Isolation and structural elucidation of a dihydroubiquinone-9 from the fungus Aureobasidium pullulans

Abstract A naturally occurring member of ubiquinone (Q) group, a dihydroubiquinone-9 (Q-9 (H₂)), has been isolated as a minor ubiquinone component from the fungus Aureobasidium pullulans . By ultraviolet absorption, mass and nuclear magnetic resonance spectrometric studies, the structure of Q-9 (H₂) was found to be 2,3-dimethoxy-5-methyl-6-IX-dihydromultiprenyl⁹-1,4-benzoquinone (I).     

Elevated polysaccharide production by mutants of the fungus Aureobasidium pullulans

Abstract Two mutants of the fungus Aureobasidium pullulans ATCC 42023 were isolated that exhibited elevated polysaccharide production. Both mutants were isolated using a combination of chemical mutagenesis and resistance to growth inhibitors. It was found that both mutants elaborated higher polysaccharide levels after 7 days of growth on corn syrup or sucrose, respectively, compared to ATCC 42023. The dry weights of the mutant cells were found not to differ greatly from those of the parent cells whether corn syrup or sucrose served as the carbon source. The pullulan content of the polysaccharide synthesized by the mutants or parent cells on sucrose was consistently lower than polysaccharide synthesized on corn syrup. Using corn syrup as a carbon source, the pullulan content of the polysaccharide elaborated by the parent was higher than either mutant. The inverse was found to occur with respect to pullulan content when the strains were grown on sucrose as a carbon source.     

Heavy metal uptake by intact cells and protoplasts of Aureobasidium pullulans

Protoplasts prepared from yeast-like cells, hyphae and chlamydospores of Aureobasidium pullulans can take up heavy metals such as Zn²⁺, Co²⁺, Cd²⁺ and Cu²⁺. In relation to intact cells, the sensitivity of protoplasts to Cu²⁺ and Cd²⁺ was increased although chlamydospore protoplasts were more tolerant than yeast-like cell protoplasts. Surface binding of metals was reduced in protoplasts as compared with intact cells and this reduction was particularly evident for chlamydospore protoplasts. At the highest concentrations used, uptake of Zn²⁺, Co²⁺ and Cd²⁺ by yeast-like cell protoplasts was greater than that observed in intact cells which may have been due to toxicity, especially for Cd²⁺, resulting in increased membrane permeability, though for Zn²⁺ and Co²⁺ some barrier effect of the cell wall could not be completely discounted. Chlamydospore protoplasts were capable of intracellular metal uptake, unlike intact chlamydospores, and for Zn²⁺, uptake appeared to be via a different system less specific than that of the other cell types. For chlamydospores, the use of protoplasts confirmed the importance of the cell wall in preventing entry of metal ions into the cell.     

Modelling of simultaneous production of polygalacturonase and exopolysaccharide by Aureobasidium pullulans ATHUM 2915

The polymorphic fungus Aureobasidium pullulans ATHUM 2915, produced significant quantities of extracellular polygalacturonase and polysaccharide when grown, under controlled conditions, in liquid medium with pectin and glucose as carbon sources and nitrogen source as limited factor. Growth, substrate consumption and products formation were simulated by a structured mathematical model, which was compared with the experimental data from batch culture in a chemostat. This model was applied successfully in the study of some essential parameters influenced the process at various pH values.     

出芽短梗霉原生质体再生变异菌株的同工酶及可溶性蛋白的研究

用聚丙烯酰胺凝胶电泳技术对出芽短梗霉的亲本菌株及其４株原生质体再生菌株的脂酶、谷氨酸脱氢酶、细胞色素氧化酶和可溶性蛋白进行了比较。发现各变异菌株之间,变异菌株与亲本菌株之间的谱带均有差异,原生质体再生菌株的变异涉及酶蛋白的改变。     

出芽短梗霉产聚苹果酸发酵条件的优化

【背景】出芽短梗霉可发酵葡萄糖生成聚苹果酸,但存在转化率和转化效率低等瓶颈,阻碍其实现商业化生产。【目的】通过优化发酵培养条件,提高出芽短梗霉的聚苹果酸产量、糖酸转化率和生产强度。【方法】采用单因素试验优化适宜出芽短梗霉BK-10菌株产生聚苹果酸的培养条件,通过Plackett-Burman法对培养基组分筛选显著性影响因素,并对其培养基中无机盐进行正交试验优化,最后进行5 L发酵罐验证。【结果】最优培养基配方和培养条件:100 g/L葡萄糖,1.5 g/L尿素,0.20 g/L KH_2PO_4,0.20 g/L ZnSO_4,0.05 g/L MgSO_4,0.75 g/L KCl,30 g/L CaCO_3,0.01%吐温-80,发酵温度26°C,250 mL摇瓶装液量50 mL。【结论】通过优化,聚苹果酸的糖酸转化率达到0.71 g/g,生产强度达到0.89 g/(L·h),较优化前分别提高了18.33%和71.15%,为发酵葡萄糖合成聚苹果酸进而生产L-苹果酸工艺的工业化生产奠定经济性基础。     

光谱法研究甲醇和盐酸对短梗霉黑色素的分离效果

应用紫外和红外光谱对甲醇和盐酸分离的短梗霉黑色素进行了分析。分析结果表明:甲醇和盐酸法分离的黑色素在紫外图谱215 nm处都有最大吸收峰,而甲醇法分离的黑色素其紫外图谱在260、280 nm处无吸收峰表明此法分离的黑色素不含核酸及蛋白质;红外图谱中,在3340 cm-1、1637 cm-1处有很强的吸收峰表现为黑色素的典型特征;同时在对照中发现盐酸多次处理后的黑色素显示较少的结构信息,说明盐酸沉降法有可能破坏黑色素的结构。由此选用甲醇作为沉淀剂有利于黑色素的纯化及确保其结构信息的完整,为进一步分析短梗霉黑色素的结构表征奠定了基础。     

Influence of starvation, surface attachment and biofilm growth on the biocide susceptibility of the biodeteriogenic yeast Aureobasidium pullulans

AIM: To investigate the effect of starvation, surface attachment and growth in a biofilm on the susceptibility of Aureobasidium pullulans to the biocides 2-n-octyl-4-isothiazolin-3-one (OIT) and sodium hypochlorite (NaOCl). METHODS AND RESULTS: Fluorescence loss from a green fluorescent protein (GFP)-transformed strain was used to monitor real-time loss in viability as previously described in situ in 96-well plates. Exponential phase, yeast-like (YL) cells were settled in the bottom of the wells as a low-density monolayer (LDM) and were susceptible to all biocide concentrations (25-100 mug ml(-1)). The exponential phase YL cells were either starved for 48 h in suspension or starved for 48 h as LDMs in the wells. Starvation in both cases led to a small reduction in susceptibility to the biocides. In contrast, 48-h biofilms grown in malt extract broth showed an apparent lack of susceptibility to 25 and 50 mug ml(-1) OIT and to 25-100 mug ml(-1) NaOCl. However, when the OIT concentration was increased to compensate for the higher cell density in the biofilm, the biofilms were found to be equally susceptible to the LDM. CONCLUSIONS: Starvation of A. pullulans YL cells either in suspension or as attached LDM resulted in a decrease in susceptibility to low concentrations of both OIT and NaOCl while the apparent reduced susceptibility of mature biofilms was due to the increase in biofilm cell density rather than true biofilm resistance per se. SIGNIFICANCE AND IMPACT OF THE STUDY: Monitoring fluorescence loss from the GFP-transformed strain of A. pullulans can be used as a fast and reliable method for monitoring cell death in real time as a response to biocide and antimicrobial challenge.     

出芽短梗霉A5产胞外多糖发酵条件的优化及产物结构鉴定

基于筛选获得能够生产分子量较高且无色素的普鲁兰多糖酵母菌株,对其进行菌株鉴定、产多糖发酵条件优化和多糖产物鉴定,旨在为工业上普鲁兰多糖发酵提供新的菌株来源。以YPD固体培养基为筛选培养基,氯霉素为筛选压力,曲利苯蓝为筛选指示剂;通过形态学,ITS间隔序列分析对筛选出的A5菌株进行鉴定。采用单因子优化A5菌株的最佳发酵条件;利用普鲁兰酶酶解并结合薄层层析法、红外光谱以及凝胶渗透色谱进行结构鉴定和分子量的测定。A5菌株鉴定为出芽短梗霉属,并被命名为出芽短梗霉A5。最优的发酵条件8%(w/v)麦芽糖,1%(w/v)酵母粉,2%(w/v)蛋白胨,0.5%(w/v)K_2HPO_4,0.06%(w/v)(NH_4)_2SO_4,0.03%(w/v)CaCl_2,pH6,7%(v/v)接种量;经过结构鉴定得知:该菌株的胞外产物是普鲁兰多糖,分子量为63.84 kDa。由此获得了一株生产普鲁兰多糖的出芽短梗霉菌株A5,产物无色素且分子量较高。经过初步的发酵条件优化,在最佳发酵条件下发酵培养后,获得普鲁兰多糖的产量为22.9 g/L。综合上述结果可知,菌株A5能够作为工业上生产普鲁兰多糖的重要候选菌株。     

The existence of a dispensable fibrillar layer on the wall surface of mycelial but not yeast cells of Aureobasidium pullulans

Abstract Wall surface ultrastructure of Aureobasidium pullulans was studied by freeze-etching. Yeast cells had a smooth wall surface as in typical yeast species. Mycelial cells and chlamydospores had an extra layer on the wall surface made mostly of fibrils. The fibrils were 20 nm in diameter, and thicker than typical major fungal wall skeletal fibrils of β-glucan and chitin. This layer was apparently easily detached from the wall proper, presumably as a result of enzymic activity or by physical means, suggesting that it is a physiologically dispensable wall component.     

A new variety of Aureobasidium pullulans characterized by exopolysaccharide structure, nutritional physiology and molecular features

The black yeast Aureobasidium pullulans (de Bary) Arnaud is known to synthesize the exopolysaccharide pullulan, a poly--1,6-maltotriose. Nine strains were found to produce additional aubasidan-like EPS, i.e. glucans with -1,4-D-, -1,6-D- and -1,3-D-glycosidic bonds. These strains had previously been found to deviate in genotypic characters. Additional physiological differences were found: the optimal nitrogen source for exopolysaccharide production in liquid medium was NaNO₃ for aubasidan-producing strains, and (NH₄)₂SO₄ for the remaining strains. A new variety, A. pullulans var. aubasidani Yurlova, is described for the strains producing aubasidan-like components. The new variety can be distinguished from A. pullulans var. pullulans by the absence of assimilation of methyl--D-glucoside and lactose.     

Pullulan production from synthetic medium by a new mutant of Aureobasidium pullulans

Pullulan with different molecular-weight could be applied in various fields. A UV-induced mutagenesis Aureobasidium pullulans UVMU6-1 was obtained from the strain A. pullulans CGMCC3.933 for the production of low-molecular-weight pullulan. First, the obtained polysaccharide from A. pullulans UVMU6-1 was purified and identified to be pullulan with thin-layer chromatography, Fourier transform infrared, and nuclear magnetic resonance. Then, culture medium and conditions for this strain were optimized by flask fermentation. Based on the optimized medium and culture conditions (pH 4, addition of 4?g/L Tween 80 for 96?hr of cultivation), continuously fermentation was performed. The highest pullulan production and dry biomass was 109 and 125?g/L after fermentation for 114?hr, respectively. The average productivity was about 1?g/L/hr, which was intensively higher than the previous reported. This study would lay foundations for the industrial production of pullulan.     

短梗霉黑色素的分离纯化及结构的初步分析

采用热碱提取、水煮酸沉法从短梗霉发酵液中提取得到黑色素粗品,再经DMSO萃取、酸性甲醇(pH=2)沉淀得到不含多糖和蛋白的短梗霉黑色素。此黑色素不溶于水及常规有机溶剂,可溶于碱性溶液和DMSO;离子交换色谱分析表明黑色素组分均一,出峰时间26±0.5 m in;紫外光谱谱图最大吸收峰为215 nm左右,未见蛋白(280 nm)与核酸(260 nm)的特征吸收峰;红外光谱谱图具有黑色素3μm和6μm的特征吸收峰,并含大量的羟基、氨基,与核磁共振和液质联机谱图结合分析推出短梗霉黑色素可能含有酚羟基、羧基和吲哚等官能团,主要结构骨架为5,6-二羟基吲哚-羧酸和多巴醌,推断该黑色素为酪氨酸酶控制合成的真黑素。     

Production of polymalic acid and malic acid by Aureobasidium pullulans fermentation and acid hydrolysis

Malic acid is a dicarboxylic acid widely used in the food industry and also a potential C4 platform chemical that can be produced from biomass. However, microbial fermentation for direct malic acid production is limited by low product yield, titer, and productivity due to end‐product inhibition. In this work, a novel process for malic acid production from polymalic acid (PMA) fermentation followed by acid hydrolysis was developed. First, a PMA‐producing Aureobasidium pullulans strain ZX‐10 was screened and isolated. This microbe produced PMA as the major fermentation product at a high‐titer equivalent to 87.6 g/L of malic acid and high‐productivity of 0.61 g/L h in free‐cell fermentation in a stirred‐tank bioreactor. Fed‐batch fermentations with cells immobilized in a fibrous‐bed bioreactor (FBB) achieved the highest product titer of 144.2 g/L and productivity of 0.74 g/L h. The fermentation produced PMA was purified by adsorption with IRA‐900 anion‐exchange resins, achieving a ～100% purity and a high recovery rate of 84%. Pure malic acid was then produced from PMA by hydrolysis with 2 M sulfuric acid at 85°C, which followed the first‐order reaction kinetics. This process provides an efficient and economical way for PMA and malic acid production, and is promising for industrial application. Biotechnol. Bioeng. 2013; 110: 2105–2113. © 2013 Wiley Periodicals, Inc.     

出芽短梗霉菌株CBS591．75和DSM2404发酵生产聚苹果酸的研究

研究了出芽短梗霉蕾株CBS591.75和DSM2404在2L发酵罐中发酵生产聚苹果酸的过程。并以CBS591．75为菌种进行了20L规模的初步放大试验。结果表明,发酵过程可以分为3个阶段,第一阶段从接种开始。到发酵液的pH上升到最高点并开始下降为止．这一阶段中细胞缓慢增长,没有聚苹果酸产生;第二阶段以pH迅速下降和聚苹果酸大量产生为特征。这一阶段中聚苹果酸的产生与细胞增长相平行;在第三阶段中,聚苹果酸产生速度减慢．pH趋于稳定。试验结果还表明。菌株CBS591．75产生聚苹果酸的能力大于DSM2404,发酵结束时,前者聚苹果酸的产量为6．9g／L,而后者为5．4g／L。CBS591．75菌种在20L规模的初步放大试验表明,发酵144h后发酵液中聚苹果酸浓度为8．Og／L,稍高于2L发酵罐的结果,为今后的放大提供了依据和参考。     

短梗霉多糖发酵条件的研究

在摇瓶发酵条件研究的基础上。于16L自控发酵罐上进行了罐上发酵条件优化研究。发现以10％淀粉水解物为碳源时,淀粉水解物的最适DE值为40－50,发酵培养基中的硫酸铵最适用量不同于摇瓶发酵时的量,种龄和接种量、通气量、罐压、搅拌速度和搅拌叶轮挡数等均对多糖的产生有较大的影响。另外还进行了发酵过程的动力学的研究。     

农杆菌介导的出芽短梗霉遗传转化及高效筛选聚苹果酸高产菌株

建立根癌农杆菌介导的出芽短梗霉遗传转化方法及T-DNA突变库,高效筛选聚苹果酸高产菌株及功能基因。通过含潮霉素和草铵磷抗性基因的农杆菌转化出芽短梗霉,抗性压力筛选及PCR验证建立根癌农杆菌介导的出芽短梗霉遗传转化方法,结合发酵液p H与聚苹果酸含量响应变化,微孔板高效筛选高产聚苹果酸的T-DNA插入突变株,基因组步移确定T-DNA插入位点及功能基因。结果获得遗传稳定的抗性基因菌株,每107个细胞可获得80-120个转化子,出芽短梗霉H27号T-DNA突变株聚苹果酸摇瓶发酵产量提高24.5%,基因组步移证实糖酵解途径磷酸甘油酸变位酶基因被破坏。成功建立了根癌农杆菌介导的出芽短梗霉遗传转化方法和T-DNA插入突变库,结合高效筛选方法为聚苹果酸合成功能基因挖掘及高产机制解析奠定基础。     

出芽短梗霉新菌株RM1603产普鲁兰多糖条件优化及多糖分析

目的:出芽短梗霉RM1603是一株高产胞外多糖新菌株,通过优化其产糖条件,鉴定其多糖结构,为进一步开发利用RM1603产胞外多糖奠定理论基础。方法:以RM1603为出发菌株,在单因素分析确定最佳氮源与无机盐的基础上,利用正交试验探究RM1603最佳发酵产糖条件;薄层层析及红外光谱分析确定胞外多糖产物结构。结果:出芽短梗霉菌RM1603的初始多糖产量为28.91g/L,发酵条件优化后产糖量达到65.213g/L,提高了约2.3倍;结构分析表明RM1603的多糖产物为普鲁兰多糖。结论:出芽短梗霉RM1603是一株具有较大产糖优势,极具开发潜力的高产普鲁兰糖新菌株;奠定了开发利用RM1603生产普鲁兰多糖的理论基础。     

Process optimization of continuous gluconic acid fermentation by isolated yeast-like strains of Aureobasidium pullulans

This study was focused on the optimization of a new fermentation process for continuous gluconic acid production by the isolated yeast-like strain Aureobasidium pullulans DSM 7085 (isolate 70). Operational fermentation parameters were optimized in chemostat cultures, using a defined glucose medium. Different optima were found for growth and gluconic acid production for each set of operation parameters. Highest productivity was recorded at pH values between 6.5 and 7.0 and temperatures between 29 and 31 degrees C. A gluconic acid concentration higher than 230 g/L was continuously produced at residence times of 12 h. A steady state extracellular gluconic acid concentration of 234 g/L was measured at pH 6.5. 122% air saturation yielded the highest volumetric productivity and product concentration. The biomass-specific productivity increased steadily upon raising air saturation. An intracellular gluconic acid concentration of about 159 g/L (0.83 mol) was determined at 31 degrees C. This is to be compared with an extracellular concentration of 223 g/L (1.16 mol), which indicates the possible existence of an active transport system for gluconic acid secretion, or the presence of extracellular glucose oxidizing enzymes. The new process provides significant advantages over the traditional discontinuous fungi operations. The process control becomes easier, thus offering stable product quality and quantity.