CN112481348A - 一种高产dha裂殖壶菌突变菌株的筛选方法 - Google Patents
一种高产dha裂殖壶菌突变菌株的筛选方法 Download PDFInfo
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Abstract
一种高产DHA裂殖壶菌突变菌株的筛选方法,具体是,将野生型裂殖壶菌菌株经过ARTP诱变处理,经烯草酮平板培养基定向初筛,磷酸香草醛法和摇瓶发酵培养测定的DHA产量为复筛,筛选后获得高产DHA菌株。本发明涉及到的高产DHA筛选方法具有操作简单,筛选成功率高,能快速获取大量目的菌株等诸多优势。
Description
技术领域
本发明涉及微生物突变体筛选技术领域,尤其涉及用于生产二十二碳六烯酸的裂殖壶菌突变株的筛选即一种高产DHA裂殖壶菌突变菌株的筛选方法。
背景技术
ω-3系列多不饱和脂肪酸对人体健康有着十分重要的影响,在食品、医药、饲料和化妆品等领域有广阔的应用前景。二十二碳六烯酸(Docosahexaenoic Acid,DHA)作为其中一种,对婴儿大脑和视网膜发育有积极作用,俗称脑黄金。我国早在2000年就已批准DHA作为营养强化剂,可以改变膳食结构,增强人体免疫力,市场潜力巨大,被誉为“21世纪功能性食品的主角”。由于它这些重要的生理功能,加快开发和利用DHA资源已被世界各国生化学家、医药研究者和营养专家等研究的热门方向之一。
裂殖壶菌(Schizochytrium)是一种类真菌类的单细胞异养原生生物,在形态、繁殖等方面与真菌非常相似。大多数裂殖壶菌在浮游植物的残渣上腐生,少数寄生在某些低等的水生生物身上。裂殖壶菌脂质中多不饱和脂肪酸组成简单,菌体本身无危害,易培养,是目前最具前景的工业化生产DHA的微生物。
为了进一步提高裂殖壶菌DHA产量,通过诱变手段获得高产菌株已经被众多科研工作者所研究。目前获得突变体的筛选方法有很多,比如苏丹黑B染色法、低温高糖处理法、流式细胞仪筛选法等,但是这些方法存在筛选效率低、操作复杂的缺点。
发明内容
针对现有技术不足,本发明一种高产DHA裂殖壶菌突变菌株的筛选方法,将野生型裂殖壶菌菌株经过诱变处理,先经烯草酮平板培养基定向初筛,再以磷酸香草醛法和摇瓶发酵培养测定的DHA产量为复筛,快速大量筛选高产DHA菌株,并且具有操作简单,筛选成功率高,能快速获取大量目的菌株等诸多优势。
一种高产DHA裂殖壶菌突变菌株的筛选方法中培养基(g/L)成分包括以下成分:
固体培养基:葡萄糖10,蛋白胨1.5,酵母膏0.1,琼脂粉20,海盐33,PH自然,1×105 Pa、115 ℃灭菌21 min。发酵培养基:葡萄糖20,蛋白胨1.5,酵母膏1.0,KH2PO4 0.25,海盐33,PH自然,1×105 Pa、115 ℃灭菌21 min。
一种高产DHA裂殖壶菌突变菌株的筛选方法,主要包括以下步骤:
(1)种子液制备:在无菌超净台中,使用接种环挑取新鲜固体培养基中的单菌落,接种在装有40 mL液体培养基的锥形瓶内,在28℃、150 rpm摇床中培养24 -36 h;
(2)菌悬液制备:取5%的上述种子液接种至发酵液体培养基中,在28℃、150 rpm摇床中培养24-36 h,取适量菌液稀释后,调整细胞OD660 值为0.3-0.4之间,用于ARTP诱变处理;
(3)ARTP诱变处理:分别取上述菌悬液10 μL均匀涂布在无菌载片上,将载片放于培养皿中并转移至ARTP诱变仪凹槽内,凹槽下方固定座固定相应的EP管,用于承接载片,EP管内含有1 mL的无菌蒸馏水。ARTP诱变仪功率设定为120 W,气量设定为10 SLM,选择致死率在90%左右的处理时间进行诱变处理;
(4)烯草酮平板初筛:将野生型裂殖壶菌在液体培养基中培养24-36 h,10倍稀释法稀释1000倍后,分别取一定体积菌液均匀涂布在烯草酮浓度为0、20、40、60、80、100 μg/mL的固体平板上,放入28℃恒温箱中避光培养2-3 d。统计生长出的单菌落个数,计算出不同浓度下烯草酮对裂殖壶菌的致死率,选择致死率在90%-95%之间的浓度进行后续筛选实验;取上述(3)中诱变后的菌悬液一定体积均匀涂布在含有烯草酮的固体平板上,放入28℃恒温箱中避光培养2-3 d。初筛出可能油脂含量高、形态大的单菌落用以后续复筛;
(5)磷酸香草醛法筛选高产油脂菌株:收集培养4 d的发酵液5 mL于离心管内,8000rpm离心10 min,蒸馏水洗涤2次,定容至5 mL,取100 μL菌液(对照取蒸馏水)于10 mL离心管内,加入18 mol/L的 H2SO4 2mL,震荡混匀,沸水浴反应10 min,常温水浴5 min;加入5 mL磷酸香草醛试剂,37℃保温15 min,常温水浴10 min,于530 nm测其OD值;
(6)生物量的测定:取10 mL培养4 d摇瓶发酵液到已称重的离心管中,8000 rpm离心15min,弃去上清液,用蒸馏水洗涤沉淀菌体2次,将盛有菌体的离心管放于-80℃冷冻1 h,再放入冷冻干燥机内干燥48 h,取出称重;
(7)摇瓶发酵测DHA产量筛选:取冻干的菌体加入4%硫酸甲醇溶液2 mL、1.0 g/L的十九烷酸内标溶液100 μL,混合均匀;置于80 ℃恒温水浴锅中1 h,取出冷却至室温,随后分别加入1 mL正己烷和1 mL超纯水,混匀离心取上层有机相,过0.22 μm滤膜后测气相得DHA含量。
一种高产DHA裂殖壶菌突变菌株的筛选方法,诱变处理后的突变体,经烯草酮平板培养基定向初筛,磷酸香草醛法和摇瓶发酵培养测定的DHA产量为复筛后,可以高效获取目的菌株;经过初筛和复筛后,最终获得两株高产突变株,其中一株菌DHA产量提高了43%,另外一株菌提高了51%,相比于常规筛选方法,本发明具有明显优势。
附图说明
图1为野生型裂殖壶菌(Schizochytrium sp.PKU#Mn4)在不同烯草酮浓度下的致死率;
图2为野生型裂殖壶菌(Schizochytrium sp.PKU#Mn4)和突变体1的生物量和DHA产量;
图3为野生型裂殖壶菌(Schizochytrium sp.PKU#Mn4)和突变体2的生物量和DHA产量。
具体实施方式
下面通过具体实例对本发明作进一步的说明,本发明的实例是为了使本领域的技术人员更好的理解本发明,并不对本发明作任何的限制。
实施案例1:筛选获取裂殖壶菌(Schizochytrium sp.PKU#Mn4)高产DHA菌株及不同烯草酮浓度致死率
(1)种子液制备:在无菌超净台中,使用接种环挑取新鲜固体培养基中的单菌落,接种在装有40 mL液体培养基的锥形瓶内,在28℃、150 rpm摇床中培养24 -36 h;
(2)菌悬液制备:取5%的上述种子液接种至发酵液体培养基中,在28℃、150 rpm摇床中培养24-36 h,取适量菌液稀释后,调整细胞OD660 值为0.3-0.4之间,用于ARTP诱变处理;
(3)ARTP诱变处理:分别取上述菌悬液10 μL均匀涂布在无菌载片上,将载片放于培养皿中并转移至ARTP诱变仪凹槽内,凹槽下方固定座固定相应的EP管,用于承接载片,EP管内含有1 mL的无菌蒸馏水。ARTP诱变仪功率设定为120 W,气量设定为10 SLM,选择致死率在90%左右的处理时间进行诱变处理;
(4)烯草酮平板初筛:将野生型裂殖壶菌在液体培养基中培养24-36 h,10倍稀释法稀释1000倍后,分别取一定体积菌液均匀涂布在烯草酮浓度为0、20、40、60、80、100 μg/mL的固体平板上,放入28℃恒温箱中避光培养2-3 d。统计生长出的单菌落个数,计算出不同浓度下烯草酮对裂殖壶菌的致死率,选择致死率在90%-95%之间的浓度进行后续筛选实验;取上述(3)中诱变后的菌悬液一定体积均匀涂布在含有烯草酮的固体平板上,放入28℃恒温箱中避光培养2-3 d。初筛出可能油脂含量高、形态大的单菌落用以后续复筛;
(5)磷酸香草醛法筛选高产油脂菌株:收集培养4 d的发酵液5 mL于离心管内,8000rpm离心10 min,蒸馏水洗涤2次,定容至5 mL,取100 μL菌液(对照取蒸馏水)于10 mL离心管内,加入18 mol/L的 H2SO4 2mL,震荡混匀,沸水浴反应10 min,常温水浴5 min;加入5 mL磷酸香草醛试剂,37℃保温15 min,常温水浴10 min,于530 nm测其OD值;
(6)生物量的测定:取10 mL培养4 d摇瓶发酵液到已称重的离心管中,8000 rpm离心15min,弃去上清液,用蒸馏水洗涤沉淀菌体2次,将盛有菌体的离心管放于-80℃冷冻1 h,再放入冷冻干燥机内干燥48 h,取出称重;
(7)摇瓶发酵测DHA产量筛选:取冻干的菌体加入4%硫酸甲醇溶液2 mL、1.0 g/L的十九烷酸内标溶液100 μL,混合均匀;置于80 ℃恒温水浴锅中1 h,取出冷却至室温,随后分别加入1 mL正己烷和1 mL超纯水,混匀离心取上层有机相,过0.22 μm滤膜后测气相得DHA含量。
烯草酮不同浓度致死率曲线如图1所示。ARTP诱变仪诱变后,经烯草酮平板培养基定向筛选后,再以磷酸香草醛法和摇瓶发酵培养测定的DHA产量为复筛,最终筛选获得一株高产突变株,其中干重为7.21 g/L,DHA产量为0.87 g/L,DHA产量比野生型裂殖壶菌提高了43%,如附图2。
实施案例2:筛选获取裂殖壶菌(Schizochytrium sp.PKU#Mn4)高产DHA菌株
(1)种子液制备:在无菌超净台中,使用接种环挑取新鲜固体培养基中的单菌落,接种在装有40 mL液体培养基的锥形瓶内,在28℃、150 rpm摇床中培养24 -36 h;
(2)菌悬液制备:取5%的上述种子液接种至发酵液体培养基中,在28℃、150 rpm摇床中培养24-36 h,取适量菌液稀释后,调整细胞OD660 值为0.3-0.4之间,用于ARTP诱变处理;
(3)ARTP诱变处理:分别取上述菌悬液10 μL均匀涂布在无菌载片上,将载片放于培养皿中并转移至ARTP诱变仪凹槽内,凹槽下方固定座固定相应的EP管,用于承接载片,EP管内含有1 mL的无菌蒸馏水。ARTP诱变仪功率设定为120 W,气量设定为10 SLM,选择致死率在90%左右的处理时间进行诱变处理。(4)烯草酮平板初筛:将野生型裂殖壶菌在液体培养基中培养24-36 h,10倍稀释法稀释1000倍后,分别取一定体积菌液均匀涂布在烯草酮浓度为0、20、40、60、80、100 μg/mL的固体平板上,放入28℃恒温箱中避光培养2-3 d。统计生长出的单菌落个数,计算出不同浓度下烯草酮对裂殖壶菌的致死率,选择致死率在90%-95%之间的浓度进行后续筛选实验;
取上述(3)中诱变后的菌悬液一定体积均匀涂布在含有烯草酮的固体平板上,放入28℃恒温箱中避光培养2-3 d。初筛出可能油脂含量高、形态大的单菌落用以后续复筛;
(5)磷酸香草醛法筛选高产油脂菌株:收集培养4 d的发酵液5 mL于离心管内,8000rpm离心10 min,蒸馏水洗涤2次,定容至5 mL,取100 μL菌液(对照取蒸馏水)于10 mL离心管内,加入18 mol/L的 H2SO4 2mL,震荡混匀,沸水浴反应10 min,常温水浴5 min;加入5 mL磷酸香草醛试剂,37℃保温15 min,常温水浴10 min,于530 nm测其OD值;
(6)生物量的测定:取10 mL培养4 d摇瓶发酵液到已称重的离心管中,8000 rpm离心15min,弃去上清液,用蒸馏水洗涤沉淀菌体2次,将盛有菌体的离心管放于-80℃冷冻1 h,再放入冷冻干燥机内干燥48 h,取出称重;
(7)摇瓶发酵测DHA产量筛选
取冻干的菌体加入4%硫酸甲醇溶液2 mL、1.0 g/L的十九烷酸内标溶液100 μL,混合均匀;置于80 ℃恒温水浴锅中1 h,取出冷却至室温,随后分别加入1 mL正己烷和1 mL超纯水,混匀离心取上层有机相,过0.22 μm滤膜后测气相得DHA含量。
ARTP诱变仪诱变后,经烯草酮平板培养基定向筛选后,再以磷酸香草醛法和摇瓶发酵培养测定的DHA产量为复筛,最终筛选获得一株高产突变株,其中干重为6.46 g/L,DHA产量为0.92 g/L,DHA产量比野生型裂殖壶菌提高了51%,如附图3。
Claims (6)
1.一种高产DHA裂殖壶菌突变菌株的筛选方法,其特征在于:ARTP诱变处理后的菌悬液,适当稀释后进行烯草酮平板初筛,于培养箱中避光保存,平板上能够生长出来形态大的菌落视为可能高产的菌株,然后以磷酸香草醛法和摇瓶发酵培养测定的DHA产量进行复筛,最终筛选出高产新菌株。
2.根据权利要求1所述的一种高产DHA裂殖壶菌突变菌株的筛选方法,其特征在于:所述的ARTP诱变处理是将对数时期的裂殖壶菌置于ARTP诱变仪内辐照处理,辐射时间为40s,功率设定为120 W,气量设定为10 SLM。
3.根据权利要求1所述的一种高产DHA裂殖壶菌突变菌株的筛选方法,其特征在于:所述的烯草酮(clethodim)是一种环己烯酮类除草剂,主要作用于乙酰辅酶A羧化酶(ACCase),当烯草酮存在时,可以高效抑制ACCase的活性,使生物脂肪酸合成受阻,导致生物生长缓慢,甚至死亡;在培养基中加入烯草酮后,诱变处理后只有突变体ACCase活性高的裂殖壶菌才能存活下来。
4.根据权利要求1所述的一种高产DHA裂殖壶菌突变菌株的筛选方法,其特征在于:所述烯草酮平板初筛是将野生型裂殖壶菌在液体培养基中培养24-36 h,10倍稀释法稀释1000倍后,分别取一定体积菌液均匀涂布在烯草酮浓度为0、20、40、60、80、100 μg/mL的固体平板上,放入28℃恒温箱中避光培养2-3 d;统计生长出的单菌落个数,计算出不同浓度下烯草酮对裂殖壶菌的致死率,选择致死率在90%-95%之间的浓度进行后续筛选实验。
5.根据权利要求1所述的一种高产DHA裂殖壶菌突变菌株的筛选方法,其特征在于:所述的磷酸香草醛法是用发酵液菌体经18 mol/L H2SO4破壁后,与磷酸香草醛反应生成有色物质,通过比较紫外分光光度计下OD530值的相对大小来筛选可能高产的菌株。
6.根据权利要求1所述的一种高产DHA裂殖壶菌突变菌株的筛选方法,其特征在于: 所述的摇瓶发酵培养测DHA产量具体为取冻干的菌体加入4%硫酸甲醇溶液2 mL、1.0 g/L的十九烷酸内标溶液100 μL,混合均匀;置于80 ℃恒温水浴锅中1 h,取出冷却至室温,随后分别加入1 mL正己烷和1 mL超纯水,混匀离心取上层有机相,过0.22 μm滤膜后测气相得DHA含量。
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