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1.
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
2.
De Felice M Medagli B Esposito L De Falco M Pucci B Rossi M Grùz P Nohmi T Pisani FM 《Extremophiles : life under extreme conditions》2007,11(2):277-282
The hyper-thermophilic archaeon Sulfolobus solfataricus possesses two functional DNA polymerases belonging to the B-family (Sso DNA pol B1) and to the Y-family (Sso DNA pol Y1).
Sso DNA pol B1 recognizes the presence of uracil and hypoxanthine in the template strand and stalls synthesis 3–4 bases upstream
of this lesion (“read-ahead” function). On the other hand, Sso DNA pol Y1 is able to synthesize across these and other lesions
on the template strand. Herein we report evidence that Sso DNA pol B1 physically interacts with DNA pol Y1 by surface plasmon
resonance measurements and immuno-precipitation experiments. The region of DNA pol B1 responsible for this interaction has
been mapped in the central portion of the polypeptide chain (from the amino acid residue 482 to 617), which includes an extended
protease hyper-sensitive linker between the N- and C-terminal modules (amino acid residues Asn482-Ala497) and the α-helices
forming the “fingers” sub-domain (α-helices R, R′ and S). These results have important implications for understanding the polymerase-switching mechanism on the damaged template
strand during genome replication in S. solfataricus. 相似文献
3.
Ha-Young Park Hye-Jung Kim Jung-Kul Lee Doman Kim Deok-Kun Oh 《World journal of microbiology & biotechnology》2008,24(8):1553-1558
A recombinant β-galactosidase from Sulfolobus solfataricus produced galactooligosaccharides (GOS) from lactose by transgalactosylation. The enzyme activity for GOS production was maximal
at pH 6.0 and 85°C. The half-lives of the recombinant β-galactosidase at 70, 75, 80, 85, and 90°C were 700, 111, 72, 43, and
2.4 h, respectively, and its deactivation energy was 213 kJ mol−1. The optimal amount of enzyme for effective GOS production was 3.6 U of enzyme ml−1. GOS production increased with increasing lactose concentration, whereas the yield of GOS from lactose was almost constant.
The rates of hydrolysis and transgalactosylation reactions increased with increasing temperature but the final concentration
of GOS was maximal at 80°C. Under the conditions of pH 6.0, 80°C, 600 g lactose l−1, and 3.6 U enzyme ml−1, 315 g GOS l−1 were obtained for 56 h with a yield of 52.5% (w/w). The β-galactosidase from S. solfataricus produced GOS with the highest concentration and yield among thermostable β-galactosidases reported to date. 相似文献
4.
Jayati Roy Choudhury Lu Rao Ulrich Bierbach 《Journal of biological inorganic chemistry》2011,16(3):373-380
A restriction enzyme cleavage inhibition assay was designed to determine the rates of DNA platination by four non-cross-linking
platinum–acridine agents represented by the formula [Pt(am2)LCl](NO3)2, where am is a diamine nonleaving group and L is an acridine derived from the intercalator 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea
(ACRAMTU). The formation of monofunctional adducts in the target sequence 5′-CGA was studied in a 40-base-pair probe containing
the EcoRI restriction site GAATTC. The time dependence of endonuclease inhibition was quantitatively analyzed by polyacrylamide gel
electrophoresis. The formation of monoadducts is approximately 3 times faster with double-stranded DNA than with simple nucleic
acid fragments. Compound 1 (am2 is ethane-1,2-diamine, L is ACRAMTU) reacts with a first-order rate constant of k
obs = 1.4 ± 0.37 × 10−4 s−1 (t
1/2 = 83 ± 22 min). Replacement of the thiourea group in ACRAMTU with an amidine group (compound 2) accelerates the rate by fourfold (k
obs = 5.7 ± 0.58 × 10−4 s−1, t
1/2 = 21 ± 2 min), and introduction of a propane-1,3-diamine nonleaving group results in a 1.5-fold enhancement in reactivity
(compound 3, k
obs = 2.1 ± 0.40 × 10−4 s−1, t
1/2 = 55 ± 10 min) compared with the prototype. Derivative 4, containing a 4,9-disubstituted acridine threading intercalator, was the least reactive compound in the series (k
obs = 1.1 ± 0.40 × 10−4 s−1, t
1/2 = 104 ± 38 min). The data suggest a correlation may exist between the binding rates and the biological activity of the compounds.
Potential pharmacological advantages of rapid formation of cytotoxic monofunctional adducts over the common purine–purine
cross-links are discussed. 相似文献
5.
Copper (II) complex of formulation [Cu–Phen–Tyr](H2O)](ClO4) (Phen = 1,10-phenanthroline, l-Tyr = l-tyrosine), has been prepared, and their induced DNA oxidative cleavage activity studied. The complex binds to DNA by intercalation,
as deduced from the absorption and fluorescence spectral data. Scatchard plots constructed from the absorption titration data
gave binding constant 2.44 × 104 M−1 of base pairs. Extensive hypochromism, broadening, and red shifts in the absorption spectra were observed. Upon binding to
DNA, the fluorescence from the DNA–ethidium bromide system was efficiently quenched by the copper (II) complex. Stern–Volmer
quenching constant 0.61 × 103 M−1 obtained from the linear quenching plots. [Cu–Phen–Tyr] complex efficiently cleave the supercoiled DNA to its nicked circular
form with gallic acid as biological reductant at appropriate complex concentration. The gallic acid as reductant could observably
improve copper (II) complex to DNA damage. The pseudo-Michaelis–Menten kinetic parameters (k
cat, K
M) were calculated to be 1.32 h−1 and 5.46 × 10−5 M for [Cu–Phen–Tyr] complex. Mechanistic studies reveal the involvement of superoxide anions and hydroxyl radical (HO·) as the reactive species under an aerobic medium. 相似文献
6.
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
7.
A thermostable esterase from the hyperthemophilic archaeonSulfolobus solfataricus was partially purified 590-fold with 16.2% recovery. The partially purified esterase had a specific activity of 29.5μmol
min−1 mg−1 when the enzyme activity was determined usingp-nitrophenyl butyrate as a substrate. The apparent molecular weight was about 100 kDa, while the optimum temperature and pH
for esterase were 75°C and 8.0, respectively. The enzyme showed high thermal stability and solvent tolerance in comparison
to its mesophilic counterpart. The enzyme also showed chiral resolution activity for (S)-ibuprofen, indicating thatS. solfataricus esterase can be used for the production of commercially important chiral drugs. 相似文献
8.
Pratibha Dheeran Sachin Kumar Yogesh K. Jaiswal Dilip K. Adhikari 《Applied microbiology and biotechnology》2010,86(6):1857-1866
A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism
was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively,
for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum
enzyme production was in exponential phase with activity 135 U ml−1 at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml−1 at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg−1 protein. The molecular mass of the purified enzyme was 97 KDa. The values of K
m
and V
max were 36 mg ml−1 and 222 μmol mg−1 protein min−1, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The
enzyme was stimulated with Mn2+, whereas it was inhibited by Hg2+, Cu2+, Zn2+, Mg2+, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance
against protease. 相似文献
9.
Kanakis CD Tarantilis PA Polissiou MG Diamantoglou S Tajmir-Riahi HA 《Cell biochemistry and biophysics》2007,49(1):29-36
In this report we are examining how the antioxidant flavonoids can prevent DNA damage and what mechanism of action is involved
in the process. Flavonoids are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these
natural products are implicated in their mechanism of actions. We study the interactions of quercetin (que), kaempferol (kae),
and delphinidin (del) with DNA and transfer RNA in aqueous solution at physiological conditions, using constant DNA or RNA
concentration 6.25 mmol (phosphate) and various pigment/polynucleotide(phosphate) ratios of 1/65 to 1 (DNA) and 1/48 to 1/8
(tRNA). The structural analysis showed quercetin, kaempferol, and delphinidin intercalate DNA and RNA duplexes with minor
external binding to the major or minor groove and the backbone phosphate group with overall binding constants for DNA adducts
K
que = 7.25 (±0.65) × 104 M−1, K
kae = 3.60 (±0.33) × 104 M−1, and K
del = 1.66 (±0.25) × 104 M−1 and for tRNA adducts K
que = 4.80 (±0.50) × 104 M−1, K
kae = 4.65 (±0.45) × 104 M−1, and K
del = 9.47 (±0.70) × 104 M−1. The stability of adduct formation is in the order of del>que>kae for tRNA and que>kae>del for DNA. Low flavonoid concentration
induces helical stabilization, whereas high pigment content causes helix opening. A partial B to A-DNA transition occurs at
high drug concentration, while tRNA remains in A-family structure. The antioxidant activity of flavonoids changes in order
delphinidin>quercetin>kaempferol. The results show intercalated flavonoids can make them strong antioxidants to protect DNA
from harmful free radical reactions. 相似文献
10.
Xin-chun Mo Chun-lan Chen Hao Pang Yi Feng Jia-xun Feng 《Applied microbiology and biotechnology》2010,87(6):2137-2146
A metagenomic library containing ca. 3.06 × 108 bp insert DNA was constructed from a rice straw degrading enrichment culture. A xylanase gene, umxyn10A, was cloned by screening the library for xylanase activity. The encoded enzyme Umxyn10A showed 58% identity and 73% similarity
with a xylanase from Thermobifida fusca YX. Sequence analyses showed that Umxyn10A contained a glycosyl hydrolase family 10 catalytic domain. The gene was expressed
in Escherichia coli, and the recombinant enzyme was purified and characterized biochemically. Recombinant Umxyn10A was highly active toward xylan.
However, the purified enzyme could slightly hydrolyze β-1,3/4-glucan and β-1,3/6-glucan. Umxyn10A displayed maximal activity
toward oat spelt xylan at a high temperature (75°C) and weak acidity (pH 6.5). The K
m
and V
max of Umxyn10A toward oat spelt xylan were 3.2 mg ml−1 and 0.22 mmol min−1 mg−1 and were 2.7 mg ml−1 and 1.0 mmol min−1 mg−1 against birchwood xylan, respectively. Metal ions did not appear to be required for the catalytic activity of this enzyme.
The enzyme Umxyn10A could efficiently hydrolyze birchwood xylan to release xylobiose as the major product and a negligible
amount of xylose. The xylanase identified in this work may have potential application in producing xylobiose from xylan. 相似文献
11.
Toxic at low concentrations, phenol is one of the most common organic pollutants in air and water. In this work, phenol biodegradation
was studied in extreme conditions (80°C, pH = 3.2) in a 2.7 l bioreactor with the thermoacidophilic archaeon Sulfolobus solfataricus 98/2. The strain was first acclimatized to phenol on a mixture of glucose (2000 mg l−1) and phenol (94 mg l−1) at a constant dissolved oxygen concentration of 1.5 mg l−1. After a short lag-phase, only glucose was consumed. Phenol degradation then began while glucose was still present in the
reactor. When glucose was exhausted, phenol was used for respiration and then for biomass build-up. After several batch runs
(phenol < 365 mg l−1), specific growth rate (μX) was 0.034 ± 0.001 h−1, specific phenol degradation rate (qP) was 57.5 ± 2 mg g−1 h−1, biomass yield (YX/P) was 52.2 ± 1.1 g mol−1, and oxygen yield factor
( \textY\textX/\textO 2 ) \left( {{\text{Y}}_{{{\text{X}}/{\text{O}}_{ 2} }} } \right) was 9.2 ± 0.2 g mol−1. A carbon recovery close to 100% suggested that phenol was exclusively transformed into biomass (35%) and CO2 (65%). Molar phenol oxidation constant
( \textY\textO 2 /\textP ) \left( {{\text{Y}}_{{{\text{O}}_{ 2} /{\text{P}}}} } \right) was calculated from stoichiometry of phenol oxidation and introducing experimental biomass and CO2 conversion yields on phenol, leading to values varying between 4.78 and 5.22 mol mol−1. Respiratory quotient was about 0.84 mol mol−1, very close to theoretical value (0.87 mol mol−1). Carbon dioxide production, oxygen demand and redox potential, monitored on-line, were good indicators of growth, substrate
consumption and exhaustion, and can therefore be usefully employed for industrial phenol bioremediation in extreme environments. 相似文献
12.
Whole-genome sequence analysis of Bacillus halodurans ATCC BAA-125 revealed an isomerase gene (rhaA) encoding an l-rhamnose isomerase (l-RhI). The identified l
-RhI gene was cloned from B. halodurans and over-expressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,257 bp capable of encoding a polypeptide of 418 amino acid residues
with a molecular mass of 48,178 Da. The molecular mass of the purified enzyme was estimated to be ∼48 kDa by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and 121 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer.
The enzyme had an optimal pH and temperature of 7 and 70°C, respectively, with a k
cat of 8,971 min−1 and a k
cat/K
m of 17 min−1 mM−1 for l-rhamnose. Although l-RhIs have been characterized from several other sources, B. halodurans
l-RhI is distinguished from other l-RhIs by its high temperature optimum (70°C) with high thermal stability of showing 100% activity for 10 h at 60°C. The half-life
of the enzyme was more than 900 min and ∼25 min at 60°C and 70°C, respectively, making B. halodurans
l-RhI a good choice for industrial applications. This work describes one of the most thermostable l-RhI characterized thus far. 相似文献
13.
Fusarium moniliforme var. subglutinans was selected from among 100 strains of fungi for producing ginsenoside F1 from ginsenoside Rg1. The enzyme responsible was purified as a single 85 kDa band with a specific activity of 136 U mg−1. It hydrolysed glucose-linked ginsenosides Rb1, Rd and Rg1 but not for other monosaccharide-linked ginsenosides, Rb2, Rc, R1, and Re. Under the optimum conditions of pH 6.0, 50°C, 30 U l−1 of enzyme, and 5 mg Rg1 ml−1, 4 mg F1 ml−1 was produced after 4 h, with a molar yield of 100% and a productivity of 1 g l−1 h−1. This represents the highest productivity and conversion yield of F1 yet reported. 相似文献
14.
T. Aravinda H. S. Bhojya Naik H. R. Prakash Naik 《International journal of peptide research and therapeutics》2009,15(4):273-279
Abstract
We present, simple approach for the accession of 1,2,3-triazole fused quinoline peptide analogues from 3-(azidomethyl)-2-chloroquinoline in a three-step mechanistic pathway. The UV–Visible absorbance plot shows dynamic interaction of parent triazole derivative with CT DNA as efficient DNA intercalator (K b = 4.6 × 10−4 M−1). Finally, the efficient DNA damage was observed on photo-irradiation at 360 nm in the presence of 2-(9H-Fluoren-9-ylmethoxycarbonylamino)-propionic acid 1-(2-chloro-quinolin-3-ylmethyl)-1H-[1,2,3]triazole-4-ylmethyl ester (6a). 相似文献15.
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
16.
17.
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
18.
Genomic DNA was extracted from wild-type Escherichia coli strains ATCC 35321 and 8677. The lac Z gene was amplified and used as a template for in vitro synthesis of β-galactosidase. In addition the enzyme was synthesized
in vitro with a C-terminal His6 tag. The enzyme expression was also induced in these strains using isopropyl-β-D-galactoside. Single enzyme molecule assays were performed using a capillary electrophoresis-based protocol on both the in
vitro and in vivo synthesized enzyme. In vivo produced enzyme from strains 35321 and 8677 showed average combined turnover
numbers for the 4 active sites of the individual enzyme molecules of 53,400 ± 18,400 (N = 139) and 34,300 ± 17,800 min−1 (N = 181) respectively. Average combined turnover numbers of 35,800 ± 20,900 (N = 302) and 31,700 ± 17,700 min−1 (N = 315) were obtained respectively for the in vitro synthesized enzyme from strain 35321 with the absence and presence of
a C-terminal His6 tag. For strain 8677, the average combined turnover numbers were 29,000 ± 17,900 (N = 288) and 25,200 ± 12,600 min−1 (N = 240) respectively for the absence and presence of a C-terminal His6 tag. The average combined turnover numbers of the enzyme from both strains synthesized in vivo and in vitro and with the
presence and absence of a His6 tag were found to differ significantly. This indicates that the in vivo and in vitro produced enzymes are not identical and
the presence of a C-terminal His6 tag alters the activity of β-galactosidase. 相似文献
19.
20.
Asiya Nazir Rohit Soni H. S. Saini R. K. Manhas B. S. Chadha 《World journal of microbiology & biotechnology》2009,25(7):1189-1197
An endoglucanase (1, 4-β-d glucan glucanohydrolase, EC 3.2.1.4) which was catalytically more active and exhibited higher affinity towards barley β-glucan,
xyloglucan and lichenin as compared to carboxymethylcellulose (CMC) was purified from Aspergillus terreus strain AN1 following ion-exchange and hydrophobic interaction chromatography and gel filtration. The purified enzyme (40-fold) that
apparently lacked a cellulose-binding domain showed a specific activity of 60 μmol mg−1 protein−1 against CMC. The purified enzyme had a molecular weight of 78 and 80 KDa as indicated by sodium dodecyl sulphate–polyacrylamide
gel electrophoresis and gel filtration, respectively, and a pI of 3.5. The enzyme was optimally active at temperature 60°C
and pH 4.0, and was stable over a broad range of pH (3.0–5.0) at 50°C. The endoglucanase activity was positively modulated
in the presence of Cu2+, Mg2+, Ca2+, Na+, DTT and mercaptoethanol. Endoglucanase exhibited maximal turn over number (K
cat) and catalytic efficiency (K
cat/km) of 19.11 × 105 min−1 and 29.7 × 105 mM−1 min−1 against barley β-glucan as substrate, respectively. Hydrolysis of CMC and barley β-glucan liberated cellobiose, cellotriose,
cellotetraose and detectable amount of glucose. The hydrolysis of xyloglucan, however, apparently yielded positional isomers
of cellobiose, cellotriose and cellotetraose as well as larger oligosaccharides. 相似文献