• KSBB Journal
• /
• v.14 no.4
• /
• pp.440-444
• /
• 1999

For the effecient production of clavulanic acid., a mutant strain Streptomyces clavuligerus KK was selected from Streptomyces clavuligerus ATCC 27064 through mutation with NTG. S. clavuligerus ATCC 27064 produced about 200 mg/L of calvulanic acid when the medium was composed of 1%(W/V) glycerol, 1.5%(W/V) soybean flour, 0.1%(W/V) $KH_2PO_4$, 0.2%(V/V) soybean oil. A selected mutant, S. clavuligerus KK, produced about 1150 mg/L of clavulanic acid in the same medium. After the addition of $MgSO_4$ to the basal medium, S. clavuligerus KK produced about 1550 mg/L of clavulanic acid, with shows about 1.3 times higher than that produced in the basal medium. In order to select the proper bioreactor for the production of clavulanic acid, a batch culture was performed in an airlift, a bubble column and an stirred tank bioreactors. In an airlift bioreactor, about 1350 mg/L of clavulanic acid was produced, in a bubble column bioreactor, about 1550 mg/L, in a stirred tank bioreactor, about 2200 mg/L, respectively. The production of clavulanic acid in stirred tank bioreactor was about 50% higer than that by an airlift and a bubble column bioreactors. According to this result, the stirred tank bioreactor was selected as a proper bioreactor.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.3
• /
• pp.417-426
• /
• 2008

The effect of increasing levels of proclavaminate amidino hydrolase (Pah) on the rate of clavulanic acid production in Streptomyces clavuligerus ATCC 27064 was evaluated by increasing dosoge of a gene (pah2) encoding Pah. A strain (SMF5703) harboring a multicopy plasmid containing the pah2 gene showed significantly retarded cell growth and reduced clavulanic acid production, possibly attributable to the deleterious effects of the multicopy plasmid. In contrast, a strain (SMF5704) carrying a single additional copy of pah2 introduced into chromosome via an integrative plasmid showed enhanced production of clavulanic acid and increased levels of pah2 transcripts. Analysis of transcripts of other genes involved in the clavulanic acid biosynthetic pathway revealed a pattern similar to that seen in the parent. From these results, it appears that clavulanic acid production can be enhanced by duplication of pah2 through integration of a second copy of the gene into chromosome. However, increasing the copy number of only one gene, such as pah2, does not affect the expression of other pathway genes, and so only modest improvements in clavulanic acid production can be expected. Flux controlled by Pah did increase when the copy number of pah2 was doubled, suggesting that under these growth conditions, Pah levels may be a limiting factor regulating the rate of clavulanic acid biosynthesis in S. clavuligerus.

• Journal of Microbiology and Biotechnology
• /
• v.8 no.5
• /
• pp.523-530
• /
• 1998

Rapid and quantitative analysis of physiological change and clavulanic acid production was studied by the combination of pyrolysis mass spectrometry (PyMS) and artificial neural network (ANN) in Streptomyces clavuligerus. Firstly, the continuous culture studies were carried out to get the physiological background and PyMS samples. Clavulanic acid production was inversely related to growth rate: Mycelium growth and $q_{cal}$ were optimum at 0.1 $h^{-1}\; and \;0.025 h^{-1}$ respectively. Changes in specific nutrient uptake rates ($q_{gly}$ and $q_{amn}$) also affected clavulanic acid production since clavulanic acid production appeared to be stimulated by the limitation of carbon and nitrogen. Fermentation broth containing mycelium taken from continuous cultures was analyzed by PyMS, and the PyMS spectra were analyzed with multivariate statistics. PCCV plots revealed that samples harvested under the same culture condition were clustered together but samples from different culture conditions formed separate clusters. To deconvolute the pyrolysis mass spectra so as to obtain quantitative information on the concentration of clavulanic acid, ANN was trained on Py MS data using a radial basis function classifier. The results showed that the physiological stages with different growth rate were successfully differentiated and it was possible to monitor the clavulanic acid production precisely and rapidly.

• Biomolecules & Therapeutics
• /
• v.7 no.1
• /
• pp.44-53
• /
• 1999

The resistant strains due to the extended-spectrum $\beta$-lactamase (ESBL) were susceptible to cefatrizine combined with clavulanic acid. The purpose of this study was to evaluate the in vitro and in vivo antibacterial activities of cefatrizine/clavulanic acid (CTRZ/CV) combination at a ratio of 2 : 1 in comparison with cefaclor (CCLO), cefuroxime (CRXM), cefuroxime axetil (CRXMA) and amoxicillin/clavulanic acid (AMXCCV). CTRZ/CV showed good activity against laboratory strains of gram-positive and gram-negative bacteria and exhibited excellent antibacterial activity against $\beta$-lactamase-producing strains. The bactericidal activity of CTRZ/CV was superior to that of CCLO and CRXM, and almost equal to that of AMXCCV against the $\beta$-lactamase-producing strains. The in vitro results were substantiated. by in vivo mouse experimental infection studies with $\beta$-lactamase-producing and non-producing strains. In mixed experimental infection due to $\beta$-lactamase-producing and non-producing strains, the therapeutic efficacy of CTRZ/CV was superior to that of CTRZ, CCLO, CRXMA and AMXCCV. In respiratory tract infection in mice due to Klebsiella pneumoniae EB4O, CTRZ/CV was more erective than CCLO, CRXMA and AMXCCV and also more efficacious than CCLO, CRXMA and AMXCCV in urinary tract infection in mice due to Escherichia coli EB13. These results indicate that CTRZ/CV is a useful drug for the treatment of infection caused by $\beta$-1actamase-producing strains including ESBL-producing strains.

• Journal of Life Science
• /
• v.16 no.1
• /
• pp.76-81
• /
• 2006

A $\gamma-butyrolactone$ autoregulator receptor has a common activity as DNA-binding transcriptional repressors controlling secondary metabolism and/or morphological differentiation in Streptomyces. A gene (scaR) encoding it was cloned from Streptomyces cravuligerus, a clavulanic acid producer, and was in vitro characterized in a previous report. In this study to clarify the in vivo function of ScaR, a $\gamma-butyrolactone$ autoregulator receptor of Streptomyces clavuligerus, we constructed a scaR-deleted strain by means of homologous recombination. No difference in morphology was found between the wild-type strain and the scaR-disruptant, but the scaR-disruptant showed higher clavulanic acid production. This indicates that the ScaR in S. clavuligerus acts as a negative regulator of the biosynthesis of clavulanic acid, but plays no role in morphological differentiation.

• Toxicological Research
• /
• v.14 no.4
• /
• pp.501-505
• /
• 1998

The acute toxicity study of combined antibiotic (Cefatrizine / Clavulanic Acid), a formulation consisting of cafatrizine and clavulanic acid in a ratio of 2 : 1, was evaluated in rats. The antibiotic was orally administered with single dose in dose levels up to 5 g/kg (0, 1.25, 2.5, 5 g/kg). Treatment-related effects were limited to soft stool excretion and caecal dilatation, but histologically no morphological changes could be detected in caecum. In hematology, serum-chemistry parameters and histopathology, no drug-related changes were found. The results of the present study indicate that cefatrizine / clavulanic acid has a low toxic potential and the oral $LD_{50}$values exceed 5 g / kg in rats

• Journal of Microbiology and Biotechnology
• /
• v.14 no.6
• /
• pp.1170-1175
• /
• 2004

The involvement of the relA and rsh genes in the morphological and physiological differentiation of Streptomyces clavuligerus was evaluated with the relA and rsh genes mutants. The morphological differentiation of S. clavuligerus was greatly affected by the disruption of the relA gene, but not very much by the disruption of the rsh gene. The altered morphological characteristics were completely restored by the complementation of the corresponding disrupted genes. Thus, it was apparent that the mycelial morphology and clavulanic acid production were severely affected by the disruption of the relA gene. Production of clavulanic acid in the submerged batch culture and glycerol-limited chemostat showed that production was inversely related to the specific growth rate in the wild-type strain. However, the production of clavulanic acid in the ${\Delta}relA$ and ${\Delta}rsh$ null mutants was completely abolished. Therefore, it seems plausible that the stringent response of S. clavuligerus to starvation for amino acids is governed mainly by ReIA, rather than Rsh, and that the (p)ppGpp synthesized immediately after the depletion of amino acids triggers the initiation of pathways for both morphological and physiological differentiation in this species.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.4
• /
• pp.590-596
• /
• 2006

Clavulanic acid (CA) produced by Streptomyces clavuligerus is degraded during the bacterial cultivation. The degradation was examined in three different aspects, including physical, chemical, and enzymatic effects, in order to understand the degradation during the cultivation. The result showed that CA was unstable in the production medium containing ammonium salts and amino acids, owing to ammonium ions and amine groups. In addition, the degradation was not only due to instability of CA by metabolites and proteins, but also enzymes from S. clavuligerus such as $\beta-lactamase$ and penicillin-binding proteins. However, the degradation caused by these enzymes was not highly significant compared with the degradation during the cultivation, owing to irreversible reactions between CA and enzymes.

• YAKHAK HOEJI
• /
• v.46 no.6
• /
• pp.387-391
• /
• 2002

In vitro $\beta$-lactamase inhibitory activity of 6-exomethylene sulbactam compounds (CH-120, 130, 140, 145, 150, 155) was compared with clavulanic acid, sulbactam and tazobactam. The inhibitory activity of CH-140 was stronger than sulbactam and clavulanic acid against Type II, III, IV, TEM enzymes and stronger than tazobactam against Type IV enzyme. The inhibitory activity of CH-145 was stronger than sulbactam and clavulanic acid against Type I, II, III, IV, TEM enzymes and stronger than tazobactam against Type III, IV enzymes. The in vitro antimicrobial activity of CH-140 and CH-145 combined with piperacillin and ceftriaxone was compared with the sulbactam and tazobactam against $\beta$-lactamase producing 31 strains. But, synergistic activity of CH-140 and CH-145 was inferior to tazobactam.

• YAKHAK HOEJI
• /
• v.47 no.6
• /
• pp.456-460
• /
• 2003

In vitro $\beta$-lactamase inhibitory activity of 6-exomethylenepenam compounds ( 1, 2, 3, 4 and 5) was compared with clavulanic acid, sulbactam and tazobactam. The inhibitory activity of compound 3 was stronger than those of sulbactam and clavulanic acid against Type I and II enzymes and stronger than tazobactam against Type III, IV, TEM enzymes. The inhibitory activity of 5 was stronger than sulbactam and clavulanic acid against Type I and II enzymes and stronger than tazobactam against Type III, and IV enzymes. The in vitro antimicrobial activity of 3, 4 and 5 combined with ampicillin and cefoperazone was compared with the sulbactam against $\beta$-lactamase producing 27 strains. But, synergistic activity of 3 and 5 was inferior to tazobactam.