Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Immobilization of penicillin G acylase on glyoxyl agarose and its further hydrophilization by physicochemical modification with ionic polymers has made it possible to perform the direct condensation between (+/-)-2-hydroxy-2-phenylethylamine [(+/-)-1] and different acyl donors in the presence of high concentrations of organic cosolvent (up to 90%) in the reaction medium. Using 50 mM phenyl acetic acid and these drastic reaction conditions, too harsh for any other PGA preparation, we have achieved an almost quantitative transformation (more than 99%) of 10 mM (+/-)-1 into the corresponding amide. Remarkably, the enantioselectivity of the enzyme immobilized on the amine was strongly dependent on the acyl donor employed. Thus, using phenylacetic acid (2), the enantioselectivity was almost negligible (1.3 favoring the S isomer), whereas using S-mandelic acid [(S)-4], the E factor reached a value of 21 (also favoring the S isomer). By using R-mandelic acid [(R)-4], we observed a different enantioselectivity (E was 3.6 favoring the R). At 4 degrees C, the E value reached a value higher than 100 when (S)-4 was used as the acyl donor. The reaction performed under these conditions allowed us to produce (2S,2'S)-N-2'-hydroxy-2'-phenyl)-2-hydroxyphenylacetamide [(2S,2'S)-7] with a diasteromeric excess higher than 98%.
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PMID:Enantioselective synthesis of phenylacetamides in the presence of high organic cosolvent concentrations catalyzed by stabilized penicillin G acylase. Effect of the acyl donor. 1517 9

The surface carboxylic groups of penicillin G acylase and glutaryl acylase were chemically aminated in a controlled way by reaction with ethylenediamine via the 1-ethyl-3-(dimethylamino-propyl) carbodiimide coupling method. Then, both proteins were immobilized on glyoxyl agarose. In both cases, the immobilization of the chemically modified enzymes improved the enzyme stability compared to the stability of the immobilized but non-modified enzyme (by a four-fold factor in the case of PGA and a 20-fold factor in the case of GA). The chemical modification presented a deleterious effect on soluble enzyme stability. Therefore, the improved stability should be related to a higher multipoint covalent attachment, involving both the lysine amino groups and also the new amino groups chemically introduced on the enzyme. Moreover, the lower pK(a) of the new amino groups permitted to immobilize the enzyme under milder conditions. In fact, the aminated proteins could be immobilized even at pH 9, while the non-modified enzymes could only be immobilized at pH over 10.
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PMID:Improved stabilization of chemically aminated enzymes via multipoint covalent attachment on glyoxyl supports. 1565 25

Considering Alcaligenes faecalis pencillin G acylase(AfPGA), which possesses the attractive characteristics for beta-lactam antibiotics conversions, the gene of PGA was cloned into an expressing vector pKKFPGA. The recombinant plasmid contained multicopy replicon(COLE 1), trc promoter, AfPGA gene, rrnB transcript terminator and ampicillin marker transformed Escherichia coli DH5alpha. As both the recombinant plasmid and the host DH5alpha had no laclq gene, the trc promoter was always active and the AfPGA could be constitutively expressed without IPTG induction in the host DH5alpha. In the shaking flask, the recombinant cell was inoculated into the fermentation medium (tryptone 10g/L, yeast extract 5g/L, MgSO4 x 7 H2O 1g, KH2 PO4 2g/L, K2HPO4 x 3H2O 5g/L, Na2HPO4 x 12H2O 7g/L, (NH4)2SO4 1.2g/L, NH4Cl 0.2 g/L, NaCl 0.1g/L, dextrin 30g/L) and cultured at 28 degrees C for 20h. The production of AfPGA reached 2,590u/L(NIPAB method), with a cell-density-specific activity of more than 300(u/L)/A600, this yield increased 432 fold higher than the native expression of Alcaligenes faecalis . Without ammonium sulphate fractionation and dialysis, the supernatant of crude extract was directly loaded on DEAE-Sepharose CL 6B column equilibrated by phosphate buffer (50mmol/L, pH7.8), and the enzyme fraction was not absorbed on the column but impurities were absorbed. Subsequently the effluent was added ammonium sulphate to 1mol/L and loaded on Butyl-Sepharose CL 4B column equilibrated by 50mmol/L phosphate buffer pH7.8-1mol/L ammonium sulphate. The enzyme was eluted as concentration of ammonium sulphate in phosphate buffer decreased to 0, PGA was eluted. After these two column chromatography, the enzyme was enriched 20 times with a 91% activity recovery. The purified enzyme had a specific activity of 68.6u/mg protein. However, the overproduction of PGA was often limited by translocation and/or periplasmic processing steps, subsequently resulted in intracellular accumulation of various types of PGA precursors and then formed inclusion bodies in the cytoplasm and/or periplasm. In this study, 5% PGA precursors formed as inclusion bodies in the cytoplasm while no inclusion bodies formed in the periplasm. It suggested most PGA precursors were transported to the periplasm and matured to active PGA and also explained why PGA gene was highly expressed in the host DH5alpha. On the other hand, inclusion bodies in the cytoplasm indicated that the maturation of PGA in the host DHSalpha was limited by the translocation step.
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PMID:[Constitutive expression and purification of Alcaligenes faecalis penicillin G acylase in Escherichia coli]. 1597

In this work, we have used supports activated with m-amino-phenylboronic groups to "reversibly" immobilize proteins under very mild conditions. Most of the proteins contained in a crude extract from E. coli could be immobilized on Eupergit C-250 L activated with phenylboronic and then fully desorbed from the support by using mannitol or SDS. This suggested that the immobilization of the proteins on these supports was not only via sugars interaction, but also by other interaction/s, quite unspecific, that might be playing a key role in the immobilization of the proteins. Penicillin acylase from E. coli (PGA) was also immobilized in Eupergit C activated with m-amino-phenylboronic groups. The enzyme could be fully desorbed with mannitol immediately after being immobilized on the support. However, longer incubation times of the immobilized preparation caused a reduction of protein elution from the boronate support in presence of mannitol. Moreover, these immobilized preparations showed a higher stability in the presence of organic solvents than the soluble enzyme; the stability also improved when the incubation time was increased (to a factor of 100). By desorbing the weakest bound enzyme molecules, it was possible to correlate adsorption strength with stabilization; therefore, it seems that this effect was due to the rigidification of the enzyme via multipoint attachment on the support.
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PMID:Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports. 1612 5

Penicillin G acylase (PGA; E.C. 3.5.1.11) is an important enzyme which has broad applications in industries of beta-lactim antibiotics production. In this study, a promising PGA gene from Alcaligenes faecalis (afpga) and another pcm gene encoding protein isoaspartate methyltransferase (PIMT) were constructed into pET43.1a((+)) and pET28a((+)), respectively. The recombinant plasmids pETAFPGA and pETPCM were transformed into the same host cell Escherichia coli BL21 (DE3). Results suggested that the two plasmids could peacefully exist in the host cell and the two genes could be efficiently expressed after induction. The product of pcm gene could function as a helper molecule for enzyme AFPGA. PIMT increased the enzymatic activities in supernatant of ferment broth (1.6 folds) and cell lysate (1.8 folds), while it did not significantly affect the expression level of penicillin G acylase.
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PMID:Enhancing enzymatic activity of penicillin G acylase by coexpressing pcm gene. 1655 Mar 78

The antioxidant and antimutagenic activity of the yeast cell-wall mannan and mannan conjugates--in particular the mannan of Saccharomyces cerevisiae (M-S.c.) and conjugates of mannan S. cerevisiae with human serum albumin (M-HSA1, M-HSA2) and the microbial enzyme penicillin G acylase (M-PGA)--were evaluated in vitro in the unicellular flagellate Euglena gracilis exposed to the genotoxic agents ofloxacin and acridine orange (AO). M-S.c., M-HSA1, M-HSA2 and M-PGA show a statistically significant, concentration-dependent protective antigenotoxic activity against both compounds. M-PGA was the most efficient inhibitor of ofloxacin- and AO-induced chloroplast DNA damage, whereas M-HSA2 and M-HSA1 were less effective and M-S.c. had the lowest antigenotoxic activity. It is suggested that different mechanisms may be involved in their protective effect--antioxidant activity in the case of ofloxacin-induced DNA damage and direct adsorption of AO on mannan conjugates as possible mechanisms of protection, based on spectrophotometric measurements. The important characteristics of yeast cell-wall mannans and mannan conjugates, such as their high water solubility, their broad spectrum of biological activity, low toxicity, stability and their antimutagenic effects via different modes of action, appear to be promising features for their practical application as antioxidants and antimutagenic agents.
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PMID:Antioxidant and antimutagenic activity of mannan neoglycoconjugates: mannan-human serum albumin and mannan-penicillin G acylase. 1667 51

Folate receptor (FR) has been proposed as a promising target for tumor drug targeting. The aim of this study was to increase the chemo-sensitivity of FR-positive cells to doxorubicin by folate-directed enzyme prodrug therapy (FDEPT). Folate conjugated penicillin-G amidase was prepared and its ability to hydrolyze N-(phenylacetyl) doxorubicin was measured by HPLC. Fluorescence and confocal image analysis revealed that Folate-PGA can be specifically delivered into FR-positive HeLa and SKOV3 tumor cells. In vitro cytotoxity assays, IC50 was reduced with N-(phenylacetyl) doxorubicin versus doxorubicin for HeLa (3.1-fold reduction; p<0.001) and SKOV3 (3.3-fold reduction; p<0.001) when Folate-PGA was specifically bound to the cells. Complete activation was confirmed in HeLa and SKOV3 cells pretreated with free folic acid (1 mM), where the combination of N-(phenylacetyl) doxorubicin with Folate-PGA did not show any significant cell toxicity to the IC50 of doxorubicin. Pharmacokinetic clearance and biodistribution studies in vivo showed that 125I-Folate-PGA was cleared from blood within 24 h and had significantly higher tumor uptake compared to 125I-PGA (p<0.05). These results demonstrate that the FDEPT approach may be a potential promising strategy to improve chemotherapy-resistant cancers therapeutic ratio and warranted future studies.
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PMID:Increase of doxorubicin sensitivity for folate receptor positive cells when given as the prodrug N-(phenylacetyl) doxorubicin in combination with folate-conjugated PGA. 1688 59

Several ionic liquids were used as reaction media for penicillin G acylase catalysis. In all the assayed ionic liquids, [bmim]PF6 proved good media for PGA-catalyzed hydrolysis. A novel [bmim]PF6/water two-phase system is provided for 6-aminopenicillanic acid (APA) production, which will be more benefical than aquous batch systems used widely in industrial production of APA.
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PMID:Penicillin acylase catalysis in the presence of ionic liquids. 1708 15

The amidase reaction of trypsin, which is a member of the serine proteinase family, is accelerated by its complexation with block ionomers containing a polycarboxylate block, such as PEG-PAA, PEG-PGA, or PEG-PMA. PEG-PAA and PEG-PGA had similar effects, causing an increase in the k(cat) value and a shift in the pH profile to a lower pH region. On the other hand, PEG-PMA showed not only an increase in the k(cat) value, but also a decrease in the activation energy; however, there was no shift in the pH dependence of the initial reaction rate. Such differences might be induced by the difference in pK(a) values of the polycarboxylate block in block ionomers.
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PMID:Effect of polycarboxylate blocks on the amidase activity of trypsin through complexation with PEG/polycarboxylate block ionomers. 1737 Feb 72

The objective of this work was to develop a hydrogel-coated monolith for the entrapment of penicillin G acylase (E. coli, PGA). After screening of different hydrogels, chitosan was chosen as the carrier material for the preparation of monolithic biocatalysts. This protocol leads to active immobilized biocatalysts for the enzymatic hydrolysis of penicillin G (PenG). The monolithic biocatalyst was tested in a monolith loop reactor (MLR) and compared with conventional reactor systems using free PGA, and a commercially available immobilized PGA. The optimal immobilization protocol was found to be 5 g l(-1) PGA, 1% chitosan, 1.1% glutaraldehyde and pH 7. Final PGA loading on glass plates was 29 mg ml(-1) gel. For 400 cpsi monoliths, the final PGA loading on functionalized monoliths was 36 mg ml(-1) gel. The observed volumetric reaction rate in the MLR was 0.79 mol s(-1) m(-3) (monolith). Apart from an initial drop in activity due to wash out of PGA at higher ionic strength, no decrease in activity was observed after five subsequent activity test runs. The storage stability of the biocatalysts is at least a month without loss of activity. Although the monolithic biocatalyst as used in the MLR is still outperformed by the current industrial catalyst (immobilized preparation of PGA, 4.5 mol s(-1) m(-3) (catalyst)), the rate per gel volume is slightly higher for monolithic catalysts. Good activity and improved mechanical strength make the monolithic bioreactor an interesting alternative that deserves further investigation for this application. Although moderate internal diffusion limitations have been observed inside the gel beads and in the gel layer on the monolith channel, this is not the main reason for the large differences in reactor performance that were observed. The pH drop over the reactor as a result of the chosen method for pH control results in a decreased performance of both the MLR and the packed bed reactor compared to the batch system. A different reactor configuration including an optimal pH profile is required to increase the reactor performance. The monolithic stirrer reactor would be an interesting alternative to improve the performance of the monolith-PGA combination.
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PMID:Hydrogel coated monoliths for enzymatic hydrolysis of penicillin G. 1842 49


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