Laccase production in solid‐state and submerged fermentation by Pleurotus ostreatus

A solid‐state fermentation (SSF) system for production of an industrially important enzyme laccase by Pleurotus ostreatus was developed by using potato dextrose yeast extract medium and polyurethane foam as a supporting material. The maximum laccase production in the SSF system was as high as 3×10⁵ U/L. Addition of inducers, such as copper and ferulic acid, further enhanced the laccase production in SSF. Moreover, the time required for the maximum laccase production was reduced to 6 days compared to 10 days reported earlier. The improvement achieved by the SSF system was investigated by comparing it to a submerged fermentation system (SmF), both experimentally and by using a standard theoretical model along with a parameter sensitivity analysis. Laccase production in SSF was found to be twice of that in SmF. One of the main reasons for higher laccase production in SSF compared to SmF was possibly due to the presence of higher proteolytic activity in SmF. Strong proteolytic activity in SmF presumably caused subsequent laccase degradation, which lowered the ultimate laccase production in SmF compared to SSF.     

丝状真菌的细胞凋亡

凋亡是一种程序性细胞死亡类型,为多细胞生物发育和维持生命所必需的,也普遍存在于细菌等原核生物和酵母、丝状真菌等真核生物中。丝状真菌既具有酵母和哺乳动物共有的凋亡同源蛋白,也具有酵母所不具备的哺乳动物凋亡同源蛋白,所以其凋亡机制较酵母更为复杂,而又较哺乳动物简单。凋亡在丝状真菌的发育、繁殖、衰老等过程中具有重要的作用。近年,丝状真菌作为新的凋亡研究的模式生物被广泛研究,而且进展迅速。综述丝状真菌的凋亡现象和检测方法,丝状真菌中凋亡的生物学功能,丝状真菌凋亡的诱导条件,以及丝状真菌凋亡相关基因的功能研究进展。     

Laccase production in bioreactor scale under saline condition by the marine-derived basidiomycete Peniophora sp. CBMAI 1063

Laccase production in saline conditions is still poorly studied. The aim of the present study was to investigate the production of laccase in two different types of bioreactors by the marine-derived basidiomycete Peniophora sp. CBMAI 1063. The highest laccase activity and productivity were obtained in the Stirred Tank (ST) bioreactor, while the highest biomass concentration in Air-lift (AL) bioreactor. The main laccase produced was purified by ion exchange and size exclusion chromatography and appeared to be monomeric with molecular weight of approximately 55 kDa. The optimum oxidation activity was obtained at pH 5.0. The thermal stability of the enzyme ranged from 30 to 50 °C (120 min). The Far-UV Circular Dichroism revealed the presence of high β-sheet and low α-helical conformation in the protein structure. Additional experiments carried out in flask scale showed that the marine-derived fungus was able to produce laccase only in the presence of artificial seawater and copper sulfate. Results from the present study confirmed the fungal adaptation to marine conditions and its potential for being used in saline environments and/or processes.     

丝状真菌蛋白质组学研究进展

丝状真菌不仅是致病菌,而且在异源表达工业酶、化学制品以及药物活性物质中发挥着越来越重要的作用。随着人类基因组计划的实施和推进,生命科学研究已进入了功能基因组时代,特别是蛋白质组学,在蛋白质水平对丝状真菌细胞生命过程中蛋白质功能和蛋白质之间的相互作用以及特殊条件下的变化机制进行研究,对生命的复杂活动进行深入而又全面的认识也为丝状真菌工业酶制剂和重组药物的开发提供广阔的创新空间。本文综述了蛋白质组学的研究内容和方法,总结了其在丝状真菌致病菌、抗生素产生菌和纤维素酶产生菌中的应用现状。不同层次的功能基因组学分析可以从各个角度掌握生物体的代谢网络和调控机制,本文还对蛋白质组学以及功能基因组学各部分内容的整合运用进行了展望。     

铅锌尾矿中耐重金属镉的丝状真菌的分离鉴定

【目的】旨在从重金属污染地分离出耐重金属镉真菌, 获得耐受重金属镉污染的高效菌株, 为重金属污染微生物修复提供菌种资源。【方法】利用稀释平板涂布法, 采用4种培养基对粤东北梅州市梅县铅锌尾矿废弃地9个样品进行分离, 并结合形态学和ITS rDNA基因序列分析, 鉴定分离到的耐镉真菌, 最后对分离到的耐镉真菌进行最小抑制浓度 (MIC) 检测。【结果】从粤东北梅州市梅县铅锌尾矿废弃地分离出72株丝状真菌, 经形态学和分子技术鉴定, 它们主要属于曲霉属(Aspergillus)、青霉属(Penicillium)、枝孢属(Cladosporium)、油瓶霉属(Lecythophora)、拟青霉属(Paecilomyces)、镰刀孢属(Fusarium)等。MIC检测发现有4株丝状真菌耐镉浓度较高, Paecilomyces lilacinus (Thom) Samson (6?20 p), Penicillium pinophilum Hedgcock (6?16 p), Penicillium rolfsii Thom (6?16 m) 和Fusarium oxysporum Schlecht. (8?11 p) 分别为200、40、25和15 mmol/L。【结论】从粤东北梅州市梅县铅锌尾矿废弃地分离到的72株丝状真菌, 不同程度耐受重金属镉, 在重金属污染的治理中有可能发挥作用。本研究为镉污染环境的微生物修复提供了重要菌株。     

Improved methods for the preparation of high molecular weight DNA from large and small scale cultures of filamentous fungi

Improved methods are described for the isolation of pure, high molecular weight DNA from small and large scale cultures of filamentous fungi. The methods depend on the extraction of DNA under conditions which prevent nuclease activity and contamination by carbohydrate. The small scale method depends on enzymatic digestion of the wall whereas the large scale method uses partial damage followed by autolysis. High yields of DNA are obtained by both methods and the DNA is suitable for restriction analysis. Southern Blotting, RFLP analysis, dot blotting and the production of gene libraries. The small scale method can be used for the simultaneous analysis of multiple cultures.     

Enantioselective reduction of 4-chromanone and its derivatives by selected filamentous fungi

Biotransformation of 4-chromanone and its derivatives in the cultures of three biocatalysts: Didymosphaeria igniaria, Coryneum betulinum and Chaetomium sp. is presented. The biocatalysts were chosen due to their capability of enantiospecific reduction of low-molecular-weight ketones (acetophenone and its derivatives and α- and β-tetralone). The substrates were reduced to the respective S-alcohols with high enantiomeric excesses, according to the Prelog's rule. In the culture of Chaetomium sp. after longer biotransformation time an inversion of configuration of the formed alcohols was also observed. The highest yield of transformation was observed for 6-methyl-4-chromanone. In all the tested cultures, the higher was the molecular weight of a chromanone, the lower conversion percent was observed.     

Glutamic protease distribution is limited to filamentous fungi

Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans, which has been linked to an increased risk for atherosclerosis-related events. In this study, we examined the effect of P. gingivalis infection on human macrophages with respect to foam cell formation, the hallmark of early atherogenesis, and the potential of P. gingivalis to induce its uptake by these cells. Human monocyte-derived macrophages were incubated with low density lipoprotein and infected with P. gingivalis FDC381 or its fimbriae deficient mutant, DPG3. Consistent with a role for fimbriae in this process, strain 381 significantly increased foam cell formation as compared to DPG3. Recovery of viable P. gingivalis in antibiotic protection experiments was significantly higher for strain 381 than for DPG3. By transmission electron microscopy, the wild-type strain was shown to adhere to and enter THP-1 cells. These results suggest that properties of P. gingivalis which render it capable of adhering to/invading other cell types may also be operative in macrophages and play an important role in its atherogenic potential.     

Flow cytometry and FACS applied to filamentous fungi

Flow cytometry is an automated, laser- or impedance-based, high throughput method that allows very rapid analysis of multiple chemical and physical characteristics of single cells within a cell population. It is an extremely powerful technology that has been used for over four decades with filamentous fungi. Although single cells within a cell population are normally analysed rapidly on a cell-by-cell basis using the technique, flow cytometry can also be used to analyse cell (e.g. spore) aggregates or entire microcolonies. Living or fixed cells can be stained with a wide range of fluorescent reporters to label different cell components or measure different physiological processes. Flow cytometry is also suited for measurements of cell size, interaction, aggregation or shape using non-labelled cells by means of analysing their light scattering characteristics. Fluorescence-activated cell sorting (FACS) is a specialized form of flow cytometry that provides a method for sorting a heterogeneous mixture of cells into two or more containers based upon the fluorescence and/or light scattering properties of each cell. The major advantage of analysing cells by flow cytometry over microscopy is the speed of analysis: thousands of cells can be analysed per second or sorted in minutes. Drawbacks of flow cytometry are that specific cells cannot be followed in time and normally spatial information relating to individual cells is lacking. A big advantage over microscopy is when using FACS, cells with desired characteristics can be sorted for downstream experimentation (e.g. for growth, infection, enzyme production, gene expression assays or ‘omics’ approaches). In this review, we explain the basic concepts of flow cytometry and FACS, define its advantages and disadvantages in comparison with microscopy, and describe the wide range of applications in which these powerful technologies have been used with filamentous fungi.     

l-leucine aminopeptidase production by filamentous Aspergillus fungi

AIMS: To screen various filamentous fungi belonging to Aspergillus spp. producing leucine and methionine aminopeptidases. METHODS AND RESULTS: Twenty-eight Aspergillus strains representing 14 species within the genus were screened for L-leucine aminopeptidase (LAP) production in two media in shake flask fermentation. Two Aspergillus sojae (NRRL 1988 and NRRL 6271) and one Aspergillus oryzae (NRRL 6270) strains were selected as the best producers for further studies. The peak LAP activities were 2.61, 2.59 and 1.30 IU ml(-1) for the three fungi on days 2, 5 and 4 respectively. In addition to LAP, L-methionine aminopeptidase (MAP) activity was also detected. Apart from submerged fermentation, the highest LAP yields by solid-state fermentation were 11.39, 17.40 and 13.02 IU g(-1) dry matter for the above fungi. The temperature and pH optimum of the enzyme was found to be in the range of 65-75 degrees C at pH 8.0-9.0 for all three fungi. Metal ions, Co(2+) and Fe(2+) in 2 mmol l(-1) concentration apparently enhanced the relative enzyme activity and heat stability. CONCLUSIONS: Two A. sojae (NRRL 1988 and NRRL 6271) and one A. oryzae (NRRL 6270) strains were found to be the best producers of LAP and MAP. The preliminary characterization studies revealed that the enzyme is considerably thermostable and belongs to the class metalloenzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: A good number of aspergilli were screened and the ability of the fungal aminopeptidase to release a particular N-terminal amino acid along with its high thermal stability, makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.     

Development of a stirred tank reactor system for the production of lignin peroxidases (ligninases) byPhanerochaete chrysosporium BKM-F-1767

Summary Lignin peroxidases produced byPhanerochaete chrysosporium have several important potential industrial applications based on their ability to degrade lignin and lignin-like compounds. A stirred tank reactor system for the production of lignin peroxidases is described here. Included in this study is an examination of the mechanics of pellet biocatalyst formation and the optimization of an acetate buffered medium. Higher levels of lignin peroxidase were obtained with acetate buffer compared to the other buffer systems tested. Concentrations of 0.05% (w/v) Tween 80 and 0.4 mM veratryl alcohol gave optimal lignin peroxidase activity in acetate buffered medium. In shake flask cultures, mycelial fragments in the inoculum aggregated into pellets during the first eight hours of incubation and thereafter increased in size through the eighth day. The agitation rate in shake flask cultures affected pellet size, the number of pellets formed, and lignin peroxidase activity. Transfer of fungal pellets from shake flask culture to a continuously oxygenated baffled stirred tank reactor (STR) resulted in production of high lignin peroxidase titres comparable to those of shake flask cultures when the agitation rate, oxygen dispersion and foaming were closely controlled.     

Biotransformation of ent-13-epi-manoyl oxides difunctionalized at C-3 and C-12 by filamentous fungi

Biotransformation of ent-3beta,12alpha-dihydroxy-13-epi-manoyl oxide with Fusarium moniliforme gave the regioselective oxidation of the hydroxyl group at C-3 and the ent-7beta-hydroxylation. The action of Gliocladium roseum in the 3,12-diketoderivative originated monohydroxylations at C-1 and C-7, both by the ent-beta face, while Rhizopus nigricans produced hydroxylation at C-7 or C-18, epoxidation of the double bond, reduction of the keto group at C-3, and combined actions as biohydroxylation at C-2/epoxidation of the double bond and hydroxylation at C-7/reduction of the keto group at C-3. In the ent-3-hydroxy-12-keto epimers, G. roseum originated monohydroxylations at C-1 and C-7 and R. nigricans originated the oxidation at C-3 as a major transformation, epoxidation of double bond and hydroxylation at C-2. Finally, in the ent-3beta-hydroxy epimer R. nigricans also originated minor hydroxylations at C-1, C-6, C-7 and C-20 and F. moniliforme produced an hydroxylation at C-7 and a dihydroxylation at C-7/C-11.     

Global nutritional profiling for mutant and chemical mode-of-action analysis in filamentous fungi

We describe a method for gene function discovery and chemical mode-of-action analysis via nutrient utilization using a high throughput Nutritional Profiling platform suitable for filamentous microorganisms. We have optimized the growth conditions for each fungal species to produce reproducible optical density growth measurements in microtiter plates. We validated the Nutritional Profiling platform using a nitrogen source utilization assay to analyze 21 Aspergillus nidulans strains with mutations in the master nitrogen regulatory gene, areA. Analysis of these data accurately reproduced expected results and provided new data to demonstrate that this platform is suitable for fine level phenotyping of filamentous fungi. Next, we analyzed the differential responses of two fungal species to a glutamine synthetase inhibitor, illustrating chemical mode-of-action analysis. Finally, a comparative phenotypic study was performed to characterize carbon catabolite repression in four fungal species using a carbon source utilization assay. The results demonstrate differentiation between two Aspergillus species and two diverse plant pathogens and provide a wealth of new data on fungal nutrient utilization. Thus, these assays can be used for gene function and chemical mode-of-action analysis at the whole organism level as well as interspecies comparisons in a variety of filamentous fungi. Additionally, because uniform distribution of growth within wells is maintained, comparisons between yeast and filamentous forms of a single organism can be performed.Electronic Supplementary Material Supplementary material is available in the online version of this article at The revised version of the PDF file was published online in January 2004. The figures are now in color.An erratum to this article can be found at     

外生菌根真菌与丝状真菌混合对红松凋落物降解效能的影响

实验以原始红松林中高频出现的丝状真菌(链格孢Alternarria sp.)和两种外生菌根真菌(细粉绒牛肝菌.Xerocomus Pulverulentus、大杯伞Clitocybe maxima)为供试菌株,以粉碎的新鲜红松凋落物为分解底物,发酵培养后测定底物的质量损失率,酶活性以及营养元素的变化情况。结果表明,AX处理(Alternaria sp.和X.Pulve Rrulentus混合菌株组合)在60天内质量损失率最高,为18.33%,而混合菌AC处理(Alternaria sp.和C.maxima混合菌株组合)由于产生拮抗作用质量损失较低,为13.27%;20天时,X处理(X.Pulverulentus)产生漆酶活性较高为0.093 U·mL^-1,C(C.maxima)和AX处理的酶活性的最高值分别为0.063 U·mL^-1和0.047 U·mL^-1,而A(Alternaria sp.)处理的纤维素酶活性为0.59 U·mL^-1,AC处理为0.57 U·mL^-1,AX为0.53 U·mL^-1,但是AX处理在50天时再次出现高峰,为0.48 U·mL^-1:AX处理底物的营养元素N含量减少幅度最为明显,减少百分率为12.21%,同时,P元素的减少幅度也最大,减少百分率25.82%。研究结果进一步揭示了森林生态系统中不同功能真菌类群对林木有机凋落物降解过程中的作用机制,及真菌类群间的相互作用关系。     

真菌通用引物Its1和Its4在丝状真菌鉴定中的价值评价

目的 对真菌通用引物Its1和Its4在丝状真菌鉴定中的价值进行评价.方法 收集中山大学附属第一医院2012年1月至2012年8月间分离的丝状真菌11株,使用真菌通用引物Its1和Its4采用PCR法扩增核糖体基因,对PCR产物进行序列测定,将测序结果与GenBank中已知标准或临床菌株DNA序列比对,确定丝状真菌的菌种;同时与传统形态学鉴定方法进行比较,从而对真菌通用引物Its1和Its4在丝状真菌鉴定中的价值进行评价.结果 从DNA提取到序列测定结束,在2个工作日内即可完成.所有菌株均测序成功,测序结果与GenBank中的序列比对,烟曲霉、杂色曲霉、橘青霉、溜曲霉可以鉴定到种;黄曲霉和米曲霉、阿姆斯特丹散囊菌和冠突散囊菌由于高度同源性无法区分鉴定到种.结论 利用真菌通用引物Its1和Its4结合PCR技术和测序技术,可快速、准确将大部分丝状真菌鉴定到种.     

Influence of the carbon source on the growth and lignocellulolytic enzyme production by Morchella esculenta strains

Growth and lignocellulolytic enzymes production by two Morchella esculenta strains (BAFC 1728 and BEL 124) growing in solid state fermentation using different lignocellulosic materials along 58 days was characterized. Both strains were able to grow on the three substrates: wheat bran, wheat bran plus corn starch, and rolled oat. The growth was characterized by measuring chitin content, reducing sugars, pH, dry weight loss, and extractable proteins, such parameters varied substantially with substrate and strain used. The maximum rate of growth in both strains was observed between 5 and 28 days. Regarding enzyme production, as a general trend strain BAFC 1728 produced the highest titres. The most evident difference was observed in laccase production by this strain on wheat bran, which exceeded that observed in strain BEL 124 by tenfold (7.45 U g⁻¹).     

丝状真菌G蛋白信号途径的研究进展

丝状真菌在工业、农业、医药等领域具有重要经济价值,一些亦可导致人类及动、植物疾病,造成经济损失.G蛋白信号途径是真核生物中普遍存在的细胞跨膜信号转导途径.近年来,丝状真菌中G蛋白信号途径的研究发展很快,相关报道表明该信号途径参与感应并传递多种胞外信号刺激,对丝状真菌的生长、分化、繁殖、致病性及真菌毒素等次生代谢产物合成有重要的调控作用.本文就丝状真菌中G蛋白信号途径的基本组成及其生理功能的研究现状进行简要综述.     

A microculture chamber and improved method for combined light and electron microscopy of filamentous fungi

A microculture chamber has been developed which enables specific fungal hyphae to be followed in their living state before being processed and longitudinally sectioned for electron microscopy. Photomicrographs and their corresponding electron micrographs are presented which illustrate the effectiveness of this approach. Examples of the application of the method are presented.     

粗毛栓菌(Trametes hirsuta) D2固态发酵山核桃蒲壳产漆酶的营养条件研究

【目的】为提高漆酶产量,降低生产成本,以山核桃蒲壳作为基质,对粗毛栓菌D2固态发酵产漆酶的营养条件进行研究。【方法】对不同碳源、氮源、碳氮比、蒲壳含量对漆酶产量的影响进行分析。【结果】山核桃蒲壳是粗毛栓菌生长的良好载体,能够促进漆酶的合成。粗毛栓菌D2漆酶固态发酵培养基干物质组成为:山核桃蒲壳40%(质量比),玉米粉24%(质量比),菜籽饼粉36%(质量比)。发酵6 d时,漆酶活性为126.8 U/g干基。【结论】粗毛栓菌固态发酵山核桃蒲壳产漆酶具有效率高,生产成本低的优点,具有潜在的工业化应用前景。