核黄素产生菌阿舒假囊酵母(Eremothecium ashbyii)抗反馈抑制突变株的选育

在调查阿舒假囊酵母(Eremothecium ashbyii)营养要求的基础上,设计了适合该菌生长的合成培养基,在合成培养基上诱变筛选得到了数株抗嘌呤拮抗物8-AG的突变株。选择其中三株U_(95-1)、U_(95-2)和U_(95-3)进行传代和摇瓶发酵试验,U_(95-3)的核黄素发酵单位低于出发菌,未经传代的U_(95-1)、U_(95-2)比出发菌株的发酵水平分别提高15.5％和9.8％,经5～10代传接,其产核黄素的遗传性状稳定。该研究表明,从代谢控制角度通过减轻核黄素合成途径中重要调节酶的反馈抑制对提高E. ashbyii的核黄素产量和稳定性是可行的,为该菌的菌种改良提供了一条遗传育种途径。     

核黄素产生菌阿舒假囊酵母(Eremothecium ashbyii)抗反馈抑制突变株的选育

在调查阿舒假囊酵母(Eremothecium ashbyii)营养要求的基础上,设计了适合该菌生长的合成培养基,在合成培养基上诱变筛选得到了数株抗嘌呤拮抗物8-AG的突变株。选择其中三株U_(95-1)、U_(95-2)和U_(95-3)进行传代和摇瓶发酵试验,U_(95-3)的核黄素发酵单位低于出发菌,未经传代的U_(95-1)、U_(95-2)比出发菌株的发酵水平分别提高15.5％和9.8％,经5～10代传接,其产核黄素的遗传性状稳定。该研究表明,从代谢控制角度通过减轻核黄素合成途径中重要调节酶的反馈抑制对提高E. ashbyii的核黄素产量和稳定性是可行的,为该菌的菌种改良提供了一条遗传育种途径。     

磁场对红酵母的生长及类胡萝卜素合成的影响

考察直流电磁场对类胡萝卜素产生前红酵母RG-98的生长及代谢的影响。结果表明,不同的磁场强度、处理时间及处理方式会产生不同的生物效应。将液体种子和发酵培养基一起置于0.10T电磁场处理2h,能使色素的发酵产量提高25％。比较经磁场处理与未经处理的发酵过程,发现磁场处理提高了红酵母对糖的代谢速率,并且在发酵后期促进生物量和色素含量持续地增加。     

利用棉籽饼粉饴糖渣发酵生产VB_2

用发酵法生产核黄素(VB_2),常用棉病囊霉(Ashbra gossypii)和阿化假囊酵母(Eremothecium ashbyii)产生菌株的代谢产物。由于VB_2的分子式是C_(17)H_(20)O_6N_4,要求在发酵培养基中有较丰富的碳源和氮源。一般要求含碳源54.2％以上、氮源14.8％以上,以适应菌体的生长、繁殖和核黄素的合成。尤其是氮源,它是构成菌体细胞蛋白质和核酸的必需元素和形成代谢产物的重要原料。在选择发酵培养基的原料时,应当充分考虑。通常是用米糠油、骨胶、玉米浆和鱼     

Eremothecium ashbyii T30发酵产脂肪酶特性的研究

研究了一株核黄素高产突变株E.ashbyii T30产脂肪酶的条件,确定了比较合适的脂肪酶发酵培养基,豆饼粉30g/L,玉米浆30ml/L,豆油5ml/L,KH2PO45g/L,MgSO4.7H2O 0.15g/L,pH消前7．5,T30在此培养基上振荡培养28h,既定条件下测脂肪酶活力可达232酶活单位／ml(较原株提高36．5％）,从一个侧面证实其突变株特性,另外,对T30所产脂肪酶的诱导酶属性及脂肪酶在T30核黄素发酵过程中的作用也进行了初步探讨。     

枯草芽孢杆菌基因修饰生产核黄素

【目的】研究枯草芽孢杆菌核黄素合成途径、木糖代谢相关基因修饰对核黄素合成的影响。【方法】单独过表达或共同过表达核黄素操纵子中的基因、过表达木糖代谢相关基因构建相应的重组菌株。通过测定和比较重组菌株摇瓶发酵的核黄素产量和生物量,表征各个基因修饰的效应。采用摇瓶和5 L罐发酵,考察木糖作为主要碳源以及木糖与蔗糖共代谢对核黄素发酵的影响。【结果】ribA基因单独过表达,使核黄素产量提高99%,但生物量降低30%,出现细胞自溶现象。ribA-ribH基因共表达,使核黄素产量提高280%,并且无细胞自溶和生物量下降现象。1.5%蔗糖与6.5%木糖作为碳源,5 L发酵罐发酵70 h,核黄素产量达到3.6 g/L,与8%蔗糖为碳源的发酵相比,核黄素产量提高80%。木糖代谢相关基因过表达,均明显降低核黄素产量。【结论】与ribA基因单独过表达相比,ribA-ribH基因共表达可有效避免细胞自溶现象,并能进一步提高核黄素产量。蔗糖与木糖共代谢,能够改善前体物供给,有利于提高核黄素产量。     

维酶素及其应用

维酶素又称粗制核黄素,是用新鲜豆渣以阿氏假囊酵母(Eremothecium ashbyii)进行固体发酵制成的一种复合制剂。维酶素中主要含有核黄素、黄素辅酶和多种微量元素。在五十年代,曾将粗制核黄素作为治疗口腔炎的药物和小儿营养助长剂,以后很少有人     

产核黄素阿舒假囊酵母的诱变效果及添加小米、肌醇的增产效果

用亚硝基胍和紫外线对产核黄素阿舒假囊酵母2.1197进行诱变处理,其核黄素发酵单位从372μg/ml提高到4660—6492μg/ml,提高幅度为25.3—74.5%;用紫外线和氮激光处理,提高62.3%;用红外线二氧化碳激光处理,提高39.7%。在该菌摇瓶发酵培养基中添加小米粉1%,发酵单位提高9.78%,添加1%小米粉和10μg/ml的肌醇,发酵单位提高16.84%。     

红酵母RY—981类胡萝卜素发酵助剂的研究

通过探索几种促进剂和前体对红酵母RY－981类胡萝卜素发酵的影响,从中选出了番茄汁和花生油2种对红酵母生长及其类胡萝卜素合成具有显著促进作用的发酵助剂,并确定了适宜的用量。应用试验和发酵过程动态分析表明,这2种发酵助剂增产效果明显（当同时添加番茄汁2.6mL/L和花生油1ml/L时,菌体量、类胡萝卜素含量和产量可分别比对照组提高53．13％、20．29％和84．07％）,且对发酵过程菌体生长及生理代谢规律无不良影响。     

维生素在丙酮酸过量合成中的重要作用

研究了烟酸、硫胺素、吡哆醇、生物素和核黄素对一株光滑球拟酵母(\%Torulopsis glabrata\%) WSH\|IP303以葡萄糖为碳源、以氯化铵为唯一氮源生产丙酮酸的影响。利用正交试验方法,确证了硫胺素是影响WSH\|IP303生产丙酮酸的最重要因素。在硫胺素浓度一定(0.01～0.015mg/L)的前提下,提高烟酸浓度有助于加快耗糖速度。当烟酸、硫胺素、吡哆醇、生物素和核黄素的浓度分别为8、0.015、0.4、0.04和01mg/L时,摇瓶发酵48h,丙酮酸产量和产率可分别达到52.4g/L和0525g/g。采用优化的维生素组合方式,进行2.5L罐分批发酵,在初糖浓度120g/L的条件下发酵57.5h,丙酮酸产量和产率分别达到69.4g/L和0593g/g,分别比摇瓶培养的最好结果提高了32.%和13%。     

核黄素生物合成研究 1.微量培养中核黄菌(Eremothecium ashbyii)发育过程中的形态变化

从本试验观察到,核黄菌的生长全部过程是自配子或菌丝在培养基中开始发育成营养菌丝,部分菌丝形成配子囊形成配子,部分菌丝衰老自溶。当菌丝发育最盛时,核黄素的形成是最多,衰老时培养基核黄素逐渐丰富起来。核黄菌在有空气和缺乏空气时,它们的发育是有区别的,即是在缺乏空气(表面下生长)时,菌丝是细长不形成配子和产生少量核黄素。     

Influence of type and concentration of flavinogenic factors on production of riboflavin by Eremothecium ashbyii NRRL 1363

A study was conducted to determine the effect of various low cost organic wastes as flavinogenic factors and the various concentrations at which they induced flavinogenecity resulting in higher yields of riboflavin. A high-yielding riboflavin strain; Eremothecium ashbyii NRRL 1363 was chosen to determine the flavinogenicity. Carbon source at 50 g l(-1) (dextrose equivalents) of molasses and nitrogen source at 50 g l(-1) (weight/volume) of peanut seed cake were found to be optimal levels to yield higher riboflavin. Among the organic wastes, (beef extract, hog casings, blood meal, fish meal) hog casings in association with fish meal supported the highest yield of riboflavin. Among the different recovery processes studied, a vacuum drying process was the most efficient allowing maximum yield, followed by drying at 90 degrees C and freeze-drying. It is apparent from this study that inexpensive or waste organic materials could induce E. ashbyii to synthesize and secrete riboflavin at higher levels in the medium and this could be purified using a vacuum drying process. This bioconversion process allows us to recycle the biomaterials and produce a value-added product of economic importance.     

Ribitol and flavinogenesis in Eremothecium ashbyii

1. Supplementation of cultures of Eremothecium ashbyii with ribitol leads to a twofold increase in riboflavin formation compared with unsupplemented cultures or those supplemented with ribose or ribulose phosphate. Addition of unlabelled ribitol decreases the incorporation of [1-(14)C]ribose into riboflavin, indicating that free ribitol is preferred to ribose for incorporation into riboflavin. 2. The enzymes ribitol kinase, d-ribose reductase, d-ribose 5'-phosphatase and GMP nucleosidase were demonstrated in the cell-free extracts. Ribitol induces the formation of ribitol kinase. The enzyme is activated in vitro by the flavinogenic purines, guanine and xanthine. d-Ribose reductase shows a specific requirement for NADPH and forms free ribitol from ribose. 3. The activities of ribitol kinase, ribose 5'-phosphatase and GMP nucleosidase reach their maximal values before riboflavin formation reaches a maximum. 4. [U-(14)C]GMP is taken up intact by the culture of E. ashbyii and is incorporated into riboflavin as well as into a blue fluorescent compound. The radioactivity from this compound is incorporated into riboflavin by the cell-free extract of E. ashbyii.     

The kinetics of riboflavin secretion by Eremothecium ashbyii nrrl 1363

For riboflavin production a highly flavinogenic strain Eremothecium ashbyii is used. Vitamins are classified as chemical substances that control and effect the physiological processes; Riboflavin is one among them. The deficiency of riboflavin in human beings results in the cracking of lips and corners of mouth (cheilosis); nerve tissues are affected. For riboflavin production a highly flavinogenic strain Eremothecium ashbyii NRRL 1363 was used. Investigations were conducted in shake flask using inexpensive and abundantly available raw materials. Among the stimulants, a combination of hog casings and beef extract stimulated the highest and promoted the maximum riboflavin yield followed by the combination of fish meal and beef extract. The fermented broth (an enriched, riboflavin concentrate) can be directly used as a feed grade riboflavin. To upgrade it to pharmaceutical grade further investigations are required.     

电视显微摄影技术在微生物教学及科研中的应用

通过对一株核黄素高产株阿舒假囊酵母Ｔ_３０的菌丝形态、孢子囊、孢子、发酵过程中出现的膨大菌体及发酵终点核黄素结晶产物的显微观察,介绍一种适用于微生物材料（如：细菌、霉菌、酵母菌等）、动植物材料（如：动物组织、植物花朵）、生物大分子物质（如：淀粉颗粒）、工业结晶（如：核黄素晶体）及金相实验、普通物理实验的显微观察、检测和记录的方法。这一技术可普遍地应用于电视教学及科研实践。     

Bioproduction of riboflavin from molasses and lentils

Fermentation studies were carried out for the bioproduction of riboflavin with an agroindustrial byproduct, molasses as the carbon source and lentils as the nitrogen source using E. ashbyii strain. With the previously recommended 1.5% (w/v) molasses, lentils at a concentration of 3% (w/v) was found to be the optimum. Acidic medium was found to be favorable for riboflavin production when the molasses to lentils ratio was greater than one and neutral medium when the ratio was one. Effect of agitation on vitamin production was also studied and it was observed that 300 rpm gives a higher yield of product.     

Changes in the enzyme activity of flavinogenesis in the process of culturing the fungus Eremothecium ashbyii

The activity of enzymes involved in the beginning (GTP cyclohydrolase) and terminal steps (riboflavin synthase EC 2.5.1.9, riboflavin kinase EC 2.7.1.26 and FMN adenyltransferase EC 2.7.7.2) of flavinogenesis was studied in the mycelium of Erenmothecium ashbyii of different age. The activity of GTP cyclohydrolas, riboflavin kinase and FMN adenyltransferase was low in the young mycelium and increased in the process of growth, which was accompanied by the acceleration of flavinogenesis. The activity of riboflavin synthase was high in the young mycelium and changed only slightly in the process of subsequent cultivation of the fungus. 8-Azaadenine and 8-hydroxyquinoline added to the young culture of E. ashbyii inhibited the flavinogenesis of the mycelium and the increase of the enzyme activity.     

8-azaguanine and flavinogenesis in Eremothecium ashbyii.

8-Azaguanine (10- minus 4 M) supplementation in synthetic medium inhibited flavinogenesis in Eremothecium ashbyii to far greater extent (68per cent) than the growth (25 per cent). That enzymes comprising the biosynthetic pathway of riboflavin are synthesized during early growth phase of the organism is supported by the data presented. 8-Azaguanine mediated inhibition in flavinogenesis was closely related with decreased levels of ribose-5'-phosphatase, ribose reductase and ribitol kinase, the enzymes involved in supplying ribitol for flavinogenesis. Addition of guanine and not ribitol during early growth phase to 8-azaguanine-added cultures released the inhibition of riboflavin synthesis and restored the enzyme levels in the presence of the antimetabolite.     

Enhanced riboflavin production by recombinant Bacillus subtilis RF1 through the optimization of agitation speed

Dissolved oxygen is one of the most important bioprocess parameters that could affect cell growth and product formation, and it is easy to control by changing agitation speed. In this work, the effects of agitation speed on the performance of riboflavin production by recombinant Bacillus subtilis RF1 was investigated in fed-batch fermentation. The lower agitation speed (600 rpm) was beneficial for cell growth and riboflavin biosynthesis in the initial phase of fermentation process. While, during the later phase, higher agitation speed (900 rpm) was favor for cell growth and riboflavin biosynthesis. Thus, a two-stage agitation speed control strategy was proposed based on kinetic analysis, in which the agitation speed was controlled at 600 rpm in the first 26 h and then switched to 900 rpm to maintain high μ for cell growth and high q _p for riboflavin production during the entire fermentation process. However, it was observed that a sharp increase of agitation speed resulted in an adverse effect on cell growth and riboflavin synthesis within a short time. To avoid this phenomenon, a multi-stage agitation speed control strategy was set up based on the two-stage control strategy, the maximum concentration of riboflavin reached 9.4 g l^?1 in 48 h with the yield of 0.051 g g^?1 by applying this strategy, which were 20.5 and 21.4 % over the best results controlled by constant agitation speeds.     

Spectrofluorimetric method for the estimation of total lipids in Eremothecium ashbyii fungal filaments using Nile blue and avoiding interference of autofluorescent riboflavin

A rapid, simple and sensitive spectrofluorimetric technique was developed for monitoring total lipids in hyphae of the riboflavin-overproducing fungus Eremothecium ashbyii using the fluorescent probe Nile blue in an aqueous system, avoiding the interference due to autofluorescent riboflavin. The existing methodologies for lipid estimation are tedious, requiring large biomass, solvent extraction and gravimetry. E. ashbyii is a hemiascomycete fungus which accumulates lipids in its mycelia prior to flavinogenesis. This study defines the conditions (wavelength selection and sensitivity) for the spectrofluorimetric quantification of lipids in situ in the macerated mycelia of this fungus in the presence of intracellular autofluorescent riboflavin without the need to extract the lipids from the mycelia. The fluorescent intensity was linear with the lipid concentration of the mycelia (by gravimetry) under three different growth conditions using glucose, olive oil and sunflower oil as carbon sources. This spectrofluorimetic method of lipid estimation can be applied to other fungi and microorganisms.