高温酵母的分离鉴定及原生质体融合构建耐高温高产酒精酵母
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摘要
从来源不同的基质中共分离得到227株耐高温酵母菌，经初筛、复筛和酒精发酵试验，获得一株耐高温且具有一定产酒精能力的酵母菌株h-1，其在42℃下72小时发酵，酒精产率为5.5％。研究了h-1的形态和生理生化特性，结果表明：h-1菌株产生1-4个土星行子囊孢子，该菌株发酵葡萄糖、蔗糖，弱发酵棉籽糖，不发酵麦芽糖、半乳糖，乳糖；同化棉籽糖、纤维二糖、木糖、柠檬酸、琥珀酸、蔗糖，不同化肌醇、核糖、赤藓糖醇、鼠李糖、核糖醇、甘露醇、可溶性淀粉、阿拉伯糖、海藻糖、半乳糖、麦芽糖、乳糖；不能同化硝酸盐；可在无维生素培养基上生长；37℃下可生长。根据《The Yeast》(第三版)，鉴定h-1菌株属于的土星汉逊酵母(Hansenula Saturnus)。
选择化学药物和紫外灭活单亲原生质体为遗传标记，测定了放线菌酮、三唑酮、多菌灵、灰酶特杀、决菌、霜霉狂杀对h-1和酿酒酵母SP-48的抑制作用，结果表明只有0.01％的三唑酮可以抑制SP-48，而不抑制h-1，选择0.01％的三唑酮作为药物标记。测定了紫外照射剂量和h-1原生质体致死率的关系，当紫外灯为20瓦，照射距离为30cm，照射时间为2分钟时，h-1原生质体的致死率为100％。因此，以0.01％的三唑酮和紫外照射h-1原生质体2分钟为遗传标记。采用PEG法将h-1酵母与不耐高温的高产酒精酵母SP-48进行原生质体融合，获得既耐高温酒精产率又较高的融合子LZ-3，LZ-3 72小时发酵，酒精产率为7.5％。融合子LZ-3遗传性状稳定，传代20次后，融合子LZ-3的酒精产率和初代相比没有明显变化。
确定了融合子LZ-3生长和发酵的最适温度和pH值；研究了温度、pH值和接种量对生长和发酵曲线的影响；测定了融合子LZ-3的耐酸碱；耐酒精；耐盐；耐糖和耐发酵副产物特性。结果表明：融合子LZ-3的最适生长温度为36℃，最适生长pH为5；最适发酵温度为34℃，最适发酵pH为5；温度和接种量对生长和发酵曲线的影响显著，pH对生长和发酵曲线的影响不显著；耐酒精能力为20％(v／v)；耐盐能力为11％；可在50％(w／w)的葡萄糖溶液中发酵；对乙酸、乳酸和甘油有一定的耐受能力。
采用四因素三水平正交试验确定了融合子LZ-3的浓醪酒精发酵工艺。浓醪发酵的最佳工艺条件是：料水比为1∶2，糖化酶用量为400IU／g原料，接种量为9％(v／v)，初始pH值为5.5，在此条件下，以玉米粉的糖化醪为培养基，发酵72小时，LZ-3的酒精产率为9.0％。
In different environments, 227 isolates out of were screened for fuel ethanol-producing yeast with high thermotolerance. Among them, the strain h-1, which was able to use maize as fermentation substate to produce higher alcohol in 42 ℃, was identified as Hansenula Saturnus based on its biophysiological and biochemical characteristics, and employed for the following fusion.
Chemical drugs and UV which exterminate singal parent's protoplasts were choosen as hereditary markers. 0.01% tradimefon could inhibit the strain SP-48, but couldn't inhibit h-1. So, tradimefon whose content was 0.01% was choosen as marker. The relation of UV's irradiated dosage and h-l's protoplasts lethal ration was identified. When UV light was 20W, the irradiated distant was 30cm, and the time was 2min, the lethal ration of h-l's protoplasts was 100%. So, tradimefon whose content was 0.01% and UV whose irradiated h-1 2min were used as hereditary markers. The protoplast yeasts of h-1 and SP-48 were fused using PEG, and fusion strains LZ-3 which could tolerate high-temperture and produced higher alcohol was achieved. After 72h fermentation, the fusion strains LZ-3 could produce alcohol's ration 7.5%. The fusion strain had stable heredity, and when inoculated 20 times, alcohol-producing ration of the fusion strain LZ-3 was not obvious changes.
The fusion yeasts LZ-3 grew the best in 36℃ and a pH of 5.0,whereas its optimum fermentation temperature and pH was 34℃ and 5.0,respectively.It was seen that temperature and initial inoculum's concentration were 2 important factors in affecting the growth and fermentation of strain 3.The strains of 3 showed good tolerances to various stresses including aciding, alkalis, and osmotic pressure. Also, the strains of LZ-3 showed .better resistances to high concentration alcohol and byproducts than the produced-commercially ADY(acid dried yeast).
If 33.3%(w/v)concentration of crude corn in fermenting material, 400 units alucoamylse per gram of raw corn, 9%(v/v) inoculum size of the yeast, and initial pH of 5.5 were fixed, orthogonal test indicated that the fusion strain LZ-3 produced the highest amount of alcohol and 9.0% after incubated at 42℃ for 72 hours when it was tested in higher temperature.
选择化学药物和紫外灭活单亲原生质体为遗传标记，测定了放线菌酮、三唑酮、多菌灵、灰酶特杀、决菌、霜霉狂杀对h-1和酿酒酵母SP-48的抑制作用，结果表明只有0.01％的三唑酮可以抑制SP-48，而不抑制h-1，选择0.01％的三唑酮作为药物标记。测定了紫外照射剂量和h-1原生质体致死率的关系，当紫外灯为20瓦，照射距离为30cm，照射时间为2分钟时，h-1原生质体的致死率为100％。因此，以0.01％的三唑酮和紫外照射h-1原生质体2分钟为遗传标记。采用PEG法将h-1酵母与不耐高温的高产酒精酵母SP-48进行原生质体融合，获得既耐高温酒精产率又较高的融合子LZ-3，LZ-3 72小时发酵，酒精产率为7.5％。融合子LZ-3遗传性状稳定，传代20次后，融合子LZ-3的酒精产率和初代相比没有明显变化。
确定了融合子LZ-3生长和发酵的最适温度和pH值；研究了温度、pH值和接种量对生长和发酵曲线的影响；测定了融合子LZ-3的耐酸碱；耐酒精；耐盐；耐糖和耐发酵副产物特性。结果表明：融合子LZ-3的最适生长温度为36℃，最适生长pH为5；最适发酵温度为34℃，最适发酵pH为5；温度和接种量对生长和发酵曲线的影响显著，pH对生长和发酵曲线的影响不显著；耐酒精能力为20％(v／v)；耐盐能力为11％；可在50％(w／w)的葡萄糖溶液中发酵；对乙酸、乳酸和甘油有一定的耐受能力。
采用四因素三水平正交试验确定了融合子LZ-3的浓醪酒精发酵工艺。浓醪发酵的最佳工艺条件是：料水比为1∶2，糖化酶用量为400IU／g原料，接种量为9％(v／v)，初始pH值为5.5，在此条件下，以玉米粉的糖化醪为培养基，发酵72小时，LZ-3的酒精产率为9.0％。
In different environments, 227 isolates out of were screened for fuel ethanol-producing yeast with high thermotolerance. Among them, the strain h-1, which was able to use maize as fermentation substate to produce higher alcohol in 42 ℃, was identified as Hansenula Saturnus based on its biophysiological and biochemical characteristics, and employed for the following fusion.
Chemical drugs and UV which exterminate singal parent's protoplasts were choosen as hereditary markers. 0.01% tradimefon could inhibit the strain SP-48, but couldn't inhibit h-1. So, tradimefon whose content was 0.01% was choosen as marker. The relation of UV's irradiated dosage and h-l's protoplasts lethal ration was identified. When UV light was 20W, the irradiated distant was 30cm, and the time was 2min, the lethal ration of h-l's protoplasts was 100%. So, tradimefon whose content was 0.01% and UV whose irradiated h-1 2min were used as hereditary markers. The protoplast yeasts of h-1 and SP-48 were fused using PEG, and fusion strains LZ-3 which could tolerate high-temperture and produced higher alcohol was achieved. After 72h fermentation, the fusion strains LZ-3 could produce alcohol's ration 7.5%. The fusion strain had stable heredity, and when inoculated 20 times, alcohol-producing ration of the fusion strain LZ-3 was not obvious changes.
The fusion yeasts LZ-3 grew the best in 36℃ and a pH of 5.0,whereas its optimum fermentation temperature and pH was 34℃ and 5.0,respectively.It was seen that temperature and initial inoculum's concentration were 2 important factors in affecting the growth and fermentation of strain 3.The strains of 3 showed good tolerances to various stresses including aciding, alkalis, and osmotic pressure. Also, the strains of LZ-3 showed .better resistances to high concentration alcohol and byproducts than the produced-commercially ADY(acid dried yeast).
If 33.3%(w/v)concentration of crude corn in fermenting material, 400 units alucoamylse per gram of raw corn, 9%(v/v) inoculum size of the yeast, and initial pH of 5.5 were fixed, orthogonal test indicated that the fusion strain LZ-3 produced the highest amount of alcohol and 9.0% after incubated at 42℃ for 72 hours when it was tested in higher temperature.
引文
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