粘性红圆酵母XJU-1产β-胡萝卜素的研究

目的:从粘性红圆酵母Rhodotorula mucilaginosaXJU-1中提取β-胡萝卜素并对其特性加以研究。方法:采用酸热法对发酵产物破壁,用丙酮∶石油醚(1∶1)萃取β-胡萝卜素,采用GB/T 5009.83-2003的HPLC方法测定β-胡萝卜素含量,以448nm波长的紫外分光光度吸收值计算出色素损失率来探讨β-胡萝卜素的稳定性。结果:该菌株在液体发酵培养基中生长72hβ-胡萝卜素含量高达389.2μg/g干重,经实验确定,葡萄糖是最有利于β-胡萝卜素生成的碳源;蛋白胨是生产β-胡萝卜素的最佳氮源。提取的β-胡萝卜素耐热性较好,需置于暗处保存。结论:该菌种β-胡萝卜素产率明显高于国内外已报道的,有很好的开发前景。     

法夫酵母β-胡萝卜素转化酶(Asy)基因的克隆及可溶性表达研究

[目的]从高产虾青素的法夫酵母(Phaffia rhodozyma) 7B12菌株中克隆β-胡萝卜素转化酶基因(β-Carotene converting enzyme gene,asy),并将该基因在大肠杆菌(Escherichia coli)中进行可溶性表达,为深入研究该酶的性质及应用提供基础.[方法]采用cDNA末端快速扩增技术,克隆得到asy基因全长cDNA序列,将其克隆到表达载体pET32a中,通过优化温度和IPTG浓度提高其可溶表达量;进一步在E.coli BL21(DE3)中共表达携带asy基因的重组质粒和pACCAR 16△crtx质粒(携带由乙酰辅酶A合成β-胡萝卜素的基因链),用液相色谱分析共转化菌株中类胡萝卜素种类的变化,鉴定可溶性表达的Asy酶活性.[结果]法夫酵母7B12菌株asy基因的cDNA序列与GenBank能检索到的唯一一条asy基因mRNA序列(Accession No.DQ002007.1)一致性达到97％,该序列总长1 971 bp,最大开放阅读框为1 614 bp,编码538个氨基酸,在E.coli BL21 (DE3)中表达的Asy融合蛋白分子量约为70 kD;条件优化后转化asy基因的重组菌在26℃、0.5 mmol/L IPTG的条件下诱导时,可溶性融合蛋白比例达85％.与pACCAR16△crtx单质粒转化相比,共转化pACCAR 16△crtx及asy基因的菌株类胡萝卜素产物的成分发生了明显的变化,其中α-胡萝卜素的含量明显减少,伴随出现了3种新的色素,根据出峰时间判断其中一种为β-胡萝卜素和虾青素的中间产物——β-隐黄质.[结论]从法夫酵母中克隆得到asy基因,通过优化诱导温度及IPTG浓度等条件提高了该基因在E.coli BL21(DE3)中的可溶性表达量,经鉴定可溶性的Asy融合蛋白具有转化β-胡萝卜素的活性.     

利用红酵母发酵生产β—胡萝卜素的研究进展

对红酵母β－胡萝卜素的提取方法以及菌株的发酵生理学条件研究作了小结,并讨论了未来的发展方向。结论认为：破壁效果是提取色素的关键,酸－热处理法是一种较好的破壁方法,但机械法应是大规模细胞玻碎更适用的方法;红酵母产β－胡萝卜素的特性（产量和产品组成）,不仅与所使用的菌株有关,还与培养条件特别是发酵生理学条件及提取方法有密切关系;超临界CO2萃取技术的应用和高产菌种的选育与构建,将是今后红酵母β－胡萝卜素实现产业化开发的主要研究方向。     

法夫酵母生物合成虾青素的研究

本文对法夫酵母是青素的生命合成进行了研究。实验结果表明,法夫酵母在ＹＥＰＤ培养基中生长时表现出二次生出现象,细胞内虾青素的合成是与细胞季开偶联的,在约６６ｈ达一在积累,３．４μｇ／ｍｌ。     

红酵母COS—5产胡萝卜素条件的研究

研究碳源、氮源和添加剂对红酵母（Rhodotorulasp．）COS－5产胡萝卜素的影响,并通过正交试验优化其产胡萝卜素的培养基组成。结果表明,COS－5产胡萝卜素的适宜培养基：蔗糖50g、蛋白胨5g、酵母膏5g、桔子皮15g、盐酸硫胺素0．002g、定容1L,pH6．0.250mL三角瓶装培养基30mL。在上述条件下28℃振荡培养96h,细胞生物量为27．5mg干重／mL发酵液,胡萝卜素含量达492．7μg／g干重细胞。COS－5胡萝卜素在474．4nm、338．6nm和310．2nm处有     

法夫红酵母主同产虾青素菌株的研究概况

虾青素是一种天然类胡萝卜素,具有较强的抗氧化作用。法夫红酵母是一种能合成虾青素的真菌。本文主要概述法夫红酵母高产虾青素菌株的育种、高产株选择、培养条件和虾青素的提取检测方法。     

流加发酵提高产胡萝卜素红酵母产量的研究

本文对产胡萝卜素的红酵母进行了间歇、恒速及变速流加发酵实验,建立了简单的数学模型控制流加。实验结果表明,变速流加可显著地提高红酵母的产量,当流加控制因子K为88×10－5,变速流水发酵比间歇发酵红酵母产量提高了56．2％。     

法夫红酵母高产虾青素菌株的研究概况

虾青素是一种天然类胡萝卜素,具有较强的抗氧化作用。法夫红酵母是一种能合成虾青素的真菌。本文主要概述法夫红酵母高产虾青素菌株的育种、高产株选择、培养条件和虾青素的提取检测方法     

一株红酵母产类胡萝卜素的研究

对自然分离得到的产类胡萝卜素菌株Ｈｍ６８进行了菌种鉴定,色素成份分析,确定其产生的类胡萝卜素中含有β胡萝卜素。该菌株在１％麦芽糖为唯一碳源的培养基中生长良好,类胡萝卜素产量达６２５μｇ／ｇ。     

红酵母细胞中β-胡萝卜素的快速分离柱高效液相色谱法测定

研究了用快速分离柱高效液相色谱法测定红酵母细胞中β-胡萝卜素的方法。发酵法得到的样品经匀浆后用四氢呋喃提取,提取液以ZORBAXStableBound(4.6×50mm,1.8μm)C18快速分离柱为固定相,乙腈-四氢呋喃体积比8020为流动相分离,流速为1.5ml/min;在该色谱条件下,β-胡萝卜素在1.0min内可达到基线分离;用紫外二极管矩阵检测器检测。方法标准回收率为98%～103%,相对标准偏差为0.46%～0.58%,可用于几种发酵样品中β-胡萝卜素的测定,结果可靠。     

红发夫酵母(phaffia rhodozyma)生产虾青素进展

本文综述了利用红发夫酵母(phaffiarhodozyma)培养生产虾青素的研究进展 。     

Transformation of Phaffia rhodozyma by electroporation

Summary The transformation of Phaffia rhodozyma by electroporation was better than the one by the lithium chloride (LiCl) or spheroplast transformation methods. The influence of several parameters on transformation efficiency was studied. Electroporation conditions of 1200 Volts, 50 Farads and 2000 Ohms were proved successful. A maximum of 1.5 × 10³ transformants per 100 ng of DNA was reached.     

Phaffia rhodozyma is polyploid

The ploidy of the red yeast Phaffia rhodozyma was evaluated using flow cytometric analyses of propidium iodide- stained cells and mutagenic inactivation kinetics. Our findings suggest that Phaffia rhodozyma is not haploid. Auxotrophic strains were generated at a high frequency following treatment of mutagenized cells with a combination of benomyl and ethyl acetate. Studies of an auxotrophic mutant using flow cytometry and UV inactivation indicated possible chromosome loss to an aneuploid state. Received 21 June 1998/ Accepted in revised form 25 September 1998     

Production of the UVB-absorbing compound mycosporine-glutaminol-glucoside by Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The ability of the basidiomycetous yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) to accumulate astaxanthin is responsible for the industrial use of this yeast as a microbial source of pigments for aquaculture. It is also hypothesized that astaxanthin accounts for its ability to thrive in highly oxidative and UV-exposed habitats. Here, we assessed the ability of this species to synthesize UV-absorbing compounds generally known as mycosporines, evaluated the effect of culture media in the production of these compounds and compared its UV growth resistance and tolerance with other yeasts. The 48 wild and collection strains screened were positive for mycosporines and a unique compound identified as mycosporine-glutaminol-glucoside (MGG) was detected. Thus, the ability of X. dendrorhous to produce MGG, as described here for the first time, is so far unique among the Cystofilobasidiales. The compound was synthesized constitutively, although growth under visible light and, to a greater extent, UVA radiation stimulated its production. Strains from UV-exposed habitats produced larger quantities and oligotrophic complex media seemed to favor MGG accumulation. UV tolerance and survival of X. dendrorhous was high and comparable to that of the polyextremophilic Rhodotorula mucilaginosa. The taxonomical and ecological implications of the production of MGG by X. dendrorhous are discussed.     

法夫酵母 JMU-MVP14菌体中类胡萝卜素成分分析

结合薄层色谱、柱色谱、以及高效液相色谱对虾青素高产菌株-法夫酵母JMU-MVP14中的类胡萝卜素成分进行初步研究。研究结果表明,硅胶柱层析和氧化镁柱层析相结合的方法对法夫酵母JMU-MVP14菌体中的类胡萝卜素成分有很好的分离效果。经过柱层析分离纯化后,各组分中类胡萝卜素的种类单一,有利于进一步通过各种波谱技术对其进行定性。此方法弥补了单纯依靠高效液相色谱（ODS 柱）对法夫酵母 JMU-MVP14菌体中类胡萝卜素分离效果不佳,可供选择的商业化类胡萝卜素标准品少,液相保留时间漂移等因素给法夫酵母JMU-MVP14菌体中类胡萝卜素定性带来的不足。     

红发夫酵母的生物学特性

红发夫酵母以单细胞为主,有时能形成假菌丝。菌落因菌体产生虾青素等类胡萝卜素而呈红色,类胡萝卜素均匀地分布于细胞脂质中。红发夫酵母为专性好氧菌,细胞产生类胡萝卜素需要大量氧气。葡萄糖和蔗糖为最佳碳源,酵母膏是最佳氮源。红发夫酵母具克拉布特里效应（Crabtree)。红发夫酵母生长温度范围为4－27℃,属于兼性嗜冷的低温型微生物。最适生长pH为6．0,色素形成的最适pH为5．0。     

虾青素摇瓶发酵条件的研究

通过对P .rhodozyma 4 - 2 6的虾青素摇瓶发酵条件包括接种比、通气量和温度等 ,以及培养基主要组分的优化 ,得到最优培养基及培养条件。在此条件下发酵 72h ,虾青素量可达 13 4 5μg/ml或 146 5μg/gCDW ,较优化前提高 1 4 8倍。     

Electrophoretic karyotypes of Phaffia rhodozyma strains

Abstract Electrophoretic karyotypes of strains from the astaxanthin-producing yeast Phaffia rhodozyma have been established. Intact chromosomal DNA molecules released from protoplasts were separated by orthogonal field alternation gel electrophoresis (OFAGE) and contour clamped homogeneous electric field (CHEF). Both small and large chromosomal DNA molecules were resolved simultaneously by optimizing the running conditions. Electrophoretic karyotypes among the Phaffia isolates examined differed significantly. Seven to thirteen chromosomal bands, ranging in size from 0.83 Mb to 3.50 Mb, were resolved, giving total genome sizes of about 15.4 to 23.2 Mb. Ribosomal DNA has been assigned to chromosomal bands using a heterologous gene probe.     

Quantification of carotenoids in cells of Phaffia rhodozyma by autofluorescence

The distribution and intensity of autofluorescence caused by carotenoids in the yeast Phaffia rhodozyma was examined by laser confocal fluorescence microscopy. Carotenoid contents of individual yeast cells could be calculated from the value of autofluorescence/unit volume of yeast cell. The average level of fluorescence in highly fluorescing yeast cells was 15 mg carotenoid g^–1 yeast which was accepted as the maximum yield of carotenoid in P. rhodozyma. The method developed in this study is useful for the research of cell biology associated with the biological functions of carotenoids in individual cells.     

Inhibition of the red yeast Phaffia rhodozyma by saponin

Summary Production of cell mass and astaxanthin by the yeast Phaffia rhodozyma was inhibited by saponin. The inhibition was partially reversed by oleic and linoleic acids and by ergosterol and cholesterol, thereby suggesting a possible interference of saponin with the yeast cell membrane.