耐盐产酯酵母的选育
摘要
本文以酿酒酵母(Saccharomyces cerevisiae)T-27(α,trp-,ura-)为出发菌株,通过紫外线和氯化锂复合诱变选育出一株耐盐突变株T-27-12,并对该突变株进行了遗传特性的初步研究。然后通过原生质体融合技术选育出一株耐盐产酯酵母应用于酱油的小型酿造试验。
以酿酒酵母T-27为出发菌株,通过紫外线和氯化锂复合诱变,得到一株耐盐突变株T-27-12,其耐盐度为16%,比出发菌株提高了60%。分别对耐盐突变株T-27-12的耐盐性产生原因、耐盐突变株的遗传决定子和耐盐基因的显隐性进行了分析和测定。试验表明,耐盐突变株的耐盐特性是由基因突变所产生,并初步确定突变株的耐盐性受核基因控制,且为显性基因控制。
对产酯酵母W的生孢条件进行了研究,最终选定培养基E(E:醋酸钠0.82%,氯化钾0.18%,葡萄糖0.1%,硫酸镁0.02%,琼脂2%,pH=6.0),采用此生孢培养基生孢最好。
对亲株耐盐酵母T-27-12和产酯酵母单倍体WS原生质体的制备条件和融合条件进行了研究:综合考虑原生质体形成率和再生率,确定原生质体制备条件为:在30℃条件下经β-巯基乙醇预处理,采用Zymolyase酶混合液,对T-27-12菌株最佳的酶浓度为1.5%,酶解时间为60min,产酯酵母WS菌株最佳酶浓度为2%,酶解时间为90min。最佳融合条件:35%的促融剂PEG中添加1.2g/L的CaCl_2促融,在30℃条件下,耐盐亲株原生质体与经灭活的产酯酵母原生质体等量恒温融合1h,融合率达到2.76×10~(-6)。通过测定亲株耐盐酵母T-27-12和产酯酵母WS和融合株耐盐产酯酵母TW-6细胞体积和DNA含量等试验结果进一步证明,融合子是经过两亲株原生质体融合并发生遗传物质重组的菌株。
对融合子进行酱油发酵试验,采用高盐稀态20d速酿工艺,添加本论文获得的耐盐产酯酵母TW-6与亲株产酯酵母相比较,酱油中的总酯含量和其主体香气成分乙酸乙酯、4-乙基愈疮木酚的含量没有较大变化;与一般生产上采用的球拟酵母相比较,酱油中的总酯含量有一定提高,总酯从0.34g/100mL提高到了0.46g/100mL。从对产物色谱结果来看,乙酸乙酯的含量也由20.46mg/L提高到28.26mg/L。4-乙基愈疮木酚由2.30mg/L提高到2.86mg/L。
The breeding of Saccharomyces cerevisiae T-27 (α,tip-, ura-) was focusedin this paper. After UV and Licl treatments, a high salt tolerant mutant—T-27-12(a, trp-, ura-) was obtained. The hereditary characteristics of the salt-tolerantmutant was also analyzed. Then through protoplast fusion to obtain Salt-Tolerantand Ester-Producing Yeast.Saccharomyces cerevisiae T-27 as starting strain, it's cells were mutated with ultraviolet radiation and Licl treatments, a high salt tolerance mutant—T-27-12 (α, trp-, ura-) was obtained. Its salt tolerance increased from 10%to 16%.The hereditary characteristics of the salt-tolerant mutant was also analyzed.The mutant T-27-12 and W-1 were applied as parent strains of protoplast fusion. Protoplast formation conditions of two parent strains were as follow: T-27-12 was treated for 60min with 1.5% Zymolyase at 30℃ to form protoplast, and WS was treated for 90min with 2% Zymolyase at 30℃. The protoplast of T-27-12 and deactivated protoplasts of WS strain fused for 60 min in 35% PEG solution. The fusion rate reached 2.76×10~(-6). Comparative studies on cell size, cell volume, DNA content per cell between fusant and its parental strains were made. These data confirm that the fusant is heterozygote of both parental strains.Then, this yeast was added in the later phase of soy sauce fermentation , with short brewing for 20d. Compared with parent strain WS the total ester and the ethyl acetate 、 4-EG was changed a little;Compared with Torulopsis, the total ester was increased by 35.29 %. From chromatogram, the ethyl acetate was increased from 20.46mg/L to 28.26mg/L, and 4-EG from 2.30mg/L to 2.86m g/L.
以酿酒酵母T-27为出发菌株,通过紫外线和氯化锂复合诱变,得到一株耐盐突变株T-27-12,其耐盐度为16%,比出发菌株提高了60%。分别对耐盐突变株T-27-12的耐盐性产生原因、耐盐突变株的遗传决定子和耐盐基因的显隐性进行了分析和测定。试验表明,耐盐突变株的耐盐特性是由基因突变所产生,并初步确定突变株的耐盐性受核基因控制,且为显性基因控制。
对产酯酵母W的生孢条件进行了研究,最终选定培养基E(E:醋酸钠0.82%,氯化钾0.18%,葡萄糖0.1%,硫酸镁0.02%,琼脂2%,pH=6.0),采用此生孢培养基生孢最好。
对亲株耐盐酵母T-27-12和产酯酵母单倍体WS原生质体的制备条件和融合条件进行了研究:综合考虑原生质体形成率和再生率,确定原生质体制备条件为:在30℃条件下经β-巯基乙醇预处理,采用Zymolyase酶混合液,对T-27-12菌株最佳的酶浓度为1.5%,酶解时间为60min,产酯酵母WS菌株最佳酶浓度为2%,酶解时间为90min。最佳融合条件:35%的促融剂PEG中添加1.2g/L的CaCl_2促融,在30℃条件下,耐盐亲株原生质体与经灭活的产酯酵母原生质体等量恒温融合1h,融合率达到2.76×10~(-6)。通过测定亲株耐盐酵母T-27-12和产酯酵母WS和融合株耐盐产酯酵母TW-6细胞体积和DNA含量等试验结果进一步证明,融合子是经过两亲株原生质体融合并发生遗传物质重组的菌株。
对融合子进行酱油发酵试验,采用高盐稀态20d速酿工艺,添加本论文获得的耐盐产酯酵母TW-6与亲株产酯酵母相比较,酱油中的总酯含量和其主体香气成分乙酸乙酯、4-乙基愈疮木酚的含量没有较大变化;与一般生产上采用的球拟酵母相比较,酱油中的总酯含量有一定提高,总酯从0.34g/100mL提高到了0.46g/100mL。从对产物色谱结果来看,乙酸乙酯的含量也由20.46mg/L提高到28.26mg/L。4-乙基愈疮木酚由2.30mg/L提高到2.86mg/L。
The breeding of Saccharomyces cerevisiae T-27 (α,tip-, ura-) was focusedin this paper. After UV and Licl treatments, a high salt tolerant mutant—T-27-12(a, trp-, ura-) was obtained. The hereditary characteristics of the salt-tolerantmutant was also analyzed. Then through protoplast fusion to obtain Salt-Tolerantand Ester-Producing Yeast.Saccharomyces cerevisiae T-27 as starting strain, it's cells were mutated with ultraviolet radiation and Licl treatments, a high salt tolerance mutant—T-27-12 (α, trp-, ura-) was obtained. Its salt tolerance increased from 10%to 16%.The hereditary characteristics of the salt-tolerant mutant was also analyzed.The mutant T-27-12 and W-1 were applied as parent strains of protoplast fusion. Protoplast formation conditions of two parent strains were as follow: T-27-12 was treated for 60min with 1.5% Zymolyase at 30℃ to form protoplast, and WS was treated for 90min with 2% Zymolyase at 30℃. The protoplast of T-27-12 and deactivated protoplasts of WS strain fused for 60 min in 35% PEG solution. The fusion rate reached 2.76×10~(-6). Comparative studies on cell size, cell volume, DNA content per cell between fusant and its parental strains were made. These data confirm that the fusant is heterozygote of both parental strains.Then, this yeast was added in the later phase of soy sauce fermentation , with short brewing for 20d. Compared with parent strain WS the total ester and the ethyl acetate 、 4-EG was changed a little;Compared with Torulopsis, the total ester was increased by 35.29 %. From chromatogram, the ethyl acetate was increased from 20.46mg/L to 28.26mg/L, and 4-EG from 2.30mg/L to 2.86m g/L.
引文
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[4] 付畅,杨传平等.酵母耐盐机制的研究进展[J].遗传,2003,25(6):757～761
[5] Seranno R. A glimpse of the mechanisms of ion hpmeostasis during salt stress[J]. Experimental Botany, 1999, 50: 1023～1036
[6] Marquez J A, Serrano R. Multiple transduction pathway regulate the sodiun-extrusion gene PMR2A/ENA1 during salt stress in yeast[J]. FEBS Lett, 1996, 392: 89～92
[7] Crepo J L, Daicho K. The GATA transcription factor GLN3 and TOR to salt stress in Saccharomyces cerevisiae[J]. Biol, 2001, 276(37): 34441～34444.
[8] Nass R. Intracelluar sequestration of sodium by a novel Na~+/H~+ exchanger in yeast is enhanced by mutation in the plasma membrane H+-ATPase[J]. Biol Chem, 1997, 272: 26145～26152
[9] Cunningham K W. Ca~(2+) transport in Saccharomyces cerevisiae[J]. Exp Biol, 1994, 196: 157～166
[10] Yoshimoto H. Genome-wide analysis of gene expression regulated by the Calcineurin/Crzlp signaling pathway in Saccharomyces cerevisiae[J]. Biol Chem2002, 277(34): 31079～31088
[11] Alejandro Ferrando. Stephen J. Regulation of Cation Transport in Saccharomyces cerevisiae by the salt tolerance gene HAL3[J]. Molecular and cellular biology, 1999, 12: 5470～5481
[12] Rois G, Ferrnado A, Serrano R. Mechanisms of the salt tolerance conferred by overexpression of the HAL1 gene in Saccharomyces cerevisiae[J]. Yeast. 1997, 13: 515～528
[13] Andre B. An overview of membrane transport proteins in Saccharomyces cerevisiae[J]. Yeast, 1995, 11: 1575～1611
[14] Hlbertyn J, Hohman Sprior BR. Characterization of the osmotic-stress response in Saccharomyces cerevisiae[J]. Curr. Genet 1994, 25: 12～18
[15] 龚继明,陈受宜.离子平衡及其相关信号传到在细胞耐盐中的作用[J].生物工程进展,1999,19(6):2～8
[16] Rebecca A. Butcher and Stuart L. S Small Molecule Suppressor of FK506 that Targets the Mitochondria and Modulates ionic Balance in Saccharomyces cerevisiae[J]. Chemistry and Biology, 2003, 10: 521～531
[17] Janet M. Treger, Thomas, R. Functional Analysis of the Stress Response Element and Its Role in the Multistress Response of Saccharomyces cerevisiae[J]. Biochemical and biophysical research communication, 1998, 243: 13～19
[18] 徐秉琦,诸葛健.酵母细胞对高渗环境的适应与胞内甘油积累[J].中国生物工程杂志,2002,23(2):25～280
[19] Thmoas Petit, Jean Francois. Accumulation of trehalose in Saccharomyces cerevisiae growing on maltose in dependent on the TPS1 gene encoding the UDP glucose-linked trehalose synthase[J]. FEBS Letters, 1994, 335: 309～313
[20] Fcarvalheiro, J. C. Roseiro and F. M. Girio. Iiteractive effects of sodium chloride and heat shock on trehalose accumulation and glycerol production by Saccharomyces cerevisiae[J]. FOOD Microbiology, 1999, 16: 543～550
[21] Murata K. Glutathione and its deriviatives: produced by recombinant Escherichia coil and Saccharomyces cerevisiae[J]. Bioprocess Technology, 1994, 159～162
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