复合诱变选育抗菌肽Sublancin高产菌株

Breeding of Antimicrobial Peptide Sublancin High-Yield Strain by Composite Mutagenesis

Bacillus subtilis BF168可以产生一种新型的抗菌肽Sublancin,Sublancin具有明显的抗革兰氏阳性菌活性,但产量较低。为了获得Sublancin高产菌株,对其产生菌B. subtilis BF168进行诱变选育研究。以B. subtilis BF168为出发菌株,采用加热、紫外(Ultraviolet,UV)、氯化锂(Li Cl)、微波(Microwave,MW)和UV-Li Cl、UV-MW、MW-LiCl处理技术分别进行单一诱变和复合诱变,不同诱变条件处理的菌悬液涂布在培养基平板上培养后获得单菌落,通过菌株致死率和正突变率确定合适的诱变条件,利用突变株摇瓶发酵效价筛选出正突变菌株。结果表明:单一诱变最佳参数分别是热诱变为80℃处理10 min;UV诱变为功率30 W、照射距离30 cm、照射时间90 s;Li Cl诱变浓度为12 g/L;MW诱变为脉冲频率2 450 MHz、功率800 W、辐射时间100 s。在不同诱变方法对比中发现,UV-Li Cl复合诱变方式可获得最高的正突变率,以突变株的发酵效价为指标的正突变率和致死率分别为21.05%和92.18%,复筛效价是出发菌株1.6倍以上的有4株,占复筛菌株的10%。经过多种方法复合诱变,最终获得一株遗传性状稳定的Sublancin高产菌株UL2,在培养温度为37℃、210 r/min的条件下,经过28~30 h培养后,该菌株的相对效价达到(204±2.83)%。实验证实UV-LiCl复合诱变可以显著提高BF168菌株的Sublancin发酵产量。

Bacillus subtilis BF168 can produce a new type of antimicrobial peptide Sublancin,which has obvious against Gram-positive bacteria activity,but its yield is low in this producing strain. In order to obtain a high-yield strain of Sublancin,the mutagenesis and breeding study was carried out with B. subtilis BF168. B. subtilis BF168 was treated by single mutagenesis including heating,ultraviolet( UV),lithium chloride( Li Cl),microwave( MW) and compound mutagenesis( UV-LiCl,UV-MW,MW-LiCl). The bacterial suspensions treated with different mutagenesis conditions were spread on the medium plates and cultured to obtain a single colony. The appropriate mutagenesis conditions are determined by the strain lethality and positive mutation rate,and the fermentation titer of the mutant strain was used to screen out positive mutant strains. The results showed that the best parameters for single mutagenesis were heat-induced mutation treatment at 80 ℃ for 10 min;UV mutation power 30 W,irradiation distance 30 cm,and irradiation time 90 s;Li Cl mutation at a mass concentration of 12 g/L;the induced pulse frequency of MW at 2 450 MHz,the power of 800 W,and the radiation time for 100 s. In comparison with different mutation methods,it was found that the UV-LiCl composite mutagenesis could obtain the highest positive mutation rate. Its positive mutation rate and lethality rate based on the fermentation titer of the mutant strain were 21. 05% and 92. 18%,respectively. The fermentation titer of 4 strains was 1. 6 times higher than that of the original strain BF168,accounting for 10% of the re-screened strains. After multiple methods of compound mutagenesis,a high-yield Sublancin strain UL2 with stable genetic traits was finally obtained. The relative titer of the strain was( 204 ± 2. 83) % at 37 °C and210 r/min for 28 ~ 30 h. These results confirmed that UV-LiCl composite mutagenesis could significantly increase the fermentation yield of Sublancin in BF168 strain.