Studies On Active Secondary Metabolites Of Actinomycete HBERC-53204

Microbial secondary metabolites are essential for the pharmaceutical research and development.Most of the current drugs were putting into market directly by natural products or indirectly by their modified structures,such as penicillin,streptomycin,kanamycin,semisynthetic tetracycline etc,which has made great contribution to the safety of human life,health and production.Soil is rich in microorganisms with complex and variable metabolic environment,and the actinomycetes genome contains a large number of gene clusters for the synthesis of secondary metabolites,so exploring soil actinomycetes resources is of special significance for seeking good bioactivity of drug source molecules.Actinomycete No.HBERC-53204 was isolated from soil by our research team.Preliminary screening showed that the fermentation broth of the strain displayed strong inhibitory effect on a variety of plant and animal pathogens.The exploitation of medicinal actinomycetes resources was described from a variety aspects of strain isolation and identification,culture,extraction and separation of active compounds,bioactivity determination,stability test and fermentation optimization.Two active compounds were isolated and identified,and the titer of the target compound Steffimycin B reached 477.26mg/L after fermentation optimization.The yield increased by 1773.08%compared with the original medium.The results of research are as follows:1.HBERC-53204 was isolated from rhizosphere soil samples collected from bamboo forest in Zhongpo National Forest Park,Huaihua City,Hunan Province,and identified as Streptomyces scabrisporus by PCR amplification and 16S r RNA sequence analysis.Two active compounds were isolated from liquid fermentation broth base on the guidance of biological activity.It was identified as Steffimycin B（SMB）,an anthracycline compound,Leptomycin B（LMB）,a fatty family compound by MS,UV and NMR.In the bioassay activities of Streptococcus suis,Staphylococcus aureus and Erysipelas erysipelas,Steffimycin B was superior to penicillin and streptomycin as the control,with MIC values of 1.56,6.25 and 1.56μg/m L respectively,and MIC values of Leptomycin B against Fusarium putrescens and Alterniella were 50,0.78μg/ml respectively.2.Response surface methodology was used to optimize the medium composition for shaker fermentation to guide fermentation tank production.The optimal medium ratio was verified as follows:glucose 36.22 g/L,peptone 8.0 g/L,yeast 8.51 g/L,acid hydrolyzed casein 1.5 g/L,Mg SO₄ 0.68 g/L,KNO₃ 1.0 g/L.The optimal tank fermentation time in fermenter is 120 hours.3.Steffimycin B in the fermentation broth showed excellent stability after UV irradiation for 1h,p H 3-9 treatment for 4h,and water bath at 40℃for 2h.It was obviously degraded under strong alkaline condition in water bath at 80℃for 2h and p H 11 for 1h.Leptomycin B showed different degrees of degradation after water bath heating at 40℃and above,but no significant degradation was observed after UV irradiation for 1h.It was found that the p H of fermentation broth at p H 7 and p H 9 were conducive to the extraction of Leptomycin B.In conclusion,a strain of Streptomyces scabrisporus HBERC-53204 was isolated and identified from rhizosphere soil of bamboo forest.It was found that the strain could metabolized and produced antibacterial products.Two active compounds were screened by activity tracing and identified as anthracycline Steffimycin B and unsaturated fat family compound Leptomycin B by organic spectroscopy.Response surface methodology（RSM）was used to optimize the HBERC-53204 fermentation medium,and SMB yield was increased to 477.26 mg/L.The stability evaluation of the two active compounds showed that SMB had the best comprehensive stability and could be applied to field production environment,while LMB showed obvious degradation at different temperatures.This paper laid the theoretical foundation and experimental work for exploring active compounds of soil actinomycetes and developing new active antibacterial drugs.