Identification of acetic-acid tolerance of Saccharomyces cerevisiae strains by microsatellite markers

The aim of this paper is to use the microsatellites to evaluate acid-tolerance in Saccharomyces(S.) cerevisiae. Microsatellites have been widely used as the molecular marker to classify and identify S. cerevisiae strains, analyze genetic relationships among strains, and reveal genetic diversity of S. cerevisiae populations. In this paper, 25 key microsatellites of S. cerevisiae from 44 industrial yeast strains are investigated in the medium withconcentration gradients of acetic acid. Based on the analysis of correlations between the key microsatellite loci repeat numbers and acid-tolerance of the strains, the allele size of 4P14 a and 10P13 is positively related to acid-tolerance(p ? 0.05), the allele size of AT-X, 4P1 a and 10P8 are significantly negatively related to acid-tolerance(p ? 0.01). The above results provide informations on the molecular biodiversity of S. cerevisiae strains and can be a theoretical guidance for molecular marker assisted selection.

灵芝液体浅层静置培养高效生产三萜的研究

三萜是灵芝Ganoderma lingzhi中重要的活性物质。本研究采用液体浅层静置培养方式(LSSC)提高灵芝三萜的产量。结果表明,在T25细胞培养瓶中的最佳培养条件为初始培养体积2mL,接种量7.0g(菌体湿重/L),在48h补加2mL培养液,发酵7d,三萜产量可达(32.95±0.51)mg/g,为摇瓶培养最高产量的2.09倍。进一步经不锈钢平盘放大研究发现,三萜最高产量为(65.91±0.84)mg/g,为摇瓶培养的4.19倍。形态学研究表明,浅层静置培养过程中形成的气生菌丝是提高三萜产量的关键因素。与深层液体发酵相比,液体浅层静置培养具有生长快,三萜产量高,发酵过程不需要搅拌等优点,具有良好的工业化应用前景。

灵芝多糖糖供体合成途径中关键酶的异源表达及其酶学性质

【背景】灵芝多糖(Ganoderma lucidum polysaccharide,GLP)是一种具有多种生物活性的大分子物质,已有研究者通过发酵调控优化或菌种改良使灵芝多糖产量得到一定的提高。但由于灵芝多糖糖供体合成途径并未完全明晰,其中关键酶特性解析也不完善,使得灵芝多糖产量的大幅度提高仍存在瓶颈。【目的】通过异源表达大量制备灵芝中含量较少的磷酸葡萄糖变位酶(Phosphoglucomutase,PGM)、UDP-葡萄糖焦磷酸化酶(UDP-glucosepyrophosphorylase,UGPG)和磷酸甘露糖异构酶(Phosphomannose isomerase,PMI),并分别探究和比较其酶学性质,深入了解灵芝多糖的糖供体合成途径中关键酶的特性信息,为灵芝多糖合成发酵策略的高效制定提供依据。【方法】以灵芝菌株CGMCC5.26的c DNA为模板,克隆得到关键酶基因gl-pgm、gl-ugpg和gl-pmi,分别在E. coli BL21(DE3)中诱导表达,产物通过Co-NTA树脂纯化后进行酶学性质研究。【结果】纯化酶GLpgm、GLugpg和GLpmi均在E. coli中实现大量表达。GLpgm的最适反应pH为8.5,GLugpg和GLpmi的最适pH同为7.5;GLpgm、GLugpg和GLpmi最适反应温度依次为35、40和30℃;1mmol/L的Ag^+和Cu^2+对3种酶均具有强烈抑制作用,Mn^2+、Mg^2+对GLpgm和GLpmi均有激活作用,其中Mn^2+对GLpgm的激活作用高达2.7倍。GLpgm、GLugpg和GLpmi的kcat/Km值分别为196.08、818.60和1 105.22 mmol/(L·s)。【结论】在最适反应pH、温度及金属离子作用方面,GLpgm、GLugpg和GLpmi与植物及真菌来源的这3种酶较为相似,对底物的催化效率相对其他来源的酶高,为基于灵芝多糖合成途径的调控提供了更完善的信息。