Cold-adapted enzymes produced by fungi from terrestrial and marine Antarctic environments

Antarctica is the coldest, windiest, and driest continent on Earth. In this sense, microorganisms that inhabit Antarctica environments have to be adapted to harsh conditions. Fungal strains affiliated with Ascomycota and Basidiomycota phyla have been recovered from terrestrial and marine Antarctic samples. They have been used for the bioprospecting of molecules, such as enzymes. Many reports have shown that these microorganisms produce cold-adapted enzymes at low or mild temperatures, including hydrolases (e.g. α-amylase, cellulase, chitinase, glucosidase, invertase, lipase, pectinase, phytase, protease, subtilase, tannase, and xylanase) and oxidoreductases (laccase and superoxide dismutase). Most of these enzymes are extracellular and their production in the laboratory has been carried out mainly under submerged culture conditions. Several studies showed that the cold-adapted enzymes exhibit a wide range in optimal pH (1.0–9.0) and temperature (10.0–70.0?°C). A myriad of methods have been applied for cold-adapted enzyme purification, resulting in purification factors and yields ranging from 1.70 to 1568.00-fold and 0.60 to 86.20%, respectively. Additionally, some fungal cold-adapted enzymes have been cloned and expressed in host organisms. Considering the enzyme-producing ability of microorganisms and the properties of cold-adapted enzymes, fungi recovered from Antarctic environments could be a prolific genetic resource for biotechnological processes (industrial and environmental) carried out at low or mild temperatures.     

Acidophilic bacteria and archaea: acid stable biocatalysts and their potential applications

Acidophiles are ecologically and economically important group of microorganisms, which thrive in acidic natural (solfataric fields, sulfuric pools) as well as artificial man-made (areas associated with human activities such as mining of coal and metal ores) environments. They possess networked cellular adaptations to regulate pH inside the cell. Several extracellular enzymes from acidophiles are known to be functional at much lower pH than the cytoplasmic pH. Enzymes like amylases, proteases, ligases, cellulases, xylanases, α-glucosidases, endoglucanases, and esterases stable at low pH are known from various acidophilic microbes. The possibility of improving them by genetic engineering and directed evolution will further boost their industrial applications. Besides biocatalysts, other biomolecules such as plasmids, rusticynin, and maltose-binding protein have also been reported from acidophiles. Some strategies for circumventing the problems encountered in expressing genes encoding proteins from extreme acidophiles have been suggested. The investigations on the analysis of crystal structures of some acidophilic proteins have thrown light on their acid stability. Attempts are being made to use thermoacidophilic microbes for biofuel production from lignocellulosic biomass. The enzymes from acidophiles are mainly used in polymer degradation.     

Acid phosphatase and invertase activities of <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Aspergillus niger is widely used as an enzyme source in industries. Considering its enzymic potential, A. niger was studied for its acid phosphatase (EC 3.1.3.2, orthophosphoric monoester phosphohydrolase), and invertase (EC 3.2.1.26, β-fructofuranoside fructohydrolase) activity in defined media supplemented with 1%, 3%, or 5% sucrose concentrations. Both these enzymes play a key role in phosphate and carbon metabolism in plants, animals, and microorganisms and hence are interesting from the standpoint of biotechnological applications. Ontogenic changes in extracellular, cytoplasmic, and wall-bound enzyme activities of A. niger were studied. Growth in terms of fresh weight showed inverse correlation with pH. At higher pH values, both enzyme activities were higher in the medium supplemented with low sucrose concentration. It was observed that the more the fresh weight of fungi decreased, the greater was the enzyme activity observed. It is suggested that these enzymes may participate in autolysis of fungi and, on the other hand, could prove to be a potential source of industrial application and exploitation.     

The 'pH optimum anomaly' of intracellular enzymes of Ferroplasma acidiphilum

A wide range of microorganisms, the so-called acidophiles, inhabit acidic environments and grow optimally at pH values between 0 and 3. The intracellular pH of these organisms is, however, close to neutrality or slightly acidic. It is to be expected that enzymatic activities dedicated to extracellular functions would be adapted to the prevailing low pH of the environment (0-3), whereas intracellular enzymes would be optimally active at the near-neutral pH of the cytoplasm (4.6-7.0). The genes of several intracellular or cell-bound enzymes, a carboxylesterase and three alpha-glucosidases, from Ferroplasma acidiphilum, a cell wall-lacking acidophilic archaeon with a growth optimum at pH 1.7, were cloned and expressed in Escherichia coli, and their products purified and characterized. The Ferroplasmaalpha-glucosidases exhibited no sequence similarity to known glycosyl hydrolases. All enzymes functioned and were stable in vitro in the pH range 1.7-4.0, and had pH optima much lower than the mean intracellular pH of 5.6. This 'pH optimum anomaly' suggests the existence of yet-undetected cellular compartmentalization providing cytoplasmic pH patchiness and low pH environments for the enzymes we have analysed.     

Use of epifluorescence microscopy for characterizing the activity of Thiobacillus Ferrooxidans on iron pyrite

The enumeration and characterization of microorganisms attached to solid surfaces have always presented significant difficulties. This is particularly true for micro organisms that are indigenous to coal mines and mineral deposits where metal sulfides are ubiquitous. The complications that arise are the result of the variety of inorganic compounds that are present in these environments, the harsh conditions under which the microorganisms proliferate, and the low cell densities to which they grow. The work presented here suggests that epifluorescence microscopy using acridine orange can be a useful probe to study acidophilic metal-leaching bacteria. Experiments involving the growth of Thiobacillus ferrooxidans on iron pyrite are described which indicate a relationship between cell fluorescence color and bacterial activity. Both attached and free-solution cell densities were determined throughout the course of the leaching process and considered along with changes in cell fluorescence color which might be associated with changes in intracellular pH. As such, epifluorescence microscopy, using acridine orange, can be used for assessing the activity of T. ferrooxidans on iron pyrite as well as resolving the controversy concerning the significance of attachment during the leaching process.     

Insights into extreme thermoacidophily based on genome analysis of Picrophilus torridus and other thermoacidophilic archaea

Thermoacidophiles are prokaryotic microorganisms with the stunning capability to survive and multiply at extremely low pH and simultaneously at high temperatures. The mechanisms by which these organisms, exclusively members of the Archaea, cope with their harsh surroundings are poorly understood. The genome sequences of several representatives of the thermoacidophilic genera Picrophilus, Thermoplasma and Sulfolobus have recently become available. Genome-wide comparison has revealed a number of features as possible facets of the overall acidophilic survival strategy of the most thermoacidophilic organisms known, such as a high ratio of secondary over primary transport systems, the composition of the respiratory chain, and the frequent genetic input via lateral gene transfer (LGT) during evolution.     

嗜酸藻类研究进展

酸性矿山废水(acid mine drainage,AMD)是硫化矿物暴露于地表,与水、大气及微生物相互作用发生氧化性溶解而形成的,其pH通常在3.5以下,并含有高浓度的金属离子,危害十分严重。因为嗜酸原核生物可通过氧化硫化矿物获得能量,从而加速酸性矿山废水的形成过程,所以一直以来都是嗜酸微生物研究的焦点。但事实上,真核生物在酸性生态系统中往往发挥着更重要的作用。尤其是光合藻类,它们在很多酸性环境都是主要的初级生产者,对酸性矿山废水的演化和修复起着关键的推动作用。本文从嗜酸藻类的常见种类和生态分布、适应极端环境的生理机制以及它们在污染治理和工业生产中的应用3个方面,综述了该领域近30年来的研究进展,并以此为基础提出了将来需要着力加强的5个研究方向。     

Oxidation and Reduction of Iron by Acidophilic Bacteria

Abstract

Redox reactions of iron in acidic environments are of economic and environmental significance, for example, for the leaching of metal ores and for the formation of acid mine drainage and acid sulfate soils. Until recently, research on microbial iron metabolism in acidic environments has mainly been focused on the role of aerobic, autotrophic ferrous iron‐oxidizing bacteria. In the present paper, recent new developments in the field of acidophilic iron metabolism are reviewed. In addition to the well‐known autotrophic ferrous iron‐oxidizing organisms, new heterotrophic isolates have been described that are capable of oxidizing ferrous iron. Microorganisms can also play an important role in the reductive part of the iron cycle. Both heterotrophic and autotrophic organisms may also be involved in this process. The contribution of heterotrophic organisms to acidophilic iron cycling can be twofold: In addition to their direct role as a catalyst, these organisms may scavenge organic compounds that inhibit their autotrophic counterparts. Detailed studies of acidophilic ecosystems are needed to assess the significance of the various types of microorganisms for the overall rate of iron cycling in these extreme environments.     

Engineering robust microorganisms for organic acid production

Organic acids are an important class of compounds that can be produced by microbial conversion of renewable feedstocks and have huge demands and broad applications in food, chemical, and pharmaceutical industries. An economically viable fermentation process for production of organic acids requires robust microbial cell factories with excellent tolerance to low pH conditions, high concentrations of organic acids, and lignocellulosic inhibitors. In this review, we summarize various strategies to engineer robust microorganisms for organic acid production and highlight their applications in a few recent examples.     

厌氧真菌纤维降解酶及其应用潜力的研究进展

反刍动物的瘤胃是已知的纤维降解能力最强的天然发酵罐,其发酵粗纤维的能力很大程度上依赖于其中栖息的各类微生物的功能。厌氧真菌作为瘤胃内的一类低丰度菌群,最先定殖到宿主动物摄入的纤维质饲料上,并通过分泌大量高效的碳水化合物活性酶降解粗纤维。然而,由于缺乏足够的基因组信息和有效的厌氧真菌遗传操作系统,目前国内外对厌氧真菌分泌的纤维降解酶及其降解机制的研究进展有限。本文对厌氧真菌的分类及已发表的基因组信息进行概述,介绍了各类纤维降解酶及纤维小体的组成结构和催化机制特点,并对纤维降解酶在生物质能、饲料处理、纺织造纸及食品加工等方面的应用进行总结。研究厌氧真菌纤维降解酶的作用特性,将有助于完善其在瘤胃环境中有效竞争资源并降解粗纤维的知识体系,也将进一步了解其生物技术应用潜力,为工业生产中应用酶制剂提供新思路。     

Bioleaching of copper from chalcopyrite ore by fungi

Microorganisms have been geologically active in mineral formation, mineral diagenesis and sedimentation via direct action of their enzymes or indirectly through chemical action of their metabolic products. This property of microorganisms is being harnessed during the recent years for extraction of metals from their ores, especially from low-grade ores. In the present study bioleaching of copper from its low-grade chalcopyrite ore using 26 isolates of acidophilic fungi is reported. Most of these fungal strains belonged to the genera Aspergillus, Penicillium and Rhizopus. The leaching experiments were conducted in Czepek Dox minimal medium containing 1% (100 mesh) ore with shaking at room temperature for 20 days. Out of these, 4 isolates exhibited significant bioleaching activities. Maximum leaching of copper (78 mg/L) was observed with Aspergillus flavus (DSF-8) and Aspergillus niger (DOF-1). Nutritional and environmental conditions for optimum bioleaching were standardized. Present study indicates the usefulness of acidophilic fungi in bioleaching of copper from its low-grade ores.     

Enzymes from extremophiles

The industrial application of enzymes that can withstand harsh conditions has greatly increased over the past decade. This is mainly a result of the discovery of novel enzymes from extremophilic microorganisms. Recent advances in the study of extremozymes point to the acceleration of this trend. In particular, enzymes from thermophilic organisms have found the most practical commercial use to date because of their overall inherent stability. This has also led to a greater understanding of stability factors involved in adaptation of these enzymes to their unusual environments.     

Biotechnological applications and potential of fungal feruloyl esterases based on prevalence,classification and biochemical diversity

Feruloyl esterases are part of the enzymatic spectrum employed by fungi and other microorganisms to degrade plant polysaccharides. They release ferulic acid and other aromatic acids from these polymeric structures and have received an increasing interest in industrial applications such as in the food, pulp and paper and bio-fuel industries. This review provides an overview of the current knowledge on fungal feruloyl esterases focussing in particular on the differences in substrate specificity, regulation of their production, prevalence of these enzymes in fungal genomes and industrial applications.     

Microbial acid-stable α-amylases: Characteristics,genetic engineering and applications

Among the wide variety of amylolytic enzymes synthesized by microorganisms, α-amylases are the most widely used biocatalysts in starch saccharification, baking industries and textile desizing. These enzymes randomly cleave the α-1,4-glycosidic linkages in starch, generating maltose and malto-oligosaccharides. The commercially available α-amylases have certain limitations, such as limited activity at low pH and Ca²⁺-dependence, and therefore, the search for novel acid-stable and thermostable amylases from extremophilic microorganisms and the engineering of the already available enzymes have been the major areas of research in this field over the years. Several attempts have been made to find suitable microbial sources of acid-stable and thermostable α-amylases. Acid-stable α-amylases have been reported in fungi, bacteria and archaea. α-Amylases that are active at elevated temperatures have been reported in bacteria as well as in archaea. α-Amylases that possess both characteristics, to the extent required for their various applications are very scarce. The developments that have been made in molecular biology, directed evolution and structural conformation studies of α-amylases for improving their properties to suit various industrial applications are discussed in this review.     

Psychrophilic and psychrotrophic fungi: a comprehensive review

This article reviews the comparative diversity of psychrophilic and psychrotrophic fungi, their adaptability mechanisms for survival and potential applications in biotechnology and pharmaceuticals. Fungi are able to grow and survive at low temperature and exist widely in polar and non-polar habitats. These cold regions are known for very low temperature, high ultra violet-B radiation, frequent freeze and thaw cycles and low water and nutrient availability. Most of the fungi adapt to such harsh conditions by evolving various strategies in their metabolism and physiology. Psychrophilic and psychrotrophic fungi are of importance in biotechnological and pharmaceutical fields due to their diverse characteristics developed or evolved due to their adaptation and survival in extreme environments, like; production of cold-active enzymes, pharmaceutical or bioactive metabolites and exo-polysaccharides, have potential for bioremediation and can also be used as biofertilizer.     

Effect of Cell Lysis (CLs) Products on Acidophilic Chemolithotrophic Microorganisms and the Role of Acidocella Species

Acidophilic chemolithotrophic microorganisms (CMs) are widely used for bioleaching of mineral resources. However, the growth of bacteria and their leaching activity are often inhibited (restricted) by organic components, e.g. lysates and exudates. The aims of this study were to examine the extent of cell lysis (CLs) inhibition on acidophilic microorganisms and to identify microorganisms that can utilize CLs products and eliminate their inhibition effect on acidophilic microorganisms. Specifically, it was revealed that Acidithiobacillus caldus was severely inhibited at 5% CLs products, whereas A. ferrooxidans and Leptospirillum ferriphilum are severely inhibited at 20%. It has been found that strains RBA and RBB of heterotrophic bacteria, isolated from anaerobic sludge, can biodegrade CLs products and when co-cultured with A. ferrooxidans, they can alleviate the toxic effect of CLs products under low pH (2–3). It has been shown that besides CLs, isolated strains can grow on glucose, glycerol, yeast extract, citric acid, and tryptone soya broth with an optimum temperature of 35°C and a pH of 3. The strains showed the ability to reduce ferric ions to ferrous ions when glycerol was used as a substrate after 2 days under both aerobic and anaerobic conditions. On the basis of morphophysiological and molecular biological studies, the isolated strains RBA and RBB were identified as Acidocella spp.     

Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella

During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i) to survive an extreme acid shock, (ii) to grow at mild acidic pH and (iii) to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection.     

Cold active microbial lipases: some hot issues and recent developments

Lipases are glycerol ester hydrolases that catalyze the hydrolysis of triglycerides to free fatty acids and glycerol. Lipases catalyze esterification, interesterification, acidolysis, alcoholysis and aminolysis in addition to the hydrolytic activity on triglycerides. The temperature stability of lipases has regarded as the most important characteristic for use in industry. Psychrophilic lipases have lately attracted attention because of their increasing use in the organic synthesis of chiral intermediates due to their low optimum temperature and high activity at very low temperatures, which are favorable properties for the production of relatively frail compounds. In addition, these enzymes have an advantage under low water conditions due to their inherent greater flexibility, wherein the activity of mesophilic and thermophilic enzymes are severely impaired by an excess of rigidity. Cold-adapted microorganisms are potential source of cold-active lipases and they have been isolated from cold regions and studied. Compared to other lipases, relatively smaller numbers of cold active bacterial lipases were well studied. Lipases isolated from different sources have a wide range of properties depending on their sources with respect to positional specificity, fatty acid specificity, thermostability, pH optimum, etc. Use of industrial enzymes allows the technologist to develop processes that closely approach the gentle, efficient processes in nature. Some of these processes using cold active lipase from C. antarctica have been patented by pharmaceutical, chemical and food industries. Cold active lipases cover a broad spectrum of biotechnological applications like additives in detergents, additives in food industries, environmental bioremediations, biotransformation, molecular biology applications and heterologous gene expression in psychrophilic hosts to prevent formation of inclusion bodies. Cold active enzymes from psychrotrophic microorganisms showing high catalytic activity at low temperatures can be highly expressed in such recombinant strains. Thus, cold active lipases are today the enzymes of choice for organic chemists, pharmacists, biophysicists, biochemical and process engineers, biotechnologists, microbiologists and biochemists.     

云南酸性土壤中度嗜酸链霉菌的多样性

[目的]了解酸性土壤环境里中度嗜酸链霉菌的多样性,调查其物种资源.[方法]用分散和差速离心法及选择性分离培养基从14份云南酸性土壤样品中分离到367株具有链霉菌培养特征的放线菌,并进行了颜色分群.从各颜色类群中选取代表菌株共97株,通过显微形态观察和pH梯度生长实验确定其中的中度嗜酸链霉菌.进一步从中筛选出16株中度嗜酸链霉菌代表菌株,进行16SrRNA基因序列的相似性和系统发育分析,并结合基因组DNA-DNA相关性数据.[结果]分离菌株归为12同的颜色类群,其中80%属于中度嗜酸链霉菌,其代表菌株在系统发育树上形成了8个距离较远且与已知种不同的进化分枝,可能代表链霉菌属内至少8个不同的新基因种.[结论]用以上方法筛选出的中度嗜酸链霉菌可归为8个不同于已知种的进化群,说明云南酸性土壤含有丰富多样的中度嗜酸链霉菌新物种.