被孢霉不饱和酸GLA高产菌株的研究

【作者】 孙继民；

【导师】 王卫卫；

【作者基本信息】 西北大学 ， 微生物学， 2005， 硕士

【摘要】 被孢霉是一种产多不饱和脂肪酸的真菌,由于多不饱和脂肪酸,在生物制药、营养保健品、饲料等领域中的特殊作用,受到了广泛关注和应用。在动植物资源缺乏的情况下,利用被孢霉生产多不饱和脂肪酸具有巨大的开发潜力。但目前,由于被孢霉的目的产量不大,工业化生产应用成本太大。因此,有必要对该菌进行诱变选育,筛选其多不饱和脂肪酸,特别是ν-亚麻酸产量高的菌株。 本文对被孢霉进行选育时,先用烟碱和紫外线对其孢子进行复合诱变,获诱变株M_F2,其菌体产量、油脂含量、GLA含量分别比出发菌株提高了34.38%、6.9%和20.83%;然后通过孢子转化法,获得突变株M_F2-70,它的菌体干重、油脂含量和GLA含量分别增加到了42.8 g/L、32.13%和9.4%;再通过原生质体的制备,用亚硝酸和激光进行复合诱变。从菌株M_F2-70中选育出一高产菌株M_F2-70-12。该高产菌株经碳饥饿和高碳培养等综合方法及遗传稳定性实验和酯酶同工酶检验,证明它是一株稳定、有变异和高产的突变株。并经摇瓶培养,获得其菌体产量、油脂含量和GLA含量分别是出发菌株地的198.6%、150.2%和260.4%。 同时,对该高产菌株影响菌体和油脂产量的培养基成分进行优化;通过单因素实验,获得如接种量、温度、PH值等对菌体的生物量、油脂产量和成分有较大的影响的最优产脂条件。具体如下:葡萄糖120g/L,尿素5g/L,（NH₄）₂SO₄2g/L,KH₂PO₄ 3 g/L,Mg SO₄·7H₂O 0.5 g/L,柠檬酸钠1 g/L,酵母膏0.5 g/L,蛋白胨0.3 g/L,微量元素液1mL/L,定容至1000mL,pH（5.0-6.0）;温度28-30℃、接种量为5%-10%等。更多还原

【Abstract】 In this article, strain screening, selection of primary screening methods, optimization of fat-producing conditions and enriching of polyunsaturated fatty acids were studied.Mortierella ramanniana is a fungi of producing poly-unsaturated fatty acid.lt was noticed and applied widely because of various action of its poly-unsaturated fatty acid in producing medicament, nutriment, health products and feedstuff et al.In deficiency badly of resource of animals and plants,it take on great development potentiality by producing effectively poly-unsaturated fatty acid.But at present,the needful output of the Mortierella is not quantum sufficient and the cost of production is too expen- sive.So It is necessary to lure and change seed selection to this fungus, screen its output of oil, especially γ-linolenic acid strain high in output.In the article,when we mutant the Mortierella ramanniana, compound to mutant to the fungi with the nicotine and ultraviolet ray at first;and obtain -ed IM_F2. its fungus body output, the content of lipid and the content of GLA are higher about 34.38%, 6.9% and 20.83% than the original strain M5;Then, DNA transform through into the spore,and obtained M_F2-70, its fungus body output, the content of lipid and the content of GLA are respectively 42.8 g/L, 32.13%和 9.4%; finally, passing the preparation of the protoplasts, compound to mutant the protoplasts with HNO2 and laser. A mutation strain of high yield M_F2-70-12 appears in the seed selection from the original strain M_F2-70. This fertile strain is selected by the comprehensive method that the lack carbon and high carbon trains etc, hereditary stability experiment, esterase isoenzyme exam-inement etc. the results show that M_F2-70-12 is a mutation strain of stable, variant, heavy producing. And it is trained in shaking bottle, and obtained its fungus body output, the content of lipid and the content of GLA are raising respectively to 198.6%, 150.2%, 260.4% by the original strain M5.At the same time, optimizing the cultivation condition of this fertile strain in influence the production of fungus body and the output of lipid and passing the singlefactor experiment, the results show: culture composition, inoculating amount, temperature, PH etc. have great influence on the fungus body output, the output of lipid and the composition of lipid. As follows: 120% glucose, 5% urea, (NH^SC^ 2 g/L, KH2PO43 g/L, Mg SO4 ? 7H2O 0.5 g/L ,1% citric acid, 0.4g/L yeast cream, 0.3g/Lpeptone,microelement lipid lmL,constant volume to 1000 mL, pH 5-6, temperature 28-30°C inoculab- ility amount 5%-10% .更多还原