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1.
The formylglycine‐generating enzyme (FGE) recognizes proteins with a specific cysteine‐containing six‐amino‐acid motif and converts this cysteine residue into formylglycine. The resulting aldehyde function provides a unique handle for selective protein labeling. We have identified two mutations in FGE from Thermomonospora curvata that increase this catalytic efficiency more than 40‐fold. The resulting activity and stability, as well as its ease of recombinant production, make this FGE variant a practical reagent for in vitro protein engineering.  相似文献   

2.
Formylglycine‐generating enzymes (FGEs) catalyze O2‐dependent conversion of specific cysteine residues of arylsulfatases and alkaline phosphatases into formylglycine. The ability also to introduce unique aldehyde functions into recombinant proteins makes FGEs a powerful tool for protein engineering. One limitation of this technology is poor in vitro activity of reconstituted FGEs. Although FGEs have been characterized as cofactor‐free enzymes we report that the addition of one equivalent of CuI increases catalytic efficiency more than 20‐fold and enables the identification of stereoselective C?H bond cleavage at the substrate as the rate‐limiting step. These findings remove previous limitations of FGE‐based protein engineering and also pose new questions about the catalytic mechanism of this O2‐utilizing enzyme.  相似文献   

3.
To produce an epoxy resin with high intrinsic self‐healing efficiency, furfurylglycidyl ether (FGE) was synthesized following a two‐step route. It carried one furan and one epoxide on each of its molecules. Having been cured using N,N′‐(4,4′‐diphenylmethane)bismaleimide and methylhexahydrophthalic anhydride, FGE was then polymerized with two types of intermonomer linkages. That is, thermally reversible Diels–Alder (DA) bonds from the reaction between furan and maleimide groups, and thermally irreversible bonds from the reaction between epoxide and anhydride groups. These two types of bonds provide the polymer with thermal remendability and load‐bearing capacity, respectively. Compared with N,N‐diglycidylfurfurylamine, which was previously developed by the authors and has a similar structure to FGE but with fewer furan rings, FGE can react with maleimide with lower activation energy and the DA bonds formed exhibit higher reversibility. Consequently, improved crack healability of the cured FGE characterized by nearly full recovery of fracture toughness was revealed using double cleavage drilled compression tests. Copyright © 2010 Society of Chemical Industry  相似文献   

4.
The substrate scope of the flavoprotein alditol oxidase (AldO) from Streptomyces coelicolor A3(2), recombinantly produced in Escherichia coli, was explored. While it has been established that AldO efficiently oxidizes alditols to D ‐aldoses, this study revealed that the enzyme is also active with a broad range of aliphatic and aromatic alcohols. Alcohols containing hydroxy groups at the C‐1 and C‐2 positions like 1,2,4‐butanetriol (Km=170 mM, kcat=4.4 s−1), 1,2‐pentanediol (Km=52 mM, kcat=0.85 s−1) and 1,2‐hexanediol (Km=97 mM, kcat=2.0 s−1) were readily accepted by AldO. Furthermore, the enzyme was highly enantioselective for the oxidation of 1,2‐diols [e.g., for 1‐phenyl‐1,2‐ethanediol the (R)‐enantiomer was preferred with an E‐value of 74]. For several diols the oxidation products were determined by GC‐MS and NMR. Interestingly, for all tested 1,2‐diols the products were found to be the α‐hydroxy acids instead of the expected α‐hydroxy aldehydes. Incubation of (R)‐1‐phenyl‐1,2‐ethanediol with 18O‐labelled water (H218O) revealed that a second enzymatic oxidation step occurs via the hydrate product intermediate. The relaxed substrate specificity, excellent enantioselectivity, and independence of coenzymes make AldO an attractive enzyme for the preparation of optically pure 1,2‐diols and α‐hydroxy acids.  相似文献   

5.
BACKGROUND: Newly synthesized composite films of P(glycidyl methacrylate85co‐vinylferrocene15)/Poly(glutaraldehyde)/ Polypyrrole [P(GMA85co‐VFc15)/PGA/PPy] and Poly(3‐methylthienyl methacrylate85co‐vinylferrocene15)/Polypyrrole [P (MTM85co‐VFc15)/PPy] were used as matrices for tyrosinase based working electrodes. Direct covalent attachment of enzyme was carried out via the pendant epoxy groups of P(GMA85co‐VFc15) film, and the entrapment of enzyme was achieved for electrode containing P(MTM85co‐VFc15) film via electropolymerization of pyrrole in the presence of enzyme. The aim of the study is amperometric determination of various phenolics and investigation of the effect of interfacial interactions between enzyme and matrices on biosensor response. RESULTS: The lowest detection limit and the highest sensitivity for a P(GMA85co‐VFc15) based working electrode was found to be 0.113 µmol L?1 for 4‐methoxyphenol, 40 nA (µmol L?1)?1 for pyrocatechol, respectively. Results showed that sensitivities were at least 8500–55000 times higher than the results in previous P(GMA‐co‐VFc) related studies. CONCLUSION: Facilitated electron transfer was achieved by means of mediator incorporated in conductive composites of VFc based redox copolymers. The effect was greater when enzyme was covalently bonded via epoxy groups due to the proximity of enzyme, mediator and electrode surface. Results showed that a multifunctional surface was provided on electrodes since the suggested copolymers could mediate an electrochemical reaction, and the multifunctional surface was capable of coating with conductive PPy. Copyright © 2011 Society of Chemical Industry  相似文献   

6.
Palladized biomass of Desulfovibrio vulgaris (Bio‐Pd(0)) reduced Cr(VI) to Cr(III) at an initial rate four‐fold higher than chemically‐prepared Pd(0) metal. Optimal Cr(VI) reduction by suspended Bio‐Pd(0) occurred at pH 3, whereas pH did not affect the rate of Cr(VI) reduction by Bio‐Pd(0) immobilized in agar beads. The rate of Cr(VI) reduction was concentration‐dependent below 300 µmol dm?3, and application of enzyme kinetics, considering Bio‐Pd(0) as an ‘artificial enzyme’, gave an apparent Km (Kmapp) of approx. 650 µmol dm?3 and Vmax of 1667 nmol h?1 mg Pd(0) for suspended Bio‐Pd(0). The potential of Bio‐Pd(0) as a method for the treatment of Cr(VI)‐wastes is discussed. Copyright © 2005 Society of Chemical Industry  相似文献   

7.
A hybrid approach was applied for the design of an inhibitor of trypsin‐like serine proteases. Compound 16 [(R,R)‐ and (R,S)‐diphenyl (4‐(1‐(4‐amidinobenzylamino)‐1‐oxo‐3‐phenylpropan‐2‐ylcarbamoyl)phenylamino)(4‐amidinophenyl)methylphosphonate hydrochloride], prepared in a convergent synthetic procedure, possesses a phosphonate warhead prone to react with the active site serine residue in a covalent, irreversible manner. Each of the two benzamidine moieties of 16 can potentially be accommodated in the S1 pocket of the target enzyme, but only the benzamidine close to the phosphonate group would then promote an irreversible interaction. The Janus‐faced inhibitor 16 was evaluated against several serine proteases and caused a pronounced inactivation of human thrombin with a second‐order rate constant (kinac/Ki) of 59 500 M ?1 s?1. With human matriptase, 16 showed preference for a reversible mode of inhibition (IC50=2.6 μM ) as indicated by linear progress curves and enzyme reactivation.  相似文献   

8.
The Rv3377c gene from the Mycobacterium tuberculosis H37 genome is specifically limited to those Mycobacterium species that cause tuberculosis. We have demonstrated that the gene product of Rv3377c is a diterpene cyclase that catalyzes the formation of tuberculosinol from geranylgeranyl diphosphate (GGPP). However, the characteristics of this enzyme had not previously been studied in detail with homogeneously purified enzyme. The purified enzyme catalyzed the synthesis of tuberculosinyl diphosphate from GGPP, but it did not bring about the synthesis of tuberculosinol. Optimal conditions for the highest activity were found to be as follows: pH 7.5, 30 °C, MgII (0.1 mM ), and Triton X‐100 (0.1 %). Under these conditions, the kinetic values of KM and kcat were determined to be 11.7±1.9 μM for GGPP and 12.7±0.7 min?1, respectively, whereas the specific activity was 186 nmol min?1 mg?1. The enzyme activity was inhibited at substrate concentrations higher than 50 μM . The catalytic activity was strongly inhibited by 15‐aza‐dihydrogeranylgeraniol and 5‐isopropyl‐N,N,N,2‐tetramethyl‐4‐(piperidine‐1‐carbonyloxy)benzenaminium chloride (Amo‐1618). The DXDTT293–297 motif, corresponding to the DXDDTA motif conserved among terpene cyclases, was mutated in order to investigate its function. The middle D295 was found to be the most crucial entity for the catalysis. D293 and two threonine residues function synergistically to enhance the acidity of D295, possibly through hydrogen‐bonding networks. The Rv3377c enzyme could also react with (14R/S)‐14,15‐oxidoGGPP to generate 3α‐ and 3β‐hydroxytuberculosinyl diphosphate. Conformational analyses were carried out with deuterium‐labeled GGPP and oxidoGGPP. We found that GGPP and (14R)‐oxidoGGPP adopted a chair/chair conformation, but (14S)‐oxidoGGPP adopted a boat/chair conformation. Interestingly, the conformations of oxidoGGPP for the A‐ring formation are the opposite of those of oxidosqualene when it is used as a substrate by squalene cyclases for the biosynthesis of hopene and tetrahymanol. (3R)‐Oxidosqualene is folded in a boat conformation, whereas (3S)‐2,3‐oxidosqualene folds into a chair conformation, for the formation of the A‐rings of the hopene and tetrahymanol skeletons, respectively.  相似文献   

9.
The NAD+‐dependent deacetylases, namely sirtuins, are involved in the regulation of a variety of biological processes such as gene silencing, DNA repair, longevity, metabolism, apoptosis, and development. An enzyme from the parasite Leishmania infantum that belongs to this family, LiSIR2RP1, is a NAD+‐dependent tubulin deacetylase and an ADP‐ribosyltransferase. This enzyme's involvement in L. infantum virulence and survival underscores its potential as a drug target. Our search for selective inhibitors of LiSIR2RP1 has led, for the first time, to the identification of the antiparasitic and anticancer bisnaphthalimidopropyl (BNIP) alkyl di‐ and triamines (IC50 values in the single‐digit micromolar range for the most potent compounds). Structure–activity studies were conducted with 12 BNIP derivatives that differ in the length of the central alkyl chain, which links the two naphthalimidopropyl moieties. The most active and selective compound is the BNIP diaminononane (BNIPDanon), with IC50 values of 5.7 and 97.4 μM against the parasite and human forms (SIRT1) of the enzyme, respectively. Furthermore, this compound is an NAD+‐competitive inhibitor that interacts differently with the parasite and human enzymes, as determined by docking analysis, which might explain its selectivity toward the parasitic enzyme.  相似文献   

10.
Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6‐anhydro‐β‐d ‐glucopyranose) in the presence of Mg2+–ATP. For the Lipomyces starkeyi LGK, we show here with real‐time in situ NMR spectroscopy at 10 °C and pH 7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of α‐d ‐glucose‐6‐phosphate in a manner reminiscent of an inverting β‐glycoside hydrolase. Kinetic characterization revealed the Mg2+ concentration for optimum activity (20–50 mm ), the apparent binding of levoglucosan (Km=180 mm ) and ATP (Km=1.0 mm ), as well as the inhibition by ADP (Ki=0.45 mm ) and d ‐glucose‐6‐phosphate (IC50=56 mm ). The enzyme was highly specific for levoglucosan and exhibited weak ATPase activity in the absence of substrate. The equilibrium conversion of levoglucosan and ATP lay far on the product side, and no enzymatic back reaction from d ‐glucose‐6‐phosphate and ADP was observed under a broad range of conditions. 6‐Phospho‐α‐d ‐glucopyranosyl fluoride and 6‐phospho‐1,5‐anhydro‐2‐deoxy‐d ‐arabino‐hex‐1‐enitol (6‐phospho‐d ‐glucal) were synthesized as probes for the enzymatic mechanism but proved inactive with the enzyme in the presence of ADP. The pyranose ring flip 4C11C4 required for 1,6‐anhydro‐product synthesis from d ‐glucose‐6‐phosphate probably presents a major thermodynamic restriction to the back reaction of the enzyme.  相似文献   

11.
Laurylfluoro glycidyl ether (FGE) was synthesized by laurylfluoro‐1‐pentanol with epichlorohydrin, and confirmed by FTIR and 13C‐NMR. The surface properties, moisture absorption, and mechanical properties of the epoxy resins modified by different content of laurylfluoro glycidyl ether acted as mono functional thinner were investigated by X‐ray photoelectron spectroscopy (XPS), universal testing machine (UTM), dynamic mechanical thermal analyzer (DMTA), etc. The fluorine content at the surface of the modified resins were enriched greatly with the increase of the content of laurylfluoro glycidyl ether, and the hydrophobic property of the resins surface increased. When the FGE content was 10%, the fluorine content at the surface of the modified epoxy resin reached to 66% and the water contact angle was 102°. The equilibrium moisture content of the resin dropped by 30% when the content of FGE was 5%. The mechanical properties of the epoxy resins modified by FGE were improved while the thermal mechanical properties changed little at low content of FGE (less than 3%). Further increase of FGE content in the epoxy resins may result in decreases of the mechanical and thermal mechanical properties of the resins. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009  相似文献   

12.
Alkaline esterase (carboxylic‐ester hydrolases; EC 3.1.1.1) extracted from germinated soybean seeds (Glycine max) was purified approximately 3.6 times by chromatography in a DEAE‐cellulose anion exchange column and filtration in Sephadex G100 gel. The molecular mass of the enzyme was estimated at 45 kDa by gel electrophoresis (SDS‐PAGE). The purified enzyme showed a specific activity of 5.6 U mg?1 using p‐nitrophenyl butyrate as substrate. The esterase showed optimal activity at 47 °C in moderately alkaline pH, low stability in temperatures higher than 50 °C, and high stability at pH values between 6 and 9.5. The Ca2+ and Co2+ ions proved to have a positive effect on enzyme activity; however, Hg2+ completely inhibited esterase activity. Using p‐nitrophenyl butyrate as substrate, the enzyme showed a Km of 0.39 mM, Vmax of 31.5 mM mg?1 min?1 and kcat 7.60 × 106 s?1. Regarding substrate affinity, the enzyme showed greater activity for substrates containing short‐chain fatty acids, especially p‐nitrophenyl acetate. Such characteristics give the enzyme great potential for application in the production of low molecular weight esters, in the food industry, and in chemical products. This enzyme is another new member of the family of lipases and esterases from vegetable seeds with high activity and stability in alkaline pH.  相似文献   

13.
Poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) p(HEMA–GMA) membrane was prepared by UV‐initiated photopolymerisation of 2‐hydroxyethyl methacrylate (HEMA) and glycidyl methacrylate (GMA) in the presence of an initiator, azobisisobutyronitrile (AIBN). Cholesterol oxidase was immobilised directly on the membrane by forming covalent bonds between its amino groups and the epoxide groups of the membrane. An average of 53 µg of enzyme was immobilised per cm2 of membrane, and the bound enzyme retained about 67% of its initial activity. Immobilisation improved the pH stability of the enzyme as well as its temperature stability. The optimum temperature was 5 °C higher than that of the free enzyme and was significantly broader. The thermal inactivation rate constants for free and immobilised preparations at 70 °C were calculated as ki (free) 1.06 × 10?1 min?1 and ki (imm) 2.68 × 10?2 min?1, respectively. The immobilised enzyme activity was found to be quite stable in the repeated experiments. © 2002 Society of Chemical Industry  相似文献   

14.
An enzyme catalysing the essential dephosphorylation of the riboflavin precursor, 5‐amino‐6‐ribitylamino‐2,4(1H,3H)‐pyrimidinedione 5′‐phosphate ( 6 ), was purified about 800‐fold from a riboflavin‐producing Bacillus subtilis strain, and was assigned as the translation product of the ycsE gene by mass spectrometry. YcsE is a member of the large haloacid dehalogenase (HAD) superfamily. The recombinant protein was expressed in Escherichia coli. It catalyses the hydrolysis of 6 (vmax, 12 μmol mg?1 min?1; KM, 54 μm ) and of FMN (vmax, 25 μmol mg?1 min?1; KM, 135 μm ). A ycsE deletion mutant of B. subtilis was not riboflavin dependent. Two additional proteins (YwtE, YitU) that catalyse the hydrolysis of 6 at appreciable rates were identified by screening 13 putative HAD superfamily members from B. subtilis. The evolutionary processes that have resulted in the handling of an essential step in the biosynthesis of an essential cofactor by a consortium of promiscuous enzymes require further analysis.  相似文献   

15.
Studies on the activity of the enzyme horseradish peroxidase (HRP) have been carried out in micellar as well as reverse‐micellar media. The activity of the enzyme was studied in the presence of different classes of surfactants – ionic as well as non‐ionic. In aqueous media, the activity of the enzyme varied depending on whether the concentration of the surfactant used was above or below the critical micellar concentration (CMC). The enzyme was also studied in reverse‐micellar systems. HRP was introduced into the reverse micellar phase by the injection method and its activity within the reverse micelles was determined. The effect of water to surfactant ratio (Wo) on activity within reverse micelles was studied, and an almost two‐fold increase in activity was seen when the enzyme was encapsulated within reverse micelles of aqueous phase fractional hold‐up (?) of 0.0072 (v/v) consisting of sodium bis‐(2‐ethylhexyl) sulfosuccinate (AOT) in isooctane at a Wo of 20. The activity of HRP was measured over a wide range of AOT concentrations having different Wo values. Back‐extraction of HRP from these reverse micelles was carried out at varying ionic strengths of phosphate buffer. Back extraction was found to be highest at pH 7.0 in 40 mol m?3 phosphate buffer and 100 mol m?3 sodium chloride. © 2001 Society of Chemical Industry  相似文献   

16.
Enzyme efficiency results from the cooperation of functional groups in the catalytic site. In order to mimic a natural enzyme, a definite 3D scaffold must be carefully designed so that the functional groups can work cooperatively. During the HIV‐1 fusion process, the gp41 N‐ and C‐terminal heptad repeat regions form a coiled‐coil six‐helical bundle (6HB) that brings the viral and target cell membranes into close proximity for fusion. We used 6HB as the molecular model for a novel scaffold for the design of an artificial enzyme, in which the modified C34 and N36 peptides formed a unique 6HB structure through specific molecular recognition, and the position and orientation of the side‐chain groups on this scaffold were predictable. The histidine modified 6HB C34H13/20/N36H15/22 showed enzyme‐like hydrolytic activity towards p‐nitrophenyl acetate (PNPA; kcat/KM=3.66 M ?1 s?1) through the cooperation of several inter‐ or intrahelical imidazole groups. Since the catalytic activity of 6HB depends on the C‐ and N‐peptide assembly, either HIV fusion inhibitors that can compete with the formation of catalytic 6HB or denaturants that can destroy the ordered structure were able to modulate its activity. Further engineering of the solvent‐exposing face with Glu?‐Lys+ salt bridges enhanced the helicity and the stability of 6HB. As a result, the population and stability of cooperative catalytic units increased. In addition, the Glu?‐Lys+‐stabilized 6HB SC35H13/20/N36H15/22 had increased catalytic efficiency (kcat/KM=6.30 M ?1 s?1). A unique 6HB system was specifically assembled and provided a scaffold sufficiently stable to mimic the function of enzymes or other biomolecules.  相似文献   

17.
The model enzyme β‐galactosidase was entrapped in chitosan gel beads and tested for hydrolytic activity and its potential for application in a packed‐bed reactor. The chitosan beads had an enzyme entrapment efficiency of 59% and retained 56% of the enzyme activity of the free enzyme. The Michaelis constant (Km) was 0.0086 and 0.011 μmol/mL for the free and immobilized enzymes, respectively. The maximum velocity of the reaction (Vmax) was 285.7 and 55.25 μmol mL?1 min?1 for the free and immobilized enzymes, respectively. In pH stability tests, the immobilized enzyme exhibited a greater range of pH stability and shifted to include a more acidic pH optimum, compared to that of the free enzyme. A 2.54 × 16.51‐cm tubular reactor was constructed to hold 300 mL of chitosan‐immobilized enzyme. A full‐factorial test design was implemented to test the effect of substrate flow (20 and 100 mL/min), concentration (0.0015 and 0.003M), and repeated use of the test bed on efficiency of the system. Parameters were analyzed using repeated‐measures analysis of variance. Flow (p < 0.05) and concentration (p < 0.05) significantly affected substrate conversion, as did the interaction progressing from Run 1 to Run 2 on a bed (p < 0.05). Reactor stability tests indicated that the packed‐bed reactor continued to convert substrate for more than 12 h with a minimal reduction in conversion efficiency. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 91: 1294–1299, 2004  相似文献   

18.
We synthesized a novel O‐glucoside of the recently reported potent factor Xa (fXa) inhibitor 1 , which bears a 5‐chlorothien‐2‐yl moiety and 1‐isopropylpiperidine as fragments that bind the S1 and S4 enzyme pockets, respectively. A β‐D ‐glucosyl unit was conjugated through an ether‐linked C3‐alkyl spacer to the central phenyl ring of 1 . The synthesized β‐D ‐glucose‐based compound 16 achieved picomolar inhibitory potency against human fXa (Ki=60 pM ) and high selectivity over thrombin and other serine proteases. In addition to the chlorothienyl S1 binder, a large gain in ΔG resulted from the addition of protonated 1‐isopropylpiperidine (ΔΔG=29.7–30.5 kJ mol?1), which should bind to the aromatic S4 pocket through efficient cation–π and C? H???π interactions. Instead, the C3‐alkyl‐linked glucose fragment, which is likely directed toward the solvent outside the enzyme binding site, improves ΔG by an average of 2.9–3.8 kJ mol?1. Compound 16 showed sub‐micromolar in vitro anticoagulant activity, as assessed by prothrombin time (PT) and activated thromboplastin time (aPTT) clotting assays in pooled human plasma (PT2 and aPTT2 equal to 0.135 and 0.389 μM , respectively). Although compound 16 was 1.4‐fold less active than parent compound 1 in the ex vivo anticoagulant assay in mice, it showed a significant (1.6‐fold) prolongation of PT relative to controls (P<0.05) 60 min after oral dosing (75 mg kg?1).  相似文献   

19.
Immobilization glucoamylase onto plain and a six‐carbon spacer arm (i.e., hexamethylene diamine, HMDA) attached poly(2‐hydroxyethylmethacrylate‐ethyleneglycol dimethacrylate) [poly(HEMA‐EGDMA] microspheres was studied. The microspheres were prepared by suspension polymerization and the spacer arm was attached covalently by the reaction of carbonyl groups of poly(HEMA‐EGDMA). Glucoamylase was then covalently immobilized either on the plain of microspheres via CNBr activation or on the spacer arm‐attached microspheres via CNBr activation and/or using carbodiimide (CDI) as a coupling agent. Incorporation of the spacer arm resulted an increase in the apparent activity of the immobilized enzyme with respect to enzyme immobilized on the plain of the microspheres. The activity yield of the immobilized glucoamylase on the spacer arm‐attached poly(HEMA‐EGDMA) microspheres was 63% for CDI coupling and 82% for CNBr coupling. This was 44% for the enzyme, which was immobilized on the plain of the unmodified poly(HEMA‐EGDMA) microspheres via CNBr coupling. The Km values for the immobilized glucoamylase preparations (on the spacer arm‐attached microspheres) via CDI coupling 0.9% dextrin (w/v) and CNBr coupling 0.6% dextrin (w/v) were higher than that of the free enzyme 0.2% dextrin (w/v).The temperature profiles were broader for both immobilized preparations than that of the free enzyme. The operational inactivation rate constants (kiop) of immobilized enzymes were found to be 1.42 × 10?5 min?1 for CNBr coupled and 3.23 × 10?5 min?1 for CDI coupled glucoamylase. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 2702–2710, 2001  相似文献   

20.
Four series of noble networks were synthesized with acrylic acid (AAc) copolymerized with varying amount of 2‐hydroxy propyl methacrylate or dodecyl methacrylate (AAc/HPMA or AAc/DMA; 5:1 to 5:5, w/w) in the presence of ethylene glycol dimethacrylate (EGDMA; 1, 5, 10, 15, and 20%, w/w) as a crosslinker and ammonium per sulfate (APS) as an initiator. Each of the networks was used to immobilize a purified lipase from Pseudomonas aeruginosa MTCC‐4713. The lipase was purified by successive salting out with (NH4)2SO4, dialysis, and DEAE anion exchange chromatography. Two of the matrices, E15a, i.e. [poly (AAc5co‐DMA1cl‐EGDMA15)] and I15c, i.e. [poly (AAc5co‐HPMA3cl‐EGDMA15)], that showed relatively higher binding efficiency for lipase were selected for further studies. I15c‐hydrogel retained 58.3% of its initial activity after 10th cycle of repetitive hydrolysis of p‐NPP, and I15c was thus catalytically more stable and efficient than the other matrix. The I15c‐hydrogel‐immobilized enzyme showed maximum activity at 65°C and pH 9.5. The hydrolytic activity of free and I15c‐hydrogel‐immobilized enzyme increased profoundly in the presence of 5 mM chloride salts of Hg2+, NH4+, Al3+, K+, and Fe3+. The immobilized lipase was preferentially active on medium chain length p‐nitrophenyl acyl ester (C:8, p‐nitrophenyl caprylate). © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 4636–4644, 2006  相似文献   

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