普鲁兰酶产生菌的诱变选育及其发酵工艺的优化

为了提高普鲁兰酶产酶酶活,以GX-6为出发菌株,采用低能离子束修饰技术对其进行诱变,并通过响应面法优化其发酵培养基。结果表明,最佳离子束诱变参数为:注入能量10 keV、诱变剂量1×10¹⁵ ions·cm^-2、诱变时间38 s,此条件下菌株正突变率比负突变率高,利用离子束诱变技术反复诱变,最终获得一株普鲁兰酶酶活较高且遗传性稳定的突变菌株GX-6-2,其酶活为2.13 U·mL^-1,较出发菌株酶活(0.65 U·mL^-1)提高了2.28倍。由Plackett-Burmen试验分析得到影响普鲁兰酶酶活的3个显著因素分别是玉米淀粉、麦芽糖和吐温-80。通过响应面试验得到最佳发酵培养参数为:玉米淀粉56.5 g·L^-1、麦芽糖11.5 g·L^-1、吐温-80 1.0 mL·L^-1、黄豆饼粉 25 g·L^-1、pH值7.0、发酵温度37℃、接种量3%、发酵时间24 h、装液量50 mL、转速180 r·min^-1,此条件下诱变菌株的酶活为2.57 U·mL^-1,较出发菌株酶活提高了2.95倍。对突变菌株的发酵特性进行初步研究发现,发酵培养24 h时,突变菌株酶活达到2.67 U·mL^-1,较出发菌株提高了3.11倍。本研究结果为利用低能离子束修饰技术诱变选育普鲁兰酶产生菌提供了一定的理论参考。

Research on Mutation and Breeding and Optimization of Fermentation Process of Pullulanase Producing Strain

In order to improve the enzymatic activity of pullulanase, the original strain named GX-6 was mutated with low energy ion beam implantation, and the fermentation medium was optimized by response surface methodology. The results showed that the strain had a higher positive rate when the implantation energy was 10 keV and the dose of N⁺implantation was 1 × 10 ¹⁵ ions·cm^-2 with implantation time of 38 s. A high-yield and high genetic stability mutant strain of GX6-2 was obtained under this mutation dose through repeated mutagenesis by using ion beam and the activity was 2.28 times higher, up to 2.13 U·mL^-1, than original strain (0.65 U·mL^-1). From the Plackett-Burmen test, three significant factors affecting the enzyme activity of pullulase were corn starch, maltose and Tween-80. The optimized fermentation parameters was shown as follows: corn starch 56.5 g·L^-1, maltose 11.5 g·L^-1, Tween-80 1.0 mL·L^-1, soybean meal 25 g·L^-1, pH 7.0, inoculation volume of 3% fermentation temperature at 37℃, 24 h fermentation, broth content 50 mL, with a spreed of 180 r·min^-1. The activity of the mutagenic strain was 2.57 U·mL^-1, which was 2.95 times higher than original strain under these conditions. The fermentation characteristics of mutant strain was also investigated. The activity of the mutant strain was 2.67 U·mL^-1, which was 3.11 times higher than original strain when the strain was cultured for 24 h. The results of this study provide some theoretical references for the mutagenesis and breeding of pullulanase-producing strain by low energy ion beam implantation.