常温常压等离子体诱变选育安普霉素高产菌株

Breeding of high-yielding strains of apramycin by atmospheric and room-temperature plasma

关键词:  安普霉素  常温常压等离子体(ARTP)  抗性筛选  高产菌株

Key words:  Apramycin  Atmospheric and room-temperature plasma (ARTP)  resistance screening  high-yielding strains

摘要:

安普霉素作为一种广谱兽用抗生素,对多种革兰氏阴性菌、阳性菌和某些支原体具有较好的抗菌作用。本论文利用常温常压等离子体(ARTP)技术结合抗性筛选方法,对安普霉素产生菌黑暗链霉菌进行诱变选育,以期得到高产菌株。以AP-520为出发菌,进行ARTP诱变处理,ARTP 最佳诱变时间是70 s,处理后的菌液在潮霉素平板上进行抗性筛选,单菌落经过48孔板大量筛选后再进行摇瓶初筛和复筛,共进行8轮筛选试验,得到一株高产菌株AP-6023,摇瓶效价达到11444 U?ml_-1^,较对照提高29 %,经过10次传代培养后仍能保持产抗能力,30 L发酵罐效价为10920 U?ml_-1^,较对照提高23 %。突变株AP-6023的获得,大大提高了安普霉素的生产水平,证实了ARTP诱变技术的有效性,试验方法为其他工业微生物的育种提供了解决思路。

Abstract:

As a broad-spectrum veterinary antibiotic, apramycin has a good antibacterial effect on a variety of Gram-negative bacteria, positive bacteria and some mycoplasmas. In order to obtain high yield strains, we used the technology of atmospheric and room-temperature plasma(ARTP) combined with resistance screening method, to mutagenesis and breeding of apramycin producing bacteria streptomyces tenebrariu. AP-520 was used as the starting bacterium for ARTP mutagenesis. The optimal mutagenesis time of ARTP was 70 s. The treated bacterial solution was screened for resistance on hygromycin plate. After a large number of single colonies were screened by 48-well plates and then subjected to primary screening and re-screening in shake flasks. A total of 8 rounds of screening tests were conducted, and a high-yield strain AP-6023 was obtained. The titer of shaking flask reached 11444 U?ml^-1, which was 29% higher than that of the control. After 10 subcultures, AP-6023 could still maintain the ability to produce antibiotics, and the titer of the 30 L fermenter was 10920 U?ml^-1, which was 23% higher than the control. The acquisition of mutant strain AP-6023 greatly improved the production level of apramycin, and confirmed the effectiveness of ARTP mutagenesis technology. The test method provides a solution for the breeding of other industrial microorganisms.