摘要
S腺苷甲硫氨酸(简称SAM),是生物体内重要的中间代谢体,具有转甲基、转硫、转氨丙基等作用,是半胱氨酸、牛磺酸、谷光甘肽、辅酶A等重要物质的前体。能治疗神经紊乱、精神抑郁、关节炎等多种疾病,有着巨大的国际市场需求。
本文对SAM产生菌进行了选育并开展发酵工艺研究,结果如下:
通过对S-腺苷甲硫氨酸生产菌Saccharomyces cerevisiae B9菌株进行分离复壮,获得一株遗传性能较为稳定的菌株SAM-39~#(1.765g/L),以SAM-39~#为出发菌株进行诱变育种,最后获得比出发菌株SAM-39~#产量高69.89%的诱变株YZ-33,其SAM含量为3.001g/L,而紫外诱变、原生质体再生育种方法提高产量的效果不明显。通过比较发现:紫外线-氯化锂复合诱变和原生质体紫外复合诱变对SAM生产菌的诱变效果较好。
以高产稳定的诱变株YZ-33为出发菌株,通过测定生长曲线,探索蜗牛酶的酶解浓度、时间、温度等因素对原生质体形成的影响,确定了制备原生质体的最佳条件为:种子液培养16h,0.1%β-巯基乙醇预处理10min,酶解浓度2.5%,酶解时间150min,用0.5mol/L的蔗糖作为渗稳剂。
针对YZ-33菌株在不改变原有培养基组成成分的基础上,对其各组成成分进行了正交实验,找到最佳配比。得出最佳培养基配方为:蔗糖12%、氯化铵0.8%,酵母粉0.8%,硫酸镁0.005%,L-甲硫氨酸1.2%,磷酸二氢钾0.4%,磷酸氢二钾0.2%。另外,对菌株YZ-33摇瓶发酵过程中的pH值、生物量、SAM含量、蔗糖含量、NH_4~+等代谢参数进行测定,为SAM发酵工艺的建立提供了参考。
还对补料工艺做了初步的研究,确定了补料工艺与最适宜补料方式。初步补料工艺为:L-Met补料终浓度0.8%,补料起始时间24h,蔗糖补料终浓度10%,补料时间48h;最适宜补料方式为:在24h加入0.6%L-Met、3%蔗糖,在48h按0.2%补加L-Met,蔗糖在48h和72h分别按2.5%补给。在此种补料条件下SAM的产量达到4.041g/L。
S-adenosyl-L-methionine(s-adenosyl-L-methionine,as SAM),is important in the middle of metabolic body,has to methyl,to sulfur,such as to aminopropyl, homocysteine is,Taurine,Gu Gan-peptide,coenzyme A,and other important precursor substances.To treat nervous disorders,depression,arthritis and other diseases,has a huge demand on the international market.
SAM of this paper have carried out a fermentation process of breeding and research results are as follows:
Through the production of S-adenosylmethionine strains of Saccharomyces cerevisiaeB9 to separate Rejuvenation,access to a relatively stable genetic strains of SAM-39~#(1.765g/L),SAM-39~# as the starting to strain mutation Breeding, finally starting strain than the SAM-39~# high yield 69.89 percent of the mutation of YZ-33,its SAM content 3.001g/L,and UV-induced,protoplast renewable breeding methods to raise production ineffective.By comparison found: ultraviolet-lithium chloride compound and protoplast UV-induced mutation of the SAM production of the mutagenic effects better.
High-yield stability to the mutation of YZ-33 as the starting strain,through the determination of the growth curve,and explore a snail's pace of the enzyme concentration,time,temperature and other factors on the formation of protoplasts,identified protoplast of the best conditions are as follows:seeds of training 16 hours,0.1%β-mercaptoethanol pretreatment 10 min,enzyme concentration of 2.5 percent,hydrolysis time 150 min,with 0.5 mol/L sucrose as the infiltration of stability.
YZ-33 against the strain does not change the original composition of the medium on the basis of its various components of a orthogonal experiment,to fred the best ratio.That the best medium for the formula:12 percent sugar,ammonium chloride 0.8%,0.8%yeast powder,magnesium sulfate 0.005%,L-methionine 1.2%,0.4%potassium dihydrogen phosphate,hydrogen phosphate 2 Potassium 0.2 percent.In addition,the YZ-33 on the fermentation process of pH,biomass,SAM content,sugar content,NH_4~+,and other metabolic parameters were measured for the fermentation process of the establishment of SAM to provide a reference.
Also,on the feeding process done a preliminary study to determine the feeding process and the most appropriate feeding methods.Initial feeding process is:L-Met-batch final concentration of 0.8 percent,the ted-start time 24h,the final concentration of sucrose feeding 10 percent,fed-time 48h;most appropriate way for the feeding:24h adding 0.6% L-met,3%sucrose,in 48h by 0.2 percent-plus L-Met,sucrose in 48h and 72h respectively by 2.5 percent recharge.In such conditions SAM-batch production totaled 4.041g/L.
引文
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