摘要
钼(Molybdenum,Mo)是动物、微生物及人体所必需的微量营养元素之一,是固氮酶、硝酸还原酶、黄嘌呤氧化酶、醛氧化酶、亚硫酸氧化酶等多种酶的重要组成成分,参与并影响机体内的多种代谢过程,如参与细胞内电子转运、机体内铁从铁蛋白的释放及铁的运输、醛氧化等。钼与乳腺癌、食道癌、心脑血管疾病、肝脏疾病、克山病、龋齿、小细胞低色素性贫血症等病症密切相关。
利用酵母富集转化钼具有结合率、利用率高和安全性好等特点,因此富钼酵母作为机体钼源的补充,尤其是作为预防和治疗癌症、心脑血管疾病和肝脏疾病药物的重要功效成分引起了人们的极大重视。本实验旨在通过诱变选育富钼能力强的菌株,分析其生物学功能和黄嘌呤氧化酶、硝酸还原酶等酶活性,为富钼酵母的生产应用奠定基础。
对十株二倍体酵母菌株进行钼酸钠液体抗性检测后,筛选出一株钼抗性高的二倍体酿酒酵母菌株Y1,其Na2MoO4抗性水平为20 mmol/L。通过Y1单倍体分离,并进行液体抗性水平检测,获得高抗单倍体菌株,将其与单倍体菌株SY10、SY11、SY12和SY14进行不同终浓度的钼酸钠液体抗性水平检测,筛选得到高抗单倍体菌株SY10(Na2MoO4抗性70 mmol/L),将其作为出发菌株。
以蜗牛酶酶解SY10制备原生质体,并对其原生质体和单倍体菌株进行紫外线诱变,最佳照射时间分别为4 min和3 min。通过抗性水平检测获得了2株诱变菌株SY10-186p和SY10-150s(Na2MoO4抗性分别为300 mmol/L和350 mmol/L),其拮抗Na2MoO4浓度分别为出发菌株的4.29倍和5.0倍。
分别将SY10-186p的原生质体和SY10-150s的单倍体进行紫外线再次诱变,最佳照射时间分别为4 min和6 min。通过抗性水平检测获得了2株诱变菌株SY10-186p-216u和SY10-150s-208u(Na2MoO4抗性均为400 mmol/L),其拮抗Na2MoO4浓度均为出发菌株的5.71倍,生物量分别为4.575 g/L和1.610 g/L,是出发菌株的1.75倍和1.94倍。通过原子吸收光谱法测定其钼含量分别为38.52 mg/L和43.95 mg/L,为出发菌株的4.86倍和5.55倍。用电感耦合等离子体发射光谱法测定其有机钼元素含量分别为15.87 mg/L和14.20 mg/L,为出发菌株的31.74倍和28.40倍。
分别将SY10-186p的原生质体和SY10-150s菌株的单倍体进行微波复合诱变,微波源为2450 MHz,功率为850 W,最佳照射时间均为5 s。通过抗性水平检测获得了2株复合诱变菌株SY10-186p-8m和SY10-150s-48m(Na2MoO4抗性均为400 mmol/L),其拮抗Na2MoO4浓度均为出发菌株的5.71倍,生物量为3.725 g/L和2.210 g/L,分别是出发菌株的1.42倍和2.66倍。通过原子吸收光谱法测定其总钼含量分别为39.13 mg/L和40.05 mg/L,为出发菌株的4.94倍和5.06倍。电感耦合等离子体发射光谱法测定其有机钼元素含量分别为15.08 mg/L和17.24 mg/L,为出发菌株的30.16倍和34.48倍。
诱变菌株SY10-186p-216u、SY10-150s-208u、SY10-186p-8m和SY10-150s-48m的黄嘌呤氧化酶和硝酸还原酶的活性提高,分别为7.679 U/L、7.380 U/L、7.679 U/L、8.876 U/L和0.784 U/L、1.040 U/L、0.525 U/L、0.691U/L,是出发菌株的15.42倍、14.82倍、15.42倍、17.82倍和2.12倍、2.81倍、1.42倍、1.87倍。
在非重金属选择压力下,50世代的遗传稳定性检测表明,4株诱变菌株SY10-186p-216u、SY10-150s-208u、SY10-186p-8m和SY10-150s-48m的遗传稳定性均保持在90 %以上。
生物学功能检测结果表明,诱变菌株SY10-186p-216u、SY10-150s-208u、SY10-186p-8m和SY10-150s-48m拮抗紫外线(UV)能力均明显高于出发菌株SY10。它们清除羟基自由基以及超氧阴离子自由基的能力也明显提高,分别达到67.26 %、73.08 %、76.55 %、62.66 %和39.8 %、41.1 %、40.5 %、43.9 %,是出发菌株的6.34倍、6.89倍、7.22倍、5.91倍和3.09倍、3.26倍、3.21倍、3.48倍。
Molybdenum is one of essential trace element of animals and human beings. It is the most important part of enzymes, such as nitrogenase, nitrate reductase, xanthine oxidase and sulfite oxidase. It influences and participates in many metabolisms of body, such as electron transfer of cell, iron metabolism and oxidation of aldehyde, etc. Molybdenum plays a good role in preventing and treating cancer, liver diseases, cardio-/cerebrovascular disease, Keshan disease, decayed tooth, hypochromic microcytic anemia and regional alopecia, etc.
Molybdenum-enriched yeast is non-toxic and has high combinability and absorptivity. So, as a complementarity of organic-molybdenum and an important component of the medicament to prevent and treat the diseases as above, molybdenum-rich yeast has attracted huge attentions. Our purpose of this research was to breed new strains of Saccharomyces cerevisiae having higher molybdenum enrichment abilities by multi-mutagenesis breeding with ultraviolet and microwave, assaying their biological functions and the activities of nitrate reductase and xanthine oxidase and applying them to production.
After the original strain Saccharomyces cerevisiae Y1 whose resistance level to Na2MoO4 was 20mmol/L was selected from 10 strains (Y1, Y19, Y21, Y22, Y23, Y24, Y26, Y27, Y30 and Y31) , Y1 was haploided. The resistance levels of its haploids as well as SY10, SY11, SY12 and SY14 were determined to select the higher resistant strain. A strain named SY10 whose resistant level is the highest, reached to 70mmol/L.
Then SY10 was lysised to get rid of cell wall by snailase. SY10 and the protoplasts obtained were mutated with ultraviolet ray. When the irradiation times were 3 min and 4 min, respectively, the highest positive mutate ratios were obtained. After tolerance assay to Na2MoO4 in liquid, mutant strains named SY10-186p and SY10-150s whose Mo6+ resistant level were 300 mmol/L and 350 mmol/L were selected, respectively. The Mo6+ resistant level of them were 4.29 and 5.0 folds higher than thatn of the original strain SY10.
Then the protoplasts of mutated strain SY10-186p and the haploid of mutated strain SY10-150s were mutated with ultraviolet ray again by the same method above. When the irradiation times were 4min and 6 min, respectively, the highest positive mutate ratios were obtained. After tolerance assay to Na2MoO4 in liquid, mutant strains named SY10-186p-216u and SY10-150s-208u whose Mo6+ resistance level were both 400 mmol/L (5.71 folds higher than the original strain) were selected, respectively, and their biomasses reached to 4.575 g/L and1.610 g/L,which were 1.75 and 1.94 folds higher than that of the original strain, respectively. Total molybdenum ions accumulated in the cells were estimated by atomic absorption spectrophotometer, which were 38.52mg/L and 43.95 mg/L, were 4.86 and 5.55 folds higher than the original strain, respectively. Organic-molybdenum accumulated in the cells were estimated by ICP-AES, which were 15.87 mg/L and 14.20 mg/L, were 31.74 and 28.40 folds higher than the original strain, respectively.
Then the protoplasts of mutated strain SY10-186p and the haploid of mutated strain SY10-150s were mutated with microwave in a microwave oven (2450 MHz, 850 W), and the optimum irradiation times were both 5 s. After tolerance assay to Na2MoO4 in liquid, mutant strains named SY10-186p-8m and SY10-150s-48m whose Mo6+ resistant level were both 400 mmol/L (5.71 folds higher than the original strain) were selected, and their biomasses reached to 3.725 g/L and 2.210 g/L,which were 1.42 and 2.26 folds higher than the original strain, respectively. Total copper ions accumulated in the cells were estimated by atomic absorption spectrophotometer, which were 39.13 mg/L and 40.05 mg/L, were 4.94 and 5.06 folds higher than the original strain, respectively. Organic-molybdenum accumulated in the cells were estimated by ICP-AES, which were 15.08 mg/L and 17.24 mg/L, were 30.16 and 34.48 folds higher than that of the original strain, respectively.
We also assayed the activities of xanthine oxidase and nitrate reductase of the four mutant strains, and the activities of them were higher in comparison with those of initial strain which were 7.679 U/L, 7.380 U/L, 7.679 U/L, 8.876 U/L and 0.784 U/L, 1.040 U/L, 0.525 U/L, 0.691U/L, about 15.42, 14.82, 15.42, 17.82-fold and 2.12, 2.81, 1.42, 1.87-fold higher than that of the original strain, respectively.
For all the measured generations, genetic stability of SY10-186p-216u, SY10-150s-208u, SY10-186p-8m and SY10-150s-48m all maintained above 90%, suggesting that the mutant strains were genetically stable after 50 generations.
The capacities of resisting UV, clearing ?OH and O2-? free radical were also compared to that of the original strain. The results showed that lethality of the mutants to UV reduced obviously and the capacities of SY10-186p-216u、SY10-150s-208u、SY10-186p-8m and SY10-150s-48m in clearing ?OH and O2-? free radical which were 67.26 %, 73.08 %, 76.55 %, 62.66 % and 39.8 %, 41.1 %, 40.5 %, 43.9 %, were 7.22, 5.91, 6.34, 6.89-fold and 3.09, 3.26, 3.21, 3.48-fold higher than the original strain, respectively.
引文
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