S-腺苷蛋氨酸和谷胱甘肽联产发酵中盐胁迫的作用

摇瓶条件下考察不同的盐(NaCl,Na2SO4,KCl,K2SO4)胁迫对产朊假丝酵母发酵联产S-腺苷蛋氨酸(SAM)和谷胱甘肽(GSH)的影响。结果发现适当浓度的Na+和K+对SAM和GSH合成具有部分促进作用,而Cl-的作用则相反。以Na2SO4为代表,考察分批发酵条件下盐胁迫的作用,结果表明:在酵母细胞生长后期(15 h)添加10 g/L Na2SO4,SAM、GSH以及二者的最大联产量为252.5、285.9和521.9 mg/L,比对照分别提高了8.8%、22.6%和13.9%。最后分别从能量代谢和发酵动力学角度对分批发酵的结果进行了分析。     

L-蛋氨酸及甲醇浓度对毕赤酵母摇瓶发酵生产S-腺苷蛋氨酸的影响

利用甲醇传感器及高效液相色谱检测毕赤酵母摇瓶发酵过程的甲醇浓度及S-腺苷蛋氨酸(SAM)浓度,发现L-蛋氨酸浓度及甲醇浓度对毕赤酵母细胞生长及合成S-腺苷蛋氨酸具有影响,据此对摇瓶发酵过程的L-蛋氨酸浓度及甲醇浓度进行优化。优化结果表明:当L-蛋氨酸浓度为7.5 g/L时,最适于SAM积累,产量达到0.83 g/L;进而利用甲醇传感器对发酵过程的甲醇浓度进行检测及控制,考察不同甲醇浓度对SAM产量的影响,毕赤酵母产SAM的最佳甲醇浓度为15 g/L,在此浓度下SAM的产量达到1.41 g/L,比对照实验增加了21%。     

S-腺苷-L-蛋氨酸(SAM)稳定性盐产品制备的研究

研究了S-腺苷-L-蛋氨酸(SAM)盐产品的制备方法,确定了甲醇沉淀结晶SAM硫酸盐的最优条件,进一步优化了脱色精制SAM双盐产品的方法。破胞液经超滤后,过D152树脂以硫酸溶液洗脱,可得到纯度(色谱纯)在98%以上的SAM硫酸盐产品,以对甲苯磺酸溶液溶解SAM硫酸盐后脱色精制得到SAM硫酸对甲苯磺酸双盐。     

离心法分离毕赤酵母活细胞和死细胞

本文以毕赤酵母为研究对象,探索出一种分离活酵母细胞的新方法。研究发现,通过改变淋巴细胞分离液和50%聚蔗糖溶液的比例,获得不同密度的酵母细胞分离液,进而通过离心分层的方法可使毕赤酵母活细胞主要存留于离心液的上层。当酵母细胞分离液的密度为1.1467 g/mL (27.5%淋巴细胞分离液+72.5%聚蔗糖溶液),分离液上下层中酵母活细胞的分配比例差别达到最大,分别为94.67%(分离液上层)和5.33%(分离液下层)。毕赤酵母细胞浓度为4.35×108~1.13×109/mL时,活细胞在分离液上下两层的分配比例约为95%和5%。低毕赤酵母细胞存活率有利于离心分离。本方法可用于有效分离培养液中的毕赤酵母活细胞。     

S-腺苷甲硫氨酸和谷胱甘肽联产发酵的环境条件

在5 L发酵罐中,研究pH、搅拌转速和温度等环境条件对产朊假丝酵母CCTCC M209298联产发酵合成S-腺苷甲硫氨酸(SAM)和谷胱甘肽(GSH)的影响,发现酵母细胞生长、SAM和GSH合成各自需要最适的pH、搅拌转速和培养温度。以SAM和GSH联产量最大化为目标,获得了较为合适的联产发酵条件:pH 5.0,搅拌转速350 r/min,温度30℃。在此环境条件下,结合不低于35%的溶氧体积分数,分批培养产朊假丝酵母24 h,最终SAM和GSH联产产量可达到579.6 mg/L。     

耦合中空纤维膜超滤分离游离细胞催化合成ATP

对耦合中空纤维膜超滤分离进行游离细胞催化合成ATP过程进行了实验研究,考察了细胞的催化效率和膜组件的操作稳定性。结果显示,中空纤维超滤膜的耦合分离能有效地截留反应液中的游离酶,其中乙醇脱氢酶(ADH)和已糖激酶(HK)的稳态截留效率在95%以上。耦合膜分离的酵母细胞催化ATP合成反应可重复使用2.5～3.0次,酶的利用率比普通分离的细胞提高2.0～2.5倍。中空纤维超滤膜于0.1Mpa工作压力下连续11批耦合分离操作,膜的渗透性无明显下降,过滤速率保持在初速率的95%以上。在稀释速率0.25h-1下,反应体系保持了连续5h的ATP高转化率合成与分离耦合的拟稳态操作。     

百部总生物碱含量测定方法的研究

分别运用酸碱滴定法和雷氏盐吸收系数法对百部中总生物碱的含量进行了测定,并加以比较。结果表明：雷氏盐吸收系数法的测定结果比酸碱滴定法的测定结果稍高,提取步骤少,操作简单,更能最大限度的减少干扰因素的影响,方法学考察均符合规定,可以用来测定百部总碱的含量．     

酵母亚细胞结构分离效率评估菌株的构建 *

背景: 真核细胞依赖其亚细胞结构高效地完成复杂的生化反应。尽管目前有一些亚细胞结构分离技术,但却缺乏评估这些分离技术的简单、有效方法。目的: 构建可以用来检测亚细胞结构分离效率的酿酒酵母菌株。方法: 通过传统的分子生物学与细胞生物学方法以酿酒酵母为背景构建了亚细胞结构分离效率评估菌株,并进行可行性检测。首先,将酵母各细胞器、自噬体及质膜的标记蛋白进行分组,用不同的蛋白标签分别标记每组蛋白。然后,以标记蛋白-GFP/RFP作为对照组通过免疫荧光法检测蛋白标签对标记蛋白的亚细胞定位是否有影响。最后,通过非连续性密度梯度离心验证该检测菌株能否用来检测亚细胞结构的分离效率。结果: 成功构建了酵母亚细胞结构分离效率评估菌株,该菌株标记了大多数酵母细胞亚细胞结构。挂标签的标记蛋白仍然定位在各自标记蛋白对应的亚细胞结构。非连续性密度梯度离心后,酵母各个亚细胞结构的标记蛋白均可以被检测到。结论: 该酵母亚细胞结构分离效率评估菌株是检测各亚细胞结构分离结果的方便工具,对今后酿酒酵母细胞生物学的研究有潜在的应用价值。     

穿心莲内酯联合S-腺苷甲硫氨酸抑制乙醇诱导肝细胞氧化应激损伤

氧化应激是酒精性肝病(ALD)发生的主要机制,因此具有抗氧化作用的活性物质对ALD具有潜在的治疗意义。在本研究当中,我们探讨了S-腺苷甲硫氨酸(SAM)和(或)穿心莲内酯(AD)对乙醇诱导肝细胞氧化应激损伤的保护作用。首先我们体外培养肝细胞L-02,用100 mmol/L乙醇作用24 h后发现,细胞的活性明显降低,胞内活性氧(ROS)含量显著增多以及脂质蓄积,同时细胞释放多种与肝病相关酶类如甘油三脂、胆固醇、转氨酶以及同型半胱氨酸等物质。而乙醇刺激前采用100μmol/L SAM或(和)30μmol/L AD处理细胞后,可明显降低细胞的氧化应激水平,同时能减少上述肝病相关标志物的的释放。此外,乙醇处理也可降低细胞内去乙酰化酶SIRT1的表达并抑制其核转位,而采用SAM或(和)AD处理后可进一步改善SIRT1的表达及活性。以上结果表明SAM或(和)AD能降低乙醇所致的氧化应激水平,两者联合应用可能对ALD具有潜在的治疗效果。     

阻断消耗途径提高毕赤酵母工程菌S-腺苷甲硫氨酸产量

【背景】S-腺苷甲硫氨酸(S-adenosyl-L-methionine, SAM)作为所有生物体内的重要中间代谢物,不仅可作为膳食补充剂,还具有良好的临床应用价值。【目的】将毕赤酵母重组菌GS115/DS16的SAM消耗途径阻断,进一步提高SAM的产量。【方法】分别敲除毕赤酵母重组菌GS115/DS16的S-腺苷同型半胱氨酸水解酶基因sah1、S-腺苷甲硫氨酸脱羧酶基因spe2和L-甲硫氨酰tRNA合酶基因msm1,构建工程菌G/Dsah、G/Dspe和G/Dmsm。检测3个工程菌的生长和SAM产量,以及L-Met添加量对SAM积累的影响。【结果】与出发菌GS115/DS16相比,工程菌G/Dsah、G/Dspe和G/Dmsm的单位菌体SAM产量分别提高了29.3%、55.6%和24.8%,其生长无显著差异。L-Met添加量优化后(0.06%),G/Dsah和G/Dmsm单位菌体的SAM产量分别提高了26.4%和28.9%。【结论】构建的毕赤酵母工程菌可用于SAM的工业化生产,该代谢工程策略可用于改进其他化学品的生产。     

Ganglioside composition of substrate-adhesion sites of normal and virally-transformed Balb/c 3T3 cells

The ganglioside composition of the so-called substrate-attached material (SAM), which remains tightly bound to the tissue culture dish after cells are detached by chelating agents, was compared with the ganglioside composition of released cell bodies in the cultures of normal and various virally-transformed Balb/c 3T3 cells. Regardless of whether the cells were untransformed or transformed, the SAM of their cultures shows a ganglioside structure characterized by a prevalence of the higher homologs, mainly GD1a, over the simpler gangliosides, even when the level of higher homologs was reduced in the cell bodies of transformed cells. This result cannot be ascribed to the presence of plasmamembranes in the SAM as shown by ganglioside analysis of the plasmamembranes of some of the cells under study. Only in a highly metastatic transformed cell line did the SAM contain the same low GD1a level as found in the cell bodies.     

In vivo hydrolysis of S-adenosyl-L-methionine in Escherichia coli increases export of 5-methylthioribose

Escherichia coli can not synthesize methionine from 5-methylthioribose (MTR) but instead exports this sulfur-containing, energy-rich molecule into the surrounding medium. Transforming E. coli with plasmids that direct expression of the cloned coliphage T3 S-adenosyl-L-methionine (SAM) hydrolase (SAMase) induces the met regulon by cleaving the SAM co-repressor to form 5'-methylthioadenosine, which is then cleaved to produce MTR. To test the effect of in vivo SAMase activity on MTR production and its fate, cultures were incubated in the presence of [35S]methionine and [methyl-3H]methionine. Cells with SAMase activity produced significantly enhanced levels (up to 40-fold in some trials) of extracellular MTR -- the only radiolabeled compound released in significant amounts -- when compared with controls. SAM synthetase (metK) mutants transformed with SAMase expression vectors did not show this increase, verifying the path through SAM as the sole route to MTR production. SAMase expression had little or no effect on intracellular MTR pools, levels of radiolabeled macromolecules, or the transfer of methyl groups to compounds that could be precipitated by trichloroacetic acid. Thus, MTR appears to be a dead-end metabolite in E. coli, begging questions about how this has evolved, the mechanism of MTR export for the cell, and whether the release of MTR is important for some other activity.     

Substrate-associated macromolecules promote cytodifferentiation of BC3H1 myogenic cells.

Differentiated mouse BC3H1 myogenic cells secrete substrate-associated macro-molecules (SAM) which restrict the proliferation of undifferentiated cells and promote both cell shape changes and expression of predominantly the vascular smooth muscle (VSM)-specific isoform of the contractile protein alpha-actin. While we previously reported that high cell density was required for stimulating maximal expression of VSM alpha-actin in BC3H1 cells (Strauch and Reeser: Journal of Biological Chemistry 264:8345-8355, 1989), the permissive effect of SAM on myoblast cytodifferentiation was not at all dependent on the formation of cell to cell contacts. This observation suggests that biogenesis of an extracellular matrix rather than the formation of physical contacts between cells may be the rate-limiting step for induction of VSM alpha-actin expression at high cell density. The biologically active moieties in SAM that promote cytodifferentiation also are expressed by mouse embryonic fibroblast cell lines and are distinctly different from a class of adheron-like macromolecules released by differentiated BC3H1 myocytes directly into the culture medium. While SAM was cell growth restrictive, reconstituted particulate material (RPM) prepared from myocyte-conditioned medium promoted the adhesion and proliferation of growth-arrested myoblasts. SAM and RPM are composed of different polypeptide subunits which collectively may establish microenvironmental conditions that are permissive for BC3H1 myogenic cell differentiation.     

补加前体L-蛋氨酸对高密度发酵生产S-腺苷-L-蛋氨酸的影响

将高密度发酵技术成功应用于S-腺苷-L-蛋氨酸的生产。考察了补加前体L-蛋氨酸的量以及补加策略对酿酒酵母G14发酵生产S-腺苷-L-蛋氨酸的影响。实验发现补加前体L-蛋氨酸能明显促进S-腺苷-L-蛋氨酸的积累。同时还发现不同的补加策略对菌体浓度以及S-腺苷-L-蛋氨酸的产量和浓度有不同的影响。确定了补加L-蛋氨酸不应低于0.7g/10g菌体干重。比较了五种不同的补加前体L-蛋氨酸的方式。结果表明在菌体干重达到高密度的情况下(120g/L)补加前体L-蛋氨酸进行转化生产S-腺苷-L-蛋氨酸能达到比较好的效果一次性补加9g L-蛋氨酸,SAM的积累量在补加后的18h达到最高,为4.31g/L;采取流加方式补加L-蛋氨酸,流加速率为2g/h,共流加5h,流加结束28h后SAM达到最高积累量后者达到4.98g/L。两者最终的生物量均可达到130g/L以上。     

发酵法生产S-腺苷蛋氨酸前体蛋氨酸补加策略

利用酿酒酵母菌株高密度发酵法生产S-腺苷蛋氨酸关键的影响因素之一是前体L-蛋氨酸的补加策略.本研究采用一支经过常规诱变处理的S-腺苷蛋氨酸优势积累菌株酿酒酵母SAM0801,通过5 L发酵罐高密度发酵实验研究,考察了6种补加策略,最终确定了L-蛋氨酸的加入时机为30h左右,当茵体干重达到100g/L时,补加量为每罐40gL-蛋氨酸,发酵58 h左右达到最高生物量干重168 g/L,产量14.48 g/L.     

Intracellular expression of Vitreoscilla hemoglobin improves S-adenosylmethionine production in a recombinant Pichia pastoris

To develop an efficient way to produce S-adenosylmethionine (SAM), methionine adenosyltransferase gene (mat) from Streptomyces spectabilis and Vitreoscilla hemoglobin gene (vgb) were coexpressed intracellularly in Pichia pastoris, both under control of methanol-inducible promoter. Expression of mat in P. pastoris resulted in about 27 times higher specific activity of methionine adenosyltransferase (SMAT) and about 19 times higher SAM production relative to their respective control, suggesting that overexpression of mat could be used as an efficient method for constructing SAM-accumulating strain. Under induction concentration of 0.8 and 2.4% methanol, coexpression of vgb improved, though to different extent, cell growth, SAM production, and respiratory rate. However, the effects of VHb on SAM content (specific yield of SAM production) and SMAT seemed to be methanol concentration-dependent. When cells were induced with 0.8% methanol, no significant effects of VHb expression on SAM content and specific SMAT could be detected. When the cells were induced with 2.4% methanol, vgb expression increased SAM content significantly and depressed SMAT remarkably. We suggested that under our experimental scheme, the presence of VHb might improve ATP synthesis rate and thus improve cell growth and SAM production in the recombinant P. pastoris.     

Increased T‐cell stimulating activity by mutated SEC2 correlates with its improved antitumour potency

Aims: To investigate the improved antitumour activity of SAM‐3 compared with recombinant staphylococcal enterotoxins C2 (rSEC2). Methods and Results: Methylthiazol tetrazolium and flow cytometry assays showed that the antitumour activity of SAM‐3 in vivo was improved because of enhanced T‐cell stimulating potency, resulting in massive activation of T cells, particularly CD4 ⁺ and CD8 ⁺ T cells, and subsequent cytokine release. Quantitative real‐time PCR assay showed that despite similar Vβ specificities induced by rSEC2 and SAM‐3, the quantities of activated T cells bearing specific Vβin vitro were different. Conclusions: The results strongly suggested that the increased SAM‐3–T‐cell receptor (TCR) binding affinity contributed to massive T‐cell activation and cytokine release, substantially amplifying antitumour immune response in vivo. Significance and Impact of the Study: This study provided evidence for the mechanism of SAM‐3 antitumour activity improvement compared with rSEC2. Results indicated that SAM‐3 could be used as a potent powerful candidate agent for tumour treatment in clinics.     

S-Adenosylmethionine-induced adipogenesis is accompanied by suppression of Wnt/β-catenin and Hedgehog signaling pathways

S-Adenosylmethionine (SAM) plays a crucial role as a methyl donor in various biological processes and has been previously shown to be involved in adipogenesis in skeletal muscle. This study was conducted to explore the mechanism of SAM inducing adipogenesis in skeletal muscle. Adipose precursor cells, 3T3-L1, and C2C12 cells, were induced into adipogenic differentiation by addition of SAM in MDI-differentiation media (0.5 mmol/L isobutylmethylxanthine, 1 μm/L dexamethasone, and 10 μg/mL insulin) to explore the role of SAM in promoting adipogenesis. Subsequently, cells were cultured with a medium containing SAM alone at the beginning of differentiation to test the relationship between SAM-induced adipogenesis and Wnt/β-catenin, and Hedgehog signaling pathways that control the cell commitment to adipogenic- or myogenic-differentiation. We found SAM possessed an additive effect with MDI in promoting adipogenesis of 3T3-L1 and C2C12 cells at the beginning of adipogenic differentiation. SAM could also individually induce cell adipogenesis in a dose-dependent manner. Moreover, the expression of Wnt/β-catenin and Hedgehog signals and their targets were suppressed by SAM (P < 0.05). These results demonstrate that SAM, as an increasingly accepted nutritional supplement, can initiate adipogenesis of adipose precursor cells derived from adipose and muscle tissues, a function at least partly correlated with the suppression of Wnt/β-catenin and Hedgehog pathways.     

拟南芥茎顶端分生组织相关突变体的表型与结构分析

以拟南芥野生型(C24)和T-DNA插入诱发的突变体(155系)为材料,通过表型分析、组织切片、GUS基因表达的组织化学定位等研究方法对155系的形态结构和生长发育进行了较为细致的观察分析,结果发现:(1)T-DNA插入诱发的155系突变体植株矮化,叶片等器官体积减小,营养生长阶段延长,发育较C24缓慢;(2)同一时期155系的茎顶端分生组织面积较C24减小,顶端平坦,细胞层数减少,两侧叶原基基部之间的距离缩短,呈现出发育迟缓、从茎顶端分生组织向花分生组织转变延迟等特征;(3)GUS基因特异性地在155系茎顶端分生组织和维管组织中表达.结果表明,T-DNA诱捕基因可能在茎顶端分生组织中发挥作用,由于T-DNA的插入使该基因的功能受到了影响,进而影响了155系中茎顶端分生组织的发育模式,产生了155系的一系列表型改变.     

Morphological characteristics and ganglioside composition of substratum adhesion sites in a hepatoma cell line (CMH5123 cells) during different phases of growth

This study compares the ganglioside composition of tissue culture substrate-attached material (SAM) with that of cell bodies in a line of transformed hepatocytes derived from the minimal deviation Morris hepatoma 5123 c (CMH5123 cells). We examined both confluent cultures (late-phase cultures) and cells which were allowed to attach for only 3 h (early-phase cultures). We also determined to what extent ganglioside compositions of SAM and cell bodies from early- and late-phase cultures of CMH5123 cells are affected by the block of complex ganglioside biosynthesis induced by treatment with chelating agents (EGTA + EDTA). The morphological characteristics of SAM were monitored by scanning electron microscopy during the different steps of this study. In early-phase cultures, SAM was composed of fragments of filopodia and small vesicles probably representing newly formed substratum adhesion sites. In contrast, SAM of late-phase cultures was made up of large pools of membranous material resulting from the breakage of thick retraction fibers connecting the cell body with broad, mature adhesion sites. SAM of early-phase cultures yielded ganglioside profiles with a higher content of GM1 and GD1 a than those of cell bodies, while in late-phase cultures there was no difference between SAM and cell body gangliosides. When cells were grown in the presence of chelating agents, SAM of early-phase cultures was composed of vesicles and filopodial fragments similar to those found in early-phase cultures grown in regular media; these morphological features also appeared in SAM of confluent cultures (in contrast to the membranous material characteristic of late-phase cultures grown in regular media). In early-phase cultures grown in the presence of chelating agents, gangliosides of SAM were enriched in complex homologs relative to their content in cell bodies. These ganglioside characteristics were also found in SAM of confluent cultures grown in the presence of chelating agents, reflecting the presence of newly formed adhesion sites. On the basis of these results, we may conclude that the molecular assembly of newly formed adhesion sites implies the preferential distribution of several surface components involved in cell adhesion, including complex gangliosides.