响应面法优化普鲁兰多糖发酵培养基

以出芽短梗霉IFO 4464为实验菌种,采用响应面法(RSM)优化了出芽短梗霉IFO 4464产普鲁兰多糖的发酵培养基。通过实验得到出芽短梗霉最佳发酵培养基为蔗糖59.8g/L,硫酸铵0.7 g/L,硫酸镁0.3 g/L,磷酸二氢钾5.0g/L,氯化钾0.5g/L,氯化钠1.5g/L,酵母浸膏2.5 g/L,多糖产量可达21.92 g/L。     

环境pH诱导出芽短梗霉细胞多形化

出芽短梗霉具有酵母状细胞、膨大细胞、菌丝、厚垣孢子、念珠状菌丝和分生组织状结构。在最适pH条件下,出芽短梗霉生长繁殖以酵母状细胞（CBS100225等4菌株）或膨大细胞（CBS249.65等4菌株）为主。pH 2.2或pH 7.0诱导全部8株出芽短梗霉形成分生组织状结构。酵母状细胞转变成膨大细胞受低pH值诱导的占75%,还受高pH诱导的占50%。膨大细胞是多形性细胞转变的中心环节,可以转变成菌丝、厚垣孢子或分生组织状结构。     

短梗霉感染文献回顾

短梗霉是一种在自然界广泛存在的暗色真菌,偶可引起人类感染,致病种有出芽短梗霉、黑色短梗霉、产酶短梗霉和曼氏短梗霉。临床表现多种多样,如真菌血症、腹膜炎、皮肤感染、脑膜炎、脾脓肿、肺炎、巩膜炎、角膜炎、淋巴结炎及甲真菌病等。短梗霉感染诊断较为困难,容易误诊。本文对Medline和中文文献数据库中的相关文献进行了系统查阅和分析,全面总结了短梗霉感染的地域分布、危险因子、临床表现形式、菌种类别、药物敏感性及其诊疗。     

短梗霉产liamocins研究进展

短梗霉真菌(Aureobasidium spp.)是一种世界性的酵母样真菌,因其产生黑色素而被称为黑酵母.短梗霉的许多菌株都能分泌细胞外脂质liamocins.Liamocins具有表面活性、良好的抗癌和抗菌活性.本文综述了分泌liamocins的短梗霉的多样性及影响其产生liamocins的因素,总结了liamoci...     

几株出芽短梗霉在不同发酵条件下产生多糖的比较

将已有的４株出芽短梗霉在摇瓶中于不同发酵条件下进行比较,考察了它们的生长情况,不同的碳源、氮源、磷酸盐、初始ｐＨ和通气量等对短梗霉多糖合成的影响,获得一株产短梗霉多糖的高产菌株,为以后工作打下良好基础。     

真菌出芽短梗霉中一个新的酚苷类化合物CSCD

真菌出芽短梗霉Aureobasidium pullulans乙酸乙酯粗提物对多种植物病原菌具有抑菌活性,结果表明对人参锈腐病病原菌Cylindrocarpon destructans具有较好的抑菌活性。因此为探究出芽短梗霉A.pullulans固体发酵物乙酸乙酯部位的化学成分,对其发酵物乙酸乙酯部位采用正相柱,反相柱,以及半制备液相色谱分离纯化,并根据核磁共振波谱数据鉴定单体化合物。分离得到4个单体化合物,经鉴定为3,4-二甲氧基-5-甲基苯-O-α-D-吡喃葡萄糖苷(1)、5′-hydroxygriseofulvin(2)、7-dechlorogriseofulvin(3)、6′-hydroxygriseofulvin(4)。以上4种化合物均为首次从该真菌发酵产物中分离得到,其中化合物1是新化合物。     

短梗茁霉胞外多糖的研究——Ⅰ.菌株的筛选

短梗茁霉为半知菌类短梗霉属,一种具有酵母型和菌丝型形态的两型真菌。近年来,人们对于利用短梗茁霉生产单细胞蛋白、细胞壁多糖、胞外多糖等方面作了大量的研究,在应用上也取得了显著的成绩。据文献报道,这种胞外多糖可用作血浆代用品,食品包装薄膜,增稠剂和鸡蛋水果的深层保鲜。为了获得产     

聚苹果酸生产菌出芽短梗霉CCTCC M2012223的全基因组测序及序列分析

【目的】解析出芽短梗霉CCTCC M2012223的基因组序列信息,分析其代谢产物聚苹果酸、黑色素、普鲁兰多糖合成相关基因,为深入研究遗传多样性和代谢工程改造提供序列背景信息。【方法】使用Illumina Hi Seq高通量测序平台对出芽短梗霉CCTCC M2012223菌株进行全基因组测序,并对测序数据进行序列拼接,基因预测与功能注释,COG/GO聚类分析,比较基因组学分析等。下载其他5株出芽短梗霉基因组序列,比较分析6株菌的种内同源基因、全基因组进化以及代谢产物合成相关基因。【结果】出芽短梗霉CCTCC M2012223基因组序列全长30756831 bp,GC含量47.49%,编码9452个基因。比较基因组分析表明出芽短梗霉CCTCC M2012223的基因组组装长度最长,6株菌的同源基因数达到7092个,普鲁兰多糖和聚苹果酸合成相关基因的蛋白序列有很高的保守性。出芽短梗霉CCTCC M2012223和Aureobasidium pullulans var.melanogenum亲缘关系最近,而这2株菌的黑色素合成相关基因的蛋白序列有一些插入和突变。【结论】本研究解析了出芽短梗霉CCTCC M2012223的基因组序列信息,获得黑色素、普鲁兰多糖和聚苹果酸合成相关基因,为后续的代谢机制解析和改造提供相关依据。     

一株出芽短梗霉全基因组序列测定及其分析

出芽短梗霉因其发酵产物种类的多样性而具有广阔的工业应用前景。本研究利用下一代测序技术,对一株高产普鲁兰多糖的出芽短梗霉菌株(Aureobasidium pullulans CCTCC M 2012259)全基因组进行测序、组装和生物信息学分析。研究表明,该菌株的基因组全长约为26.37 Mb,共包含36条scaffolds和76 contigs,Gen Bank登录号:PRJNA350822。利用Gene Mark-ES软件对该基因组进行基因预测,共得到10 069个编码蛋白的基因。使用Blastp将其与Uniprot KB数据库中所有已知真菌蛋白进行比对,发现有6 218个预测蛋白与Uniprot KB数据库中的4 925个已知蛋白高度相似。利用DAVID工具对这些蛋白进行GO基因功能注释、KEGG通路注释和蛋白酶分析,分别注释得到4 444条GO功能条目、1 566条KEGG通路条目和1 740条蛋白酶信息。测定与分析为今后针对出芽短梗霉的功能基因挖掘以及分子遗传改造等工作的开展奠定了坚实的理论基础。     

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。     

Monitoring and control of pullulan production using vision sensor

The production of the polysaccharide pullulan by the yeast-like fungi, Aureobasidium pullulans, is accompained by cellular morphogenetic changes. High productivity and yield of the process have been found to correlate with high concentration of yeast-like cells in the culture. The morphogenetic changes of A. pullulans cells depend on the culture conditions, e.g., dissolved oxygen, shear rate and medium composition. In order to improve the productivity of the process, a novel control law was formulated. A feeding strategy dependent on the culture cellular composition was designed and aimed to keep the yeast-like cell concentration high. The culture morphogenetic composition during the process was monitored by a recently developed vision sensor. Feeding was actuated when the yeast-like cell concentration decreased below a threshold. The proposed control strategy improved pullulan production by increasing both productivity and yield of the cells by 67% and 80%, correspondingly. The results point to the advantage and the potential of using the monitoring and control system and algorithm to increase productivity and yield in cellular bioprocesses.     

Development of an oligonucleotide probe for Aureobasidium pullulans based on the small-subunit rRNA gene.

Aureobasidium pullulans, a cosmopolitan yeast-like fungus, colonizes leaf surfaces and has potential as a biocontrol agent of pathogens. To assess the feasibility of rRNA as a target for A. pullulans-specific oligonucleotide probes, we compared the nucleotide sequences of the small-subunit rRNA (18S) genes of 12 geographically diverse A. pullulans strains. Extreme sequence conservation was observed. The consensus A. pullulans sequence was compared with other fungal sequences to identify potential probes. A 21-mer probe which hybridized to the 12 A. pullulans strains but not to 98 other fungi, including 82 isolates from the phylloplane, was identified. A 17-mer highly specific for Cladosporium herbarum was also identified. These probes have potential in monitoring and quantifying fungi in leaf surface and other microbial communities.     

The use of solid medium to study effects of cadmium, copper and zinc on yeasts and yeast-like fungi: applicability and limitations

A defined solid medium has been used to examine the responses of Aureobasidium pullulans, Saccharomyces cerevisiae and Sporobolomyces roseus to cadmium, copper and zinc. Experiments where aliquot volumes of metal salt solutions were added to wells in the centre of agar plates revealed marked differences between these organisms. The yeast-like fungus A. pullulans was the least sensitive but S. cerevisiae was more sensitive to cadmium than zinc: the reverse was true for S. roseus. Quantitative data, which complemented the qualitative results, were obtained by measurement of metal concentrations in the plates.     

The halophilic fungus Hortaea werneckii and the halotolerant fungus Aureobasidium pullulans maintain low intracellular cation concentrations in hypersaline environments

Hortaea werneckii and Aureobasidium pullulans, black yeast-like fungi isolated from hypersaline waters of salterns as their natural ecological niche, have been previously defined as halophilic and halotolerant microorganisms, respectively. In the present study we assessed their growth and determined the intracellular cation concentrations of salt-adapted and non-salt-adapted cells of both species at a wide range of salinities (0 to 25% NaCl and 0 to 20% NaCl, respectively). Although 5% NaCl improved the growth of H. werneckii, even the minimal addition of NaCl to the growth medium slowed down the growth rate of A. pullulans, confirming their halophilic and halotolerant nature. Salt-adapted cells of H. werneckii and A. pullulans kept very low amounts of internal Na+ even when grown at high NaCl concentrations and can be thus considered Na+ excluders, suggesting the existence of efficient mechanisms for the regulation of ion fluxes. Based on our results, we can conclude that these organisms do not use K+ or Na+ for osmoregulation. Comparison of cation fluctuations after a hyperosmotic shock, to which nonadapted cells of both species were exposed, demonstrated better ionic homeostasis regulation of H. werneckii compared to A. pullulans. We observed small fluctuations of cation concentrations after a hyperosmotic shock in nonadapted A. pullulans similar to those in salt-adapted H. werneckii, which additionally confirmed better regulation of ionic homeostasis in the latter. These features can be expected from organisms adapted to survival within a wide range of salinities and to occasional exposure to extremely high NaCl concentrations, both characteristic for their natural environment.     

Fungal colonization and biodeterioration of plasticized polyvinyl chloride

Significant substratum damage can occur when plasticized PVC (pPVC) is colonized by microorganisms. We investigated microbial colonization of pPVC in an in situ, longitudinal study. Pieces of pPVC containing the plasticizers dioctyl phthalate and dioctyl adipate (DOA) were exposed to the atmosphere for up to 2 years. Fungal and bacterial populations were quantified, and colonizing fungi were identified by rRNA gene sequencing and morphological characteristics. Aureobasidium pullulans was the principal colonizing fungus, establishing itself on the pPVC between 25 and 40 weeks of exposure. A group of yeasts and yeast-like fungi, including Rhodotorula aurantiaca and Kluyveromyces spp., established themselves on the pPVC much later (after 80 weeks of exposure). Numerically, these organisms dominated A. pullulans after 95 weeks, with a mean viable count +/- standard error of 1,000 +/- 200 yeast CFU cm(-2), compared to 390 +/- 50 A. pullulans CFU cm(-2). No bacterial colonization was observed. We also used in vitro tests to characterize the deteriogenic properties of fungi isolated from the pPVC. All strains of A. pullulans tested could grow with the intact pPVC formulation as the sole source of carbon, degrade the plasticizer DOA, produce extracellular esterase, and cause weight loss of the substratum during growth in vitro. In contrast, several yeast isolates could not grow on pPVC or degrade DOA. These results suggest that microbial succession may occur during the colonization of pPVC and that A. pullulans is critical to the establishment of a microbial community on pPVC.     

The use of solid medium to study effects of cadmium, copper and zinc on yeasts and yeast-like fungi: applicability and limitations

A defined solid medium has been used to examine the responses of Aureobasidium pullulans, Saccharomyces cerevisiae and Sporobolomyces roseus to cadmium, copper and zinc. Experiments where aliquot volumes of metal salt solutions were added to wells in the centre of agar plates revealed marked differences between these organisms. The yeast-like fungus A. pullulans was the least sensitive but S. cerevisiae was more sensitive to cadmium than zinc: the reverse was true for S. roseus. Quantitative data, which complemented the qualitative results, were obtained by measurement of metal concentrations in the plates.     

Secretion of β-galactosidases by Aureobasidium pullulans and Penicillium brevicompactum on polygalacturonate medium

An assay has been developed to detect production of extracellular β -galactosidases by fungi during growth on a defined medium containing polygalacturonate. Using this method strains of the yeast-like fungus Aureobasidium pullulans and the filamentous fungus Penicillium brevicompactum which secrete β -galactosidase have been isolated. Seventy strains of yeast were tested but none secrete detectable extracellular β -galactosidase during growth on polygalacturonate agar medium.     

耐辐射黑色酵母状真菌的筛选和特性研究

【目的】确定辐射污染土样中分离筛选获得的一批黑色酵母状真菌的分类学地位和抗逆特性。【方法】通过菌落形态和菌丝特征观察,结合LSU rDNA D1/D2区序列的系统发育分析,并进行了菌株的60Coγ射线照射、紫外线照射和对等重金属离子以及NaCl生长压力实验。【结果】上述菌株均为Aureobasidium属菌,其中菌株F99和F134均与A.pullulans var.subglaciale CBS 123388T具有最大同源性,为100%;菌株F19与A.pullulans var.melaogenum CBS 105.22T具有最大同源性,为99.8%;上述菌株均具有极强的抗逆特性。【结论】为了解耐辐射真菌抗逆机理提供了重要的研究材料。     

Salt stress and plasma-membrane fluidity in selected extremophilic yeasts and yeast-like fungi

We have investigated changes in plasma-membrane fluidity in relation to NaCl concentrations in yeasts and yeast-like fungi that were isolated from either subglacial ice or hypersaline waters. In both of these natural environments, these organisms are exposed to low water activity, due to either high NaCl concentrations or low temperatures. Our data indicate that the fluidity of the plasma membrane can be used as an indicator of fitness for survival in extreme environments. Fungi that can survive in such extreme environments, such as Hortaea werneckii in the hypersaline waters of salterns, and Cryptococcus liquefaciens in subglacial environments, showed similar profiles of plasma-membrane fluidity in response to raised salinity. The same was seen for ubiquitous fungi, which are generally adapted for different types of stress, such as Aureobasidium pullulans and Rhodotorula mucilaginosa. Representatives of both of these groups modulated their plasma-membrane fluidity differently. When salinity exceeded their optimal range, the ubiquitous stress-tolerant species (A. pullulans, Rh. mucilaginosa) showed increased plasma-membrane fluidity, whereas in the dominant extremophiles (H. werneckii, Cr. liquefaciens), it decreased. On the other hand, the plasma membranes of the fungi with a narrow ecological amplitude (Arctic A. pullulans and Rhodosporium diobovatum) showed different responses.     

Chemical properties and NMR spectroscopic identification of certain fungal siderophores

Siderophores of six fungi viz. Aspergillus sp. ABp4, Aureobacidium pullulans, Penicillium oxalicum, P. chrysosporium, Mycotypha africana and Syncephalastrum racemosum were examined for their (1) electrophoretic mobilities to determine the acidic, basic or neutral charge; (2) Fe (III) binding nature viz., mono-, di-, or trihydroxamate; (3) amino acid composition; and (4) NMR (nuclear magnetic resonance) spectroscopy to determine their structure. Electrophoretic mobilities of siderophores of 3 fungi (P. oxalicum, P. chrysosporium, and M, africana) exhibited net basic charge, siderophores of 2 fungi (Aspergillus sp. ABp4 and S. racemosum) were acidic and 1 fungus (A. pullullans) was neutral. Electrophoresis of ferrated siderophore at pH 2 and colour of the spots indicated that siderophores of Aspergillus sp. ABp4 and P. oxalicum and A. pullulans were trihydroxamates, whereas siderophore of P. chrysosporium was dihydroxamate. Amino acid composition of siderophores purified by XAD-2 column chromatography, revealed the presence of asparagine, histidine, and proline in Aspergillus sp. ABp4, serine and alanine in P. chrysosporium, and valine in M. africana. The structure of purified siderophores as revealed by NMR spectroscopy identified siderophore of AB - 2670 (A. pullulans) as asperchrome F1, and AB-513 (M. africana) as rhizoferrin. The peak obtained for siderophore AB-5 (Aspergillus sp. ABp4) did not show resemblance to any known siderophore, therefore may be an exception.