Production of extracellular hydrolase enzymes by fungi from King George Island

Fungi are known to produce a range of extracellular enzymes and other secondary metabolites. Investment in extracellular enzyme production may be an important element of the survival strategy of these fungi in maritime Antarctic soils. This study focuses on fungi that were isolated from ornithogenic, undisturbed and human-impacted soils collected from the Fildes Peninsula, King George Island, Antarctica, during the austral summer in February 2007. We (1) describe fungal diversity based on molecular approaches, (2) describe the thermal characteristics of the fungal isolates, and (3) screen extracellular hydrolase enzyme production (amylase and cellulase) by the isolates. Soil samples were cultured using the Warcup soil plating technique and incubated at 4 and 25 °C to allow basic thermal classification. In total, 101 isolates were obtained. All the isolates were screened at culture temperatures of 4 and 25 °C in order to detect activity of extracellular hydrolase enzymes. At 25 °C, ornithogenic penguin rookery soils recorded the lowest diversity of fungi, with little difference in diversity apparent between the other soils examined. At 4 °C, an undisturbed site recorded the lowest and a human-impacted site the highest diversity of fungi. The majority of the fungi identified in this study were in the mesophilic thermal class. Six strains possessed significant activity for amylase and 13 for cellulase at 25 °C. At 4 °C, four strains showed significant amylase and 22 significant cellulase activity. The data presented increase our understanding of microbial responses to environmental temperature.     

Mutants ofXanthomonas campestris defective in secretion of extracellular enzymes

Summary Mutants ofXanthomonas campestris B 1459 were isolated that are defective in secretion of both cellulase and amylase. Both enzymes accumulated in the periplasmic space. The defects in secretion of cellulase or amylase were partly overcome by introducing into the mutants specific multiple copies of DNA cloned fromX. campestris, and presumed to code for cellulase or amylase enzymes. The mutant strains also showed reduced amounts of extracellular pectinase and protease activities, as if the mutants were generally defective for secretion of extracellular enzymes. The mutants showed reduced pathogenesis for turnip seedlings. The secretion-defective mutants may allow production of xanthan gum with reduced cellulose, pectin, protein and starch-degrading enzyme activities, thereby allowing more widespread mixing of microbially produced xanthan gum with these commercially important water-soluble polymers.     

Desert soil fungi isolated from Saudi Arabia: cultivable fungal community and biochemical production

Desert soils harbor fungi that have survived under highly stressed conditions of high temperature and little available moisture. This study was designed to survey the communities of cultivable fungi in the desert soils of the Arabian Peninsula and to screen the fungi for the potentially valuable antioxidants (flavonoids, phenols, saponins, steroids, tannins, terpenoids, and alkaloids) and enzymes (cellulase, laccase, lipase, protease, amylase, and chitinase). Desert soil was sampled at 30 localities representing different areas of Saudi Arabia and studied for physico-chemical soil properties. Five types of soil texture (sand, loamy sand, sandy loam, silty loam, and sandy clay loam) were observed. A total of 25 saprotrophic species was identified molecularly from 68 isolates. Our survey revealed 13 culturable fungal species that have not been reported previously from Arabian desert soils and six more species not reported from Saudi Arabian desert soils. The most commonly recorded genera were Aspergillus (isolated from 20 localities) and Penicillium (6 localities). The measurements of biochemicals revealed that antioxidants were produced by 49 and enzymes by 52 isolates; only six isolates did not produce any biochemicals. The highest biochemical activity was observed for the isolates Fusarium brachygibbosum and A. phoenicis. Other active isolates were A. proliferans and P. chrysogenum. The same species, for instance, A. niger had isolates of both high and low biochemical activities. Principal component analysis gave a tentative indication of a relationship between the biochemical activity of fungi isolated from soil and soil texture variables namely the content of silt, clay and sand. However, any generalizable relation between soil properties and fungal biochemical activities cannot be suggested. Each fungal isolate is probable to produce several antioxidants and enzymes, as shown by the correlation within the compound groups. Desert soil warrants further research as a promising source of biochemicals.     

Antagonistic and plant growth promoting activities of endophytic and soil actinomycetes

The objective of this study was the isolation and screening of actinomycete isolates for antagonistic potential and plant growth promoting activities. A total of 321 isolates were recovered from different plants, their rhizospheric soils and non-rhizospheric soils of Punjab and Himachal Pradesh regions. Out of these, 62 were endophytic, 156 were rhizospheric and 103 were non-rhizospheric isolates. In primary screening (dual culture assay), 83 isolates antagonised one or more test phytopathogenic fungi. From these active isolates, 20 were found to be antagonistic in well diffusion assay (secondary screening) and most of them demonstrated broad spectrum inhibitory activity against five to six test fungi. Studies on plant growth promoting activities revealed that 12 showed abilities to produce indole acetic acid, 10 produced siderophores and 12 showed ammonia production. Phosphate solubilisation was observed in five isolates and four fixed atmospheric N₂. In addition, production of hydrolytic enzymes such as chitinase, amylase, cellulase and protease was demonstrated by five, twenty, eleven and eleven isolates, respectively. The results of this study indicate that these isolates may be used as biocontrol and plant growth promoting agents. Morphological and chemotaxonomic studies revealed that all the active isolates belonged to the genus Streptomyces     

Role of Melanin in Release of Extracellular Enzymes and Selection of Aggressive Isolates of Bipolaris sorokiniana in Barley

Eighteen barley isolates of Bipolaris sorokiniana belonging to wild and clonal type of black, mixed and white subpopulations were quantitatively assayed for their melanin content and aggressiveness with respect to production of some of the extracellular enzymes such as cellulase, pectinase, amylase and protease. Cellulase and pectinase constituted major portion of the enzymes recovered from the black, mixed and white isolates. Enzyme production and aggressiveness were relatively higher in melanin devoid or low melanin isolates. The melanin deficient isolates were also differentiated from black and mixed isolates on the basis of variation in internal transcribed spacer region of the ribosomal DNA. Higher enzyme productions positively correlated with area under disease progress curve (AUDPC) and lesion development. Melanin content was negatively correlated with extracellular enzymes and aggressiveness of the isolates. Based on melanin content, lesion size, AUDPC and extracellular enzymes, the isolates were grouped in two major clusters (I and II) with further division of cluster II into two sub-clusters (II-A and II-B). The results appears to indicate a possible role of melanin in release of extracellular enzymes and hence in evolution and selection of aggressive isolates of B. sorokiniana in barley.     

Functional and phylogenetic diversity of root-associated bacteria of Ajuga bracteosa in Kangra valley

Present study investigates the cultivable diversity of root-associated bacteria from a medicinal plant Ajuga bracteosa in the Kangra valley, in order to determine their plant growth promoting (PGP) and biotechnological potential. The plant was found to exhibit a positive rhizosphere effect of 1.3-1.5. A total of 123 morphologically different bacteria were isolated from the rhizospheric soil and roots of the plant. Medium composition was found to have significant effect on the composition of isolated bacterial populations. Majority of the rhizospheric soil isolates belonged to α- and γ-Proteobacteria, with Pseudomonas constituting the most dominant species. Endophytic bacterial community, on other hand, consisted almost exclusively of Firmicutes. Majority of the isolates showed PGP activity by producing siderophores and indole acetic acid. Several isolates were found to exhibit very high antioxidant activity in the culture medium. A significant proportion of isolates also demonstrated other ecologically important activities like phosphate solubilization, nitrogen fixation, and production of hydrolytic enzymes including amylase, protease, lipase, chitinase, cellulase, pectinase and phosphatase. Firmicutes were found to be metabolically the most versatile group and performed multiple enzyme activities. This is the first systematic study of culturable bacterial community from the rhizosphere of A. bracteosa, particularly in the Kangra valley region.     

Effect of herbizid and touchdown herbicides on soil fungi and on production of some extracellular enzymes

Glucophilic and cellulose-decomposing fungi were significantly reduced in soil samples treated with 0.019-0.152 mg a.i./kg soil of the herbicides Herbizid and Touchdown. The decrease was regularly correlated with the doses of the two herbicides and persisted till the end of the experiment (12 weeks). The isolated fungi were found to be able to produce hydrolytic extracellular enzymes in solid media but with variable capabilities. The ability to produce enzymes was adversily affected by the incorporation of herbicides in culture media. Lower doses of herbicides were occasionally promotive to enzyme production and mycelial growth of some fungi. Incorporation of 50 ppm of Herbizid and Touchdown significantly activated amylase production and mycelial dry weight in cultures of Fusarium oxysporum, Mucor hiemalis and Penicillium chrysogenum. There was a significant increase in C1-cellulase produced by F. oxysporum and P. aurantiogriseum when cultures were treated with 50, 100 and 200 ppm of Herbizid which induced also more Cx-cellulase production by P. chrysogenum. Lipase and protease production was always lower in treated than in control fungal cultures.     

内蒙古鄂尔多斯一古盐湖真菌多样性及其功能酶的初步筛选

【目的】探索内蒙古鄂尔多斯一古盐湖土壤沉积物和湖水样品中的真菌多样性,并初步筛选出产功能酶的活性菌株。【方法】采用含有2.5%、5%、10%及15%NaCl的3种分离培养基,利用稀释平板法分离可培养真菌,基于形态学和ITS序列分析对获得菌株进行系统分类学分析。利用6种筛选培养基定性检测盐湖真菌的产酶活性。【结果】共分离到2 121株真菌,共45个形态种,根据形态学研究和ITS序列的系统分类学分析将其归类为27个属,优势属为曲霉属(Aspergillus)。功能酶筛选结果表明,45个形态种中有22个形态种具有产酶活性,其中6个形态种只产蛋白酶,6个只产纤维素酶,2个只产复合酶,1个只产淀粉酶。有4个形态种可同时产3种酶,有3个可同时产2种酶。【结论】内蒙古鄂尔多斯地区盐湖中真菌多样性丰富,产酶特性良好,研究工作为耐(嗜)盐真菌资源的进一步开发和利用提供依据。     

春季树麻雀体内几种消化酶活性研究

应用酶学分析法测定了树麻雀Passer montanus春季的腺胃、肌胃、小肠、大肠、肝脏和胰脏组织中淀粉酶、蛋白酶和纤维素酶的活力.结果表明,不同组织中的消化酶活力差异显著,淀粉酶和蛋白酶以胰脏中活力最高,腺胃次之,纤维素酶均较低;同一组织中不同消化酶的活力差异显著,淀粉酶活性最高,蛋白酶次之,纤维素酶活力最低.这些差异提示消化酶活力大小与器官分化有关,并受食物组成的影响,因此产生了不同的酶活力分布.这是树麻雀长期适应东北地区寒冷环境的生存策略之一.     

Fungal isolates from natural pectic substrates for polygalacturonase and multienzyme production

Pectin rich wastes and waste dump yard soils were screened and eighty pectinolytic fungal isolates were obtained by enrichment culturing and ruthenium red plate assay. Eight isolates with higher zones of pectin hydrolysis were selected and tested for polygalacturonase production. One isolate identified as Aspergillus awamori MTCC 9166 with highest polygalacturonase activity was tested for utilization of raw pectins for enzyme production. Polygalacturonase production was high in raw pectin sources like Orange peel (16.8 U/ml) Jack fruit rind (38 U/ml) Carrot peel (36U/ml) and Beet root peel (24U/ml). Selected Aspergillus awamori MTCC 9166 was found to be having good polygalacturonase, xylanase, cellulase and weak amylase and protease activities. This isolate with multi-enzyme production could have application for enzymes production and degradation of fruit and vegetable waste in the process of urban waste disposal.     

Effect of garlic bulb extract on the growth and enzymatic activities of rhizosphere and rhizoplane fungi

Eighteen fungal species were isolated from rhizospheric soil and rhizoplane samples of three plant crops in southern Iraq. The fungal isolates were examined for the activities of four enzymes (amylase, cellulase, phenoloxidase, and protease), as well as their growth, against crude garlic extract added to the culture agar medium. A high reduction or inhibition of enzymatic activities was observed for the fungi treated with garlic extract compared with untreated fungal cultures. However, most of the species showed inhibition of enzymes due to the effect of garlic extract. The growth of the fungal species was also remarkably reduced by the garlic extract.This revised version was published online in October 2005 with corrections to the Cover Date.     

Assessment of biotechnological potentials of strains isolated from repasso olive pomace in Tunisia

The agri-food industry generates significant amounts of byproducts, among them repasso olive pomace (ROP) which are rejected and can constitute a serious environmental problem. Our study aimed to investigate the diversity of microbiota isolated from ROP and screen for their biotechnological potentials. A collection of 102 strains (88 bacteria and 14 fungi) was obtained from a ROP sample. The diversity of the bacterial collection was evaluated by amplification of the internal transcribed spacers between the 16S and the 23S rRNA genes (ITS-PCR) and by 16S rRNA sequencing. Fungal identification was performed by polymerase chain reaction (PCR) amplification of the ITS1–5.8S–ITS2 ribosomal DNA region. The specific enzymatic screening of the detected microorganisms was tested. Partial 16S rRNA gene sequencing performed on 44 isolates showed a high level of identity with known strains. Fungal strains identification showed that they belong to four families: Trichocomaceae, Pleurostomataceae, Mucoraceae, and Bionectriaceae. Our results demonstrated that Gram-positive bacteria were mostly active, particularly for protease, lipase, amylase, cellulase laccase, and for biosurfactant production. From the 88 isolated bacteria, Firmicutes were the most prevalent and microdiverse. Bacillus and Paenibacillus, together with some other Gram-negative bacteria such as Pseudocitrobacter anthropi, and Acinetobacter johnsonii showed significant hydrolytic activities and biosurfactant production. The 14 isolated fungi showed a high capacity of enzyme production. This is the first study in Tunisia describing the microbial diversity in ROP as well as the isolation of Bacillus mojavensis producing lipase. Microorganisms especially fungi present in the repasso olive cake produce diverse hydrolytic enzymes of industrial interest.     

秦岭地区野生细鳞鲑肠道微生物多样性及产酶活性分析

为探究秦岭地区野生细鳞鲑(Brachymystax lenok)肠道细菌组成多样性,筛选出产胞外酶菌株,利用传统分离培养并分子鉴定的方法和基于16S r RNA基因克隆的现代分子生物技术相结合测定秦岭野生细鳞鲑肠道细菌菌群多样性并构建系统发育树,利用淀粉酶、蛋白酶、纤维素酶及脂肪酶4种胞外酶筛选培养基筛选出产上述酶的细菌。细菌传统分离培养并分子鉴定法从细鳞鲑肠道获得18个属的细菌类群,分别归属于变形菌门、拟杆菌门和厚壁菌门,其中,气单胞菌属(Aeromonas)为优势菌群。基于16S r RNA基因克隆的现代分子方法获得22个属的细菌类群,分别归属于变形菌门、拟杆菌门、厚壁菌门和放线菌门,其中,鞘氨醇杆菌属(Sphingomonas)为优势菌群。4种胞外酶筛选获得53株细菌产胞外酶,其中21株可在低温(10℃)环境下产胞外酶。结果表明,传统分离培养法与基于16S r RNA基因克隆的现代分子生物技术相结合能够更有效全面地分析细鳞鲑鱼肠道微生物的多样性,并且细鳞鲑肠道微生物具有一定的产酶活性。     

阿特拉津降解菌Acinetobacter sp. DNS32对无机氮源的响应

【目的】研究Acinetobacter sp.DNS32的生长、阿特拉津降解能力和降解基因转录水平的表达对无机氮素的响应关系,为菌株的工程应用提供指导与理论基础。【方法】以Acinetobactersp.DNS32为对象,采用摇瓶法研究菌株在阿特拉津培养基中菌株生长情况及降解能力对外加硝态氮与铵态氮的响应关系,利用荧光定量PCR技术检测DNS32降解基因表达量对外加无机氮源的响应关系。【结果】外加无机氮源可以促进DNS32菌株的生长,提高阿特拉津降解能力,无机氮源对DNS32菌株的trzN、atzB和atzC 3种降解基因表达均有促进作用,加入无机氮源的试验处理中DNS32菌株trzN基因的表达量最高可达对照的11.252±2.408倍,推断DNS32菌株的这3种降解基因所编码的酶是稳定表达的组成酶。【结论】DNS32降解阿特拉津不受"氮饥饿"诱导机制调控,且无机氮源的存在对菌株的生长与降解有促进作用,因此菌株在土壤修复实践中具有广阔的应用前景。     

Enzymatic activity of fungi endophytic on five medicinal plant species of the pristine sacred forests of Meghalaya,India

Fungal species that establish an endophytic role inside the tissues of medicinal plants are known to produce a wide range of biologically active metabolites and enzymes. In the present study, the most dominant and representative endophytic fungal species of five ethno-medicinal plants prevalent in the pristine sacred forests of Meghalaya, were screened for their ability to produce amylase, cellulase, protease, lipase, and xylanase. Each of endophytic fungal isolates showed a wide range of enzyme activity. Mycelial biomass generation and root colonization, in addition to the enzyme activity of the endophytic fungal isolates, provided insights into their probable origin and ecological roles within the plant host.     

The effect of burning on soil enzyme activities in natural grasslands in southern India

Soil amylase, cellulase, invertase and phosphatase activities were determined following wildfire in a savanna type grassland in southern India. Activities of these enzymes increased substanially in burned soils compared to those in adjacent unburned soils. Surface soil (0–10 cm) exhibited a greater increase in enzyme activities than subsurface soil (10–20 cm). Amylase activity was more pronounced following fire than the other enzyme activities.     

Effect of two systemic fungicides on cellulose decomposing fungi of tomato plants and on some enzymatic activities

Kocide 101 (77% cupric hydroxide) and Ridomil plus (15% metalaxyl and 35% copper oxychloride) were used in the treatment of tomato plants. The two fungicides exerted a depressive effect on the total counts and on the individual cellulose decomposing fungal species associated with the roots and shoots of tomato. When these fungicides were incorporated in the liquid culture medium specified for growth and extracellular enzyme production by some selected fungal species, there was a significant reduction in mycelial growth as well as in amylase, cellulase, lipase and protease production by the fungi tested, particularly at the higher doses (200-400 ppm). Exceptions were observed with lower doses (50 and 100 ppm) especially in case of Aspergillus flavus, Cunninghamella echinulata, Penicillium chrysogenum and Fusarium oxysporum grown for amylase or cellulase production.     

Exocellular enzyme activity of dermatophytes and other fungi isolated from ruminants in Southern Iraq

Sixteen fungal species were isolated from 182 specimens collected from four ruminants (buffalo, camel, cattle and sheep) in Southern Iraq. Fungi represented by five species of dermatophytes and eleven species of other fungi were screened for the activity of four enzymes; keratinase, proteinase, lipase and amylase. Keratinase was found to be produced by all of the dermatophytes and non-dermatophytes, except for Paecillomyces variottii and Scytalidium lignicola. However, high keratinase activity was expressed by the dermatophytic species particularly by Trichophyton mentagrophytes var. erinacei and Microsporum gypseum. Three dermatophytes viz. M. gypseum, T. verrucosum and T. mentagrophytes var. nodulare were capable of producing protease, lipase and amylase. Although, T. mentagrophytes var. erinacei showed high protease activity, it did not produce lipase and amylase. On the contrary most of the non-dermatophytic species revealed protease and lipase activities higher than the dermatophytes. The Curvularia spp. isolates showed the highest protease and amylase activity, while Aspergillus parasiticus revealed the highest activity of lipase and amylase. No correlation was observed between enzyme activity and the growth rate of the examined fungi. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studies on the ecology of actinomycetes in an agricultural soil amended with organic residues: II. Assessment of enzymatic activities of <Emphasis Type="Italic">Actinomycetales</Emphasis> isolates

The main objective of this investigation was to study the enzymatic activities of Actinomycetales strains isolated from an agricultural soil amended with different amounts of municipal solid waste compost (MSWC) or farmyard manure (FM). For this purpose, the hydrolytic activities of carboxymethyl cellulase, xylanase, pectinase, amylase, chitinase and protease were tested for 75 isolates of Sterptomyces, Amycolatopsis and Nocardioides from different sources (unamended soil, amended soil with FM or MSWC, FM and MSWC) at temperature ranging between 30 and 50°C. It was shown that the highest rate of enzymes producer’s strains was registered at 30°C, and decreased gradually to annul at 50°C, with the exception of the MSWC strains origin. It was also shown that the percentage of strains producers of enzymes isolated from soil amended with MSWC appeared higher than the one registered for those isolated from control and amended with FM soils. Application of MSWC increases the number of enzymes producer-actinomycetes in the soil and then it improves its fertility.     

Fungi from koala (Phascolarctos cinereus) faeces exhibit a broad range of enzyme activities against recalcitrant substrates

Aims:  Identification of fungi isolated from koala faeces and screening for their enzyme activities of biotechnological interest.
Methods and Results:  Thirty-seven fungal strains were isolated from koala faeces and identified by the amplification and direct sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA. The fungi were screened for selected enzyme activities using agar plates containing a single substrate for each target class of enzyme. For xylanase, endoglucanase, ligninase (ligninolytic phenoloxidase) and protease over two-thirds of the isolates produced a clearing halo at 25°C, indicating the secretion of active enzyme by the fungus, and one-third produced a halo indicating amylase, mannanase and tannase activity. Some isolates were also able to degrade crystalline cellulose and others displayed lipase activity. Many of the fungal isolates also produced active enzymes at 15°C and some at 39°C.
Conclusions:  Koala faeces, consisting of highly lignified fibre, undigested cellulose and phenolics, are a novel source of fungi with high and diverse enzyme activities capable of breaking down recalcitrant substrates.
Significance and Impact of the Study:  To our knowledge, this is the first time fungi from koala faeces have been identified using ITS sequencing and screened for their enzyme activities.