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1.
β-Glucosidase catalyzes the sequential breakdown of cyanogenic glycosides in cyanogenic plants. The β-glucosidase from Prunus armeniaca L. was purified to 8-fold, and 20% yield was obtained, with a specific activity of 281 U/mg protein. The enzyme showed maximum activity in 0.15 M sodium citrate buffer, pH 6, at 35 °C with p-nitrophenylglucopyranoside as substrate. The β-glucosidase from wild apricot was used successfully for the saccharification of cellobiose into D-glucose. This enzyme has a Vmax of 131.6 μmol min−1 mg−1 protein, Km of 0.158 mM, Kcat of 144.8 s−1, Kcat/Km of 917.4 mM−1 s−1, and Km/Vmax of 0.0012 mM min mg μmole−1, using cellobiose as substrate. The half-life, deactivation rate coefficient, and activation energy of this β-glucosidase were 12.76 h, 1.509 × 10−5 s−1, and 37.55 kJ/mol, respectively. These results showed that P. armeniaca is a potential source of β-glucosidase, with high affinity and catalytic capability for the saccharification of cellulosic material. 相似文献
2.
《Neurochemistry international》2009,54(6-8):317-324
A novel neutral aminopeptidase (NAP-2) was found exclusively in the rat central nervous system (CNS). It was separated from the ubiquitous puromycin-sensitive aminopeptidase (PSA) and the neuron-specific aminopeptidase (NAP) by an automated FPLC-aminopeptidase analyzer. The activity of the neuronal aminopeptidase enriched in the synaptosomes is different from NAP and PSA in distribution and during brain development. The enzyme was purified 2230-fold to apparent homogeneity from rat brain cytosol with 4% recovery by ammonium sulfate fractionation, followed by column chromatography successively on Phenyl-Sepharose, Q-Sepharose, Sephadex G-200, and Mono Q. The single-chain enzyme with a molecular mass of 110 kDa has an optimal pH of 7.0 and a pI of 5.6. It splits β-naphthylamides of amino acid with aliphatic, polar uncharged, positively charged, and aromatic side chain. Leucyl β-naphthylamide (Leu βNA) is the best substrate with the highest hydrolytic coefficiency followed by Met βNA = Arg βNA = Lys βNA > Ala βNA > Tyr βNA > Phe βNA. The cysteine-, metallo-, glyco-aminopeptidase releases the N-terminal Tyr from Leu-enkephalin with a Km 82 μM and a kcat of 1.08 s−1, and Met-enkephalin with a Km of 106 μM and a kcat of 2.6 s−1. The puromycin-sensitive enzyme is most susceptible to amastatin with an IC50 of 0.05 μM. The data indicate that the enzyme is a new type of NAP found in rodent. Its possible function in neuron growth, neurodegeneration, and carcinomas is discussed. 相似文献
3.
A solvent-tolerant bacterium Burkholderia ambifaria YCJ01 was newly isolated by DMSO enrichment of the medium. The lipase from the strain YCJ01 was purified to homogeneity with apparent molecular mass of 34 kDa determined by SDS-PAGE. The purified lipase exhibited maximal activity at a temperature of 60 °C and a pH of 7.5. The lipase was very stable below 55 °C for 7 days (remaining 80.3% initial activity) or at 30 °C for 60 days. PMSF significantly inhibited the lipase activity, while EDTA had no effect on the activity. Strikingly, the lipase showed distinct super-stability to the most tested hydrophilic and hydrophobic solvents (25%, v/v) for 60 days, and different optimal pH in contrast with the alkaline lipase from B. cepacia S31. The lipase demonstrated excellent enantioselective transesterification toward the S-isomer of mandelic acid with a theoretical conversion yield of 50%, eep of 99.9% and ees of 99.9%, which made it an exploitable biocatalyst for organic synthesis and pharmaceutical industries. 相似文献
4.
Sanjay Kumar Singh Santosh Kumar Singh Vinayak Ram Tripathi Satyendra Kumar Garg 《Process Biochemistry》2012,47(10):1479-1487
A thermoalkaline protease with a molecular weight of 22 kDa was purified from the Bacillus cereus SIU1 strain using a combination of Q-Sepharose and Sephadex G-75 chromatography. The kinetic analyses revealed the Km, Vmax and kcat to be 1.09 mg ml?1, 0.909 mg ml?1 min?1 and 3.11 s?1, respectively, towards a casein substrate. The protease was most active and stable at pH 9.0 and between a temperature range of 45–55 °C. It was fully stable at 0.0–2.0% and moderately stable at 2.5–10.0% (w/v) sodium chloride. Phenyl methyl sulfonyl fluoride, ethylene diamine tetra acetic acid and ascorbic acid were inhibitory with regard to enzyme activity, whereas cysteine, β-mercaptoethanol, calcium, magnesium, manganese and copper at concentration of 1.0 mM increased enzyme activity. Sodium dodecyl sulfate, Triton X-100, Tween 80, hydrogen peroxide and sodium perborate significantly enhanced protease activity at 0.1 and 1.0% concentrations. In the presence of 0.1 and 1.0% (w/v) detergents, the protease was fairly stable and retained 50–76% activity. Therefore, it may have a possible application in laundry formulations. An initial analysis of the circular dichroism (CD) spectrum in the ultraviolet range revealed that the protease is predominantly a β-pleated structure and a detailed structural composition showed ~50% β-sheets. The CD-based conformational evaluation of the protease after incubation with modulators, metal ions, detergents and at different pH values, revealed that the change in the β-content directly corresponded to the altered enzyme activity. The protease combined with detergent was able to destain blood stained cloth within 30 min. 相似文献
5.
Ariosvana Fernandes Lima Kenia Franco Cavalcante Maria de Fátima Matos de Freitas Tigressa Helena Soares Rodrigues Maria Valderez Ponte Rocha Luciana Rocha Barros Gonçalves 《Process Biochemistry》2013,48(3):443-452
An investigation was conducted on the production of β-galactosidase (β-gal) by different strains of Kluyveromyces, using lactose as a carbon source. The maximum enzymatic activity of 3.8 ± 0.2 U/mL was achieved by using Kluyveromyces lactis strain NRRL Y1564 after 28 h of fermentation at 180 rpm and 30 °C. β-gal was then immobilized onto chitosan and characterized based on its optimal operation pH and temperature, its thermal stability and its kinetic parameters (Km and Vmax) using o-nitrophenyl β-d-galactopyranoside as substrate. The optimal pH for soluble β-gal activity was found to be 6.5 while the optimal pH for immobilized β-gal activity was found to be 7.0, while the optimal operating temperatures were 50 °C and 37 °C, respectively. At 50 °C, the immobilized enzyme showed an increased thermal stability, being 8 times more stable than the soluble enzyme. The immobilized enzyme was reused for 10 cycles, showing stability since it retained more than 70% of its initial activity. The immobilized enzyme retained 100% of its initial activity when it was stored at 4 °C and pH 7.0 for 93 days. The soluble β-gal lost 9.4% of its initial activity when it was stored at the same conditions. 相似文献
6.
《Journal of biochemical and biophysical methods》2008,70(3):471-479
The kinetic data obtained from the action of a cathepsin D-like enzyme from Biomphalaria glabrata hepatopancreas (digestive gland) on MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNp)-D–Arg-NH2, was studied as a data prototype, generated by means of a fluorogenic substrate. An initial fluorescence, due to incomplete energy transfer, of about 8% of the values attained after complete substrate hydrolysis; a non-linear standard curve even at μM concentrations and an exponential decay of the steady state fluorescence of reaction product of the order of 10− 4 × s− 1 were the main analytical problems encountered. The standard curves for fluorescence of the substrate reaction product after 48 h of hydrolysis, and the reference compound MOCAc-Pro-Leu-Gly-NH2, were fitted by polynomial approximation and the point derivates used as calibration factors. Time dependence of the calibration factor for the reaction product was − 2.96 × 10− 4 a.u μM− 1 × s− 1 that is, in the same order of observed enzymic reaction rates. A mathematical treatment was devised for obtaining rates corrected for errors derived from the three analytical problems indicated. The method is of general application in continuous fluorometric assays, irrespective of the particular enzyme used, but of special value for substrates that present significant initial fluorescence. The reaction rates were 11% higher; as calculated by means of the calibration factor [substrate] ÷ (final − initial fluorescence intensities), which is the prevalent procedure in the literature; leading to underestimation of Km and overestimation of Vmax. 相似文献
7.
The phytase of Sporotrichum thermophile was purified to homogeneity using acetone precipitation followed by ion-exchange and gel-filtration column chromatography. The purified phytase is a homopentamer with a molecular mass of ~456 kDa and pI of 4.9. It is a glycoprotein with about 14% carbohydrate, and optimally active at pH 5.0 and 60 °C with a T1/2 of 16 h at 60 °C and 1.5 h at 80 °C. The activation energy of the enzyme reaction is 48.6 KJ mol?1 with a temperature quotient of 1.66, and it displayed broad substrate specificity. Mg2+ exhibited a slight stimulatory effect on the enzyme activity, while it was markedly inhibited by 2,3-butanedione suggesting a possible role of arginine in its catalysis. The chaotropic agents such as guanidinium hydrochloride, urea and potassium iodide strongly inhibited phytase activity. Inorganic phosphate inhibited enzyme activity beyond 3 mM. The maximum hydrolysis rate (Vmax) and apparent Michaelis–Menten constant (Km) for sodium phytate were 83 nmol mg?1 s?1 and 0.156 mM, respectively. The catalytic turnover number (Kcat) and catalytic efficiency (Kcat/Km) of phytase were 37.8 s?1 and 2.4 × 105 M?1 s?1, respectively. Based on the N-terminal and MALDI–LC–MS/MS identified amino acid sequences of the peptides, the enzyme did not show a significant homology with the known phytases. 相似文献
8.
Tannase production by Bacillus subtilis PAB2, was investigated under solid state fermentation using tamarind seed as sole carbon source and it was found as the highest titer (73.44 U/gds). The enzyme was purified to homogeneity, which showed the molecular mass around 52 kDa (Km = 0.445 mM, Vmax = 125.8 mM/mg/min and Kcat = 2.88 min–1). The enzyme was found stable in a range of pH (3.0–8.0) and temperature (30–70 °C) with an optimal activity at pH 5.0, pI of 4.4 and at 40 °C temperature. It exhibited half-life (t1/2) of 4.5 h at 60 °C. The enzyme comprised a typical secondary structure containing α-helix (9.3%), β-pleated sheet (33.6%) and β-turn (17.2%). The native conformation of the enzyme was alike a 44 nm spherical nanoparticle upon aggregation. Thermodynamic parameters of tannase revealed that it was stable at 40 °C and showed Q10, ΔGd and ΔSd values of 2.08, 99.37 KJ/mol and 252.38 J mol−1 K−1, respectively. Organic solvents were stimulatory with regard to enzyme activity. Moreover, the altered enzyme activity was determined to be correlated with the changes in structural conformation in presence of inducer and inhibitor. Tannase was explored to have no cytotoxicity on Vero cell line as well as rat model study. 相似文献
9.
Sergio Martínez-Rodríguez Ana Isabel Martínez-Gómez Josefa María Clemente-Jiménez Felipe Rodríguez-Vico Juan Ma García-Ruíz Francisco Javier Las Heras-Vázquez Jose Antonio Gavira 《Journal of structural biology》2010,169(2):200-208
The recombinant dihydropyrimidinase from Sinorhizobium meliloti CECT4114 (SmelDhp) has been characterised and its crystal structure elucidated at 1.85 Å. The global architecture of the protein is reminiscent of that of the amidohydrolase superfamily, consisting of two domains; an (α/β)8 TIM-like barrel domain, where the catalytic centre is located, and a smaller β-sheet sandwich domain of unknown function. The c-terminal tails of each subunit extend toward another monomer in a swapping-like manner, creating a hydrogen bond network which suggests its implication in protein oligomerisation. Mutational and structural evidence suggest the involvement of a conserved tyrosine in the reaction mechanism of the enzyme. SmelDhp presents both hydantoinase and dihydropyrimidinase activities, with higher affinity for the natural six-membered ring substrates. For the five-membered ring substrates, affinity was greater for those with aliphatic and apolar groups in the 5th carbon atom, with the highest rates of hydrolysis for d-5-methyl and d-5-ethyl hydantoin (kcat/Km = 2736 ± 380 and 944 ± 52 M?1 s?1, respectively). The optimal conditions for the enzyme activity were found to be 60 °C of temperature at pH 8.0. SmelDhp retains 95% of its activity after 6-hour preincubation at 60 °C. This is the first dihydropyrimidinase used for the hydrolytic opening of non-natural 6-monosubstituted dihydrouracils, which may be exploited for the production of β-amino acids. 相似文献
10.
Masahiro Nakajima Mamoru Nishimoto Motomitsu Kitaoka 《Enzyme and microbial technology》2010,46(3-4):315-319
We characterized a glycoside hydrolase family 112 protein from Opitutus terrae (Oter_1377 protein). The enzyme phosphorolyzed d-galactosyl-β1→4-l-rhamnose (GalRha) and also showed phosphorolytic activity on d-galactosyl-β1→3-d-glucose as a minor substrate. In the reverse reaction, the enzyme showed higher activity on l-rhamnose derivatives than on d-glucose derivatives. The enzyme was stable up to 45 °C and at pH 6.0–7.0. The values of kcat and Km of the phosphorolytic activity of the enzyme on GalRha were 60 s?1 and 2.1 mM, respectively. Thus, Oter_1377 protein was identified as d-galactosyl-β1→4-l-rhamnose phosphorylase (GalRhaP). The presence of GalRhaP in O. terrae suggests that genes encoding GalRhaP are widely distributed in different organisms. 相似文献
11.
l-Amino acid oxidases (LAAOs) are useful catalysts for the deracemisation of racemic amino acid substrates when combined with abiotic reductants. The gene nadB encoding the l-aspartate amino acid oxidase from Pseudomonas putida (PpLASPO) has been cloned and expressed in E. coli. The purified PpLASPO enzyme displayed a KM for l-aspartic acid of 2.26 mM and a kcat = 10.6 s−1, with lower activity also displayed towards l-asparagine, for which pronounced substrate inhibition was also observed. The pH optimum of the enzyme was recorded at pH 7.4. The enzyme was stable for 60 min at up to 40 °C, but rapid losses in activity were observed at 50 °C. A mutational analysis of the enzyme, based on its sequence homology with the LASPO from E. coli of known structure, appeared to confirm roles in substrate binding or catalysis for residues His244, His351, Arg386 and Arg290 and also for Thr259 and Gln242. The high activity of the enzyme, and its promiscuous acceptance of both l-asparagine and l-glutamate as substrates, if with low activity, suggests that PpLASPO may provide a good model enzyme for evolution studies towards AAOs of altered or improved properties in the future. 相似文献
12.
Sara Caramia Angela Gala Morena Gatius Fabrizio dal Piaz Denis Gaja Alejandro Hochkoeppler 《Biochemistry and Biophysics Reports》2017
The activity of Prunus dulcis (sweet almond) β-glucosidase at the expense of p-nitrophenyl-β-d-glucopyranoside at pH 6 was determined, both under steady-state and pre-steady-state conditions. Using crude enzyme preparations, competitive inhibition by 1–5 mM imidazole was observed under both kinetic conditions tested. However, when imidazole was added to reaction mixtures at 0.125–0.250 mM, we detected a significant enzyme activation. To further inspect this effect exerted by imidazole, β-glucosidase was purified to homogeneity. Two enzyme isoforms were isolated, i.e. a full-length monomer, and a dimer containing a full-length and a truncated subunit. Dimeric β-glucosidase was found to perform much better than the monomeric enzyme, independently of the kinetic conditions used to assay enzyme activity. In addition, the sensitivity towards imidazole was found to differ between the two isoforms. While monomeric enzyme was indeed found to be relatively insensitive to imidazole, dimeric β-glucosidase was observed to be significantly activated by 0.125–0.250 mM imidazole under pre-steady-state conditions. Further, steady-state assays revealed that the addition of 0.125 mM imidazole to reaction mixtures increases the Km of dimeric enzyme from 2.3 to 6.7 mM. The activation of β-glucosidase dimer by imidazole is proposed to be exerted via a conformational transition poising the enzyme towards proficient catalysis. 相似文献
13.
Characterization and directed evolution of BliGO,a novel glycine oxidase from Bacillus licheniformis
Glycine oxidase (GO) has great potential for use in biosensors, industrial catalysis and agricultural biotechnology. In this study, a novel GO (BliGO) from a marine bacteria Bacillus licheniformis was cloned and characterized. BliGO showed 62% similarity to the well-studied GO from Bacillus subtilis. The optimal activity of BliGO was observed at pH 8.5 and 40 °C. Interestingly, BliGO retained 60% of the maximum activity at 0 °C, suggesting it is a cold-adapted enzyme. The kinetic parameters on glyphosate (Km, kcat and kcat/Km) of BliGO were 11.22 mM, 0.08 s−1, and 0.01 mM−1 s−1, respectively. To improve the catalytic activity to glyphosate, the BliGO was engineered by directed evolution. With error-prone PCR and two rounds of DNA shuffling, the most evolved mutant SCF-4 was obtained from 45,000 colonies, which showed 7.1- and 8-fold increase of affinity (1.58 mM) and catalytic efficiency (0.08 mM−1 s−1) to glyphosate, respectively. In contrast, its activity to glycine (the natural substrate of GO) decreased by 113-fold. Structure modeling and site-directed mutation study indicated that Ser51 in SCF-4 involved in the binding of enzyme with glyphosate and played a crucial role in the improvement of catalytic efficiency. 相似文献
14.
Alessio Innocenti Claudiu T. Supuran 《Bioorganic & medicinal chemistry letters》2010,20(21):6208-6212
Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to α-, β-, γ- and ζ-classes and from various organisms, ranging from the bacteria, archaea to eukarya domains, were investigated for their esterase/phosphatase activity with 4-nitrophenyl acetate, 4-nitrophenyl phosphate and paraoxon as substrates. Only α-CAs showed esterase/phosphatase activity, whereas enzymes belonging to the β-, γ- and ζ-classes were completely devoid of such activity. Paraoxon, the metabolite of the organophosphorus insecticide parathione, was a much better substrate for several human/murine α-CA isoforms (CA I, II and XIII), with kcat/KM in the range of 2681.6–4474.9 M?1 s?1, compared to 4-nitrophenyl phosphate (kcat/KM of 14.9–1374.4 M?1 s?1). 相似文献
15.
《Bioorganic & medicinal chemistry》2014,22(5):1667-1671
We investigated the catalytic activity and inhibition of the β-class carbonic anhydrase (CA, EC 4.2.1.1) CahB1, from the relict cyanobacterium Coleofasciculus chthonoplastes (previously denominated Microcoleus chthonoplastes). The enzyme showed good activity as a catalyst for the CO2 hydration, with a kcat of 2.4 × 105 s−1 and a kcat/Km of 6.3 × 107 M−1 s−1. A range of inorganic anions and small molecules were investigated as inhibitors of CahB1. Perchlorate and tetrafluoroborate did not inhibit the enzyme (KIs >200 mM) whereas selenate and selenocyanide were ineffective inhibitors too, with KIs of 29.9–48.61 mM. The halides, pseudohalides, carbonate, bicarbonate, trithiocarbonate and a range of heavy metal ions-containing anions were submillimolar–millimolar inhibitors (KIs in the range of 0.15–0.90 mM). The best CahB1 inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with KIs in the range of 8–75 μM, whereas acetazolamide inhibited the enzyme with a KI of 76 nM. This is the first kinetic and inhibition study of a cyanobacterial CA. As these enzymes are widespread in many cyanobacteria, being crucial for the carbon concentrating mechanism which assures substrate to RubisCO for the CO2 fixation by these organisms, a detailed kinetic/inhibition study may be essential for a better understanding of this superfamily of metalloenzymes and for potential biotechnological applications in biomimetic CO2 capture processes. 相似文献
16.
《Process Biochemistry》2010,45(6):887-891
For efficient production of (R)-(−)-mandelic acid, a nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli. After simple optimization of the culture conditions, the biocatalyst production was greatly increased from 500 to 7000 U/l. The recombinant E. coli whole cells showed strong tolerance against a high substrate concentration of up to 200 mM, and the concentration of (R)-(−)-mandelic acid after only 4 h of transformation reached 197 mM with an enantiomeric excess (eep) of 99%. In a fed-batch reaction with 600 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic acid after 17.5 h of conversion reached 520 mM. The recombinant E. coli cells could also be repeatedly used in the biotransformation, retaining 40% of the initial activity after 10 batches of reaction. The highly substrate/product tolerable and enantioselective nature of this recombinant nitrilase suggests that it is of great potential for the practical production of optically pure (R)-(−)-mandelic acid. 相似文献
17.
Ganoderma lucidum is a saprotrophic white-rot fungus which contains a rich set of cellulolytic enzymes. Here, we screened an array of potential 1,4-β-endoglucanases from G. lucidum based on the gene annotation library and found that one candidate gene, GlCel5A, exhibits CMC-hydrolyzing activity. The recombinant GlCel5A protein expressed in Pichia pastoris is able to hydrolyze CMC and β-glucan but not xylan and mannan. The enzyme exhibits optimal activity at 60 °C and pH 3–4, and retained 50% activity at 80 and 90 °C for at least 15 and 10 min. The crystal structure of GlCel5A and its complex with cellobiose, solved at 2.7 and 2.86 Å resolution, shows a classical (β/α)8 TIM-barrel fold as seen in other members of glycoside hydrolase family 5. The complex structure contains a cellobiose molecule in the +1 and +2 subsites, and reveals the interactions with the positive sites of the enzyme. Collectively, the present work provides the first comprehensive characterization of an endoglucanase from G. lucidum that possesses properties for industrial applications, and strongly encourages further studying in the cellulolytic enzyme system of G. lucidum. 相似文献
18.
The LAC4 gene of Kluyveromyces lactis encoding for β-galactosidase was overexpressed in the yeast Arxula adeninivorans to produce the enzyme, which can be used for the synthesis of β-d-galactosides. These compounds play a major role as precursors for the synthesis of glycolipids and glycoproteins in medicine or for the production of tensides.The Xplor®2 transformation/expression platform was used because it enabled stable integration of the gene in the Arxula genome and the production of high levels of the enzyme. The recombinant β-galactosidase, fused with C-terminal His-tag region (Lac4-6hp), was purified by precipitation with ammonium sulphate and FPLC using hydroxylapatite. The enzyme exhibited optimal activity at 37 to 40 °C, pH 6.5 in 50 mM sodium acetate buffer. Activity was measured by the formation of p-nitrophenol at 405 nm from the hydrolyzed chromogenic substrate, p-nitrophenyl-β-d-gal. Biochemical characterization included the calculation of KM and apparent kcat values of the enzyme. The formation of benzyl β-d-gal by 0.1 U enzyme from A. adeninivorans with transgalactosylation was six times higher than that for the prokaryotic enzyme from E. coli. Moreover, the partially purified enzyme was used for the selective hydrolysis of allyl β-d-gal in a mixture of allyl β- and allyl α-d-gal, with 4 g l−1 being hydrolysed within one day by 1 U ml−1. Thus, the recombinant β-galactosidase produced in A. adeninivorans is of potential interest for the enzymatic synthesis of benzyl β-d-gal and other galactosides as well as the selective hydrolysis of anomeric mixtures and could be used to replace difficult chemical procedures. 相似文献
19.
An industrial enzyme, alkaline serine endopeptidase, was immobilized on surface modified SBA-15 and MCF materials by amide bond formation using carbodiimide as a coupling agent. The specific activities of free enzyme and enzyme immobilized on SBA-15 and MCF were studied using casein (soluble milk protein) as a substrate. The highest activity of free enzyme was obtained at pH 9.5 while this value shifted to pH 10 for SBA-15 and MCF immobilized enzyme. The highest activity of immobilized enzymes was obtained at higher temperature (60 °C) than that of the free enzyme (55 °C). Kinetic parameters, Michaelis–Menten constant (Km) and maximum reaction velocity (Vmax), were calculated as Km = 13.375, 11.956, and 8.698 × 10?4 mg/ml and Vmax = 0.156, 0.163 and 0.17 × 10?3 U/mg for the free enzyme and enzyme immobilized on SBA-15 and MCF, respectively. The reusability of immobilized enzyme showed 80% of the activity retained even after 15 cycles. Large pore sized MCF immobilized enzyme was found to be more promising than the SBA-15 immobilized enzyme due to the availability of larger pores of MCF, which offer facile diffusion of substrate and product molecules. 相似文献
20.
Semra Isik Feray Kockar Meltem Aydin Oktay Arslan Ozen Ozensoy Guler Alessio Innocenti Andrea Scozzafava Claudiu T. Supuran 《Bioorganic & medicinal chemistry》2009,17(3):1158-1163
The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, has been cloned, purified, characterized kinetically and investigated for its inhibition with a series of sulfonamides and one sulfamate. The enzyme showed high CO2 hydrase activity, with a kcat of 9.4 × 105 s?1, and kcat/KM of 9.8 × 107 M?1 s?1. Simple benzenesulfonamides substituted in 2-, 4- and 3,4-positions of the benzene ring with amino, alkyl, halogeno and hydroxyalkyl moieties were weak scCA inhibitors with KIs in the range of 0.976–18.45 μM. Better inhibition (KIs in the range of 154–654 nM) was observed for benzenesulfonamides incorporating aminoalkyl/carboxyalkyl moieties or halogenosulfanilamides; benzene-1,3-disulfonamides; simple heterocyclic sulfonamides and sulfanilyl-sulfonamides. The clinically used sulfonamides/sulfamate (acetazolamide, ethoxzolamide, methazolamide, dorzolamide, topiramate, celecoxib, etc.) generally showed effective scCA inhibitory activity, with KIs in the range of 82.6–133 nM. The best inhibitor (KI of 15.1 nM) was 4-(2-amino-pyrimidin-4-yl)-benzenesulfonamide. These inhibitors may be useful to better understand the physiological role of β-CAs in yeast and some pathogenic fungi which encode orthologues of the yeast enzyme and eventually for designing novel antifungal therapies. 相似文献