应用代谢通量法分析花生四烯酸的合成过程

代谢通量分布分析已经成为研究发酵过程特性的有效方法.今建立了花生四烯酸(AA)在高山被孢霉ME-1(Mortierella alpina ME-1)体内合成的代谢通量模型,求解不同氮源浓度下发酵各时期的碳流分布.充足氮源发酵时,指数生长期、减速期、稳定期流向AA的碳流分别占总碳流的3.28%,8.80%,6.97%.而通过限制性氮源发酵并在96 h补加0.05%的NaNO3成功地引导了发酵碳流迁移,将各时期流向AA的碳流提高至3.95%,19.21%,39.29%,并最终实现AA产量从1.3 g·L-1提高到3.5 g·L-1.这些结果表明限制性氮源发酵并在稳定期补加低浓度的氮源能显著提高AA产量.     

超临界CO_2萃取被孢霉菌体油脂及花生四烯酸的研究

采用超临界CO2 萃取技术提取被孢霉菌体油脂 ,研究在超临界状态下 ,温度、压力、夹带剂与时间对油脂萃取率的影响 ,并分析对油脂成分的影响 ,从实际结果确定的优惠工艺条件为 :萃取压力为 13MPa ,萃取温度为 32℃ ,时间为 70min ,甲醇 10 %。此时油脂得率达到 37 4 5 % ,花生四烯酸含量达到 15 6 8%。     

高山被孢霉△5脱饱和酶基因的分离与验证

原节录：花生四烯酸（arachidonicacid）是人体的必需脂肪酸，具有独特的生物活性。A5脱饱和酶是花生四烯酸生物合成途径上的关键酶，以二高γ亚麻酸为底物，催化其第5位碳脱氢形成花生四烯酸。从花生四烯酸高产菌株高山被孢霉M6（Mortierellaalpina）中通过RTPCR分离了可能的编码△5脱饱和酶完整的cDNA，其大小为1366bp，编码446个氨基酸。推导出的蛋白质含有A5脂酰脱饱和酶中保守的特征性结构域，包括该蛋白质N端典型的细胞色素b5结构域，以及3个保守的组氨酸盒。为验证该蛋白质的功能，将该基因片段克隆到穿梭表达载体pPIC9K中，获得的重组载体pPIC9KD5通过电转化法转化到巴斯德毕赤酵母GS115中，     

尿素包合法纯化油脂中花生四烯酸的工艺研究

采用尿素包合法从被孢霉菌体油脂中提取花生四烯酸。试验运用正交设计，考察了包合反应中尿素、溶剂的量等因素对产品中花生四烯酸含量的影响，优化了工艺条件．花生四烯酸的纯度由粗油中的23．21％提高到61．52％。     

花生四烯酸的研究进展

花生四烯酸(AA)是人体必需脂肪酸,具有极高的保健价值。对菌种培育、发酵条件优化进行了分析,综述了该研究领域的国内外发展现状、动态及存在的问题,并对其发展趋势作了展望。     

一株利用稻草秸秆发酵产油脂菌株的筛选及其发酵条件的初步研究

从土壤中筛选到一株能在纤维素刚果红培养基上生长的真菌,经形态学和ITS全序列分析鉴定该菌株为长孢被孢霉PFY。菌株在发酵过程中不需额外添加纤维素酶就可直接利用预处理后的稻草秸秆进行发酵生产油脂。利用气质联用技术分析了所产油脂成分,该油脂中含有较多的不饱和组分,其中油酸和亚油酸百分含量为31.70%和27.30%。对其利用预处理后的秸秆进行发酵因素的考察,适宜发酵条件为底物浓度20g/L,初始pH6,温度28℃,接种量2%,添加0.2%KH2PO4和0.05%ZnSO4。在适宜条件下发酵后油脂得率为8.15%。     

长孢被孢霉PFY降解木质素的初步研究

为了研究高效降解木质素类化合物,利用木质素磺酸钠为唯一碳源,采用苯胺蓝和愈创木酚平板法从土壤分离、筛选得到一株具有高降解木质素活性的丝状真菌,经鉴定为长孢被孢霉PFY(Mortierella elongataPFY)。在添加0.5g/L葡萄糖,最适氮源及浓度为1.5g/L硫酸铵,底物浓度为2g/L,30℃,pH=7.0的条件下,木质素降解率可达52.4%。通过对木质素降解特性的初步分析表明,在该降解过程中主要起作用的是木质素过氧化物酶和漆酶。这为以稻草秸秆为代表的生物质废弃物资源化提供了借鉴,也有利于造纸黑液中木质素的回收利用。     

阿魏酸对皮状丝孢酵母细胞生长与油脂积累影响的研究

本研究首次研究了阿魏酸对皮状丝孢酵母细胞生长与油脂积累的影响。研究表明,低浓度下,阿魏酸对皮状丝孢酵母细胞生长影响不大,且会对其油脂积累略有促进。但在高浓度下会对皮状丝孢酵母的细胞生长与油脂积累产生明显的抑制。阿魏酸对皮状丝孢酵母糖代谢的影响部分解释了其对皮状丝孢酵母细胞生长与油脂积累的影响。此外,阿魏酸对皮状丝孢酵母的油脂脂肪酸组成也会造成一定影响。     

光诱导作用对木霉菌产孢量的影响研究

通过固体发酵和液体发酵,对比研究了光诱导作用对8株木霉菌菌株产孢量的影响。结果表明,在固体发酵条件下,50～200 Lux蓝光照射能显著提高菌株Tr673、TL02、Tr25、Tr775的产孢量(P≤0.01);菌株Tr673、TL02、Tr25、Tr775在避光处理36 h后再进行蓝光照射的产孢量较持续光照显著降低(P≤0.01)。在液体发酵条件下,菌株Tr673、TL02、Tr25、Tr775在100～200 Lux白光或蓝光照射下的产孢量较避光处理提高数倍(P≤0.01),较其它光源照射提高2～8倍;100～200 Lux蓝光照射能显著提高Tr673、TL02、Tr775的产孢量(P≤0.01),但对Tr25的产孢量提高不明显。     

深黄被孢霉Δ5-脱饱和酶的原核表达及纯化

目的原核表达并纯化深黄被孢霉Δ5-脱饱和酶。方法采用RT-PCR技术扩增深黄被孢霉Δ5-脱饱和酶基因,插入表达载体pET-30a(+)中,构建重组表达质粒pET-D5D,转化入大肠埃希菌(E.coli)BL21(DE3)中,IPTG诱导表达。表达的重组蛋白经Ni2+-NTA纯化后,进行Western blot鉴定。结果重组表达质粒pET-D5D经双酶切(NotⅠ/SalⅠ)和测序鉴定,证明构建正确;表达的重组蛋白相对分子质量约为55 000,诱导6 h表达量较高,主要以包涵体形式表达;纯化的重组蛋白可与兔抗Δ5-脱饱和酶多克隆抗体特异性结合。结论成功在E.coli中表达了深黄被孢霉Δ5-脱饱和酶,纯化后的重组蛋白反应原性良好,为进一步研究Δ5-脱饱和酶的功能奠定了基础。     

Lipid characterization of an arachidonic acid-rich oil producing fungus Mortierella alpina

Mortierel a alpina has been considered as the most effective producer of arachidonic acid (ARA)-rich oil. It was found that several methods could improve the percentage of ARA in total lipids successful y, as they activated the desaturation system on the endoplasmic reticulum. Additionally, in M. alpina the ARA exists in several forms, such as triacylglycerol (TAG), and diacylglycerol (DAG). These forms are caused by different acyltransferases and they determine the nutrient value of the microbial oil. However, few works revealed de-tailed fatty acid distribution among lipid classes, which to some extent impeded the accurate regulation in ARA accumulation. Herein, this paper gives information on the accumulation process of main lipid classes and the changes of fatty acid composition in these lipids during ARA accumulation period in M. alpina. The result dem-onstrates that TAG was the dominant component of the total lipids, and it is the main form for ARA storage. The ARA enrichment stage occurred during 168–192 h when the amount of total lipids maintained steady. Further analysis indicated that the newly formed ARA-TAG might come from the incorporation and modification of sat-urated and monounsaturated fatty acids in other lipid classes. This work could be helpful for further optimization of ARA-rich TAG production.     

Enhancement of arachidonic acid production by Mortierella alpina 1S-4

The effect of mineral addition on arachidonic acid (AA) production by Mortierella alpina 1S-4 was evaluated. At first, the addition of minerals such as sodium, potassium, calcium, and magnesium was examined in flask cultures, and then the addition of phosphorus with the optimal amounts of the minerals was investigated in a 10-L jar-fermenter. As a result, 1.5% soy flour medium with the addition of 0.3% KH₂PO₄, 0.1% Na₂SO₄, 0.05% CaCl₂·2H₂O, and 0.05% MgCl₂·6H₂O was found to enhance the AA yield 1.7-fold over that without mineral addition. When 1% yeast extract with the above mineral mixture was used, the AA yield was enhanced 1.35-fold over that without minerals. We also verified that an increase in the polar lipid content occurred in the case of only KH₂PO₄ addition, and that the above-mentioned increase in the AA yield was due to the minerals themselves, not a pH buffer effect.     

Process for production of arachidonic acid concentrate by a strain of mortierella alpina

Various Mortierella alpina fungi were screened for their capacities to produce arachidonic acid. A strain of M. alpina was found to show the highest productivity. Arachidonic acid content of biomass and overall yield per litre of culture was highest in soya flour supplemented medium which produced dispersed mycelium. When the glucose concentration in the medium was varied from 30 to 100 g/L, biomass, lipid, arachidonic acid content of biomass and arachidonic acid yield increased with increasing glucose concentration. Several natural oils, when added to the growth medium, stimulated arachidonic acid production. After fermentation in a 20-L fermenter under optimal culture conditions, the arachidonic acid yield was 5.3 g/L, representing 34.2% w/w of total fatty acids and 13.7% w/w of biomass. An extract containing 72.5% w/w arachidonic acid was prepared from the recovered mycelium.     

Effects of mineral addition on the growth morphology of and arachidonic acid production by Mortierella alpina 1S-4

The culture conditions for arachidonic acid (AA) production by Mortierella alpina 1S-4 were investigated by means of a morphological study with the aims of obtaining a high AA yield and scale-up. In a 50-L jar fermentor study, a medium containing 3.1% soy flour and 1.8% glucose with 0.3% KH₂PO₄, 0.1% Na₂SO₄, 0.05% CaCl₂·2H₂O and 0.05% MgCl₂·6H₂O was found to be optimum. The AA yield reached 9.8 g/L/7 d, and the major morphology was small pellets (1–2 mm). However, in the case of the only addition of KH₂PO₄, the major morphology was filaments. The apparent viscosity increased to 2240 cp, thereby requiring a high agitation speed to maintain adequate oxygen tension, which caused mycelial damage due to shear stress and therefore a decrease in the AA yield. When a medium with Na₂SO₄, CaCl₂, and MgCl₂ was used, the major morphology was large pellets (2–3 mm), and mass transfer limitation through the pellet wall caused a decrease in the AA yield. Based on these results, a scale-up study was carried out under the optimal conditions described above. An AA yield of 10.9 g/L/8 d was obtained in a 10-kL industrial fermentor, and the major morphology was small pellets.     

Production of the Quorum-Sensing Molecules N-Acylhomoserine Lactones by Endobacteria Associated with Mortierella alpina A-178

Gram‐negative bacteria communicate with one another using N‐acylhomoserine lactones (AHLs) as signaling molecules. This mechanism, known as quorum sensing (QS), is needed to develop pathogenicity, as well as symbiotic interactions with eukaryotic hosts, such as animals and plants. Increasing evidence indicates that certain bacteria, namely endobacteria, also inhabit fungal cells and establish symbiotic relationships with their hosts. However, it has not been clear whether bacterial QS acts in developing the relationships. Here we describe the isolation and identification of N‐heptanoylhomoserine lactone and N‐octanoylhomoserine lactone from the culture broth of the zygomycete fungus Mortierella alpina A‐178. This suggested the presence of endobacteria in the fungus, as was confirmed by PCR, fluorescence in situ hybridization, and transmission electron microscopy. Two major bands obtained by PCR‐denaturing gradient gel electrophoresis showed sequence identity to genes in the β‐proteobacterium Castellaniella defragrans (100 %) and the Gram‐positive bacterium Cryobacterium sp. (99.8 %). The production of AHLs depended on the presence of endobacteria and was induced in response to the increase in the concentration of AHLs, suggesting that the bacterium conducts AHL‐mediated QS in the fungus. This paper is the first to report the production of AHLs by endofungal bacteria and raises the possibility that QS plays roles in the development of fungus–endobacterium symbiosis.     

Optimization of arachidonic acid production by Mortierella alpina Wuji-H4 isolate

A fungal isolate Wuji-H4 with a dense-lobe rosette growth pattern on malt extract agar was identified as Mortierella alpina Peyronel. It was capable of producing 504 mg/L of arachidonic acid (AA) in the screening medium. Its AA content accounted for 42.4% of the total fatty acids. The AA yield was raised to 1,817 mg/L by a step-by-step approach, which uncovered that the preferred carbon source, nitrogen source, and temperature for fungal growth and lipid production were soluble starch, urea, and 24°C, respectively. Productivity was further optimized by exploiting the interactions between the constituents of the medium by the response surface method. A partial factorial design, followed by steepest ascent analysis, was carried out to locate the general vicinity of the optimal level of each nutrient. The response surface of AA production in this optimal region was then approximated with a full quadratic equation obtained from a three-factor/five-level central composite rotatable design. Maximum AA yield was predicted to occur in a medium that contained 99.7 g/L of soluble starch, 12.6 g/L of yeast extract, and 3.0 g/L of KH₂PO₄. Upon verification, the average experimental yield of AA (3,885 mg/L) was not significantly different from the predicted AA yield (3,940 mg/L), indicating that the response surface method had succeeded in exploiting the AA production potential of this new fungal isolate.     

Production of Polyunsaturated Fatty Acids by Mortierella alpina Using Submerse and Solid State Fermentation

A new isolate of Mortierella alpina, > 98 % identical with M. alpina ATCC 16266, was cultivated in a defined glucose‐based medium with three organic nitrogen sources (glycine, urea and Na‐L‐glutamate) at three different concentrations in shaking flasks at 20 °C. The results were compared to the cultivation in complex medium with yeast extract as nitrogen source. In the defined media, high yields of polyunsaturated fatty acids (PUFAs) and arachidonic acid (ARA), respectively, were obtained with Na‐L‐glutamate. However, the absolute highest yields of PUFA and ARA were measured with the yeast extract medium. An optimized yeast extract complex medium was used for a submerse bioreactor cultivation in a 45‐L scale. Furthermore, M. alpina was cultivated in a solid state fermenter, using an oat bran water mixture as substrate.     

Production of 8,11,14,17-cis-eicosatetraenoic acid by Δ5 desaturase-defective mutants of an arachidonic acid-producing fungus, Mortierella alpina

Δ5 Desaturase-defective mutants of an arachidonic acid-producing fungus, Mortierella alpina 1S-4, accumulate large amounts of 8,11,14,17-cis-eicosatetraenoic acid (20:4ω3) when grown with linseed oil. One of the mutants, the S14 strain, produced 1.65 mg of 20:4ω3 per mL of culture medium (corresponding to 66.0 mg/g dry mycelia and 11.6% of total cellular fatty acids) when grown in a medium containing 1% glucose, 1% yeast extract, and 4% linseed oil methyl ester at 28°C for 2 d, and then at 16°C for 7 d. In a bench-scale fermentation in a 5-L jar fermenter, 20:4ω3 production reached 1.60 g/L of culture medium on the eighth day (corresponding to 77.3 mg/g dry mycelia and 26.0% of total cellular fatty acids). The cellular lipids of the S14 strain comprised 75.8% triacylglycerol (TG), 6.7% diacylglycerol, and 13.3% phospholipids (PL). The percentage of 20:4ω3 was higher in PL than in TG, and highest in phosphatidylcholine (32.6%).     

Production of arachidonic acid by filamentous fungus, Mortierella alliacea strain YN-15

A filamentous fungus producing significant levels of arachidonic acid (AA, C20∶4n−6) was isolated from a freshwater pond sample and assigned to the species Mortierella alliacea. This strain, YN-15, accumulated AA mainly in the form of triglyceride in its mycelia. An optimized culture in 25 L of medium containing 12% glucose and 3% yeast extract yielded 46.1 g/L dry cell weight, 19.5 g/L total fatty acid, and 7.1 g/L AA by 7-d cultivation in a 50-L jar fermenter. Assimilation of soluble starch by YN-15 was notably enhanced by the addition of oleic acid, soybean oil, ammonium sulfate, or potassium phosphate to a starch-based medium. Using starch as a main carbon source in the pre-pilot scale cultivation improved the production of AA by up to 5.0 g/L. Mortierella alliacea strain YN-15 is therefore a promising fungal isolate for industrial production of AA and other polyunsaturated fatty acids.