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Showing papers on "Penicillin amidase published in 2006"


Journal ArticleDOI
TL;DR: This review focuses specially on aspects of the reactions' kinetics that may affect the performance of the enzymatic reactor.

86 citations


Journal ArticleDOI
TL;DR: In this paper, a composite support polyethyleneimine (PEI) was grafted onto the surface of silica gel particles via the coupling effect of γ-chloropropyl trimethoxysilane (CP), and the novel composite support PEI/SiO2 was prepared.

47 citations


Journal ArticleDOI
TL;DR: The immobilization on ionic polymeric beds allowed a significant increase in enzyme stability against the inactivation and inhibitory effects of organic solvents, very likely by the promotion of a certain partition of the organic solvent out of the enzyme environment.

45 citations


Journal ArticleDOI
TL;DR: In this article, porous hollow silica nanotubes (PHSNTs) were synthesized via a sol-gel route using nano-sized needle-like CaCO3 the inorganic templates were employed as a support for immobilization of penicillin G acylase (PGA) biocatalyst.
Abstract: In this study, porous hollow silica nanotubes (PHSNTs) synthesized via a sol–gel route using nano-sized needle-like CaCO3 the inorganic templates were employed as a support for immobilization of penicillin G acylase (PGA) biocatalyst. The produced PHSNTs were characterized by BET and transmission electron microscopy (TEM). Effect of various factors such as loading temperature and ratio of carries to free PGA (g/mL) on the catalytic activity of the immobilized PGA was also investigated by unitary factor testing method. The results show that under optimized conditions the relative loading amount and the total activity yield of immobilized enzyme (IME) amounts to 97.20% and 88.80%, respectively. Several advantages, i.e. the rapid immobilization of PGA onto PHSNTs, the high tolerability to the pH, the less sensitivity to the temperature and the improved storage stability render PHSNTs potential support materials for enzyme immobilization.

45 citations


Journal ArticleDOI
TL;DR: 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.
Abstract: 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.

34 citations


Journal ArticleDOI
TL;DR: The best carriers for penicillin acylase immobilization are crosslinked with DEGDA as they display preferential sorption and then immobilization of Penicillin Acylase, what leads to highly active preparations.

33 citations


Journal ArticleDOI
TL;DR: In this article, four polysaccharides (dextran, mannan, potassium pectate and sodium alginate) were used for derivatization of penicillin G acylase (PGA).

32 citations


Journal ArticleDOI
TL;DR: In this article, a recombinant strain Bacillus subtilis WB600 (pMA5) was used to produce Alcaligenes faecalis penicillin G acylase (PGA).

26 citations


Journal ArticleDOI
TL;DR: Penicillin G acylase from Kluyvera citrophila immobilized on Amberzyml was used for enantioselective hydrolysis of N-phenylacetylated-dl-tert-leucine to produce l-Tle to investigate the effects of various organic cosolvents on hydrolytic activity.
Abstract: Penicillin G acylase (PGA) from Kluyvera citrophila immobilized on Amberzyml was used for enantioselective hydrolysis of N-phenylacetylated-DL-tert-leucine (N-Phac-DL-Tle) to produce L-tert-leucine (L-Tle). The effects of various organic cosolvents on hydrolysis of N-Phac-DL-Tle have been investigated in aqueous-cosolvent medium. It was founded that the rate of PGA-catalyzed reaction was significantly affected by the presence of 2% (v/v) organic cosolvent concentration. The initial rate fell with increasing logP of the cosolvent, but for logP values less than -0.24 the rate was faster than in purely aqueous medium. Additionally, the relative rate increases with the increase of dielectric constant (epsilon) of organic cosolvents. The yields of L-Tle in all aqueous-cosolvent systems were above 95% with the enantiomeric excess (ee) of >99%.

24 citations


Journal ArticleDOI
TL;DR: The activity of penicillin G acylase from Alcaligenes faecalis increased 7.5-fold when cells were permeabilized with 0.3% (w/v) CTAB and about 65% enzyme activity was retained at the end of the 31th cycle.
Abstract: The activity of penicillin G acylase from Alcaligenes faecalis increased 7.5-fold when cells were permeabilized with 0.3% (w/v) CTAB. The treated cells were entrapped by polyvinyl alcohol crosslinked with boric acid, and crosslinked with 2% (v/v) glutaraldehyde to increase the stability. The conversion yield of penicillin G to 6-aminopenicillanic acid was 75% by immobilized system in batch reaction. No activity was lost after 15 cycles and about 65% enzyme activity was retained at the end of the 31th cycle.

24 citations


Journal ArticleDOI
TL;DR: Enzymatic hydrolysis of penicillin G in a discrete semi-batch mode, which simulates a semi-continuous process, envisages a completely eco-friendly, sustainable and efficient process for production of 6-aminopenicillanic acid.
Abstract: Enzymatic hydrolysis of penicillin G by immobilized penicillin acylase in a nonionic surfactant mediated cloud point system was presented. The effect of the operation parameters on equilibrium pH of this enzymatic hydrolysis process without pH control was examined. A relatively high equilibrium pH in cloud point system without pH control can be obtained. The feasibility of recycling utilization of the nonionic surfactant, a novel green solvent, was also investigated experimentally. Enzymatic hydrolysis of penicillin G in a discrete semi-batch mode, which simulates a semi-continuous process, envisages a completely eco-friendly, sustainable and efficient process for production of 6-aminopenicillanic acid.

Journal ArticleDOI
TL;DR: A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri of industrial applicability and the efficiency of the immobilized rPACP.rett was evaluated by studying the kinetically controlled synthesis of β‐lactam antibiotics (cephalexin), which is a crucial parameter for the feasibility of the process.
Abstract: A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri(rPAC(P.rett))of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEP4 gene encoding for proteinase A, resulting in an increased rPAC(P.rett) activity of approximately 40% (3.8 U/mL vs. 2.7 U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPAC(P.rett) purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPAC(P.rett) expressed in P.pastoris were fully exploited and emphasized through the covalent immobilization of rPAC(P.rett). The enzyme was immobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPAC(P.rett) performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability >3 years and specific activity ranging from 237 to 477 U/g (dry) based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPAC(P.rett) was finally evaluated by studying the kinetically controlled synthesis of beta-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process.

Journal ArticleDOI
TL;DR: A new approach for predicting the selectivity of penicillin G amidase (PGA) is described, which allows one to develop a whole predicting model in a few hours, once a small set of experimental data is made available.
Abstract: A new approach for predicting the selectivity of penicillin G amidase (PGA) - expressed as k cat / K M - is here described. Regression models were constructed correlating the experimentally determined k cat /K M of a limited number of substrates to molecular descriptors calculated by using methods generally employed in drug discovery for quantitative structure-activity relationship (3D-QSAR methods). Two different methods for the calculation of molecular descriptors have been tested, namely GRIND and Volsurf. The real predictions, made on molecules not used for constructing the models, had an accuracy sufficient for being useful in the experimental practice. Both approaches led to models able to predict substrate selectivity even without modelling the enzyme-substrate complex, whereas the prediction of enantioselectivity was feasible only by combining the GRIND approach with the conformational analysis of the substrates inside the enzyme's active site. The present approach represents an actual alternative to screening procedures since it allows one to develop a whole predicting model in a few hours, once a small set of experimental data is made available.

Journal ArticleDOI
TL;DR: The modeled structure of penicillin acylase from Alcaligenes faecali (AFPGA) was constructed by comparative modeling with the Modeller program and was the first structure-based genetic modification of AFPGA.
Abstract: The modeled structure of penicillin acylase from Alcaligenes faecali (AFPGA) was constructed by comparative modeling with the Modeller program. Candidate positions that could be replaced with cysteine were estimated by scanning the modeled structure of AFPGA with the program MODIP (modeling disulfide bond in protein). The mutant Q3C/P751C had a higher optimum temperature by three degrees than that of the wild type AFPGA. The half life of the double mutant Q3C/P751C at 55 degrees C was increased by 50%. To our knowledge, this was the first structure-based genetic modification of AFPGA.

Journal ArticleDOI
TL;DR: The present work aimed to improve the production of penicillin G acylase (PGA) and reduce the β‐lactamase activity through acridine orange (AO) induced mutation in Escherichia coli.
Abstract: Aims: The present work aimed to improve the production of penicillin G acylase (PGA) and reduce the β-lactamase activity through acridine orange (AO) induced mutation in Escherichia coli. Methods and Results: Three wild E. coli strains BDCS-N-FMu10, BDCS-N-S21 and BDCS-N-W50, producing both the enzymes PGA and β-lactamase were treated by AO. Minimum inhibitory concentration of AO was 10 μg ml−1 and it was noted that bacterial growth was gradually suppressed by increasing the concentration of AO from 10 to 100 μg ml−1. The highest concentration that gave permissible growth rate was 50 μg ml−1. The isolated survivals were screened on the bases of PGA and β-lactamase activities. Among the retained mutants, the occurrence of β-lactamase deficient ones (91%) was significantly higher than penicillin acylase deficient ones (27%). Conclusions: In seven of the mutants, PGA activity was enhanced with considerable decrease in β-lactamase activity. One of the mutant strains (BDCS-N-M36) exhibited very negligible expression of β-lactamase activity and twofold increase in PGA activity [12·7 mg 6-amino-penicillanic acid (6-APA) h−1 mg−1 wet cells] compared with that in the wild-type strain (6·3 mg 6-APA h−1 mg−1 wet cells). Significance and Impact of the Study: The treatment of E. coli cells with AO resulted in mutants with enhanced production of PGA and inactivation of β-lactamase. These mutants could be used for industrial production of PGA.

Journal ArticleDOI
TL;DR: Results suggested that the two plasmids could peacefully exist in the host cell and the two genes could be efficiently expressed after induction, and the product of pcm gene could function as a helper molecule for enzyme AFPGA.
Abstract: Penicillin G acylase (PGA; E.C. 3.5.1.11) is an important enzyme which has broad applications in industries of β-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.

Journal ArticleDOI
TL;DR: TFH is able to degrade specific polyesters such as poly (ethylene terephthalate) (PET) or poly (butylene tereplet) (PBT), which are hither toregarded as 'non-biodegradable' plastics.
Abstract: DSM43793(TFH)) were used to further investigate and improve thesystem. Penicillin amidase is applied in the synthesis ofsemisynthetic penicillins. TFH is able to degrade specificpolyesters such as poly (ethylene terephthalate) (PET) orpoly (butylene terephthalate) (PBT), which are hither toregarded as 'non-biodegradable' plastics [3].

Journal ArticleDOI
TL;DR: Penicillin G amidase activity of MNNG-37 appeared during an early stage of growth, whereas PCSIR-102 did not exhibit PGA activity due to the presence of penicillinase enzyme which inhibits the activity of enzyme PGA.
Abstract: The penicillin G amidase (PGA) activity of a parent strain of E. coli (PCSIR-102) was enhanced by chemical mutagenization with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). After screening and optimization, a penicillinase deficient mutant (MNNG-37) was isolated and found effective for the production of penicillin G amidase as compared to the parent strain of E. coli (PCSIR-102). Penicillin G amidase activity of MNNG-37 appeared during an early stage of growth, whereas PCSIR-102 did not exhibit PGA activity due to the presence of penicillinase enzyme which inhibits the activity of enzyme PGA. However, MNNG-37 gave a three-fold increase in enzyme activity (231 IU mg−1) as compared to PCSIR-102 (77 IU mg−1) in medium containing 0.15 and 0.1% concentrations of phenylacetic acid, respectively which was added after 6 h of cultivation. The difference in K m values of the enzyme produced by parent strain PCSIR-102 (0.26 mM) and mutant strain MNNG-37 (0.20 mM) is significant (1.3-fold increase in K m value) which may show the superiority of the latter in terms of better enzyme properties.

Patent
23 Aug 2006
TL;DR: In this paper, the authors performed culture in shake flask and extending fermenting tank with gene engineering bacteria of fecal alkaligenous bacillus penicillin amidase as target.
Abstract: The present invention performs culture in shake flask and extending fermenting tank with gene engineering bacteria of fecal alkaligenous bacillus penicillin amidase as target. Under proper fermentation condition, the shake flask has fecal alkaligenous bacillus penicillin amidase yield of 54726 U/L and the extending culture has yield up to 77181 U/L (PDAB process). The fecal alkaligenous bacillus penicillin amidase has specific activity up to 11.249 U/mg proteins, and may be immobilized.