共查询到20条相似文献,搜索用时 31 毫秒
1.
We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino
acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble α-D-glucan from sucrose (K
M 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted
in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0–6.2. The enzyme was quickly inactivated
at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal inactivation between
room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-d-glucan revealed that it had approximately equal amounts of α(1 → 3)-linked and α(1 → 6)-linked d-glucopyranosyl units and a low degree of branching. 相似文献
2.
Tony Marcio da Silva Alexandre Maller André Ricardo de Lima Damásio Michele Michelin Richard John Ward Izaura Yoshico Hirata João Atilio Jorge Héctor Francisco Terenzi Maria Lourdes T. M. de Polizeli 《Journal of industrial microbiology & biotechnology》2009,36(12):1439-1446
A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40°C. The enzyme was purified by DEAE-Fractogel
and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a
molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400
gel filtration, respectively. The pH optimum was 5.0–5.5, and the enzyme remained stable for at least 2 h in the pH range
of 4.0–9.5. The temperature optimum was 65°C and retained 100% activity after 240 min at 60°C. The glucoamylase remained completely
active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed,
confirming that the enzyme was a glucoamylase (1,4-alpha-d-glucan glucohydrolase). The K
m was calculated as 0.32 mg ml−1. Circular dichroism spectroscopy estimated a secondary structure content of 33% α-helix, 17% β-sheet and 50% random structure,
which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi,
A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications. 相似文献
3.
I-Son Ng Chen-Wei Li Yi-Fang Yeh Po Ting Chen Jiun-Ly Chir Chin-Hua Ma Su-May Yu Tuan-hua David Ho Chii-Gong Tong 《Extremophiles : life under extreme conditions》2009,13(3):425-435
A thermophilic Geobacillus bacterium secreting high activity of endo-glucanase (EC 3.2.1.4) was isolated from rice straw compost supplemented with pig
manure. A full-length gene of 1,104 bp, celA, encoding this glycosyl hydrolase family 5 endo-glucanase of 368 amino acids was isolated. No related gene from Geobacillus has been reported previously. The recombinant CelA expressed in Escherichia coli had an optimal activity at 65°C and pH 5.0, and it exhibited tenfold greater specific activity than the commercially available
Trichoderma reesei endo-glucanase. CelA displayed activity over a broad temperature range from 45 to 75°C and was a thermostable enzyme with
90% activity retained after heating at 65°C for 6 h. Interestingly, CelA activity could be enhanced by 100% in the presence
of 2 mM MnSO4. CelA had high specific activity over β-d-glucan from barley and Lichenan, making it a potentially useful enzyme in biofuel and food industries. 相似文献
4.
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
5.
J. Xu N. Takakuwa M. Nogawa H. Okada Y. Morikawa 《Applied microbiology and biotechnology》1998,49(6):718-724
A third xylanase (Xyn III) from Trichoderma reesei PC-3–7 was purified to electrophoretic homogeneity by gel filtration and ion-exchange chromatographies. The enzyme had a
molecular mass of 32 kDa, and its isoelectric point was 9.1. The pH optimum of Xyn III was 6.0, similar to that of Xyn II,
another basic xylanase of T. reesei. The purified Xyn III showed high activity with birchwood xylan but no activity with cellulose and aryl glycoside. The hydrolysis
of birchwood xylan by Xyn III produced mainly xylobiose, xylotriose and other xylooligosaccharides. The amino acid sequences
of the N-terminus and internal peptides of Xyn III exhibited high homology with the family F xylanases, showing that they
were distinct from those of Xyn I and Xyn II of T. reesei, which belong to family G. These results reveal that Xyn III is a new specific endoxylanase, differing from Xyn I and Xyn
II in T. reesei. It is noteworthy that this novel xylanase was induced only by cellulosic substrates and l-sorbose but not by xylan and its derivarives. Furthermore, T. reesei PC-3-7 produced Xyn III in quantity when grown on Avicel or lactose as a carbon source, while T. reesei QM9414 produced little or no Xyn III.
Received: 7 November 1997 / Received last revision: 2 February 1988 / Accepted: 23 February 1998 相似文献
6.
A putative N-acyl-d-glucosamine 2-epimerase from Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was identified as a cellobiose 2-epimerase by the analysis of the activity for substrates, acid-hydrolyzed
products, and amino acid sequence. The cellobiose 2-epimerase was purified with a specific activity of 35 nmol min–1 mg–1 for d-glucose with a 47-kDa monomer. The epimerization activity for d-glucose was maximal at pH 7.5 and 75°C. The half-lives of the enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 142, 71, 35,
18, and 4.6 h, respectively. The enzyme catalyzed the epimerization reactions of the aldoses harboring hydroxyl groups oriented
in the right-hand configuration at the C2 position and the left-hand configuration at the C3 position, such as d-glucose, d-xylose, l-altrose, l-idose, and l-arabinose, to their C2 epimers, such as d-mannose, d-lyxose, l-allose, l-gulose, and l-ribose, respectively. The enzyme catalyzed also the isomerization reactions. The enzyme exhibited the highest activity for
mannose among monosaccharides. Thus, mannose at 75 g l–1 and fructose at 47.5 g l–1 were produced from 500 g l–1 glucose at pH 7.5 and 75°C over 3 h by the enzyme. 相似文献
7.
The codon modified neutral endo-β-1,4-glucanase gene celEn, originating from the anaerobic fungus Orpinomyces sp. strain PC-2, was inserted between the strong promoter Pcel7A and the terminator Tcel7A from Trichoderma reesei. The resulting expression cassette was ligated to the pCAMBIA1300 Agrobacterium binary vector to construct pCB-hE that also contains a hygromycin B resistance marker. pCB-hE was introduced into T. reesei ZU-02 through an Agrobacterium tumefaciens–mediated transformation procedure that has been modified with an improved transformation efficiency of 12,500 transformants
per 107 conidia. Stable integration of the celEn gene into the chromosomal DNA of T. reesei ZU-02 was confirmed by PCR. After 48 h fermentation in shaking flasks, the endo-β-1,4-glucanase activities increased to 55–70 IU ml−1 in transgenic strains, which were about 6–7 times higher than that of the original ZU-02 strain (9.5 IU ml−1). When the avicel was added in fermentation medium, the endo-β-1,4-glucanase activity in the transgenic strains could be
further increased to 193.6 IU ml−1 after 84 h fermentation. Transgenic T. reesei strains with high neutral endo-β-1,4-glucanase activity will be particularly suitable for certain applications in textile
industry. The improved procedures for overproduction and secretion of heterologous proteins in transgenic T. reesei can also be used to generate similar recombinant proteins for research or industrial purposes. 相似文献
8.
Amore A Amoresano A Birolo L Henrissat B Leo G Palmese A Faraco V 《Applied microbiology and biotechnology》2012,94(4):995-1006
An α-l-arabinofuranosidase produced by Pleurotus ostreatus (PoAbf) during solid state fermentation on tomato pomace was identified and the corresponding gene and cDNA were cloned and
sequenced. Molecular analysis showed that the poabf gene carries 26 exons interrupted by 25 introns and has an open reading frame encoding a protein of 646 amino acid residues,
including a signal peptide of 20 amino acid residues. The amino acid sequence similar to the other α-l-arabinofuranosidases indicated that the enzyme encoded by poabf can be classified as a family 51 glycoside hydrolase. Heterologous recombinant expression of PoAbf was carried out in the
yeasts Pichia pastoris and Kluyveromyces lactis achieving the highest production level of the secreted enzyme (180 mg L−1) in the former host. rPoAbf produced in P. pastoris was purified and characterized. It is a glycosylated monomer with a molecular weight of 81,500 Da in denaturing conditions.
Mass spectral analyses led to the localization of a single O-glycosylation site at the level of Ser160. The enzyme is highly specific for α-l-arabinofuranosyl linkages and when assayed with p-nitrophenyl α-l-arabinofuranoside it follows Michaelis–Menten kinetics with a K
M of 0.64 mM and a k
cat of 3,010 min−1. The optimum pH is 5 and the optimal temperature 40°C. It is worth noting that the enzyme shows a very high stability in
a broad range of pH. The more durable activity showed by rPoAbf in comparison to the other α-l-arabinofuranosidases enhances its potential for biotechnological applications and increases interest in elucidating the molecular
bases of its peculiar properties. 相似文献
9.
Verónica Beatriz Rajal Alicia Graciela Cid Guillermo Ellenrieder Carlos Mario Cuevas 《World journal of microbiology & biotechnology》2009,25(6):1025-1033
Penicillium ulaiense is a post-harvest pathogenic fungus that attacks citrus fruits. The objective of this work was to study this microorganism
as an α-l-rhamnosidase producer and to characterize it from P. ulaiense. The enzyme under study is used for different applications in food and beverage industries. α-l-Rhamnosidase was produced in a stirred-batch reactor using rhamnose as the main carbon source. The kinetic parameters for
the growth of the fungi and for the enzyme production were calculated from the experimental values. A method for partial purification,
including (NH4)2SO4 precipitation, incubation at pH 12 and DEAE-sepharose chromatography yielded an enzyme with very low β-glucosidase activity.
The pH and temperature optima were 5.0 and 60°C, respectively. The Michaelis–Menten constants for the hydrolysis of p-nitrophenyl-α-l-rhamnoside were V
max = 26 ± 4 IU ml−1 and K
m
= 11 ± 2 mM. The enzyme showed good thermostability up to 60°C and good operational stability in white wine. Co2+ affected positively the activity; EDTA, Mn2+, Mg2+, dithiotreitol and Cu2+ reduced the activity by different amounts, and Hg2+ completely inhibited the enzyme. The enzyme showed more activity on p-nitrophenyl-α-l-rhamnoside than on naringin. According to these results, this enzyme has potential for use in the food and pharmacy industries
since P. ulaiense does not produce mycotoxins. 相似文献
10.
Kiril Tishinov Nikolina Stambolieva Svetla Petrova Boris Galunsky Peter Nedkov 《Acta Physiologiae Plantarum》2009,31(1):199-205
The sunflower seed (Helianthus annuus L.) major peptidase was purified to molecular homogeneity. It is an 80 kDa enzyme with pI of 4.6 and optimal activity at
pH 7.5–8.0 and 45–50°C. It is a thiol-dependent aminopeptidase hydrolyzing peptides in a step-by-step manner as cleaving after
the N-terminal amino acid residue of the substrate. It requires substrate acyl parts with a free amino group in either α-
or β-position and l-configuration of the adjacent carbon atom. The enzyme prefers amino acid residues with bulky hydrophobic side chains at P1-position and its catalytic efficacy is affected by the structure of both P1 and P1′ parts of the substrate. 相似文献
11.
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
12.
Mohamed Guerfali Moncef Chaabouni Ali Gargouri Hafedh Belghith 《Applied microbiology and biotechnology》2010,85(5):1361-1372
This study is an application of an experimental design methodology for the optimization of the culture conditions of α-l-arabinofuranosidase production by Talaromyces thermophilus. Wheat bran and yeast extract were first selected as the best carbon and nitrogen sources, respectively, for enzyme production.
A Plackett–Burman design was then used to evaluate the effects of eight variables. Statistical analyses showed that while
pH had a negative effect on α-l-arabinofuranosidase production, wheat bran and MgSO4 had a significantly positive effect. The values of the latter three parameters were further optimised using a central composite
design and a response surface methodology. The experimental results were fitted to a second-order polynomial model that yielded
a determination coefficient of R
2 = 0.91. The statistical output showed that the linear and quadric terms of the three variables had significant effects. Using
optimal conditions, the experimental value of α-l-arabinofuranosidase activity produced was very close to the model-predicted value. The optimal temperature and pH of enzyme
activity were 55 °C and 7.0, respectively. This enzyme was very stable over a considerable pH range from 4 to 9. The crude
enzyme of T. thermophilus rich in α-l-arabinofuranosidase was also used for saccharification of lignocellulosic materials and arabinose production. 相似文献
13.
Kazuyoshi Ohta Hidenori Tanaka Daisuke Yamakawa Hironori Hamasuna Hirohisa Fujimoto 《Journal of industrial microbiology & biotechnology》2011,38(8):967-973
An extracellular xylanase XynI of glycoside hydrolase family 11 from the dimorphic fungus Aureobasidium pullulans ATCC 20524 possesses an N-terminal extension of 34 amino acids (Ohta et al., J. Biosci. Bioeng. 92:262–270, 2001). The N-terminal
extension includes three sites (Ala-X-Ala-X-Ala-X-Ala) that are potentially cleavable by signal peptidase I of Escherichia
coli. The A. pullulans
xynI signal sequence was fused in frame to the mature protein region of the equivalent xylanase gene xynA from the filamentous fungus Penicillium citrinum. The gene fusion xynI::A was inserted into the plasmid pET-26b(+) to yield pEXP401. An E. coli BL21(DE3) transformant harboring the pEXP401 exhibited xylanase activity (per ml of the culture) of 16.8 U in the fraction
of culture supernatant as well as 4.29 U in the fraction of cell-free extract after 12 h of growth with isopropyl-β-d-thiogalactopyranoside at 30°C. N-terminal amino acid sequence analysis of the secreted recombinant proteins revealed cleavage
at four distinct sites within the N-terminal extension of XynI, two of which conformed to the Ala-X-Ala motif prior to the
cleavage site. The XynA proteins secreted into the culture medium showed high specific activities from 506 to 651 U/mg, which
were twofold higher than that of the native enzyme. 相似文献
14.
Subramanian Mohan Raj Chelladurai Rathnasingh Woo-Chel Jung Sunghoon Park 《Applied microbiology and biotechnology》2009,84(4):649-657
The stability and specific activity of endo-β-1,4-glucanase III from Trichoderma reesei QM9414 was enhanced, and the expression efficiency of its encoding gene, egl3, was optimized by directed evolution using error-prone PCR and activity screening in Escherichia coli RosettaBlue (DE3) pLacI as a host. Relationship between increase in yield of active enzyme in the clones and improvement
in its stability was observed among the mutants obtained in the present study. The clone harboring the best mutant 2R4 (G41E/T110P/K173M/Y195F/P201S/N218I)
selected in via second-round mutagenesis after optimal recombinating of first-round mutations produced 130-fold higher amount
of mutant enzyme than the transformant with wild-type EG III. Mutant 2R4 produced by the clone showed broad pH stability (4.4–8.8)
and thermotolerance (entirely active at 55°C for 30 min) compared with those of the wild-type EG III (pH stability, 4.4–5.2;
thermostability, inactive at 55°C for 30 min). k
cat of 2R4 against carboxymethyl-cellulose was about 1.4-fold higher than that of the wild type, though the K
m became twice of that of the wild type. 相似文献
15.
Jiro Arima Masanori Chiba Tsuyoshi Ichiyanagi Yukinori Yabuta Nobuhiro Mori Tadanori Aimi 《Applied microbiology and biotechnology》2010,87(5):1791-1801
An aminopeptidase that has peptide bond formation activity was identified in the cell-free extract of carpophore of Pleurotus eryngii. The enzyme, redesignated as eryngase, was purified for homogeneity and characterized. Eryngase had a molecular mass of approximately
79 kDa. It showed somewhat high stability with respect to temperature and pH; it was inhibited by iodoacetate. Among hydrolytic
activities toward aminoacyl-p-nitroanilides (aminoacyl-pNAs), eryngase mainly hydrolyzed hydrophobic l-aminoacyl-pNAs and exhibited little activity toward d-Ala-pNA and d-Leu-pNA. In terms of peptide bond formation activity, eryngase used various aminoacyl derivatives as acyl donors and acceptors.
The products were all dipeptidyl derivatives. Investigation of time dependence on peptide synthesis revealed that some peptides
that are not recognized as substrates for hydrolytic activity of eryngase could become good targets for synthesis. Furthermore,
eryngase has produced opioid dipeptides––l-kyotorphin (l-Tyr-l-Arg) and d-kyotorphin (l-Tyr-d-Arg)––using l-Tyr-NH2 and d- and l-Arg-methyl ester respectively as an acyl donor and acceptor. Yield evaluation of kyotorphin synthesis indicated that the
conversion ratio of substrate to kyotorphin was moderate: the value was estimated as greater than 20%. 相似文献
16.
Sampietro D Quiroga E Sgariglia M Soberón J Vattuone MA 《Antonie van Leeuwenhoek》2012,102(2):257-267
An α-galactosidase was isolated from a culture filtrate of Lenzites elegans (Spreng.) ex Pat. MB445947 grown on citric pectin as carbon source. It was purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration chromatography and anion-exchange chromatography. The relative molecular mass of the native purified enzyme was 158 kDa determined by gel filtration and it is a homodimer (Mr subunits = 61 kDa). The optimal temperature for enzyme activity was in the range 60–80 °C. This α-galactosidase showed a high thermostability, retaining 94 % of its activity after preincubation at 60 °C for 2 h. The optimal pH for the enzyme was 4.5 and it was stable from pH 3 to 7.5 when the preincubation took place at 60 °C for 2 h. It was active against several α-galactosides such as p-nitrophenyl-α-d-galactopyranoside, α-d-melibiose, raffinose and stachyose. The α-galactosidase is a glycoprotein with 26 % of structural sugars. Galactose was a non-competitive inhibitor with a Ki = 22 mM versus p-nitrophenyl-α-d-galactoside and 12 mM versus α-d-melibiose as substrates. Glucose was a simple competitive inhibitor with a Ki = 10 mM. Cations such as Hg2+ and p-chloromercuribenzoate were also inhibitors of this activity, suggesting the presence of –SH groups in the active site of the enzyme. On the basis of the sequence of the N-terminus (SPDTIVLDGTNFALN) the studied α-galactosidase would be a member of glycosyl hydrolase family 36 (GH 36). Given the high optimum temperature and heat stability of L. elegans α-galactosidase, this fungus may become a useful source of α-galactosidase production for multiple applications. 相似文献
17.
Satomura T Zhang XD Hara Y Doi K Sakuraba H Ohshima T 《Applied microbiology and biotechnology》2011,89(4):1075-1082
The activity of a dye-linked l-proline dehydrogenase (dye-l-proDH) was found in the crude extract of an aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis JCM 11548, and was purified 163-fold through four sequential chromatography steps. The enzyme has a molecular mass of about
108 kDa and is a homodimer with a subunit molecular mass of about 46 kDa. The enzyme retained more than 90% of its activity
after incubation at 100 °C for 120 min (pH 7.5) or after incubation at pHs 4.5–9.0 for 30 min at 50 °C. The enzyme catalyzed
l-proline dehydrogenation to Δ1-pyroline-5-carboxylate using 2,6-dichloroindophenol (DCIP) as the electron acceptor and the Michaelis constants for l-proline and DCIP were 1.67 and 0.026 mM, respectively. The prosthetic group on the enzyme was identified as flavin adenine
dinucleotide by high-performance liquid chromatography. The subunit N-terminal amino acid sequence was MYDYVVVGAG. Using that
sequence and previously reported genome information, the gene encoding the enzyme (Pcal_1655) was identified. The gene was
then cloned and expressed in Escherichia coli and found to encode a polypeptide of 415 amino acids with a calculated molecular weight of 46,259. The dye-l-proDH gene cluster in P. calidifontis inherently differs from those in the other hyperthermophiles reported so far. 相似文献
18.
A putative d-lyxose isomerase from Dictyoglomus turgidum was purified with a specific activity of 19 U/mg for d-lyxose isomerization by heat treatment and affinity chromatography. The native enzyme was estimated as a 42 kDa dimer by
gel-filtration chromatography. The activity of the enzyme was highest for d-lyxose, suggesting that it is a d-lyxose isomerase. The maximum activity of the enzyme was at pH 7.5 and 75°C in the presence of 0.5 mM Co2+, with a half-life of 108 min, K
m of 39 mM, and k
cat of 3,570 1/min. The enzyme is the most thermostable d-lyxose isomerase among those characterized to date. It converted 500 g d-xylulose/l to 380 g d-lyxose/l after 2 h. This is the highest concentration and productivity of d-lyxose reported thus far. 相似文献
19.
Sunshine C. Silver Tilak Chandra Egidijus Zilinskas Shourjo Ghose William E. Broderick Joan B. Broderick 《Journal of biological inorganic chemistry》2010,15(6):943-955
Spore photoproduct lyase (SP lyase), a member of the radical S-adenosylmethionine superfamily of enzymes, catalyzes the repair of 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)],
a type of UV-induced DNA damage unique to bacterial spores. The anaerobic purification and characterization of Clostridium acetobutylicum SP lyase heterologously expressed in Escherichia coli, and its catalytic activity in repairing stereochemically defined synthetic dinucleotide SPs was investigated. The purified
enzyme contains between 2.3 and 3.1 iron atoms per protein. Electron paramagnetic resonance (EPR) spectroscopy reveals an
isotropic signal centered at g = 1.99, characteristic of a [3Fe–4S]+ cluster accounting for 3–4% of the iron in the sample. Upon reduction, a nearly axial signal (g = 2.03, 1.93 and 1.92) characteristic of a [4Fe–4S]+ cluster is observed that accounts for 34–45% of total iron. Addition of S-adenosylmethionine to the reduced enzyme produces a rhombic signal (g = 2.02, 1.93, 1.82) unique to the S-adenosyl-l-methionine complex while decreasing the overall EPR intensity. This reduced enzyme is shown to rapidly and completely repair
the 5R diastereomer of a synthetic dinucleotide SP with a specific activity of 7.1 ± 0.6 nmol min−1 mg−1, whereas no repair was observed for the 5S diastereomer. 相似文献
20.
Attila Vig Attila Igloi Nora Adanyi Gyongyi Gyemant Csaba Csutoras Attila Kiss 《Bioprocess and biosystems engineering》2010,33(8):947-952
An amperometric detector and an enzymatic reaction were combined for the measurement of l-ascorbic acid. The enzyme cell (containing immobilized ascorbate oxidase) was connected to a flow injection analyzer (FIA)
system with a glassy carbon electrode as an amperometric detector. During optimization and measurements two sample injectors
were used, one before and one after the enzyme cell, thus eliminating the background interferences. Subtraction of the signal
area given in the presence of enzyme from the one given in the absence of enzyme was applied for measuring analyte concentrations
and calibration at 400 mV. Analysis capacity of system is 25 samples/hour. The relative standard deviation (RSD) was below
5% (5 times repeated, 400 μmol/L conc.), linearity up to 400 μmol/L, limit of detection (LOD) 5 μmol/L, fitting of calibration
curve in 25–400 μmol/L range was R
2 = 0.99. 相似文献