Bacillus brevis로 부터 생산되는 항세균물질의 분리 및 정제

임정수, 김성호, 김승욱

고려대학교 화학공학과 생물공정연구실

Separation  and Purification of Antibacterial Compound Produced

from Bacillus brevis

Jung-Soo Lim, Sung-Ho Kim, Seung-Wook Kim

Laboratory of Bioprocess Engineering, Department of Chemical Engineering,

Korea University,

Introduction

Methicillin-resistant strains of Staphylococcus aureus (MRSA) are resistant to essentially all □-lactam antibiotics. Many are also resistant to other antibacterial agents, including aminoglycosides. Because of the relatively high frequencies of resistance of Staphylcocci to third-generation cephems, in hospitals where these drugs are used extensively, the MRSA strains have a selective advantage in colonizing patients and spreading throughout the hospital. Resistance has emerged to all traditional anti-MRSA antibiotics including teicoplanin, leaving vancomycin as the only antimicrobial agent to which MRSA has remained susceptible. However, in-vitro selection for vancomycin resistance is possible and resistance in clinical isolates may be encountered in the future. The fluoroquinolones were reported to have useful anti-MRSA activity when introduced in the early 1980s, but soon resistance was reported. Cross resistance between fluoroquinolones is usually reported. The more recent quinolones such as sparfloxacin and clinafloxacin, and the MICs of these antibiotics for ciprofloxacin-resistant strains are often in the susceptible range. Minocycline and the new streptogramin, RP 59500 (quinupristin/dalfopristin) are potential alternative anti-MRSA antibiotics. In this study, by using Bacillus brevis strain collected in soil, antibacterial compound which has activity against MRSA and P. aeruginosa was separated and purified. The culture conditions for the production of antibacterial compound from Bacillus brevis were optimized.

Theory

MRSA is characterized by its acquisition of the mecA gene which encodes for MRSA-specific penicillin-binding protein, PBP2'. The intrinsic resistance caused by PBP2' renders all the □-lactam antibiotics ineffective in the treatment of the infected patients as well as the hospital personnel colonized by MRSA using antibiotics. Pseudomonas aeruginosa is a clinically significant pathogen with intrinsic resistance to various antimicrobial agents. Although this organism has an outer membrane with low permeability, this alone does not adequately explain its intrinsic resistance. An additional mechanism that interferes with access of the agents to their targets is suspected to exist. MexA-MexB-OprM, MexC-MexD-OprJ, and MexE-MexF-OprN are multidrug efflux systems of P. aeruginosa. Each of these systems consists of three components. The MexAB-OprM system expressed in wild-type strains contributes to the intrinsic resistance of P. aeruginosa to most □-lactams and many other structually unrelated antimicrobial agents by actively excruding them out of the cells. In fact, inactivation of this system by an artificial mutation causes hypersusceptibility to the agents in this bacterium. The chromosomal AmpC □-lactamase can also contribute to the intrinsic resistance of P. aeruginosa to □-lactams by inactivating the agents via hydrolysis. Although the respective contributions of the efflux system and □-lactamase have been well characterized, there is little information on the interplay between the two.

Experiments

1. Strain

Bacillus brevis was isolated in soil. Soil sample was collected in Munchon, Kyungkido.

2. Media and culture condition

The stock cultures were maintained by transferring the organism monthly on nutrient agar slants. Basal seed medium consisted of 1% tryptone, 0.05% MgSO4·7H2O, 0.05%KCl, 0.05%NaCl. The 2days inocula were inoculated into the optimal medium. Optimal medium consisted of 3% mannitol, 1% tryptone, 0.05% MgSO4·7H2O, 0.05%KCl, 0.05%NaCl. pH was adjusted to 7.0 with 1N NaOH prior to sterilization. Seed and main cultures were carried out in 250ml Erlenmeyer flasks containing 50ml of medium. Flask cultures were operated at 250rpm, 30℃, on a rotary shaking incubator.

3. Cell mass

Cell mass was measured with dry cell weight(DCW).

4. Antibacterial activity test

Compounds that have antibacterial activity were analyzed with three different methods.

5. HPLC

Compounds were assayed by Micro-high performance liquid chromatography(HPLC) using a reverse phase column, and UV detector at 214nm. Mobile phase was acetonitrile-water(0.1% TFA)buffer. The elution mixture was 50% acetonitrile and 50%water(0.1% TFA) with a flow of 10□/min.

6. Preparative HPLC

Waters(U.S.A)□-Bondapak C18 column(30×300mm) was used for preparative HPLC. As the eluent, 50% acetonitrile(0.1% TFA) was used. Flow rate was 10ml/min.

7. Purification of antibacterial compounds

Culture broth 1L was sonicated to break the cell because it is important to recover intracellular proteins, and 40% Ammonium sulfate dissoved in culture broth and precipitated at 4℃ for 1 day. After precipitates were centrifuged at 22,000×g for 10min, and extracted with 100% IPA for 2 hrs in sonicator. With same method as described previously, simultaneous extraction with 100% IPA was carried out for 2 hrs each in sonicator, and then precipitates were extracted with 50% IPA three times for 2 hrs in sonicator. Residue was collected and IPA was recovered by evaporator at 40℃. Residue was passed through column which packed with 20ml Diaion HP-20, and antibacterial compounds were adsorbed in the resin. Column was washed with 50ml water. Antibacterial material was eluted with 50% IPA. Eluate was concentrated and antibacterial compound was separated using prepative HPLC.

8. Antibacterial spectrum and antibacterial activity

To investigate antibacterial spectrum of the compound which produced by Bacillus brevis, Minimal Inhibitory Concentrations(MICs) were determined by a broth dilution method using YM broth. MIC was employed as MIC95 which could inhibit growth of test organism to 95%.

Results and Disccussion

Organism which was isolated from soil has good activity for test organisms. Microorganism should has not only good antibacterial activity but also ability of good growth to be utilized for industrial purpose. Fig. 1 shows culture profile of Bacillus brevis in optimal medium. Antibacterial compound which was separated with HP-20 column chromatography still had the impurities. It is necessary that compounds should be purified more than 98% for the analysis of NMR. Fraction  containing antibacterial compounds was concentrated and purified by using preparative HPLC.  Fig. 2 shows preparative HPLC profile of the compound. Table. 1 shows antibacterial spectrum of the compound. Purified compound showed a weak antibacterial activity against Gram negative bacteria, and strong activity against Gram positive bacteria. But it showed a strong activity against Pseudomonas aeruginosa ATCC 10145, which is Gram negative bacteria. Pseudomonas aeruginosa is a fatal pathogen which causes pus when burnt patient is infected. Moreover, this compound showed a strong activity against MRSA. Against MRSA, this compound showed higher MICs than that of vancomycin(MIC 1.0□/□). In conclusion, this compound has a potential for alternatives of vancomycin. Table. 2 shows the molecular weight of peptide produced from Bacillus brevis. Comparing with table, molecular weight of this compound was different from that of other compounds. This result suggested the probability that this compound would be new peptide produced from Bacillus brevis.

Fig.2 Preparative HPLC profile of the compound.

Peptides	Molecular weight
Gramicidin D	2000
Gramicidin J1	831.5
Gramicidin J2	684.4
Gramicidin S	1140.7
Tyrocidine A	1268.6
Tyrocidine B	1308.7

Table 1. Antibacterial spectrum of the     Table 2. Molecular weight of peptide                compound                                               produced from Bacillus brevis.

Test organisms	MIC95 (□/□)
Escherichia coli ATCC 10586 (-)	28.75
Proteus mirabilis ATCC 25933 (-)	30.00
Providencia rettgeri ATCC 9919 (-)	>100
Pseudomonas aeruginosa ATCC 10145 (-)	3.593
Staphylococcus aureus ATCC 29213 (+)	3.015
Bacillus subtilis ATCC 6633 (+)	1.796
Staphylococcus epidermidis ATCC 12228 (+)	1.796
MRSA	4.132

References

1. Brumfitt, W., Hamilton-Miller, J. (1989), □Methicillin-resistant Staphylococcus aureus.□ N. Engl. J. Med. 320, 1188-1196.

2. Cohen, M.L. (1992),□Epidemiology of drug resistance:  implications for a post-antimicrobial era.□ Science 257, 1050-1055.

3. Ohno, A., Yamaguchi. K. (1991), □Infectious diseases caused by MRSA, I. From bacteriological viewpoint.□ Nippon Yakuzaishikai Zasshi 43, 1181-1188.