【作者】 梁艳；

【导师】 闻建平；

【作者基本信息】 天津大学 ， 生物化工， 2007， 硕士

【摘要】 多杀菌素是一种新型生物杀虫剂,具有以下优良特性:杀虫谱广,作用方式独特,杀虫活性高,半衰期短,残留低,无抗药性,对人畜无害,环境污染少等。多杀菌素在农牧业生产中具有广阔的应用前景,该产品的开发具有良好的经济效益和社会效益。多杀菌素的两种活性组分spinosyn A和spinosyn D,是刺糖多孢菌经有氧发酵产生的。目前,筛选高产菌株和优化发酵条件是提高多杀菌素产量的主要方法,也是国内外学者研究的热点。本文通过筛选多杀菌素高产菌株和优化发酵条件,使多杀菌素的产量提高了438.5%。本论文根据多杀菌素的生物合成途径和代谢调节机理,对刺糖多孢菌进行理性化选育。以刺糖多孢菌原始菌株为出发菌株,经过紫外诱变,以2 mg/L鼠李糖和1.5%丙酸钠为筛选剂,筛选出抗鼠李糖和丙酸钠的多杀菌素高产突变株U-4213,通过前体正丙醇的添加实验,证明突变株对前体的耐受性增加,产素能力提高,产量达到120 mg/L,较出发菌株(产量为32.5 mg/L)提高了269.2%,且遗传性状稳定。以突变株U-4213为出发菌株,通过一系列的单因子实验、Plackeet-Burman筛选实验和中心组合实验对培养基组分和发酵工艺条件进行优化,最终确定最佳的培养条件:葡萄糖68.5 g/L,麦芽糖10 g/L,棉籽蛋白26.0 g/L,玉米浆17.8 g/L,牛肉膏2.0 g/L,硫酸锌0.2 g/L,硫酸铵1.5 g/L,CaCO3 5 g/L,接种量10%,初始pH 7.0,摇床转速220 rpm,培养7天。在此条件下进行多杀菌素的发酵实验,得到多杀菌素的产量为175 mg/L,与优化前(120 mg/L)相比产量提高了45.8%。最后建立了刺糖多孢菌原始菌株和突变菌株U-4213分批发酵培养的菌体生物量,多杀菌素浓度和底物消耗的动力学方程,方程的拟和值与实验值吻合很好,其相关系数(R2)分别为:0.9961,0.9955和0.9944以及0.9984,0.9934和0.9938,该结果表明,所选的动力学模型可以很好的描述多杀菌素的发酵行为,所建立的动力学方程能以高的准确度模拟刺糖多孢菌原始菌株发酵过程中的细胞生物量、产物多杀菌素浓度和底物浓度,为放大性实验研究提供基础。更多还原

【Abstract】 Spinosad, a novel kind of biopesticide, has following excellent characteristics: effective on a wide variety of crops pest, unique mode of action, active to economically important pests, short half-life, low residue, non-drug resistant, large margins of safety to mammals, mankind and environmental profile. Spinosad will be widely used in agriculture and stockbreeding, and its exploitation has good economic and societal benefit. Spinosyn A and spinosyn D are the most active compositions of spinosad, which are producted by the aerobic fermentation of Saccharopolyspora spinosa. Nowadays, breeding high-producing strains and optimizing culture conditions are the research focus in the field of increasing the yield of spinosad. In this paper, the productivity of spinosad was improved by 438.5% through breeding high-producing strains and optimizing culture conditions.A rational selection procedure was developed based on the biosynthetic pathway and metabolic regulation of spinosad. The wild type strain of Saccharopolyspora spinosa was treated with UV irradiation. A new mutant U-4213 was obtained using 2mg/L rhamnose and 1.5% sodium propionate as screening agents by resistant selection strategies in series. The results of experiment on tolerance to propyl alcohol showed that, in the mutant strain, the tolerance to precursor was improved and the level of producing spinosad was increased, the productivity of spinosad reached 120 mg/L, which was improved by 269.2% compared with that in the original strain (32.5 mg/L).The optimization of the fermentation conditions for U-4213 was investigated by series single factor experiments, Plackeet-Burman selective experiment and central composite design experiment. The final obtained optimal condition was: glucose 68.5 g/L, maltose 10 g/L, cottonseed protein 26.0 g/L, corn steep liquor 17.8 g/L, beef extract 2.0 g/L, zinc sulfate 0.2 g/L, ammonium sulfate 1.5 g/L, calcium carbonate 5 g/L, the liquid volume 30 mL in 250 mL shaking flask, the inoculum ratio 10%, the initial pH 7.0,shaker speed 220 rpm, cultivate for 7 days. The productivity of spinosad under this optimal fermentation condition by shaking-flask batch fermentation reached 175 mg/L, which was increased by 45.8% compared with that under the initial fermentation condition (120 mg/L). The kinetic models for biomass, substrate consumption and spinosad production were studied for the mutant strain U-4213 and the wild strain by conducting batch fermentation. The models performed well in simulating the cell, spinosad and carbohydrate concentrations of both wild strain and mutant strain with high accuracy (the correlative coefficient R2 were 0.9961, 0.9955, 0.9944 and 0.9984, 0.9934, 0.9938 respectively for wild strain and mutant strain). The result suggests that the kinetic models can describe well the process of spinosad fermentation, and will be a basic for the research of scaling up.更多还原