKa 9.2 and 8.8); m7Guo consists of an equilibrium mixture of the cationic and zwitterionic forms (pKa 7.0); and Pi and SPi exhibit equilibria between monoanionic and dianionic forms (pKa 6.7 and 5.4, respectively). The phosphorolysis of m7Guo (at saturated concentration) with both enzymes exhibits Michaelis kinetics with SPi, independently of pH. With Pi, the human enzyme shows Michaelis kinetics only at pH ∼5. However, in the pH range 5–8 for the bacterial enzyme, and 6–8
for the human enzyme, enzyme kinetics with Pi are best described by a model with high- and low-affinity states of the enzymes, denoted as enzyme-substrate complexes with
one or two active sites occupied by Pi, characterized by two sets of enzyme-substrate dissociation constants (apparent Michaelis constants, K
m1 and K
m2) and apparent maximal velocities (V
max1 and V
max2). Their values, obtained from non-linear least-squares fittings of the Adair equation, were typical for negative cooperativity
of both substrate binding (K
m1 < K
m2) and enzyme kinetics (V
max1/K
m1 > V
max2/K
m2). Comparison of the pH-dependence of the substrate properties of Pi versus SPi points to both monoanionic and dianionic forms of Pi as substrates, with a marked preference for the dianionic species in the pH range 5–8, where the population of the Pi dianion varies from 2 to 95%, reflected by enzyme efficiency three orders of magnitude higher at pH 8 than that at pH 5.
This is accompanied by an increase in negative cooperativity, characterized by a decrease in the Hill coefficient from n
H ∼1 to n
H ∼0.7 for Guo with the human enzyme, and to n
H ∼0.7 and 0.5 for m7Guo with the E. coli and human enzymes, respectively. Possible mechanisms of cooperativity are proposed. Attention is drawn to the substrate properties
of SPi in relation to its structure. 相似文献
12.
Park SY Kim JT Kang SG Woo JH Lee JH Choi HT Kim SJ 《Applied microbiology and biotechnology》2007,77(1):107-115
Vibrio sp. GMD509, a marine bacterium isolated from eggs of the sea hare, exhibited lipolytic activity on tributyrin (TBN) plate, and the gene representing lipolytic activity was cloned. As a result,
an open reading frame (ORF) consisting of 1,017 bp (338 aa) was found, and the deduced amino acid sequence of the ORF showed
low similarity (<20%) to α/β hydrolases such as dienelactone hydrolases and esterase/lipase with G–X1–S–X2–G sequence conserved. Phylogenetic analysis suggested that the protein belonged to a new family of esterase/lipase together
with various hypothetical proteins. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme (Vlip509) showed the best hydrolyzing activity toward p-nitrophenyl butyrate (C4) among various p-nitrophenyl esters (C2 to C18), and optimal activity of Vlip509 occurred at 30°C and pH 8.5, respectively. Kinetic parameters toward p-nitrophenyl butyrate were determined as K
m (307 μM), k
cat (5.72 s−1), and k
cat/K
m (18.61 s−1 mM−1). Furthermore, Vlip509 preferentially hydrolyzed the S-enantiomer of racemic ofloxacin ester. Despite its sequence homology to dienelactone hydrolase, Vlip509 showed no dienelactone
hydrolase activity. This study represents the identification of a novel lipolytic enzyme from marine environment. 相似文献
13.
Canakci S Kacagan M Inan K Belduz AO Saha BC 《Applied microbiology and biotechnology》2008,81(1):61-68
The gene, AbfAC26Sari, encoding an α-l-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari, was isolated, cloned, sequenced, and characterizated. On the basis of amino acid sequence similarities, this 57-kDa
enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. Characterization of the purified recombinant
α-l-arabinofuranosidase produced in Escherichia coli BL21 revealed that it is active at a broad pH range (pH 4.5 to 9.0) and at a broad temperature range (45–85°C) and it has
an optimum pH of 5.5 and an optimum temperature of 65°C. Kinetic experiment at 65°C with p-nitrophenyl α-l-arabinofuranoside as a substrate gave a V
max and K
m values of 1,019 U/mg and 0.139 mM, respectively. The enzyme had no apparent requirement of metal ions for activity, and its
activity was strongly inhibited by 1 mM Cu2+ and Hg2+. The recombinant arabinofuranosidase released l-arabinose from arabinan, arabinoxylan, oat spelt xylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose.
Endoarabinanase activity was not detected. These findings suggest that AbfAC26Sari is an exo-acting enzyme. 相似文献
14.
The starch-binding domain of Bacillus sp. strain TS-23 α-amylase was introduced into the C-terminal end of Bacillus kaustophilus leucine aminopeptidase (BkLAP) to generate a chimeric enzyme (BkLAPsbd) with raw-starch-binding activity. BkLAPsbd, with an apparent molecular mass of approximately 65 kDa, was overexpressed in Escherichia coli M15 cells and purified to homogeneity by nickel–chelate chromatography. Native PAGE and chromatographic analyses revealed that the purified fusion protein has a hexameric structure. The half-life for BkLAPsbd was 12 min at 70°C, while less than 20% of wild-type enzyme activity retained at the same heating condition. Compared with the wild-type enzyme, the 60% decrease in the catalytic efficiency of BkLAPsbd was due to a 91% increase in K
m value. Starch-binding assays showed that the K
d and B
max values for the fusion enzyme were 2.3 μM and 0.35 μmol/g, respectively. The adsorption of the crude BkLAPsbd onto raw starch was affected by starch concentration, pH, and temperature. The adsorbed enzyme could be eluted from the adsorbent by 2% soluble starch in 20 mM Tris–HCl buffer (pH 8.0). About 49% of BkLAPsbd in the crude extract was recovered through one adsorption–elution cycle with a purification of 11.4-fold. 相似文献
15.
Gulelat D. Haki Alfredo J. Anceno Sudip K. Rakshit 《World journal of microbiology & biotechnology》2008,24(11):2517-2524
Bacillus sp. GRE1 isolated from an Ethiopian hyperthermal spring produced raw-starch digesting, Ca2+-independent thermostable α-amylase. Enzyme production in shake flask experiments using optimum nutrient supplements and environmental
conditions was 2,360 U l−1. Gel filtration chromatography yielded a purification factor of 33.6-fold and a recovery of 46.5%. The apparent molecular
weight of the enzyme was 55 kDa as determined by SDS-PAGE. Presence or absence of Ca2+ produced similar temperature optima of 65–70°C. The optimum pH was in the range of 5.5–6.0. The enzyme maintained 50% of
its original activity after 45 min of incubation at 80°C and was stable at a pH range of 5.0–9.0. The V
max and K
m values for soluble starch were 42 mg reducing sugar min−1 and 4.98 mg starch ml−1, respectively. Strong inhibitors of enzyme activity included Cu2+, Zn2+ and Fe2+. The enzyme coding gene and the deduced protein translation revealed a characteristic but markedly atypical homology to Bacillus species α-amylase sequences. The enzyme hydrolyzed wheat, corn and tapioca starch granules efficiently below their gelatinization
temperatures. Rather than the higher oligosaccharides normally produced by Bacillus α-amylases operating at high temperatures, maltose was the major hydrolysis product with the present enzyme. 相似文献
16.
I. V. Melnick 《Neurophysiology》2011,42(4):239-243
Upon application of a long-lasting rectangular stimulus, neurons of the substantia gelatinosa (SG) display three main types of intrinsic firing behavior, tonic, adapting, and delayed onset. The electrical landmark of delayed-firing
neurons (DFNs), i.e., a significant delay before initiation of action potentials (APs), is believed to result from activation
of subthreshold A-type K+ current (KA). We checked out this hypothesis by comparing the voltage dependence of the firing delay with steady-state inactivation of
KA in spinal cord slices of 3- to 5-week-old rats. The delay strongly decreased with membrane depolarization and disappeared
at ~ –60 mV; herewith the discharge pattern was transformed to either a tonic or an adapting one. This correlated well with
inactivation of KA recorded in a whole-cell mode in low-Cl– intracellular solution; inactivation was nearly complete at –60 mV (voltage of half-maximum inactivation, V
1/2 ~ –74.5 mV). Unexpectedly, it was found that filling the cells with high-Cl– solution, to minimize the liquid junction potential, produced at least a 10 mV-difference between voltage dependences of
the firing delay and KA inactivation; the latter shifted toward negativity (V
1/2 ~ –88.3 mV). The results suggest that the KA and its inactivation properties determine the appearance and voltage dependence of the firing delay in SG neurons; the apparent influence of intracellular Cl– on inactivation properties needs further investigation. 相似文献
17.
Cloning,sequence analysis,and expression of a gene encoding <Emphasis Type="Italic">Chromobacterium</Emphasis> sp. DS-1 cholesterol oxidase 总被引:1,自引:1,他引:0
Noriyuki Doukyu Kanpei Shibata Hiroyasu Ogino Martin Sagermann 《Applied microbiology and biotechnology》2009,82(3):479-490
Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence
of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase.
The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid
residues. The amino acid sequence of the product showed significant similarity (53–62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an
active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1.
K
m and V
max values of the cholesterol oxidase were estimated from Lineweaver–Burk plots. The V
max/K
m ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral
analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures. 相似文献
18.
Enzyme access, kinetic behavior, and protein–protein interactions are critical for explaining reaction of the metabolites
contained within the myriad compartments of biological systems. To explore these relationships, the reaction kinetics of oil
bodies versus oil emulsions as substrates for lipolytic reactions were measured. The initial rate of hydrolysis for the oil
body system was comparatively very low due to a brief latency period. However, the complete activation of the lipase at the
interface resulted in an enzyme–membrane complex that was catalytically enhanced 3–15-fold over the emulsion system for substrate
concentrations in the measured range of approximately 1–5.5 mM. This disparity is explained by the availability of substrate
to the enzyme active site (defined as the availability parameter “A”) which varies between the two substrates by 40-fold. A simple hyperbolic kinetic mechanism is proposed with K
m replaced by the parameter, A, to account for this phenomenon, leading to a maximum rate of approximately 1450 IU/mg protein. The interaction is verified
through separation of the enzyme–membrane complex which shows nearly double the activity towards an emulsified soybean oil
substrate (activity ratio of 5:3) when compared to the native enzyme. 相似文献
19.
An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The Km and Vmax values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg−1 protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity
was strongly inhibited by Cu2+ and Hg2+ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose. 相似文献
20.
Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) catalyses the reduction of the monodehydroascorbate (MDHA) radical to ascorbate,
using NADH or NADPH as an electron donor, and is believed to be involved in maintaining the reactive oxygen scavenging capability
of plant cells. This key enzyme in the ascorbate-glutathione cycle has been studied here in the moss Physcomitrella patens, which is tolerant to a range of abiotic stresses and is increasingly used as a model plant. In the present study, three
cDNAs encoding different MDHAR isoforms of 47 kDa were identified in P. patens, and found to exhibit enzymic characteristics similar to MDHARs in vascular plants despite low-sequence identity and a distant
evolutionary relationship between the species. The three cDNAs for the P. patens MDHAR enzymes were expressed in Escherichia coli and the active enzymes were purified and characterized. Each recombinant protein displayed an absorbance spectrum typical
of flavoenzymes and contained a single non-covalently bound FAD coenzyme molecule. The K
m and k
cat values for the heterologously expressed PpMDHAR enzymes ranged from 8 to 18 μM and 120–130 s−1, respectively, using NADH as the electron donor. The K
m values were at least an order of magnitude higher for NADPH. The K
m values for the MDHA radical were ∼0.5–1.0 μM for each of the purified enzymes, and further kinetic analyses indicated that
PpMDHARs follow a ‘ping–pong’ kinetic mechanism. In contrast to previously published data, site-directed mutagenesis indicated
that the conserved cysteine residue is not directly involved in the reduction of MDHA. 相似文献