• Journal of Microbiology and Biotechnology
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• v.7 no.3
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• pp.194-196
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• 1997

Using the polyclonal antibody for Xanthomonas citri ampicillin acylase raised in Pseudomonas-free Balb/c mice, the immunochemical similarity of several types of penicillin acylases including Erwinia aroideae penicillin V acylase, Escherichia coli penicillin G acylase, Pseudomonas melanogenum and Acetobacter turbidans ampicillin acylases, and Pseudomonas cephalosporin acylase was examined. Among tested, only P. melanogenum ampicillin acylase showed the cross-reactivity with the antibody.

• Microbiology and Biotechnology Letters
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• v.8 no.1
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• pp.33-39
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• 1980

In order to invertigate penicillin acylase produced by a strain of Genus Escherichia, 47 strains of Genus Escherichia were isolated and examined to the extend of the inactivation of penicillins and the ability of 6-amino penicillanic acid (6-APA) production. Among them, 12 strains were found to produce penicillin acylase and to form 6-APA. The strain No. 11 of the isolates was selected to be the most excellent one producing the acylase. Optimum culture conditions for the production of the acylase of the strain were found as follows : pH at 7.6~8.0, time on 18 hrs, temperature at 34 to 38$^{\circ}C$. And effective levels of the medium were found to be contained 0.3% phenylacetic acid, 1.0％ yeast extract, 1.0％ peptone and 0.3％ L-glutamate. And, the production of the acylase by the isolate was strongly inhibited by 1% glycerol and the growth was remarkably retarded by the addition of 1.0% n-butylacetate. The acylase was extracted from the isolate and the crude enzyme of the acylase was prepared and the characteristies of the enzyme were primary examined in optimum pH, temperature, stabilities and reaction time.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.62-67
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• 2004

The biosynthesis of cephalexin was carried out in the aqueous two-phase systems using penicillin acylase as a catalyst, and 7-aminodeacetoxicephalosporanic acid (7-ADCA) and phenylglycine methyl ester (PGME) as substrates. 20% PEG400-l7.5% ${(NH_4)}_2SO_4$ containing 0.5 M NaCl and 1.5 M methanol aqueous two-phase systems (ATPS) were selected as reaction medium, and 53% product yield was obtained using immobilized penicillin acylase as a catalyst. 20% PEG400-l5% $MgSO_4$ ATPS was also used for the synthesis of cephalexin, and 60-62% product yield was obtained by using free penicillin acylase as a catalyst. When batch reactions were repeated in the ATPS, the cephalexin yields decreased during the reactions due to deactivation, loss, and product inhibition of penicillin acylase. The effect of different ratio of phenylglycine methyl ester to 7-ADCA on the product yield was investigated, and high cephalexin yield was obtained at a high molar ratio.

• Korean Journal of Microbiology
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• v.32 no.3
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• pp.215-221
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• 1994

The complete nucleotide sequence of the cloned pga gene encoding the penicillin G acylase of Bacillus megaterium ATCC 14945 and its 5'- and 3'-flanking regions was determined. The sequence revealed only one large open reading frame (2,406 hp) of the penicillin G acylase (pga) gene. Upstream from ATG of the pga gene, there was a putative ribosome binding site, Shine-Dalgarno sequence. The promoter-like structure, - 10 and - 35 sequences, was also found. Following the stop codon, TAG, a structure reminiscent of the E. coli rho-independent transcription terminator was present. The amino acid sequence was deduced from the nucleotide sequence. The molecular mass of the polypeptide was 91,983 Da. There was a potential signal sequence in its amino-terminal region. A comparison of its deduced amino acid sequence with other characterized penicillin G acylases and the result of SDS-polyacrylamide gel electrophoresis of the purified enzyme showed that a precursor polypeptide of 92 kDa was processed into two dissimilar ${\alpha}$ and ${\beta}$-subunits of 25 and 61 kDa.

• Korean Journal of Microbiology
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• v.21 no.2
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• pp.95-102
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• 1983

The penicillin G acylase(pga) gene was cloned in the vector plasmid pKM $300(Ar^r,\;Tc^r,\;6.33kb)$ for the study of the structure and expression of the pga gene. This recombinant plasmid pPAKS-1 DNA(24.5 Kb) was cleaved into 2 fragments by restriction enzyme Eco R1.1fragment by BamH1, 4fragments by Hind III, and 2 fragments by Pst I. The pga gene was located on the Eco R1.Hind III-C fragement of pPAKS-1. The recombinant plasmids pPAKS-1 and pPAKS-2, in which the Hind III-B and Hind III-D fragments pPAKS-1 are deleted, are characterized. The results are summarized as follows : 1. Doubling times of bacterial strain bearing pPAKS-1 and pPAKS-2 are 90 and 60 minutes, respectively. 2. pPAKS-1 and pPAKS-2 are present at about 16-32 and 70 copies per cell, respectively, are 0.66 and 5.5 units, respectively, which represent 2-fold and 20-fold higher enzyme 4. pPAKS-1 is very unstable, but pPAKS-2 is stable.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.231-236
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• 2004

The inexpensive large-scale production of pure PGA (Penicillin G Acylase) has been a commercial goal. PGA has been used as a model enzyme in the development of simple one-step purification methods. In this study, the purification of poly-His tagged PGA protein secreted into the periplasmic space was carried out by using immobilized metal-ion affinity chromatography (IMAC). The PGA gene was obtained from E. coli ATCC 11105. Codons encoding histidines were fused at the C-terminus of the PGA gene by PCR. E. coli JM109 harboring pPGA-HIS6 vector produced active his-tagged acylases in the presence of lac promoter during cultivation at $26^{\circ}C$. The maximum specific activity of the acylase purified by using one-step chromatography after osmotic shock was 38.5 U/mg and was recovered with the yield of 70％. Both 23 kDa ($\alpha$) and 62 kDa ($\beta$) subunits were recovered by using IMAC with just C-terminus tagging of the $\beta$ subunit. The purification of the periplasmic fraction by osmotic shock and that of purified acylase was increased by 2.6-fold and 19-fold, respectively, compared to the crude extract.

• Journal of Microbiology and Biotechnology
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• v.26 no.5
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• pp.829-836
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• 2016

Penicillin G acylase (PGA) was immobilized on magnetic Fe₃O₄@chitosan nanoparticles through the Schiff base reaction. The immobilization conditions were optimized as follows: enzyme/support 8.8 mg/g, pH 6.0, time 40 min, and temperature 25 ℃. Under these conditions, a high immobilization efficiency of 75% and a protein loading of 6.2 mg/g-support were obtained. Broader working pH and higher thermostability were achieved by the immobilization. In addition, the immobilized PGA retained 75% initial activity after ten cycles. Kinetic parameters V_max and K_m of the free and immobilized PGAs were determined as 0.113 mmol/min/mg-protein and 0.059 mmol/min/mg-protein, and 0.68 mM and 1.19 mM, respectively. Synthesis of amoxicillin with the immobilized PGA was carried out in 40% ethylene glycol at 25 ℃ and a conversion of 72% was obtained. These results showed that the immobilization of PGA onto magnetic chitosan nanoparticles is an efficient and simple way for preparation of stable PGA.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.331.3-332
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• 2002

A novel penicillin G acylase (PGA)-producing bacterial strain was isolated from soil by using the Serratia marcescens overlay technique. The isolated strain was identified as Leclercia adecarboxylata based on the analyses of the biochemical characteristics (API 20E). the cellular fatty acid profile. and the 16S rDNA sequences. The gene encoding the PGA (pac gene) was cloned into the pHSG399 vector and the recombinant E. coliHB101 clones harboring the pac gene were isolated on agar plates containing phenylacetyl-L -leucine and penicillin G. (omitted)

• YAKHAK HOEJI
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• v.27 no.3
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• pp.185-205
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• 1983

Penicillins and cephalosporins are biosynthesized from L-.alpha.-aminoadipic acid, L-cysteine and L-valine. A tripeptide, LLD-$\delta$-($\alpha$-aminoadipyl)cysteinylvaline(LLD-ACV) was isolated from fermentation broths of Cephalosporium acremonium as well as of Penicillium chrysogenum and it was proved that the LL-$\delta$-($\alpha$-aminoadipyl cysteine was formed first in mycelia, to which valine would be connected to give LLD-ACV. However, several points are still unsolved; first, what mechanism is involved in the configurational change from L-valine to D-valine, second, what kind of cyclization mechanism gives a $\betha$-lactam ring and a thiazolidine ring and third, what is the pathways for the ring expansion from penicillins to cephalosporins. At present, it seems clear that LLD-ACV is cyclized to give isopenicillin N, which is transformed to penicillin N and further to cepbalosporin C. Other hydrophobic penicillins, including benzyl penicillin and penicillin V, are formed from isopenicillin N by acyl-exchange reactions catalyzed by penicillin transferase, rather than by acylation reaction on 6-aminopenicillanic acid(6-APA), which was isolated from the fermentation broth of P. chrysogenum and which would be formed by hydrolysis of $\delta-(\alpha$-amincadipyl)amido moiety at the C-6 position in isopenicillin N or penicillin N by penicillin acylase. Acylation of 6-APA is catalyzed also by penicillin acylase, but the reaction is proved not to be involved in penicillin biosynthesis. Understanding the biosynthesis of penicillins and cephalsoporins would provide solutions to increase in fermentation yields of penicillins, especially of cephalosporins and a solution to biological production of 7-aminocepbalosporanic acid (7-ACA) which is of importance in pharmaceutical industry. Still regulation mechanisms in penicillin and cephalosporin biosynthesis are unveiled at all.

• Journal of Microbiology and Biotechnology
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• v.29 no.6
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• pp.913-922
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• 2019

Magnetic $Ni_{0.7}Co_{0.3}Fe_2O_4$ nanoparticles that were prepared via the rapid combustion process were functionalized and modified to obtain magnetic $Ni_{0.7}Co_{0.3}Fe_2O_4@SiO_2-CHO$ nanocomposites, on which penicillin G acylase (PGA) was covalently immobilized. Selections of immobilization concentration and time of fixation were explored. Catalytic performance of immobilized PGA was characterized. The free PGA had greatest activity at pH 8.0 and $45^{\circ}C$ while immobilized PGA's activities peaked at pH 7.5 and $45^{\circ}C$. Immobilized PGA had better thermal stability than free PGA at the range of $30-50^{\circ}C$ for different time intervals. The activity of free PGA would be 0 and that of immobilized PGA still retained some activities at $60^{\circ}C$ after 2 h. $V_{max}$ and $K_m$ of immobilized PGA were 1.55 mol/min and 0.15 mol/l, respectively. Free PGA's $V_{max}$ and $K_m$ separately were 0.74 mol/min and 0.028 mol/l. Immobilized PGA displayed more than 50% activity after 10 successive cycles. We concluded that immobilized PGA with magnetic $Ni_{0.7}Co_{0.3}Fe_2O_4@SiO_2-CHO$ nanocomposites could become a novel example for the immobilization of other amidohydrolases.