• Journal of Microbiology and Biotechnology
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• 제7권3호
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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.

• 한국미생물·생명공학회지
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• 제8권1호
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• pp.33-39
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• 1980

6-APA의 생산에 사용할 Penicillin acylase를 생산하는 야생균주를 분리하여 생리화학적특성을 조사하여 Escherichia속을 동정하였다. Penicillin acylase 생산성이 가장 높은 균주를 선별하여 배양조건을 조사하고 균체를 생산하여 효소를 추출하여 효소의 반응특성을 조사한 결과는 다음과 같다. 1. 분리한 121주의 생리화학적 특성에 의해 47주가 Escherichia 속으로 동정되고 이들중 12주가 penicillin acylase 활성을 나타냈다. 2. 12주중 가장 강력한 P. acylase 활성을 나타낸 균주가 Escherichia No.11 이였다. 3. 이 균주의 효소생산조건은 탄소원은 0.3%-phenylacetic acid, 질소원은 1%-peptone과 1%-yeast extract, amino 산으로는 0.3%, L-glutamic acid가 영향을 주었다. 그리고 배지의 pH는 7.6~8.0, 배양온도는 34~38$^{\circ}C$, 배양시간은 18시간이 최적 생산조건 이였다. 4. 효소는 균체를 초음파에 의해 추출하여 원심 분리하여 상등액을 $0^{\circ}C$에서 보존하여 효소원으로 하였다. 5. 추출효소의 반응특성은 최적 반응온도는 38$^{\circ}C$, PH는 8.0이였고, 1% penicillin G를 기질로 37$^{\circ}C$에서 반응시켰을 때 4시간의 반응에서 이론식의 56%가 6-APA로 변화되었다.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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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.

• 미생물학회지
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• 제32권3호
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• pp.215-221
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• 1994

Bacillus megaterium ATCC 14945의 penicillin G acylase 유전자의 염기배열을 결정하였다. 이 유전자에는 2,406 염기쌍으로 이루어진 하나의 open reading frame이 존재하는데, 개시코돈의 5' 위쪽에서 Shine-Dalgarno 배열과 promoter로 여겨지는 부분을 발견하였으며, 종결코돈의 3' 아래쪽에서 rho-independent한 전사종결체와 dby사한 구조를 발견하였다. 염기배열로부터 폴리펩티드의 아미노산 배열을 유추하였다. 이 폴리펩티드의 분자량은 91,983 Da이었으며, 아미노 말단 부이에 signal sequence가 존재하였다. 이 아미노산 배열을 여러 다른 penicillin G acylase의 아미노산 배열과 비교하고 분리 정제한 효소를 SDS-polyacrylamide gel 전기영동으로 분석한 결과로부터 이 효소는 92kDa의 전구체로 해독된 후 processing 과정을 거쳐 각각 25kDa과 61kDa의 ${\alpha}$-, ${\beta}$-단위체로 구성됨을 알 수 있었다.

• 미생물학회지
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• 제21권2호
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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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• 제14권2호
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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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• 제26권5호
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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.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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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)

• 약학회지
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• 제27권3호
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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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• 제29권6호
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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.