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Applied Microbiology and Biotechnology (v.85, #5)

Genetic engineering of macrolide biosynthesis: past advances, current state, and future prospects by Sung Ryeol Park; Ah Reum Han; Yeon-Hee Ban; Young Ji Yoo; Eun Ji Kim; Yeo Joon Yoon (pp. 1227-1239).

Polyketides comprise one of the major families of natural products. They are found in a wide variety of bacteria, fungi, and plants and include a large number of medically important compounds. Polyketides are biosynthesized by polyketide synthases (PKSs). One of the major groups of polyketides are the macrolides, the activities of which are derived from the presence of a macrolactone ring to which one or more 6-deoxysugars are attached. The core macrocyclic ring is biosynthesized from acyl-CoA precursors by PKS. Genetic manipulation of PKS-encoding genes can result in predictable changes in the structure of the macrolactone component, many of which are not easily achieved through standard chemical derivatization or total synthesis. Furthermore, many of the changes, including post-PKS modifications such as glycosylation and oxidation, can be combined for further structural diversification. This review highlights the current state of novel macrolide production with a focus on the genetic engineering of PKS and post-PKS tailoring genes. Such engineering of the metabolic pathways for macrolide biosynthesis provides attractive alternatives for the production of diverse non-natural compounds. Other issues of importance, including the engineering of precursor pathways and heterologous expression of macrolide biosynthetic genes, are also considered.

Keywords: Genetic engineering; Macrolide biosynthesis; Post-PKS modification; Heterologous expression

Carbohydrate-binding domains: multiplicity of biological roles by Daniel Guillén; Sergio Sánchez; Romina Rodríguez-Sanoja (pp. 1241-1249).

Insoluble polysaccharides can be degraded by a set of hydrolytic enzymes formed by catalytic modules appended to one or more non-catalytic carbohydrate-binding modules (CBM). The most recognized function of these auxiliary domains is to bind polysaccharides, bringing the biocatalyst into close and prolonged vicinity with its substrate, allowing carbohydrate hydrolysis. Examples of insoluble polysaccharides recognized by these enzymes include cellulose, chitin, β-glucans, starch, glycogen, inulin, pullulan, and xylan. Based on their amino acid similarity, CBMs are grouped into 55 families that show notable variation in substrate specificity; as a result, their biological functions are miscellaneous. Carbohydrate or polysaccharide recognition by CBMs is an important event for processes related to metabolism, pathogen defense, polysaccharide biosynthesis, virulence, plant development, etc. Understanding of the CBMs properties and mechanisms in ligand binding is of vital significance for the development of new carbohydrate-recognition technologies and provide the basis for fine manipulation of the carbohydrate–CBM interactions.

Keywords: Carbohydrate-binding domains; Carbohydrate-active proteins; Glucoside hydrolases; Carbohydrate targeting; Expansins; Lectins

Molecular genetic tools to infer the origin of forest plants and wood by Reiner Finkeldey; Ludger Leinemann; Oliver Gailing (pp. 1251-1258).

Most forest tree species exhibit high levels of genetic diversity that can be used to trace the origin of living plants or their products such as timber and processed wood. Recent progress to isolate DNA not only from living tissue but also from wood and wood products offers new opportunities to test the declared origin of material such as seedlings for plantation establishment or timber. However, since most forest tree populations are weakly differentiated, the identification of genetic markers to differentiate among spatially isolated populations is often difficult and time consuming. Two important fields of “forensic” applications are described: Molecular tools are applied to test the declared origin of forest reproductive material used for plantation establishment and of internationally traded timber and wood products. These applications are illustrated taking examples from Germany, where mechanisms have been developed to improve the control of the trade with forest seeds and seedlings, and from the trade with wood of the important Southeast Asian tree family Dipterocarpaceae. Prospects and limitations of the use of molecular genetic methods to conclude on the origin of forest plants, wood, and wood products are discussed.

Keywords: DNA marker; Genetic fingerprint; Forensic application; Forest reproductive material; Tropical timber; Dipterocarpaceae

Light regulation of metabolic pathways in fungi by Doris Tisch; Monika Schmoll (pp. 1259-1277).

Light represents a major carrier of information in nature. The molecular machineries translating its electromagnetic energy (photons) into the chemical language of cells transmit vital signals for adjustment of virtually every living organism to its habitat. Fungi react to illumination in various ways, and we found that they initiate considerable adaptations in their metabolic pathways upon growth in light or after perception of a light pulse. Alterations in response to light have predominantly been observed in carotenoid metabolism, polysaccharide and carbohydrate metabolism, fatty acid metabolism, nucleotide and nucleoside metabolism, and in regulation of production of secondary metabolites. Transcription of genes is initiated within minutes, abundance and activity of metabolic enzymes are adjusted, and subsequently, levels of metabolites are altered to cope with the harmful effects of light or to prepare for reproduction, which is dependent on light in many cases. This review aims to give an overview on metabolic pathways impacted by light and to illustrate the physiological significance of light for fungi. We provide a basis for assessment whether a given metabolic pathway might be subject to regulation by light and how these properties can be exploited for improvement of biotechnological processes.

Keywords: Light response; Metabolic pathways; Strain improvement; Biotechnological processes

Galacto-oligosaccharide production using microbial β-galactosidase: current state and perspectives by Ah-Reum Park; Deok-Kun Oh (pp. 1279-1286).

Galacto-oligosaccharides have become the focus of a great deal of attention in the field of functional foods, owing to their known health benefits and potential to improve the quality of many foods. Because of these properties, they are currently used as low-calorie sweeteners in fermented milk products, confectioneries, breads, and beverages. In this article, the characterization and product inhibition of many microbial β-galactosidases with transgalactosylation activities, their galacto-oligosaccharide production from lactose, and the application of galacto-oligosaccharides are reviewed. A method for increasing galacto-oligosaccharide production by reducing product inhibition properties via the immobilization and structure modification of the enzymes is also introduced herein.

Keywords: Galacto-oligosaccharides; β-Galactosidase; Product inhibition; Characterization; Transgalactosylation

Multifunctional properties of phosphate-solubilizing microorganisms grown on agro-industrial wastes in fermentation and soil conditions by Maria Vassileva; Mercedes Serrano; Vicente Bravo; Encarnación Jurado; Iana Nikolaeva; Vanessa Martos; Nikolay Vassilev (pp. 1287-1299).

One of the most studied approaches in solubilization of insoluble phosphates is the biological treatment of rock phosphates. In recent years, various techniques for rock phosphate solubilization have been proposed, with increasing emphasis on application of P-solubilizing microorganisms. The P-solubilizing activity is determined by the microbial biochemical ability to produce and release metabolites with metal-chelating functions. In a number of studies, we have shown that agro-industrial wastes can be efficiently used as substrates in solubilization of phosphate rocks. These processes were carried out employing various technologies including solid-state and submerged fermentations including immobilized cells. The review paper deals critically with several novel trends in exploring various properties of the above microbial/agro-wastes/rock phosphate systems. The major idea is to describe how a single P-solubilizing microorganism manifests wide range of metabolic abilities in different environments. In fermentation conditions, P-solubilizing microorganisms were found to produce various enzymes, siderophores, and plant hormones. Further introduction of the resulting biotechnological products into soil-plant systems resulted in significantly higher plant growth, enhanced soil properties, and biological (including biocontrol) activity. Application of these bio-products in bioremediation of disturbed (heavy metal contaminated and desertified) soils is based on another important part of their multifunctional properties.

Keywords: Agro-wastes; Fermentations; Microbial properties; Phosphate solubilization; Soil-plant systems

Methodologies to increase the transformation efficiencies and the range of bacteria that can be transformed by Trond Erik Vee Aune; Finn Lillelund Aachmann (pp. 1301-1313).

Methods for transfer of exogenous DNA into cells are essential for genetics and molecular biology, and the lack of effective methods hampers research on many different species of bacteria which have shown to be particularly recalcitrant to transformation. This review presents the progress on the development of methods for artificial transformation of bacteria with emphasis on different methodologies and the range of bacteria that can be transformed. The methods' strengths and weaknesses are described.

Keywords: DNA; Plasmid; Bacteria; Competent cell and transformation

Pyrazines: occurrence, formation and biodegradation by Rudolf Müller; Sugima Rappert (pp. 1315-1320).

Pyrazines are a class of compounds that occur almost ubiquitously in nature. Pyrazines can be synthesised chemically or biologically, and are used as flavouring additives. The major formation of pyrazines occurs during heating of food. There is very little information available on the degradation of these compounds. In humans and animals, pyrazines are excreted as glucuronates or bound to glutathione via the kidney after hydroxylation, but the pyrazine ring is not cleaved. Bacteria have been isolated, which are able to use various substituted pyrazines as a sole carbon and energy source. In a few cases, the initial metabolites have been characterised; however, the mechanism of ring cleavage and the further degradation pathways are still unknown and await further investigation.

Keywords: Pyrazine; Alkylpyrazines; Maillard reaction; Odour; Biodegradation

Cultivation of Pleurotus ostreatus and other edible mushrooms by Carmen Sánchez (pp. 1321-1337).

Pleurotus ostreatus is the second most cultivated edible mushroom worldwide after Agaricus bisporus. It has economic and ecological values and medicinal properties. Mushroom culture has moved toward diversification with the production of other mushrooms. Edible mushrooms are able to colonize and degrade a large variety of lignocellulosic substrates and other wastes which are produced primarily through the activities of the agricultural, forest, and food-processing industries. Particularly, P. ostreatus requires a shorter growth time in comparison to other edible mushrooms. The substrate used for their cultivation does not require sterilization, only pasteurization, which is less expensive. Growing oyster mushrooms convert a high percentage of the substrate to fruiting bodies, increasing profitability. P. ostreatus demands few environmental controls, and their fruiting bodies are not often attacked by diseases and pests, and they can be cultivated in a simple and cheap way. All this makes P. ostreatus cultivation an excellent alternative for production of mushrooms when compared to other mushrooms.

Keywords: Pleurotus ostreatus ; Mushroom cultivation; Edible mushrooms

Bench to batch: advances in plant cell culture for producing useful products by Pamela J. Weathers; Melissa J. Towler; Jianfeng Xu (pp. 1339-1351).

Despite significant efforts over nearly 30 years, only a few products produced by in vitro plant cultures have been commercialized. Some new advances in culture methods and metabolic biochemistry have improved the useful potential of plant cell cultures. This review will provide references to recent relevant reviews along with a critical analysis of the latest improvements in plant cell culture, co-cultures, and disposable reactors for production of small secondary product molecules, transgenic proteins, and other products. Some case studies for specific products or production systems are used to illustrate principles.

Keywords: Bioreactor; Secondary metabolism; Co-cultures; Disposable reactors

Greater enhancement of Bacillus subtilis spore yields in submerged cultures by optimization of medium composition through statistical experimental designs by Zhen-Min Chen; Qing Li; Hua-Mei Liu; Na Yu; Tian-Jian Xie; Ming-Yuan Yang; Ping Shen; Xiang-Dong Chen (pp. 1353-1360).

Bacillus subtilis spore preparations are promising probiotics and biocontrol agents, which can be used in plants, animals, and humans. The aim of this work was to optimize the nutritional conditions using a statistical approach for the production of B. subtilis (WHK-Z12) spores. Our preliminary experiments show that corn starch, corn flour, and wheat bran were the best carbon sources. Using Plackett–Burman design, corn steep liquor, soybean flour, and yeast extract were found to be the best nitrogen source ingredients for enhancing spore production and were studied for further optimization using central composite design. The key medium components in our optimization medium were 16.18 g/l of corn steep liquor, 17.53 g/l of soybean flour, and 8.14 g/l of yeast extract. The improved medium produced spores as high as $$ 1.52 pm 0.06 imes {10^{10}}{ ext{spores}}/{ ext{ml}} $$ under flask cultivation conditions, and $$ 1.56 pm 0.07 imes {10^{10}}{ ext{spores}}/{ ext{ml}} $$ could be achieved in a 30-l fermenter after 40 h of cultivation. To the best of our knowledge, these results compared favorably to the documented spore yields produced by B. subtilis strains.

Keywords: Bacillus subtilis ; Spore yields; Plackett–Burman design; Central composite design

Improvement of α-l-arabinofuranosidase production by Talaromyces thermophilus and agro-industrial residues saccharification by Mohamed Guerfali; Moncef Chaabouni; Ali Gargouri; Hafedh Belghith (pp. 1361-1372).

This study is an application of an experimental design methodology for the optimization of the culture conditions of α-l-arabinofuranosidase production by Talaromyces thermophilus. Wheat bran and yeast extract were first selected as the best carbon and nitrogen sources, respectively, for enzyme production. A Plackett–Burman design was then used to evaluate the effects of eight variables. Statistical analyses showed that while pH had a negative effect on α-l-arabinofuranosidase production, wheat bran and MgSO₄ had a significantly positive effect. The values of the latter three parameters were further optimised using a central composite design and a response surface methodology. The experimental results were fitted to a second-order polynomial model that yielded a determination coefficient of R ² = 0.91. The statistical output showed that the linear and quadric terms of the three variables had significant effects. Using optimal conditions, the experimental value of α-l-arabinofuranosidase activity produced was very close to the model-predicted value. The optimal temperature and pH of enzyme activity were 55 °C and 7.0, respectively. This enzyme was very stable over a considerable pH range from 4 to 9. The crude enzyme of T. thermophilus rich in α-l-arabinofuranosidase was also used for saccharification of lignocellulosic materials and arabinose production.

Keywords: Response surface methodology; Optimization; α-l-arabinofuranosidase; Arabinose; Talaromyces thermophilus

The effect of flavin electron shuttles in microbial fuel cells current production by Sharon B. Velasquez-Orta; Ian M. Head; Thomas P. Curtis; Keith Scott; Jonathan R. Lloyd; Harald von Canstein (pp. 1373-1381).

The effect of electron shuttles on electron transfer to microbial fuel cell (MFC) anodes was studied in systems where direct contact with the anode was precluded. MFCs were inoculated with Shewanella cells, and flavins used as the electron shuttling compound. In MFCs with no added electron shuttles, flavin concentrations monitored in the MFCs' bulk liquid increased continuously with FMN as the predominant flavin. The maximum concentrations were 0.6 μM for flavin mononucleotide and 0.2 μM for riboflavin. In MFCs with added flavins, micro-molar concentrations were shown to increase current and power output. The peak current was at least four times higher in MFCs with high concentrations of flavins (4.5–5.5 μM) than in MFCs with low concentrations (0.2–0.6 μM). Although high power outputs (around 150 mW/m²) were achieved in MFCs with high concentrations of flavins, a Clostridium-like bacterium along with other reactor limitations affected overall coulombic efficiencies (CE) obtained, achieving a maximum CE of 13%. Electron shuttle compounds (flavins) permitted bacteria to utilise a remote electron acceptor (anode) that was not accessible to the cells allowing current production until the electron donor (lactate) was consumed.

Keywords: Microbial fuel cell; Electron shuttle (mediator); Flavins; Shewanella oneidensis MR-1; Clostridium

Coupled bioconversion for preparation of N-acetyl-d-neuraminic acid using immobilized N-acetyl-d-glucosamine-2-epimerase and N-acetyl-d-neuraminic acid lyase by Shiyuan Hu; Jun Chen; Zhongyi Yang; Lijun Shao; Hua Bai; Jiali Luo; Weihong Jiang; Yunliu Yang (pp. 1383-1391).

N-Acetyl-d-neuraminic acid (Neu5Ac) can be produced from N-acetyl-d-glucosamine (GlcNAc) and pyruvate by a chemoenzymatic process in which an alkaline-catalyzed epimerization transforms GlcNAc to N-acetyl-d-manosamine (ManNAc). ManNAc is then condensed biocatalytically with pyruvate in the presence of N-acetyl-d-neuraminic acid lyase (NAL) or by a two-step, fully enzymatic process involving bioconversions of GlcNAc to ManNAc and ManNAc to Neu5Ac using N-acetyl-d-glucosamine 2-epimerase (AGE) and NAL. There are some drawbacks to this technique, such as lengthy reaction time, and the low conversion rate when the soluble forms of the enzymes are used in the two-step enzymatic process. In this study, the Escherichia coli-expressed AGE and NAL in the supernatant were purified by FP-based affinity chromatography and then immobilized on Amberzyme oxirane resin. These two immobilized enzymes, with a specific activity of 78.18 U/g for AGE and 69.30 U/g for NAL, were coupled to convert GlcNAc to Neu5Ac directly in one reactor. The conversion rate of the two-step reactions from GlcNAc to Neu5Ac was ∼73% within 24 h. Furthermore, the immobilized AGE and NAL could both be used up to five reaction cycles without loss of activity or significant decrease of the conversion rate.

Keywords: N-acetyl-d-neuraminic acid; N-acetyl-d-glucosamine 2-epimerase; N-acetyl-d-neuraminic lyase; Immobilized enzyme; Coupled reaction

Establishment of a simple and effective isolation method for cyanophycin from recombinant Saccharomyces cerevisiae by Anna Steinle; Alexander Steinbüchel (pp. 1393-1399).

An efficient, time-saving, and cost-effective method for isolation of the polyamide cyanophycin from recombinant Saccharomyces cerevisiae was established. Due to its simple procedure, this isolation method may be also applicable at industrial scale and also to other intracellular compounds in this yeast. Production of cyanophycin gained preferential interest in the past, as degradation products thereof are of pharmaceutical and technical interest. Recently, it was shown that Saccharomyces cerevisiae represents a putative candidate for cyanophycin synthesis at industrial scale. For identification of optimal isolation procedures, several parameters such as heat stress, freeze drying, and freeze/thaw cycles of transgenic yeast cells were compared for their effectiveness of cyanophycin isolation. Additionally, optimal resuspension solutions for the applied cells and minimal required materials or chemicals were determined to make the process most environmentally and economically friendly. Maximal cyanophycin granule polypeptide yields of 21% (w/w) were obtained after incubation of dry cells at 70 °C or 80 °C and precipitation of the polymer with two volumes of ethanol.

Keywords: Cyanophycin; Saccharomyces cerevisiae ; Cyanophycin isolation

Effects of pH profiles on nisin fermentation coupling with foam separation by Wei Liu; Huijie Zheng; Zhaoliang Wu; Yinfeng Wang (pp. 1401-1407).

Online foam separation was proposed to recover nisin during fermentation of Lactococcus lactis subsp. lactis ATCC 11454. Firstly, the optimal pH profile of nisin fermentation was investigated including different realkalization set values and pH drop gradients. Then the selected pH profiles of 5.75 ± 0.05 and 6.25–5.75 (±0.02) were used to perform nisin fermentation coupling with foam separation. The results showed that pH profile of 5.75 ± 0.05 was better than that of 6.25–5.75 (±0.02) for online foam separation. With the optimal pH profile, an aeration of 20 ml min⁻¹ that started at 8 h of incubation and lasted for 2 h resulted in 6.6 times higher specific productivity than that of the fermentation without aeration. Nisin synthesis was therefore prolonged with low sucrose concentration in the culture broth, which indicated that the feedback inhibition of nisin is more influential than the substrate limitation of sucrose in the late phase of nisin fermentation. Total nisin production (4,870 ± 180 IU ml⁻¹) was increased by 30.3% with online foam separation. This effective online recovery method for nisin production could be easily scaled up due to the facile operation of foaming process.

Keywords: Feedback inhibition; Foam separation; Lactococcus lactis ; Nisin; Online; pH profile

Production of diosgenin from Dioscorea zingiberensis tubers through enzymatic saccharification and microbial transformation by Yu-Ling Zhu; Wen Huang; Jin-Ren Ni; Wei Liu; Hui Li (pp. 1409-1416).

In order to develop a clean and effective approach for producing the valuable drug diosgenin from Dioscorea zingiberensis tubers, two successive processes, enzymatic saccharification and microbial transformation, were used. With enzymatic saccharification, 98.0% of starch was excluded from the raw herb, releasing saponins from the network structure of starch. Subsequently, the treated tubers were fermented with Trichoderma reesei under optimal conditions for 156 h. During microbial transformation, glycosidic bonds, which link β-d-glucose or α-l-rhamnose with aglycone at the C-3 position in saponins, were broken down effectively to give a diosgenin yield of 90.6 ± 2.45%, 42.4% higher than that obtained from bioconversion of raw tubers directly. Scaled up fermentation was conducted in a 5.0-l bioreactor and gave a diosgenin yield of 91.2 ± 3.21%. This is the first report on the preparation of diosgenin from herbs through microbial transformation as well as utilizing other available components in the raw material, providing an environmentally friendly alternative to diosgenin production.

Keywords: Diosgenin yield; Enzymatic saccharification; Microbial transformation; Saponins; Trichoderma reesei

Cloning and functional expression of a nitrile hydratase (NHase) from Rhodococcus equi TG328-2 in Escherichia coli, its purification and biochemical characterisation by Kamila Rzeznicka; Sebastian Schätzle; Dominique Böttcher; Joachim Klein; Uwe T. Bornscheuer (pp. 1417-1425).

The nitrile hydratase (NHase, EC 4.2.1.84) genes (α and β subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (α subunit, M_r 23 kDa) and 218 amino acids (β subunit, M_r 24 kDa) and the NHase activator of 413 amino acids (M_r 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity was observed for aromatic compounds only with E values ranging 5–17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30°C and showed the highest stability at 4°C in thermal inactivation studies between 4°C and 50°C.

Keywords: Cloning; Nitrile hydratase; Recombinant expression; Nitriles; Rhodococcus equi

Effects of galactose and glucose on the hydrolysis reaction of a thermostable β-galactosidase from Caldicellulosiruptor saccharolyticus by Ah-Reum Park; Deok-Kun Oh (pp. 1427-1435).

A recombinant β-galactosidase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 211 U mg⁻¹ by using heat treatment and His-trap affinity chromatography. The native enzyme was an 80-kDa trimer with a molecular mass of 240 kDa. Maximum activity was observed at pH 6.0 and 80ºC, and the half-life at 70ºC was 48 h. The enzyme exhibited hydrolytic activity for p-nitrophenyl-β-d-galactopyranoside (pNPGal), oNPGal, or lactose, whereas no activity for p-nitrophenyl-β-d-glucopyranoside (pNPGlu), oNPGlu, or cellobiose. The catalytic residues E150 and E311 of β-galactosidase from C. saccharolyticus were completely conserved in all aligned glycoside hydrolase family 42 β-galactosidases. The results indicated that the enzyme was a β-galactosidase. Galactose uncompetitively inhibited the enzyme. Glucose inhibition of the enzyme was the lowest among β-galactosidases. When 50 g l⁻¹ galactose was added, the enzyme activity for pNPGal was reduced to 26%. When 400 g l⁻¹ glucose instead of galactose was added, the activity was reduced to 82%. When adding galactose (200 g l⁻¹), only 14% of the lactose was hydrolyzed after 180 min. In contrast, the addition of glucose (400 g l⁻¹) did not affect lactose hydrolysis, and more than 99% of the lactose was hydrolyzed after 120 min.

Keywords: β-Galactosidase; Caldicellulosiruptor saccharolyticus ; Galactose inhibition; Glucose inhibition; Lactose hydrolysis

Characterisation of a detergent-stable alkaline protease from a novel thermophilic strain Paenibacillus tezpurensis sp. nov. AS-S24-II by Sudhir K. Rai; Jetendra K. Roy; Ashis K. Mukherjee (pp. 1437-1450).

An alkaline-protease-producing bacterial strain (AS-S24-II) isolated from a soil sample in Assam is a Gram-stain-positive, catalase-positive, endospore-forming rod and grows at temperatures ranging from 30 °C to 60 °C and salinity ranging from 0% to 7% (w/v) NaCl. Phenotypic characterisation, chemotaxonomic properties, presence of Paenibacillus-specific signature sequences, and ribotyping data suggested that the strain AS-S24-II represents a novel species of the genus Paenibacillus, for which the name Paenibacillus tezpurensis sp. nov. (MTCC 8959) is proposed. Phylogenetic analysis revealed that P. lentimorbus strain DNG-14 and P. lentimorbus strain DNG-16 represent the closest phylogenetic neighbour of this novel strain. Alkaline protease production (598 × 10³ U l⁻¹) by P. tezpurensis sp. nov. in SmF was optimised by response surface method. A laundry-detergent-stable, Ca²⁺-independent, 43-kDa molecular weight alkaline serine protease from this strain was purified with a 1.7-fold increase in specific activity. The purified protease displayed optimum activity at pH 9.5 and 45–50 °C temperature range and exhibited a significant stability and compatibility with surfactants and most of the tested commercial laundry detergents at room temperature. Further, the protease improved the wash performance of detergents, thus demonstrating its feasibility for inclusion in laundry detergent formulations.

Keywords: Alkaline protease; Detergent compatibility; Paenibacillus tezpurensis ; Response surface; Serine protease; Submerged fermentation

Molecular cloning and characterization of a bifunctional xylanolytic enzyme from Neocallimastix patriciarum by Cheng-Kang Pai; Zong-Yuan Wu; Ming-Ju Chen; Yi-Fang Zeng; Jr-Wei Chen; Chung-Hang Duan; Ming-Liang Li; Je-Ruei Liu (pp. 1451-1462).

A cDNA encoding a bifunctional acetylxylan esterase/xylanase, XynS20E, was cloned from the ruminal fungus Neocallimastix patriciarum. A putative conserved domain of carbohydrate esterase family 1 was observed at the N-terminus and a putative conserved domain of glycosyl hydrolase family 11 was detected at the C-terminus of XynS20E. To examine the enzyme activities, XynS20E was expressed in Escherichia coli as a recombinant His₆ fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling combined with central composite design and regression analysis was then applied to determine the optimal temperature and pH conditions of the recombinant XynS20E. The optimal conditions for the highest xylanase activity of the recombinant XynS20E were observed at a temperature of 49°C and a pH of 5.8, while those for the highest carbohydrate esterase activity were observed at a temperature of 58°C and a pH of 8.2. Under the optimal conditions for the enzyme activity, the xylanase and acetylxylan esterase specific activities of the recombinant XynS20E toward birchwood xylan were 128.7 and 873.1 U mg⁻¹, respectively. To our knowledge, this is the first report of a bifunctional xylanolytic enzyme with acetylxylan esterase and xylanase activities from rumen fungus.

Keywords: Rumen; Neocallimastix patriciarum ; Xylanase; Acetylxylan esterase

Geranylgeranyl diphosphate synthase genes in entomopathogenic fungi by Suthitar Singkaravanit; Hiroshi Kinoshita; Fumio Ihara; Takuya Nihira (pp. 1463-1472).

Based on comparative amino-acid sequence alignment of geranylgeranyl diphosphate (GGPP) synthase from filamentous fungi, degenerated oligonucleotide primers were designed for searching GGPP synthase gene(s) in entomopathogenic fungi. Polymerase chain reaction with the designed primers amplified GGPP synthase homologues from five representative entomopathogenic fungi: Metarhizium anisopliae, Beauveria bassiana, Verticillium lecanii, Paecilomyces farinosus, and Nomuraea rileyi. Sequence comparison of the amplified of GGPP synthase homologue fragments revealed that M. anisopliae and B. bassiana have at least two different types of the GGPP synthase gene homologues. The first type (designated as ggs1), which is highly conserved among the five strains, has a unique Ser-rich region, SSXSSVSGSSS (X refers to L, A, V, or S), and is constitutively expressed throughout growth. In contrast, the second type of GGPP synthase gene homologue (ggs2) was discovered only in some strains, and genes of this type possessed high similarity to each other but showed relatively weak similarity to the ggs1 genes, with no detectable transcription under the cultivation conditions applied in this experiment. The ggs1 cloned from M. anisopliae, which encoded a putative protein of 359 amino acid residues, was heterologously expressed in E. coli. The recombinant protein showed activity to synthesize GGPP from farnesyl diphosphate and isopentenyl diphosphate. These results strongly suggested that the ggs1 gene encodes a GGPP synthase involved in primary metabolism.

Keywords: Geranylgeranyl diphosphate synthase; Metarhizium anisopliae

Improvement of extracellular production of a thermophilic subtilase expressed in Escherichia coli by random mutagenesis of its N-terminal propeptide by Nan Fang; Chuan-Qi Zhong; Xiaoliang Liang; Xiao-Feng Tang; Bing Tang (pp. 1473-1481).

Limited secretion capacity remains a drawback of using Escherichia coli as the host for the production of recombinant proteins. In this report, random mutagenesis was performed within the N-terminal propeptide of thermostable WF146 protease, a subtilase from thermophilic Bacillus sp. WF146, generating a variant named WBM^MT with improved capacity for extracellular production when expressed in E. coli. Two mutations, L(-57)Q and E(-10)D, were identified within the N-terminal propeptide. The amount of WBM^MT in the culture medium was found to be about three times higher than that of wild type. Besides, the introduction of mutations L(-57)Q/E(-10)D into the N-terminal propeptide also accelerated the maturation of the enzyme. Biochemical analysis indicated that the thermostability and the catalytic activity of mature WBM^MT were similar to those of wild type. Far-UV CD spectra analysis and limited proteolysis experiments suggested that the mutations L(-57)Q/E(-10)D resulted in a structural change in the N-terminal propeptide of the proform, and the N-terminal propeptide became more flexible, which might be beneficial for the proform to keep in a translocation-competent state. Our result indicates that N-terminal propeptide engineering may be a valuable approach for improving extracellular production of recombinant subtilases expressed in E. coli.

Keywords: N-terminal propeptide; Subtilase; Extracellular secretion; E. coli

Cloning, characterization, and antifungal activity of an endo-1,3-β-d-glucanase from Streptomyces sp. S27 by Pengjun Shi; Guoyu Yao; Peilong Yang; Ning Li; Huiying Luo; Yingguo Bai; Yaru Wang; Bin Yao (pp. 1483-1490).

An endo-1,3-β-d-glucanase gene, designated as bglS27, was cloned from Streptomyces sp. S27 and successfully expressed in Escherichia coli BL21 (DE3). The full-length gene contains 1,362 bp and encodes a protein of 453 amino acids with a calculated molecular mass of 42.7 kDa. The encoded protein comprises a catalytic module of glycosyl hydrolase family 16, a short glycine linker region, and a family 13 carbohydrate-binding module. The purified recombinant enzyme (BglS27) showed optimal activity at 65°C and pH 5.5 and preferentially catalyzed the hydrolysis of glucans with a β-1,3-linkage using an endolytic mode of action. The specific activity and K _m value of BglS27 for laminarin were 236.0 U mg^–1 and 1.89 mg ml^–1, respectively. In antifungal assay, BglS27 had the ability to inhibit the growth of phytopathogenic fungi Rhizoctonic solani and Fusarium oxysporum and some mycotoxin-producing fungi Fusarium crookwellense and Paecilomyces variotii. These favorable properties make BglS27 a good candidate for utilization in biotechnological applications such as plant protection, feed, and food preservation.

Keywords: Streptomyces sp. S27; Endo-1,3-β-d-glucanase; Antifungal protein

Novel strategy for yeast construction using δ-integration and cell fusion to efficiently produce ethanol from raw starch by Ryosuke Yamada; Tsutomu Tanaka; Chiaki Ogino; Hideki Fukuda; Akihiko Kondo (pp. 1491-1498).

We developed a novel strategy for constructing yeast to improve levels of amylase gene expression and the practical potential of yeast by combining δ-integration and polyploidization through cell fusion. Streptococcus bovis α-amylase and Rhizopus oryzae glucoamylase/α-agglutinin fusion protein genes were integrated into haploid yeast strains. Diploid strains were constructed from these haploid strains by mating, and then a tetraploid strain was constructed by cell fusion. The α-amylase and glucoamylase activities of the tetraploid strain were increased up to 1.5- and tenfold, respectively, compared with the parental strain. The diploid and tetraploid strains proliferated faster, yielded more cells, and fermented glucose more effectively than the haploid strain. Ethanol productivity from raw starch was improved with increased ploidy; the tetraploid strain consumed 150 g/l of raw starch and produced 70 g/l of ethanol after 72 h of fermentation. Our strategy for constructing yeasts resulted in the simultaneous overexpression of genes integrated into the genome and improvements in the practical potential of yeasts.

Keywords: Cell fusion; δ-integration; Ethanol fermentation; Raw starch

Expression of phenol oxidase and heat-shock genes during the development of Agaricus bisporus fruiting bodies, healthy and infected by Lecanicillium fungicola by Michèle L. Largeteau; Camille Latapy; Nathalie Minvielle; Catherine Regnault-Roger; Jean-Michel Savoie (pp. 1499-1507).

The fungal pathogen Lecanicillium fungicola (formerly Verticillium fungicola) is responsible for severe losses worldwide in the mushroom (Agaricus bisporus) industry. Infected crops are characterised by masses of undifferentiated tissue (bubbles) growing in place of sporophores. The expression of three laccase genes (lcc1, lcc2 and lcc3), two tyrosinase genes (AbPPO1 and AbPPO2) and the hspA gene encoding a heat-shock protein known to be potentially associated with host–pathogen interaction was investigated in mycelial aggregates and during the development of healthy sporophores and bubbles of a susceptible cultivar. The lcc3, AbPPO2 and hspA genes were each expressed at different levels at the different stages of sporophore morphogenesis, whilst they showed a stable expression throughout bubble development. The transcript levels were similar in bubbles and at the first developmental stage of healthy fruiting bodies, both showing no tissue differentiation. These observations suggest that lcc3, AbPPO2 and hspA are associated with A. bisporus morphogenesis. Comparing the expression of the hspA gene in three susceptible and three tolerant strains showed that the latter displayed a higher level of transcript in the primordium, which is the stage receptive to the pathogen. The six strains exhibited a comparable expression in the vegetative mycelium, non-receptive to L. fungicola.

Keywords: Fungal pathogen; Mushroom; Gene expression; HSP70; Laccase; Tyrosinase

Overexpression of Newcastle disease virus (NDV) V protein enhances NDV production kinetics in chicken embryo fibroblasts by Juno Jang; Sung-Hwan Hong; Dongwon Choi; Kang-Seuk Choi; Seongman Kang; Ik-Hwan Kim (pp. 1509-1520).

Newcastle disease virus (NDV) is not only one of the most economically important pathogen of poultry but also has a potential as anticancer virotherapy. The role of NDV V protein in virus-production kinetics was investigated using DF-1 cell-based production system. The presence of an anti-interferon (IFN)-alpha antibody resulted in enhanced NDV production kinetics in a dose-dependent manner by blocking binding of NDV-induced IFN to its receptor. To prepare DF-1 cell whose cellular IFN signaling is blocked efficiently, stable cell lines expressing either lentogenic or velogenic NDV V protein known as an IFN antagonist were established. The overexpression of NDV V protein enhanced NDV production kinetics and expedited the rate of NDV production, while it had no effect on Japanese encephalitis virus production. NDV V protein functions as an IFN antagonist by inhibiting the increase in type I IFNs by NDV infection. The IFN signals in cells expressing NDV V protein were weakened by decreased activation or expression of the dsRNA-activated enzymes. These IFN antagonist activities enhance rapid virus replication and spread in the early phase of viral infection and will be useful in improving the production of viral vaccine strains.

Keywords: Newcastle disease virus (NDV); Vaccine production; Interferon (IFN) antagonist; V protein; Innate immunity

New approach for the detection of non-ribosomal peptide synthetase genes in Bacillus strains by polymerase chain reaction by Arthur Tapi; Marlène Chollet-Imbert; Bart Scherens; Philippe Jacques (pp. 1521-1531).

Bacillus strains produce non-ribosomal lipopeptides that can be grouped into three families: surfactins or lichenysins, iturins and fengycins or plispastatins. These biosurfactants show a broad spectrum of biological activities. To detect strains able to produce these lipopeptides, a new polymerase chain reaction screening approach was developed using degenerated primers based on the intraoperon alignment of adenylation and thiolation nucleic acid domains of all enzymes implicated in the biosynthesis of each lipopeptide family. The comparative bioinformatics analyses of each operon led to the design of four primer pairs for the three families taking into account the differences between open reading frames of each synthetase gene. Tested on different Bacillus sp. strains, this technique was used successfully to detect not only the expected genes in the lipopeptide producing strains but also the presence of a plispastatin gene in Bacillus subtilis ATCC 21332 and a gene showing a high similarity with the polyketide synthase type I gene in the B. subtilis ATCC 6633 genome. It also led to the discovery of the presence of non-ribosomal peptide synthetase genes in Bacillus thuringiensis serovar berliner 1915 and in Bacillus cereus LMG 2098. In addition, this work highlighted the differences between the fengycin and plipastatin operon at DNA level.

Keywords: Bacillus sp.; Non-ribosomal peptide synthetase; PCR; Lipopeptide; Degenerated primers

Intercrossing of phage genomes in a phage cocktail and stable coexistence with Escherichia coli O157:H7 in anaerobic continuous culture by Hiroya Kunisaki; Yasunori Tanji (pp. 1533-1540).

The emergence of phage-resistant cells is the most serious problem for realizing phage therapy and is observed frequently if only one phage strain is used against a particular bacterium. By contrast, using multiple phages (phage cocktail) can delay or control the appearance of phage-resistant cells. Anaerobic continuous culturing of Escherichia coli O157:H7 and a cocktail of EP16, PP17, and SP22 phages were conducted. Comparison of the restriction fragment length polymorphism (RFLP) pattern of each phage genome showed a pattern different from wild type. Furthermore, the RFLP pattern of mutant phages consisted of fragments of PP17 and SP22 genome, suggesting both phages had infected the same host simultaneously (superinfection) and exchanged genomic DNA. Through observation of the binding of SYBR Gold-stained mutant phage to individual phage-resistant cells (RC), we found that clonal RC cultures were heterogeneous in their ability to bind mutant phage. The ratio of susceptibility was a few percent, which suggested that a minority of the RC population was susceptible to phage, and this heterogeneity contributes to the stable coexistence of RC and chimeric phages. The ratio of susceptible cells did not change appreciably from bacterial generation to generation.

Keywords: Bacteriophage; Phage therapy; Phage resistance; E. coli O157:H7

Strain improvement of Sporolactobacillus inulinus ATCC 15538 for acid tolerance and production of D-lactic acid by genome shuffling by Huijie Zheng; Jixian Gong; Tao Chen; Xun Chen; Xueming Zhao (pp. 1541-1549).

Improvement of acid tolerance and production of D-lactic acid by Sporolactobacillus inulinus ATCC 15538 was performed by using recursive protoplast fusion in a genome shuffling format. The starting population was generated by ultraviolet irradiation, diethyl sulfate mutagenesis, and pH-gradient filter and then, subjected for the recursive protoplast fusion. The concentration of lysozyme, time, and temperature for enzyme treatment were optimized by response surface methodology based on the central composite design. Based on contour plots and variance analysis, the model predicted a maximum Y (multiply protoplasts formation ratio by protoplasts regeneration ratio), 60.4%, and the corresponding above used values were 7.75 mg/ml lysozyme, 1.59 h, and 38°C. A pH-5-resistant recombinant, F3-4, was obtained after three rounds of genome shuffling and its production of D-lactic acid reached 93.4 g/l in a 5 L bioreactor, which was increased by 39.8% and 119% in comparison with that of UV generated strain and the original strain S. inulinus ATCC 15538, respectively. The subculture experiments indicated that F3-4 was genetically stable.

Keywords: D-lactic acid; Sporolactobacillus inulinus ATCC 15538; Genome shuffling; Protoplast fusion; Response surface methodology (RSM); Breeding

Inhibition of Candida albicans growth by brominated furanones by Miao Duo; Mi Zhang; Yan-Yeung Luk; Dacheng Ren (pp. 1551-1563).

Candida albicans is the most virulent Candida species of medical importance, which presents a great threat to immunocompromised individuals such as HIV patients. Currently, there are only four classes of antifungal agents available for treating fungal infections: azoles, polyenes, pyrimidines, and echinocandins. The fast spread of multidrug resistant C. albicans strains has increased the demand for new antifungal drugs. In this study, we demonstrate the antifungal activity of brominated furanones on C. albicans. Studying the structure and activity of this class of furanones reveals that the exocyclic vinyl bromide conjugated with the carbonyl group is the most important structural element for fungal inhibition. Furthermore, gene expression analysis using DNA microarrays showed that 3 μg/mL of 4-bromo-5Z-(bromomethylene)-3-butylfuran-2-one (BF1) upregulated 32 C. albicans genes with functions of stress response, NADPH dehydrogenation, and small-molecule transport, and repressed 21 genes involved mainly in cell-wall maintenance. Interestingly, only a small overlap is observed between the gene expression changes caused by the representative brominated furanone (BF1) in this study and other antifungal drugs reported in literature. This result suggests that brominated furanones and other antifungal drugs may target different fungal proteins or genes. The existence of such new targets provides an opportunity for developing new agents to control fungal pathogens which are resistant to currently available drugs.

Keywords: Brominated furanone; Candida albicans ; Growth inhibition; DNA microarray

Precursor for biosynthesis of sugar moiety of doxorubicin depends on rhamnose biosynthetic pathway in Streptomyces peucetius ATCC 27952 by Bijay Singh; Chang-Beom Lee; Jae Kyung Sohng (pp. 1565-1574).

The doxorubicin biosynthetic gene cluster in Streptomyces peucetius ATCC 27952 contains a TDP-D-glucose 4,6-dehydratase gene, dnmM, that is putatively involved in the biosynthesis of daunosamine, but the gene contains a frameshift in the DNA sequence that would cause premature termination of translation. In pursuit of another TDP-D-glucose 4,6-dehydratase in S. peucetius, a homologue gene, rmbB, was found, whose deduced product exhibits high sequence similarity to a number of TDP-D-glucose 4,6-dehydratases. The gene was located within a putative rhamnose biosynthetic gene cluster at another locus in the genome. RmbB was verified to be a functional TDP-D-glucose 4,6-dehydratase by enzyme assay as it catalyzed the conversion of TDP-D-glucose into TDP-4-keto-6-deoxy-D-glucose. Inactivation of rmbB in the S. peucetius genome abolished the production of doxorubicin while complementation of the same gene in an rmbB knockout mutant restored the doxorubicin production. Hence, rmbB provides TDP-4-keto-6-deoxy-D-glucose as a nucleotide sugar precursor for the biosynthesis of doxorubicin.

Keywords: Daunosamine; Doxorubicin; TDP-D-glucose 4,6-dehydratase; Rhamnose; Streptomyces peucetius

Isolation of the exoelectrogenic denitrifying bacterium Comamonas denitrificans based on dilution to extinction by Defeng Xing; Shaoan Cheng; Bruce E. Logan; John M. Regan (pp. 1575-1587).

The anode biofilm in a microbial fuel cell (MFC) is composed of diverse populations of bacteria, many of whose capacities for electricity generation are unknown. To identify functional populations in these exoelectrogenic communities, a culture-dependent approach based on dilution to extinction was combined with culture-independent community analysis. We analyzed the diversity and dynamics of microbial communities in single-chamber air-cathode MFCs with different anode surfaces using denaturing gradient gel electrophoresis based on the 16S rRNA gene. Phylogenetic analyses showed that the bacteria enriched in all reactors belonged primarily to five phylogenetic groups: Firmicutes, Actinobacteria, α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria. Dilution-to-extinction experiments further demonstrated that Comamonas denitrificans and Clostridium aminobutyricum were dominant members of the community. A pure culture isolated from an anode biofilm after dilution to extinction was identified as C. denitrificans DX-4 based on 16S rRNA sequence and physiological and biochemical characterizations. Strain DX-4 was unable to respire using hydrous Fe(III) oxide but produced 35 mW/m² using acetate as the electron donor in an MFC. Power generation by the facultative C. denitrificans depends on oxygen and MFC configuration, suggesting that a switch of metabolic pathway occurs for extracellular electron transfer by this denitrifying bacterium.

Keywords: Comamonas denitrificans ; Exoelectrogen; Denitrifying bacteria; Microbial community; Dilution to extinction; Microbial fuel cell

Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols by Paul D’Abzac; François Bordas; Eric Van Hullebusch; Piet N. L. Lens; Gilles Guibaud (pp. 1589-1599).

The characteristics of the extracellular polymeric substances (EPS) extracted with nine different extraction protocols from four different types of anaerobic granular sludge were studied. The efficiency of four physical (sonication, heating, cationic exchange resin (CER), and CER associated with sonication) and four chemical (ethylenediaminetetraacetic acid, ethanol, formaldehyde combined with heating, or NaOH) EPS extraction methods was compared to a control extraction protocols (i.e., centrifugation). The nucleic acid content and the protein/polysaccharide ratio of the EPS extracted show that the extraction does not induce abnormal cellular lysis. Chemical extraction protocols give the highest EPS extraction yields (calculated by the mass ratio between sludges and EPS dry weight (DW)). Infrared analyses as well as an extraction yield over 100% or organic carbon content over 1 g g⁻¹ of DW revealed, nevertheless, a carry-over of the chemical extractants into the EPS extracts. The EPS of the anaerobic granular sludges investigated are predominantly composed of humic-like substances, proteins, and polysaccharides. The EPS content in each biochemical compound varies depending on the sludge type and extraction technique used. Some extraction techniques lead to a slightly preferential extraction of some EPS compounds, e.g., CER gives a higher protein yield.

Keywords: Anaerobic granular sludge; Extracellular polymeric substances (EPS); Extraction; Biochemical composition

Potential cause of aerobic granular sludge breakdown at high organic loading rates by Sunil S. Adav; Duu-Jong Lee; Juin-Yih Lai (pp. 1601-1610).

Aerobic sludge granules are compact, strong microbial aggregates that have excellent settling ability and capability to efficiently treat high-strength and toxic wastewaters. Aerobic granules disintegrate under high organic loading rates (OLR). This study cultivated aerobic granules using acetate as the sole carbon and energy source in three identical sequencing batch reactors operated under OLR of 9–21.3 kg chemical oxygen demand (COD) m⁻³ day⁻¹. The cultivated granules removed 94–96% of fed COD at OLR up to 9–19.5 kg COD m⁻³ day⁻¹, and disintegrated at OLR of 21.3 kg COD m⁻³ day⁻¹. Most tested isolates did not grow in the medium at >3,000 mg COD l⁻¹; additionally, these strains lost capability for auto-aggregation and protein or polysaccharide productivity. This critical COD regime correlates strongly with the OLR range in which granules started disintegrating. Reduced protein quantity secreted by isolates was associated with the noted poor granule integrity under high OLR. This work identified a potential cause of biological nature for aerobic granules breakdown.

Keywords: Aerobic granule; Breakdown; OLR; Proteins; Isolates

Growth condition and bacterial community for maximum hydrolysis of suspended organic materials in anaerobic digestion of food waste-recycling wastewater by Man Deok Kim; Minkyung Song; Minho Jo; Seung Gu Shin; Jee Hyeong Khim; Seokhwan Hwang (pp. 1611-1618).

This paper reports the effects of changing pH (5–7) and temperature (T, 40–60 °C) on the efficiencies of bacterial hydrolysis of suspended organic matter (SOM) in wastewater from food waste recycling (FWR) and the changes in the bacterial community responsible for this hydrolysis. Maximum hydrolysis efficiency (i.e., 50.5% reduction of volatile suspended solids) was predicted to occur at pH 5.7 and T = 44.5 °C. Changes in short-chain volatile organic acid profiles and in acidogenic bacterial communities were investigated under these conditions. Propionic and butyric acids concentrations increased rapidly during the first 2 days of incubation. Several band sequences consistent with Clostridium spp. were detected using denaturing gel gradient electrophoresis. Clostridium thermopalmarium and Clostridium novyi seemed to contribute to butyric acid production during the first 1.5 days of acidification of FWR wastewater, and C. thermopalmarium was a major butyric acid producer afterward. C. novyi was an important propionic acid producer. These two species appear to be important contributors to hydrolysis of SOM in the wastewater. Other acidogenic anaerobes, Aeromonas sharmana, Bacillus coagulans, and Pseudomonas plecoglossicida, were also indentified.

Keywords: Acidogen; Denaturing gradient gel electrophoresis; Food waste-recycling wastewater; Hydrolysis; Microbial community structure; Particulate organic materials

Electrical protein array chips for the detection of staphylococcal virulence factors by Annett Quiel; Britta Jürgen; Gundula Piechotta; Anne-Pascale Le Foll; Anne-Kathrin Ziebandt; Christian Kohler; Daniela Köster; Susanne Engelmann; Christian Erck; Rainer Hintsche; Jürgen Wehland; Michael Hecker; Thomas Schweder (pp. 1619-1627).

A new approach for the detection of virulence factors of Staphylococcus aureus and Staphylococcus epidermidis using an electrical protein array chip technology is presented. The procedure is based on an enzyme-linked sandwich immunoassay, which includes recognition and binding of virulence factors by specific capture and detection antibodies. Detection of antibody-bound virulence factors is achieved by measuring the electrical current generated by redox recycling of an enzymatically released substance. The current (measured in nanoampere) corresponds to the amount of the target molecule in the analyzed sample. The electrical protein chip allows for a fast detection of Staphylococcus enterotoxin B (SEB) of S. aureus and immunodominant antigen A homologue (IsaA homologue) of S. epidermidis in different liquid matrices. The S. aureus SEB virulence factor could be detected in minimal medium, milk, and urine in a concentration of 1 ng/ml within less than 23 min. Furthermore, a simultaneous detection of SEB of S. aureus and IsaA homologue of S. epidermidis in a single assay could be demonstrated.

Keywords: Electrical protein array chips; Sandwich immunoassay; Staphylococcus aureus ; Staphylococcus epidermidis ; Virulence factors; Diagnostic

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Applied Microbiology and Biotechnology (v.85, #5)

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Applied Microbiology and Biotechnology (v.85, #5)