• 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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• 제50권4호
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• pp.548-556
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• 2022

Quorum quenching 활성을 나타내는 acylase 효소는 생물막 형성에 중요한 영향을 미친다. 본 연구에서는 acylase 처리 조건(acylase 처리 시기 및 기간)이 다종 생물막(multispecies biofilm) 형성에 미치는 영향을 규명하였다. 서로 다른 속(genus)에 속하는 9종의 박테리아로 구성된 컨소시엄을 사용하여 acylase 처리 조건에 따른 10가지 episode에서 다양한 acylase 농도(1, 5, 10, 20, 50 mg·l^-1)에 따라 5일 동안 생물막을 형성시켰다. 각 농도별로 5일 동안 acylase를 처리한 조건에서 acylase의 농도가 증가함에 따라 생물막 형성은 억제되었다(linear regression, Y = -0.05· x + 2.37, p < 0.05, R² = 0.88). 모든 acylase 농도 조건에서 acylase를 생물막이 형성된 후에 처리한 경우(L1-L4)에 비해서 생물막 형성 시작 단계에 처리한 경우(B1-B4) 생물막 형성이 더 효과적으로 억제되었다(p < 0.05). ANOVA 결과에 따르면 acylase 10 mg·l^-1 이상 농도에서 acylase 처리 기간(period)은 acylase 처리 시기(application timings, beginning or later)에 상관없이 생물막 형성에 영향을 미쳤다(p < 0.05). 각 농도별 처리 시기(L1-L4 또는 B1-B4)에서 처리 기간과 생물막 형성 사이의 선형 회귀 분석 결과에 따르면 acylase 10 mg·l^-1 이상 농도에서 acylase 처리 기간이 증가함에 따라 생물막 형성은 억제되었다(p < 0.05, 20 mg·l^-1 농도의 L1-L4 제외). 시간에 따른 생물막 형성 결과에 따르면 모든 10가지 episode에서 생물막은 시간에 따라 점차 증가했으며(p < 0.05), 배양 시간과 생물막 형성 사이의 선형 회귀 분석 기울기 값은 acylase를 생물막 형성 시작 단계에 처리했을 때 더 낮게 나타났다(p < 0.05). 본 연구 결과는 생물막 형성 억제에 대한 acylase의 처리 시기 및 기간의 중요성을 시사한다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권6호
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• pp.510-515
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• 2005

Pseudomonas cepacia BY21 was found to produce glutaryl acylase that is capable of deacylating glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) to 7-aminocephalosporanic acid (7-ACA), which is a starting material for semi-synthetic cephalosporin antibiotics. Amino acids of the reported glutaryl acylases from various Pseudomonas sp. strains show a high similarity (>93% identity). Thus, with the known nucleotide sequences of Pseudomonas glutaryl acylases in GenBank, PCR primers were designed to clone a glutaryl acylase gene from P. cepacia BY21. The unknown -subunit gene of glutaryl acylase from chromosomal DNA of P. cepacia BY21 was cloned successfully by PCR. The -subunit amino acids of P. cepacia BY21 acylase (GenBank accession number AY948547) were similar to those of Pseudomonas diminuta KAC-1 acylase except that Asn408 of P. diuminuta KAC-1 acylase was changed to Leu408.

• Journal of Microbiology
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• 제37권4호
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• pp.200-205
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• 1999

7-Aminocephalosporanic acid (7-ACA) is the initial compound in preparation of cephalosporin antibiotics widely used in clinical treatment. Bacteria producing glutaryl 7-ACA acylase, which convert cephalosporin C to 7-ACA, has been screened in soil samples. A bacterial strain exhibiting high glutaryl 7-ACA acylase activity, designated KAC-1, was isolated and identified as a strain of Pseudomonas diminuta by characterizing its morphological and physiological properties. The screening procedures include culturing on enrichment media containing glutaric acid, glutamate, and glutaryl 7-aminocephalosporanic acid as selective carbon sources. To enhance enzyme production, optimal cultivation conditions were investigated. This strain grew optimally at pH 7 to 9 and in temperatures of 20 to 40 C, but acylase production was higher when the strain was grown at 25 C. Glutaric acid, glutamate and glucos also acted as inducers for acylase production. In a jar fermenter culture, P. diminuta KAC-1 produce acylase in a growth-associated manner. The substrate specificity of KAC-1 acylase by cell extract showed that this enzyme had specificity toward glutaryl 7-ACA, glutaryl 7-ADCA, but not cephalosporin C.

• 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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• 제11권3호
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• pp.452-457
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• 2001

The glutaryl 7-aminocephalosporanic acid (glutaryl 7-ACA) acylase was purified from Pseudomonas diminuta KAC-1 cells isolated from soil, and characterized. The acylase was purified by procedures including ammonium sulfate fractionation and column chromatographies on DEAE-Sepharose, Phenyl-Sepharose, Q-Sepharose, and Superose 12H/R. The negative acylase was found to be composed of two subunits with molecular masses of approximately 55 kDa and 17 kDa, respectively. The isoelectric point of the enzyme was 4.0. The specific activities of the purified acylase were 8.0 and 7.0 U/mg on glutaryl 7-ACA and glutaryl 7-aminodesacetoxy cephalosporanic acid (glutaryl 7-ADCA), respectively, and $K_m$ values were 0.45 mM for glutaryl 7-ADCA and 0.67 mM for glutaryl 7-ADCA. The enzyme had a pH optimum at 8.0 and a tmperature optimum at $40^{\circ}C$. The acylase catalyzed the synthesis of glutaryl 7-ACA from glutaric acid and 7-ACA as well as the hydrolysis of glutaryl 7-ADCA, although the reaction rate of the synthesis was slower than that of the hydrolysis. In addition, it was found that the enzyme had a glutaryl transferase activity, thereby transferring the glutaryl group from one cephalosporin nucleus to another.

• Biotechnology and Bioprocess Engineering:BBE
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• 제2권2호
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• pp.105-108
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• 1997

A search was undertaken to screen microorganisms that produce an enzyme capable of deacylating glutary1-7-amincephalosporanic acid to 7-aminocephalosporanic acid in soil samples. The screening was carried out by preparing enrichment cultures containing glutary-7ACA and cephalosporin C as selective carbon sources. A non-${\beta}$-lactam model compound,, glutary-p-nitroanilide, was synthesized as a substrate suitable for the rapid screening of microorganisms isolated from the enrichment cultures. Two isolates exhibiting acylase activity, designated BY7.4 and BY8.1, were identified as strains of Pseudomonas species. Pseudomonas BY8.1 showed higher acylase activity toward G1-7ACA than Pseudomonas BY7.4. Environmental conditions for the optimal acylase activity of Pseudomonas BY8.1 were shown to be pH9 and 30$^{\circ}C$.

• 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.

• 한국미생물·생명공학회지
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• 제23권5호
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• pp.559-564
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• 1995

Twenty microbial strains producing the acylase were isolated from soil by using Micrococcus luteus ATCC 9341 as an indicator strain, using either D-($\alpha $)-phenylglycine methylester and 7-aminocephalosporanic acid (7-ACA) or glutaric acid dimethylester and 7-ACA as substrates. Among the isolates, only one strain was turned out to be the 7-ACA producer from either cephalosporin C or glutaryl 7-ACA as the substrates by using the overlay of 7-ACA sensitive strain (SS5). 7-ACA produced from cephalosporin C by an isolate (APS20) was detected by high performance liquid chromatography. The isolated strain (APS20) was identified to Bacillus macerans on the basis of cellular fatty acid profile by gas chromatography. Bacillus macerans APS20 had no $\beta $-lacta-mase activity on cephalosporin C, and that is very important for the enzymatic production process of 7-ACA. However, this strain was resistant up to 100 $\mu $g/ml of cephalosporin C.

• 미생물학회지
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• 제30권6호
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• pp.421-424
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• 1992

Bacillus megaterium ATCC14945의 페니실린 지 아실라제 유전자를 갖는 Escherichia coli JM83이 한 종류의 단백질을 대량으로 만들어 내었다(전체 세포 단백질 양의 20%이상). 이 단백질은 아미노 말단의 아미노산 서열 분석을 통해서 E. coli heat protein인 GroEL로 확인되었다. 또한 이 groEL 단백질은 외래의 페니실린 지 아실라제가 발현됨으로써 27과 37도 두 온도에서 생산됨을 알았다.