Yarrowia lipolytica发酵生产γ-癸内酯工艺的初步研究

目的:研究生物转化方法生产天然γ-癸内酯。方法:在单因素实验的基础上,对Yarrowia lipolytica AS2.1405菌株以蓖麻油为底物发酵生产γ-癸内酯的培养基配方进行优化。结果:在较为适宜的培养基中培养48h后,γ-癸内酯发酵产量达到0.6g/L。结论:实验结果可为γ-癸内酯发酵工艺改进提供依据。     

原生质体融合法构建γ-癸内酯高产菌株

该文确定了亲本原生质体制备的最佳条件为:采用0.3%β-巯基乙醇和0.1mol/L EDTA-Na2的复合预处理剂于28℃预处理10min,高渗稳定剂为1 mol/L pH 6.0的山梨醇,蜗牛酶浓度为2%、酶解温度为35℃、时间为2h.经过原生质体融合实验,成功获得了多株高产菌株,其中以QY30 γ-癸内酯产量最高为1.4852g/L,比亲本Y1-2提高了2 82倍.该菌株遗传性状基本稳定.     

γ-癸内酯的生物法制取

γ 癸内酯 (GDL)是香料工业由生物技术得到的主要产品。采用生物技术可以通过生物合成、生物转化和生物催化得到GDL。生物合成法通过真菌和酵母在静止期能合成和积累对于细胞生长非必需的作为次生代谢产物的内酯 ;生物转化法以羟基脂肪酸、非羟基脂肪酸和脂肪酸酯底物 ,经过微生物体内酶转化成γ 羟基脂肪酸 ,然后转化成内酯 ;生物催化法是利用脂肪酶 (如EC3 1 1 3)催化脂肪的水解反应、酯化反应和酯交换反应 ,包括催化羟基脂肪酸形成内酯。     

利用Yarrowia lipolytica基因重组菌株发酵产γ-癸内酯的初步研究

对基因重组菌株发酵生产γ-癸内酯的能力进行研究。对实验室构建的Yarrowia lipolytica基因重组菌株TP12与野生型菌株的生长能力及生成γ-癸内酯的能力进行比较。之后对重组菌株进行单因素条件优化,并在单因素条件优化的基础上进行正交实验优化。在最优发酵条件下,经过84h的发酵,γ-癸内酯产量最高得到3.337g/L。基因重组菌株TP12产γ-癸内酯的能力较野生型有大幅度的提高,为更高产γ-癸内酯基因重组菌的构建及其条件优化提供了参考。     

γ-癸内酯发酵条件的初步研究

通过对筛选出的一株酵母Y 1产GDL的发酵培养基组成及发酵工艺条件的摸索 ,并经正交实验 ,得到较优培养基配方为 :蛋白胨 3 5g/L ,酵母浸汁 2 5 g/L ,蓖麻油 5 % ,KH2 PO4 0 5 g/L ,Na2 HPO4 1 2 g/L ,MgSO4 ·7H2 O 2 4 g/L ,表面活性剂 0 4 % ,起始 pH7 0。在此条件下 ,发酵液中GDL含量可达 4 0 0mg/L。     

复合诱变选育γ-癸内酯生产菌株

以紫外诱变和化学诱变相结合的复合诱变方法,对γ-癸内酯产生菌株Yarrowia lipolytica酵母进行诱变。经过一次紫外诱变后,γ-癸内酯产量为过去的2.5倍,但多次诱变后其产量并没有进一步提高,且稳定性不好。其后采用硫酸二乙酯进行化学诱变稳定其产量。最终γ-癸内酯产量为出发菌株的2.3倍。     

田口设计优化耶氏酵母生产γ-癸内酯

通过田口设计对产γ-癸内酯的培养基成分进行优化。根据单因素实验结果,利用Minitab软件设计正交实验,采用田口设计方法分析正交实验中各因素水平的均值情况和信噪比情况。实验结果表明,复合氮源即酵母膏和（NH4）2HPO4对γ-癸内酯产量影响极显著,MgSO.47H2O对γ-癸内酯产量影响显著。软件预测培养基的最优组合为麸皮20g/L、酵母膏7.5g/L、（NH4）2HPO416.5g/L、KH2PO46g/L、MgSO4.7H2O0.5g/L,预测产量为2.49g/L,信噪比为9.04dB,经验证γ-癸内酯产量为2.65g/L,信噪比为9.68dB,优化方案达到了预期效果,具有较好的稳定性。      

耶罗维亚酵母(Yarrowia lipolytica)发酵蓖麻油制备γ-癸内酯的研究

γ-癸内酯是一种内酯类香味物质,主要用于配制奶油、桃子、柑橘和椰子等型香精.本研究的目的是探讨其微生物发酵制备方法.对耶罗威亚解脂酵母(Yarrowia lipolytica BTBU-205)以蓖麻油为底物产生γ-癸内酯的可行性、发酵条件、提取条件和发酵过程中各种因素的改变对产物的影响进行了研究,最终确定了发酵γ-癸内酯的培养基组成,即葡萄糖2%,玉米浆6%,磷酸氢二钠0.24%,硫酸镁1.2%,吐温-80 0.3%,蓖麻油50 g/L;发酵条件装液量为150mL发酵液/500 mL三角瓶,发酵温度27 ℃,pH6.5.在此条件下,γ-癸内酯的发酵产量达到1.9 g/L.试验结果表明,γ-癸内酯的生物制备方法是可行的.     

基因组改组技术提高γ-癸内酯产量

应用基因组改组技术提高耶罗维亚酵母γ-癸内酯的产量.耶罗维亚酵母原生质体在成串脉冲电场频率1000kHz,融合电压强度6kV/cm,脉冲个数4个,成串脉冲电压40V,脉冲宽度20μs的条件下进行电融合,原生质体的存活率能达到61.6％,其中一株改组菌株GDL产量为1.237g/L,是出发菌株产量的8倍.     

γ-癸内酯高产菌株的诱变选育和发酵条件的研究

以Yarrowia lipolytica As 2.1405为出发菌株,经紫外线-亚硝基胍复合诱变,筛选到遗传性状稳定的一株高产γ-癸内酯的突变菌株C9,并对该菌株的发酵培养基配方进行优化,γ-癸内酯平均产量提高至2.04 g/L。     

Production of proteases by psychrotrophic microorganisms.

Six milk-derived psychrotrophic microbial cultures were screened for the ability to grow at refrigerated temperatures and produce proteases in reconstituted skim milk. Of these, two cultures, Pseudomonas fluorescens M3/6 and Pseudomonas fragi K122, produced extracellular protease(s) beginning 7 d postinoculation when the cultures had entered late log or early stationary phases of growth. Further work with these two cultures showed that intracellular proteases were present after only 20-h incubation, before detection of the extracellular proteases. Using H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide (S-2251), a sensitive substrate for plasmin activity, P. fluorescens was shown to have greater intracellular proteolytic activity than extracellular activity at 20 h of incubation. The intracellular enzyme activity remained constant while the extracellular and periplasmic activities increased over the remaining 6-d incubation period. The proteases in crude extracellular extracts from both cultures were characterized and were heat stable with broad temperature (7 to 52 degrees C) and pH (pH 5.5 to 8.5) ranges for activity and were inhibited by the metal chelator, EDTA, indicating that they were metalloproteases.     

Production of indole by wine-associated microorganisms under oenological conditions

A high concentration of indole has been linked to ‘plastic-like’ off-flavour in wines, predominantly in wines produced under sluggish fermentation conditions. The purpose of this study was to determine the ability of yeast and bacteria to form indole and whether tryptophan was required for indole accumulation during winemaking. Wine-associated yeast and bacteria species (Saccharomyces cerevisiae, Saccharomyces bayanus, Candida stellata, Hanseniaspora uvarum, Kluyveromyces thermoloterans, Oenococcus oeni, Lactobacillus lindneri, Pediococcus cerevisiae and Pediococcus parvulus) were screened for their potential to generate indole during alcoholic or malolactic fermentation. Tryptophan was required for the accumulation of indole in chemically defined medium, and all yeast and bacteria fermentations were able to accumulate indole. C. stellata showed the greatest potential for indole formation (1033 μg/L) and among the bacteria, the highest concentration was generated by L. lindneri (370 μg/L). Whether primary fermentation is the principle cause of indole formation remains to be determined. We hypothesise that during an efficient fermentation, indole is removed through catabolic metabolism, but, when a sluggish fermentation arises, non-Saccharomyces species might produce excess indole that is still present by end of fermentation.     

Production of aldehyde oxidases by microorganisms and their enzymatic properties

In order to establish an efficient process to decompose environmentally toxic aldehydes, dioxygen-dependent aldehyde oxidase (ALOD) from microorganisms was first sought, and some bacteria and actinomycetes were found to produce the enzyme in their cells. Methylobacillus sp., Pseudomonas sp. and Streptomyces moderates were selected as the representative ALOD-producing strains and their enzymes were partially purified and characterized. The three ALODs could oxidize a wide range of aldehydes including formaldehyde, aliphatic aldehydes, and aromatic aldehydes, though their preferences differ depending on their producing strains. The other enzymatic properties were also determined with regard to their producing strains. Methylobacillus sp. ALOD had the most acidic optimum pH for its activity and stability and Pseudomonas sp. ALOD had the highest stability against heat treatment. Three native ALODs had molecular weights ranging from 140 to 148 kDa and were composed of three subunits of different sizes: large (85 to 88 kDa), medium-sized (37 to 39 kDa) and small (18 to 23 kDa).     

Production of poly-beta-hydroxybutyric acid by microorganisms accumulated from river water using a two-stage perfusion culture system

The perfusion culture system using a shaken ceramic membrane flask (SCMF) was employed to accumulate microorganisms separated from river water and to produce poly-beta-hydroxybutyric acid (PHB). Using a two-step culture method with a single SCMF, river microorganisms were cultured by separately feeding four representative carbon sources, n-propanol, lactic acid, methanol, and formic acid. After 140 h culture, the cell concentration and PHB content respectively reached 43 g/l and 35% when a propanol medium was fed. Using a two-stage perfusion culture with twin SCMFs, the seed cell mass was increased in the first SCMF and then supplied to the second flask for PHB production. As a consequence, the cellular PHB content rose to 51% in the second SCMF, while the cell concentration gradually increased to 25 g/l after 175 h perfusion culture. These results demonstrated the utility of the two-stage perfusion culture system for developing a cheap means of producing PHB coincident with wastewater treatment.     

微生物凝乳酶的研究进展

介绍了凝乳酶的分子特性和凝乳机理,综述了微生物凝乳酶的研究进展,最后介绍了利用基因工程生产凝乳酶和蛋白质工程改造凝乳酶的研究状况.并对凝乳酶的未来生产与开发作了展望.     

Biotechnological production of zeaxanthin by microorganisms

BackgroundZeaxanthin is a natural xanthophyll carotenoid that is widely produced by plants, algae and microorganisms and plays a critical role in the prevention of age-related eye diseases, such as macular degeneration and cataracts. Zeaxanthin is also used in the food, pharmaceutical and nutraceutical industries because of its strong antioxidant and anti-cancer properties. To date, zeaxanthin has been primarily produced by extraction from natural resources, especially plants, which is costly and environmentally unfriendly. The biosynthesis of zeaxanthin by microorganisms has been reported in lots of works to provide another potential route for zeaxanthin production.Scope and approachIn this review, we discuss the zeaxanthin biosynthetic pathway, naturally occurring zeaxanthin-accumulating microorganisms containing bacteria and microalgae, the optimization of fermentation conditions using these microorganisms, and zeaxanthin production using microbial cells factory constructed by metabolic engineering. The different metabolic engineering strategies and the zeaxanthin-accumulating level of the reviewed wild and engineered microorganisms are also considered. Furthermore, this work presents perspectives concerning the microbial production of zeaxanthin, especially the trends to construct the metabolically engineered microorganisms for zeaxanthin production.Key findings and conclusionsTo date, all the reported wild zeaxanthin-accumulating microorganisms belong to either bacteria or microalgae, while most of the reported engineering microorganisms for zeaxanthin production are Escherichia coli or yeast. A feasible strategy for zeaxanthin production is the use of metabolic engineering to construct a zeaxanthin-accumulating microbial cells factories followed by the optimization of fermentation with the engineered strain. Besides the simple overexpression of the biosynthesizing genes, the dynamic regulation of the constructed pathway has also been used for zeaxanthin production by metabolic engineering. Construction of better microbial cells factories which produce more zeaxanthin will profit from the breakthrough of the following fields: Introduction of higher plant zeaxanthin biosynthesizing genes into microorganisms; Characterization of novel zeaxanthin pathway genes from the wild microorganisms producing high level of zeaxanthin; Deep investigation of the farnesyl diphosphate formation pathway; Construction of microbial host with weak antioxidative capacity.     

食用微生物色素的研究进展

随着人们对食品添加剂的广泛热议,食用色素也备受关注。由于现代生活水平的提高,人们开始追求更加健康的生活方式,故合成食用色素已经不能满足人们的要求。因此,天然来源的食用色素已经成为当今食用色素发展的主流。食用天然色素是指由天然资源中获得的食用色素,主要是指从动物和植物组织及微生物发酵液中提取的色素。但是植物来源的色素会存在来源不易保证、经济花费高和技术方面的问题,而微生物产食用色素则刚好可以解决这些问题。微生物产色素具有生产周期短、产量高的特点,利用微生物产色素则成为当今天然色素的发展趋势。本文将综述微生物色素的分类研究、有食用价值的聚酮类色素及其生产菌株的研究进展,并对其可能产生的毒素进行探讨。