Streptomyces clavuligerus mutant로부터 클라블란산 생산을 위한 배지 최적화

이순덕, 오경근*, 김승욱

고려대학교 화학공학과, 단국대학교 공업화학과*

Optimization of fermentation medium for the production of clavulanic acid by Streptomyces clavulgerus mutant

Soon-Duk Lee, Keong-keun Oh*, Seung-Wook Kim

Department of Chemical Engineering, Korea University,

Department of Industrial Chemistry, Dankook University*

Introduction

Clavulanic acid is a naturally occurring substance first detected in Streptomyces clavuligerus and shown to be a fused bicyclic □-lactam structually different from the penicillins and cephalosporins in having an oxygen atom in place of sulphur. It was found to inhibit the activity of □-lactamase enzymes which are produced by many penicillin and cephalosporin-resistant clinical pathogens. Clavulanic acid appears to be active against a wide spectrum of Gram-positive and Gram-negative bacteria. The level of observed activity is, however, quite low. It has been coformulated with other broad spectrum antibiotics. This study was carried out for the development of high calvulanic acid-proucing strain and optimization of clavulanic acid fermentation medium to increase the productivity of clavulanic acid.

Theory

In fermentation technology, improvement in the productivity of the microbial metabolite is carried out by improving the strain genetically and by manipulating the nutritional and physical parameters of fermentation. Strain development from antibiotic production has been essential prerequisite to developing an efficient antibiotic production process. Mutagenesis can be induced in microorganisms by physical agents or by radiation. Ultraviolet light is a fairly potent mutagen and its radiation mainly induces base substitutions(60 to 75%)followed by single-base frameshifts, deficiencies and duplications. In the particular case of secondary metabolites, the interaction between growth metabolism and product secretion is critically influenced by growth limiting nutrient concentrations. Medium optimization approached either empirically or by statistical methods. Empirical method has several limitations towards complete optimization. Experimental designs and response surface methodology as statistical methods are very useful tools for the optimization of independent variables to be considered in the fermentation process. They can provide statistical models. From these models, the relative influence of various factors can be determined and their optimal concentration can also be calculated for maximal growth or metabolite production.

Experiments

1. Microorganism

Streptomyces clavuligerus ATCC 27064 supplied by KCTC was used as the wild- type strain. S. clavuligerus ATCC 27064 was treated with ultraviolet light and S. clavuligerus mutant 15 was selected.

2. Ultraviolet mutagenesis

The suspension of S. clavuligerus was filtered through glass-wool for the spore mutation. The spore suspension was irradiated with ultraviolet light in time interval 60s. After serial dilution, it was spreaded on the Bennett agar and incubated at 28℃ untill colonies were formed. To measure the □-lactamase inhibitory activity, agar diffusion method was carried out using the nutrient agar containing 10□/ml of penicillin G and penicillin-resistant Klebsiella aerogenes ATCC 15380.

3. Medium optimization

For the three levels, three variables, one-third fractional factorial design, a set of 9 experiments was carried at various conditions, with 6 star points and 4 replicates at the settle point for the application of statistical methods. The ressults were statistically analyzed to estimate the regression equation for the biomass and clavulanic acid production by using RSREG(SAS; Statistical Analysis System).

Results and Disccussion

When UV was irradiated for 4-5minutes, 1% of survival rate was obtained. One hundred of colonies of each strain were selected by growth observation. Among these colonies which have larger inhibition zone than wild strain were chosen and these colonies were cultivated in submerged culture. Three strains were chosen by bioassay. Fig. 1 shows the production of clavulanic acid for wild strain and three mutated strains. The highest level of clavulanic acid was obtained from mutant 15.

Productivity of mutant 15 was investigated in the several industrial fermentation media. Mutant 15 showed the highest level of clavulanic acid in the medium including soybean flour, rice bran oil(RBO) and dextrin (Fig. 2)

Fig 1. Clavulanic acid produced by           Fig 2. The level of clavulanic acid of

wild type and mutants.                      mutant 15 in the several media.

To determine the concentration of each ingredient, response surface methodology was used and the response equation was obtained for biomass and clavulanic production separately, and the contour graphs which show the relation between ingredients were represented. As shown in Fig. 3-5, for clavulanic acid production,  interaction between soybean flour and RBO are significant, but dextrin is not insignificant. Also, the response surface plots show that the biomass was closely related to soybean flour and RBO (Fig. 6).

Fig. 3 The effect of soybean flour and RBO      Fig. 4 The effect of RBO and dextrin on

on the production of clavulanic acid.             the production of clavulanic acid.

Fig. 5 The efffect of soybean flour and dextrin     Fig. 6 The effect of soybean flour and RBO

on the production of clavulanic acid.              on the biomass.

References

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