• Korean Journal of Microbiology
• /
• v.32 no.3
• /
• pp.232-237
• /
• 1994

Microorganism producing glutaryl 7-aminodeacetoxycephalosporanic acid (GL-7-ADCA) acylase was screened from soil. The microorganism was identified as Alcaligenes sp. J-421 by its morphology and biochemical properties. Cultural conditions of Alcaligenes sp. J-421 were investigated for the production of GL-7-ADCA acylase. Optimum medium composition was 1% glucose, 1% beef extract, 0.5% yeast extract, 0.2% monosodium L-glutamate, 0.1% glutaric acid, 0.2% NaCl, 0.5% $K_2$ $HPO_4$, and 0.05% $CuSO _4{\cdot}5H_2O$. Optimum cultivation conditions for the production of the enzyme in 5 l jar fermentor were $37^{\circ}C$, tip speed 300 rpm, aeration 1 vvm. Optimum reaction pH of the enzyme was 8.0 and the enzyme was stable at pH7.0-11.0.

• Journal of Microbiology and Biotechnology
• /
• v.11 no.2
• /
• pp.329-332
• /
• 2001

It has been known that, in enzymatic synthesis of cephalexin, the conversion yield was reduced by high loading of ampicillin acylase. In order to elucidate this phenomena, pH-controlled synthesis of cephalexin was examined using a purified Acetobacter turbidans acylase. When the pH of the reaction mixture was maintained at $6.20{\pm}0.04$, the reduction of the maximal conversion rate was not observed even with high enzyme loading. The kinetic parameters also suggest that pH drop during the enzymatic synthesis of cephalexin was mainly attributed to the rapid hydrolysis of D-${\alpha}$-phenylglycine methyl ester to D-${\alpha}$-phenylglycine, rather than the disappearance of 7-amino-3-deacetoxycephalosporanic acid for cephalexin synthesis. At higher molar ratio of two substrates, [D-${\alpha}$-phenylglycine methyl ester]/[7-amino-3-deacetoxycephalosporanic acid], the conversion rate was also elevated under pH-controlled enzymatic synthesis, which implies that the main reason for the pH drop is due to the production of D-${\alpha}$-phenylglycine methyl easter, the effect of a water-methanol cosolvent system on the ester, the effect of a water-methanol cosolvent system on the conversion profile was also examined. Even the though the conversion rate was increased in 10% methanol solution, a higher than 16% methanol in the reaction mixture caused an inactivation of enzyme.

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

• Korean Journal of Microbiology
• /
• v.21 no.2
• /
• pp.95-102
• /
• 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
• /
• v.6 no.6
• /
• pp.375-380
• /
• 1996

The gene coding for glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase was cloned from Pseudomonas sp. GK16 and some of its characteristics were analyzed. The complete nucleotide sequence revealed that the putative open reading frame is 2160 bases long and encodes 720 amino acids. By SDS-PAGE three proteins, approximately corresponding to 70, 54 and 16 kDa of molecular weight, were detected in E. coli cells carrying pGAP18. The largest protein should be a precursor which is not processed yet, while the other two proteins must be derived from the precursor by the proteolytic processing.

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

• The Microorganisms and Industry
• /
• v.20 no.1
• /
• pp.46-49
• /
• 1994

• Journal of Microbiology and Biotechnology
• /
• v.6 no.5
• /
• pp.336-339
• /
• 1996

Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. SY-77-1 was immobilized with oxiran acrylic beads for the production of 7-aminocephalosporanic acid (7-ACA) from glutaryl 7-aminocephalosporanic acid (GL 7-ACA). The immobilized enzyme maintained its activity at a constant level for 7 days, but lost 30$%$ of its activity after 20 days. Optimal reaction conditions for the synthesis of 7-ACA were found to be $30^{\circ}C$ and pH 8.0 using the immobilized enzyme. For the economic production of 7-ACA, substrate and enzyme concentrations were optimized to 60 mM and 0.5 g wet weight per 10 $m\ell$ of reaction volume, respectively. Under optimized conditions, 50 mM 7-ACA was produced from 60mM GL 7-ACA within 8 h, resulting in a conversion yield of 83$%$.

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

• Microbiology and Biotechnology Letters
• /
• v.27 no.2
• /
• pp.96-101
• /
• 1999

Various side-chains are introduced to the 7-amino position of 7-aminocepha-losporanic acid (7-ACA) to make semi-synthetic cephalosporin antibiotics. In order to convert cephalosporin C (CPC) to 7-ACA, two enzymatic reactions are generally imployed. Glutary1-7-aminocephalosporanic acid (Gl-7-ACA) acylase is involved in the second step where the reaction intermediate, Gl-7-ACa is converted into 7-ACA. It was recently reported that CPC amidase can convert CPC directly into 7-ACA in a single enzymatic reaction. A study was undertaken to screen microorganisms conferring enzyme activity to convert Gl-7-ACA or CPC into 7-ACA by one or two enzymatic reactions. In order to screen the microorganisms rapidly, a non-$\beta$-lactam model compund, glutaryl-$\rho$-nitroanilide, was utilized in an early stage, thereafter the selected microorganisms were examined with real substrates. One microorganism exhibiting both Gl-7-ACA acylase and CPC amidase activities was obtained by the colorimetry method and HPLC assay, and was identified as a strain of Serratia species, designated as Serratia sp. N14.4. The optimal fermentation conditions for Serratia sp. N14.4 was pH9.0 and 3$0^{\circ}C$.