酒醅来源酵母菌合成异戊醇能力与途径解析

【背景】异戊醇是酵母菌在白酒发酵过程中通过氨基酸合成代谢途径和氨基酸分解代谢途径合成的主要高级醇,其含量影响白酒饮用的舒适度。【目的】分析和比较分离自浓香型白酒酒醅中的酵母菌合成异戊醇的能力,揭示酵母菌合成异戊醇的途径。【方法】从酒醅中分离具有异戊醇合成能力的酵母菌株,比较不同生长时期酵母菌合成异戊醇的能力,通过前体物代谢分析它们合成异戊醇的途径。【结果】分离自酒醅的5株酵母的异戊醇合成能力从强到弱依次为Naumovozyma castellii JP3-1、Saccharomyces cerevisiae JP3、Pichia fermentans JP22、Pichia kudriavzevii JP1和Naumovozyma dairenensis CBS421。这些酵母合成异戊醇的时期主要在对数生长期,N. castellii JP3-1、P. fermentans JP22和N. dairenensis CBS421在稳定生长期也合成异戊醇。S. cerevisiae JP3、N. castellii JP3-1和N. dairenensis CBS421在整个生长时期主要通过Harris途径合成异戊醇;P. kudriavzevii JP1在整个时期主要通过Ehrlich途径合成异戊醇;P. fermentans JP22在对数生长期通过Harris途径和Ehrlich途径合成异戊醇的能力接近,在稳定生长期主要通过Harris途径合成异戊醇。【结论】本研究揭示了酒醅来源5个属种酵母合成异戊醇的途径、能力与其生长时期的关系,研究结果可为解析浓香型白酒发酵过程异戊醇合成、积累机制及实施白酒发酵过程异戊醇合成的精准调控提供理论依据。     

盐析法提取烟粉虱基因组DNA

所提供的提取烟粉虱基因组DNA的新方法-盐析法,同氯仿-异戊醇抽提法相比,无需利用研磨棒,提取基因组DNA所用时间更短并且有效地避免了氯仿-异戊醇对实验人员的身体伤害.经检测,利用盐析法可以有效的提取B型烟粉虱的卵、伪蛹和成虫、Q型、ZHJ-1型与ZHJ-2型烟粉虱成虫的基因组DNA,并且适用于RAPD、COI、SSR分子标记的PCR扩增.     

微藻油脂生产的现状和发展

随着社会生产力的提高,对油脂在"用"和"吃"的方面的需求也越大。如何获取更高产和更优质的油脂是各国研究人员都在思考和探讨的问题。本文探讨和归纳总结了国内外微藻生产油脂的菌种特点、培养方法及油脂提取加工等最新研究成果及其优缺点。     

在大肠杆菌中表达酮酸脱羧酶产异戊醇

酮酸脱羧酶作为异戊醇生物合成的关键酶,不存在于大肠杆菌中。以乳酸乳球菌的基因组DNA为模板,经过PCR扩增得到酮酸脱羧酶基因kivD(rbs),插入到大肠杆菌高效表达载体pET-28a(+)上形成pET-kivD(rbs),重组质粒热击转化进大肠杆菌BL21(DE3)中,其成功表达了酮酸脱羧酶。对发酵产物进行分析,检测到了微量的目标产物—异戊醇。     

全媒体时代医院舆情管理危机的应对措施

随着科学技术的快速发展,各类新媒体的出现让部分医疗事件有了更高的关注度。近年来医疗纠纷日益严重,在全媒体时代的背景下,医院舆情危机频繁发生,对医院声誉和医患关系产生较大的不良影响。如何正确的引导网络舆情、降低医院舆情危机带来的不利影响成为急需解决的问题。本研究就全媒体时代医院舆情管理危机展开探讨,从医院舆情管理现状、医院舆情管理所存在的问题及相关的应对措施这三个方面入手,旨在为医院管理者在全媒体背景下控制医院舆情、化解医院突发事件、改善医患关系提供参考。     

湖泊沉积物基因组DNA的提取方法比较研究

水体沉积物为一新型微生物种质资源库,不依赖于培养的菌种多样性研究的首要条件是获取优质的基因组DNA。本研究以改进的氯仿异戊醇法、蛋白酶K+SDS法、玻璃珠+蛋白酶K+SDS法提取湖泊基因组DNA,并与试剂盒提取结果进行比较,为沉积物分子生态学的研究提供参考和借鉴。结果表明,改进后的氯仿异戊醇法提取的DNA纯度较低,腐殖质明显,纯化后PCR产量较低;蛋白酶K+SDS法获得的DNA完整性较好,浓度高,PCR扩增应用效果最好;玻璃珠+蛋白酶K+SDS法提取的基因组DNA效果次之;试剂盒法提取的基因组DNA浓度较低,片段长度略小。蛋白酶K+SDS法为除试剂盒外适合于湖泊沉积物基因组DNA提取的较好方法。     

绿豆核DNA的快速提取方法研究

为缩短提取核DNA的时间,提高DNA的得率,对绿豆核DNA的提取条件进行了优化,用4.00ml细胞提取液,2.50ml饱和KCl溶液,0.30倍样液体积的酚/氯仿/异戊醇（21：28：1）溶液,0.30倍样液体积的氯仿/异戊醇溶液,0.90倍体积的异丙醇,可从1.0g绿豆芽中提得4.0mg纯DNA。     

植物细胞培养物已成为有益药物及稀有植物的库源

目前,上百万胡萝卜胚、脱毒甘蔗、抗癌化合物紫杉酚(一种抗细胞微管解聚化合物)及抗龋齿剂血根碱均可由当今的植物细胞培养技术所生产。 研究人员正在探索和利用复杂的植物细胞培养技术方法。这些方法同天然物质生产过程相比较,可生产一些有用的化合物、植物繁殖速度更快、更一致、产量更高。此外,植物细胞培养物可防止稀有植物因过量收获而使物种灭绝。未来的植物细胞工程为医用药物和其他治疗剂的生产、植物的育种与选择、杀虫剂与杀菌剂的生产以及食品加工和食品风味的改良展现了广阔的前景。     

酿酒酵母类丙酮酸脱羧酶基因缺失对高级醇生成量的影响

【目的】通过构建酿酒酵母类丙酮酸脱羧酶基因(YDL080C)缺失的工程菌株,研究该基因对酿酒酵母浓醪发酵产高级醇特别是异戊醇的影响。【方法】以酿酒酵母工业菌株AY-15的单倍体a-8或α-22的基因组DNA为模板,PCR分别扩增YDL080C上下游非编码区片段YA和YB;以pUG6质粒为模板,PCR扩增KanMX抗性基因片段。分别将YA、YB和KanMX片段连入pUC19载体,构建重组质粒pUC-YABK;并以其为模板,PCR扩增YA-KanMX-YB重组盒,分别电转化单倍体a-8和α-22。将转化子和亲本分别进行酒精浓醪发酵,发酵结束后测定其发酵性能和高级醇的生成量。【结果】筛选获得了YDL080C基因缺失突变株。酒精发酵后发酵性能和高级醇测定结果显示,转化子的异戊醇及总高级醇生成量与对应的单倍体亲本相比没有明显变化,但酒精度分别比亲本提高了0.6(%,v/v)和0.4(%,v/v)。【结论】YDL080C基因缺失对降低酿酒酵母发酵产高级醇特别是异戊醇没有明显作用,但会使酒精度有所提高。     

脱落酸抗代谢与光稳定性类似物研究进展

脱落酸是一种广泛存在于植物体内的抑制性植物激素, 具有诱导种子休眠、抑制种子萌发、控制气孔关闭和增强植物抗逆性等生物活性, 在植物生长发育的各个阶段起着独特而重要的生理作用。但是较高的生产成本、在植物体内快速代谢失活和侧链2-位顺式双键的光异构化失活限制了脱落酸在农业生产中的应用。因此, 合成并筛选出活性更高、更稳定的脱落酸类似物, 是备受关注的研究领域。该文综述了脱落酸抗代谢与光稳定性研究的新进展, 介绍了脱落酸抗代谢与光稳定性类似物研究中存在的问题并对今后的研究方向进行了展望。     

Engineering the leucine biosynthetic pathway for isoamyl alcohol overproduction in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Isoamyl alcohol can be used not only as a biofuel, but also as a precursor for various chemicals. Saccharomyces cerevisiae inherently produces a small amount of isoamyl alcohol via the leucine degradation pathway, but the yield is very low. In the current study, several strategies were devised to overproduce isoamyl alcohol in budding yeast. The engineered yeast cells with the cytosolic isoamyl alcohol biosynthetic pathway produced significantly higher amounts of isobutanol over isoamyl alcohol, suggesting that the majority of the metabolic flux was diverted to the isobutanol biosynthesis due to the broad substrate specificity of Ehrlich pathway enzymes. To channel the key intermediate 2-ketosiovalerate (KIV) towards α-IPM biosynthesis, we introduced an artificial protein scaffold to pull dihydroxyacid dehydratase and α-IPM synthase into the close proximity, and the resulting strain yielded more than twofold improvement of isoamyl alcohol. The best isoamyl alcohol producer yielded 522.76 ± 38.88 mg/L isoamyl alcohol, together with 540.30 ± 48.26 mg/L isobutanol and 82.56 ± 8.22 mg/L 2-methyl-1-butanol. To our best knowledge, our work represents the first study to bypass the native compartmentalized α-IPM biosynthesis pathway for the isoamyl alcohol overproduction in budding yeast. More importantly, artificial protein scaffold based on the feature of quaternary structure of enzymes would be useful in improving the catalytic efficiency and the product specificity of other enzymatic reactions.     

Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei

The effects of important reaction parameters for enhancing isoamyl acetate formation through lipase-catalyzed esterification of isoamyl alcohol were investigated in this study. Increase in substrate (acid) concentration led to decrease in conversions. A critical enzyme concentration of 3 g l(-1) was detected for a substrate concentration of 0.06 M (each of alcohol and acid). Solvents with partition coefficient higher than 1000 (log P>3.0) supported enzyme activity to give high conversions. Acetic acid at higher concentrations could not be esterified easily probably owing to its role in lowering the microaqueous pH of the enzyme. Extraneous water/buffer addition decreased the isoamyl acetate yields slightly ( approximately 10%) at 0.005-0.01% v/v of the reaction mixture and drastically (>40%) at above 0.01% v/v. Buffer saturation of the organic solvent employed improved esterification (upto two-fold), particularly at moderately higher substrate concentrations (>0.18 M). Employing acetic anhydride instead of acetic acid resulted in a two-fold increase in the yields (at 0.25 M substrate). Use of excess nucleophile (alcohol) concentration by increasing the alcohol/acid molar ratio resulted in higher conversions in shorter duration (upto eight-fold even at 1.5 M acetic acid). Yields above 80% were achieved with substrate concentrations as high as 1.5 M and more than 150 g l(-1) isoamyl acetate concentrations were obtained employing a relatively low enzyme concentration of 10 g l(-1). The operational stability of lipase was also observed to be reasonably high enabling ten reuses of the biocatalyst.     

Lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate

Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.     

Glycerol triacetate as solvent and acyl donor in the production of isoamyl acetate with Candida antarctica lipase B

Glycerol triacetate was successfully used as a green solvent and as the acyl donor in the transesterification of isoamyl alcohol to produce isoamyl acetate using free and immobilized Candida antarctica lipase B. Immobilized lipase was more catalytically active than free lipase and could be easily separated from the reaction mixture by filtration. In addition, it was found that increasing either the reaction temperature or the enzyme to substrate ratio increased the conversion of isoamyl alcohol. Using triacetin as the solvent also enabled the separation of product by simple extraction with petroleum ether and catalyst recycling.     

A kinetic study of isoamyl acetate synthesis by immobilized lipase-catalyzed acetylation in n-hexane

The objective of this work was to propose a reaction mechanism and to develop a rate equation for the synthesis of isoamyl acetate by acylation of the corresponding alcohol with acetic anhydride using the lipase Novozym 435 in n-hexane. The reaction between isoamyl alcohol and acetic anhydride occurred at high rate in first place. Then, if excess alcohol was used, produced acetic acid further reacted with remaining alcohol, leading to yields higher than 100% (based on initial acetic anhydride content). This reaction was much slower and took place only when acetic anhydride had been totally consumed. Optimal pH for Novozym 435 was 7.7. Acetic acid strongly inactivated the enzyme but it was partially caused by the pH drop in the biocatalyst aqueous microenvironment. Acetic anhydride also showed an important inhibition effect. On the contrary, isoamyl alcohol and isoamyl acetate had no negative effect on the lipase. The analysis of the initial rate data showed that reaction followed a Ping-Pong Bi-Bi mechanism with inhibition by acetic anhydride. The kinetic constants were obtained by multiple regression analysis of experimental findings. Equation predictions and experimental reaction rate values matched very well at conditions where acetic acid concentration in the medium was low.     

Short-chain aliphatic alcohols increase rat-liver microsomal membrane fluidity and affect the activities of some microsomal membrane-bound enzymes

n-Butyl and isoamyl alcohols decrease the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and enhance the efficiency of pyrene excimer formation when these probes are incorporated in rat-liver microsomal membrane, suggesting an increase in rotational and translational mobilities. Neither alcohol modifies NADH-ferricyanide reductase activity but both increase NADH-cytochrome c reductase activity. This was interpreted as an increase in the rate of lateral diffusion of the proteins cytochrome b5 and cytochrome b5 reductase as a consequence of the enhanced membrane lipid phase fluidity. Microsomal delta 9 and delta 6 desaturase activities in the presence of isoamyl alcohol were also studied. This alcohol decreases delta 9 desaturation when it is measured at a low substrate concentration (13 microM palmitic acid), but it is not modified when it is measured at a high substrate concentration (66 microM palmitic acid). delta 6 desaturation is diminished by isoamyl alcohol when it is measured with both 13 microM and 66 microM linoleic acid. The influence of isoamyl alcohol on the glucose-6-phosphatase system activity was also studied. In non-detergent-treated microsomes, isoamyl alcohol enhances glucose-6-phosphatase activity. However, if microsomes are previously treated with 0.1% Triton X-100 isoamyl alcohol does not modify this activity. The enhancement of the glucose 6-phosphate transport rate is not due to membrane permeability barrier disruption, since isoamyl alcohol does not modify mannose-6-phosphohydrolase latency. This would suggest that an increase in membrane lipid phase fluidity specifically activates glucose 6-phosphate transport across the membrane.     

Metabolic engineering of the anaerobic central metabolic pathway in Escherichia coli for the simultaneous anaerobic production of isoamyl acetate and succinic acid

An in vivo method of producing isoamyl acetate and succinate simultaneously has been developed in Escherichia coli to maximize yields of both high value compounds as well as maintain the proper redox balance between NADH and NAD⁺. Previous attempts at producing the ester isoamyl acetate anaerobically did not produce the compound in high concentrations because of competing pathways and the need for NAD⁺ regeneration. The objective of this study is to produce succinate as an example of a reduced coproduct to balance the ratio of NADH/NAD⁺ as a way of maximizing isoamyl acetate production. Because the volatility of the two compounds differs greatly, the two could be easily separated in an industrial setting. An ldhA, adhE double mutant strain (SBS110MG) served as the control strain to test the effect of an additional ackA‐pta mutation as found in SBS990MG. Both strains overexpressed the two heterologous genes pyruvate carboxylase and alcohol acetyltransferase (for ester production). The triple mutant SBS990MG was found to produce higher levels of both isoamyl acetate and succinate. At the optimal condition of 25°C, the culture produced 9.4 mM isoamyl acetate and 45.5 mM succinate. SBS990MG produced 36% more ester and over 700% more succinate than SBS110MG. In addition, this study demonstrated that a significantly higher isoamyl acetate concentration can be attained by simultaneously balancing the carbon and cofactor flow; the isoamyl acetate concentration of 9.4 mM is more than seven times higher than an earlier report of about 1.2 mM. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009