Check out our New Publishers' Select for Free Articles

Applied Microbiology and Biotechnology (v.91, #4)

Modelling as a tool of enzyme reaction engineering for enzyme reactor development by Durda Vasić-Rački; Zvjezdana Findrik; Ana Vrsalović Presečki (pp. 845-856).

Strategy of the development of model for enzyme reactor at laboratory scale with respect to the modelling of kinetics is presented. The recent literature on the mathematic modelling on enzyme reaction rate is emphasized.

Keywords: Modelling of enzyme kinetics; Enzyme kinetics; Development of biocatalytic reaction; Enzyme reactor development

Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria—mini review by Valerii Z. Akhverdyan; Evgueni R. Gak; Irina L. Tokmakova; Nataliya V. Stoynova; Yurgis A. V. Yomantas; Sergey V. Mashko (pp. 857-871).

The advantages of phage Mu transposition-based systems for the chromosomal editing of plasmid-less strains are reviewed. The cis and trans requirements for Mu phage-mediated transposition, which include the L/R ends of the Mu DNA, the transposition factors MuA and MuB, and the cis/trans functioning of the E element as an enhancer, are presented. Mini-Mu(LR)/(LER) units are Mu derivatives that lack most of the Mu genes but contain the L/R ends or a properly arranged E element in cis to the L/R ends. The dual-component system, which consists of an integrative plasmid with a mini-Mu and an easily eliminated helper plasmid encoding inducible transposition factors, is described in detail as a tool for the integration/amplification of recombinant DNAs. This chromosomal editing method is based on replicative transposition through the formation of a cointegrate that can be resolved in a recombination-dependent manner. (E-plus)- or (E-minus)-helpers that differ in the presence of the trans-acting E element are used to achieve the proper mini-Mu transposition intensity. The systems that have been developed for the construction of stably maintained mini-Mu multi-integrant strains of Escherichia coli and Methylophilus methylotrophus are described. A novel integration/amplification/fixation strategy is proposed for consecutive independent replicative transpositions of different mini-Mu(LER) units with “excisable” E elements in methylotrophic cells.

Keywords: Chromosomal editing; Dual-component system; Enhancer element; Excisable marker; L/R ends; MuA transposase; Plasmid-less recombinant strain; Replicative transposition

Review of mass transfer aspects for biological gas treatment by Norbertus J. R. Kraakman; Jose Rocha-Rios; Mark C. M. van Loosdrecht (pp. 873-886).

This contribution reviews the mass transfer aspects of biotechnological processes for gas treatment, with an emphasis on the underlying principles and technical feasible methods for mass transfer enhancements. Understanding of the mass transfer behavior in bioreactors for gas treatment will result in improved reactor designs, reactor operation, and modeling tools, which are important to maximize efficiency and minimize costs. Various methods are discussed that show the potential for a more effective treatment of compounds with poor water solubility.

Keywords: Mass transfer limitation; Bioavailability; Biofiltration; Biotrickling filters; Biofilters; Partitioning bioreactor; Capillary channels; Taylor flow

Versatility and application of anaerobic ammonium-oxidizing bacteria by Da-Wen Gao; Yu Tao (pp. 887-894).

With the unique cell compartmentalization and the ability to simultaneously oxidize ammonium and reduce nitrite into nitrogen gas, anaerobic ammonium-oxidizing (anammox) bacteria have challenged our recognitions of microorganism. The research conducted on these bacteria has been extended from bench-scale tryouts to full-scale reactor systems. This review addresses the recently discovered versatile properties of anammox bacteria and the applications and obstacles of implementing the anammox process in ammonia-rich wastewater treatment. We also discuss the merits and drawbacks of traditional and anammox-based processes for nitrogen removal and suggest areas for improvement.

Keywords: Anammox bacteria; Versatile metabolism; Enrichment; Full-scale application

Poly(aspartate) hydrolases: biochemical properties and applications by Tomohiro Hiraishi; Mizuo Maeda (pp. 895-903).

Thermally synthesized poly(aspartate) (tPAA) shows potential for use in a wide variety of products and applications as a biodegradable replacement for non-biodegradable polycarboxylates, such as poly(acrylate). The tPAA molecule has unnatural structures, and the relationship between its biodegradability and structures has been investigated. Two tPAA-degrading bacteria, Sphingomonas sp. KT-1 and Pedobacter sp. KP-2, were isolated from river water; from them, two PAA-hydrolyzing enzymes, PAA hydrolases-1 and -2, were purified and biologically and genetically characterized. Interestingly, not only are PAA hydrolases-1 from those two strains novel in terms of structural genes and substrate specificities (they specifically cleave the amide bond between β-aspartate units in tPAA), they also probably play a central role in tPAA biodegradation by both strains. In green polymer chemistry, one active area of research is the use of purified enzymes for the enzyme-catalyzed synthesis of polypeptides by taking advantage of their substrate specificities. Recently, β-peptides have attracted academic and industrial interest as functional materials as they possess both functions of α-peptides and excellent metabolic stability. As one of the attractive applications of PAA hydrolases, we report here the enzyme-catalyzed synthesis of poly(α-ethyl β-aspartate), which is composed of only β-linkages and belongs to β-peptides, using the unique substrate specificity of the enzyme from Pedobacter sp. KP-2.

Keywords: Poly(aspartate); Poly(aspartate) hydrolase; β-amide linkage; Enzyme-catalyzed polymerization

LC-PUFA from photosynthetic microalgae: occurrence, biosynthesis, and prospects in biotechnology by Inna Khozin-Goldberg; Umidjon Iskandarov; Zvi Cohen (pp. 905-915).

Microalgae offer potential for numerous commercial applications, among them the production of long-chain polyunsaturated fatty acids (LC-PUFAs). These valuable fatty acids are important for a variety of nutraceutical and pharmaceutical purposes, and the market for these products is continually growing. An appropriate ratio of LC-PUFA of the ω-3 and ω-6 groups is vital for “healthy” nutrition, and adequate dietary intake has strong health benefits in humans. Microalgae of diverse classes are primary natural producers of LC-PUFA. This mini-review presents an introductory overview of LC-PUFA-related health benefits in humans, describes LC-PUFA occurrence in diverse microalgal classes, depicts the major pathways of their biosynthesis in microalgae, and discusses the prospects for microalgal LC-PUFA production.

Keywords: Arachidonic acid; Desaturase; Docosahexaenoic acid; Eicosapentaenoic acid; LC-PUFA; Microalgae

Post-mortem volatiles of vertebrate tissue by Sebastian Paczkowski; Stefan Schütz (pp. 917-935).

Volatile emission during vertebrate decay is a complex process that is understood incompletely. It depends on many factors. The main factor is the metabolism of the microbial species present inside and on the vertebrate. In this review, we combine the results from studies on volatile organic compounds (VOCs) detected during this decay process and those on the biochemical formation of VOCs in order to improve our understanding of the decay process. Micro-organisms are the main producers of VOCs, which are by- or end-products of microbial metabolism. Many microbes are already present inside and on a vertebrate, and these can initiate microbial decay. In addition, micro-organisms from the environment colonize the cadaver. The composition of microbial communities is complex, and communities of different species interact with each other in succession. In comparison to the complexity of the decay process, the resulting volatile pattern does show some consistency. Therefore, the possibility of an existence of a time-dependent core volatile pattern, which could be used for applications in areas such as forensics or food science, is discussed. Possible microbial interactions that might alter the process of decay are highlighted.

Keywords: Post-mortem decay; Post-mortem volatiles; Decaying vertebrates; Volatile emission; Biochemical volatile formation; Volatiles of microbial metabolism

Scleroglucan: biosynthesis, production and application of a versatile hydrocolloid by Jochen Schmid; Vera Meyer; Volker Sieber (pp. 937-947).

Since its first description in the early 1960s, scleroglucan attracted much attention from both academia and industry. Scleroglucan is an exopolysaccharide secreted by the basidiomycete Sclerotium rolfsii and appreciated as a multipurpose compound applicable in many industrial fields, including oil industry, food industry and pharmacy. In this review, the current knowledge on scleroglucan chemistry, genetics, biosynthesis and production will be summarized and different application possibilities will be discussed. The biosynthesis of scleroglucan in S. rolfsii will be highlighted by recent transcriptomic data and linked to physiological data to better understand the biogenesis of scleroglucan and its link to the phytopathologic lifestyle of S. rolfsii.

Keywords: Scleroglucan; Biosynthesis; Sclerotium rolfsii ; Fermentation; Transcriptome; Exopolysaccharide

Metabolic engineering of flavonoids in plants and microorganisms by Yechun Wang; Steven Chen; Oliver Yu (pp. 949-956).

Over 9,000 flavonoid compounds have been found in various plants, comprising one of the largest families of natural products. Flavonoids are an essential factor in plant interactions with the environment, often serving as the first line of defense against UV irradiation and pathogen attacks. Flavonoids are also major nutritional compounds in foods and beverages, with demonstrated health benefits. Some flavonoids are potent antioxidants, and specific flavonoid compounds are beneficial in many physiological and pharmacological processes. Therefore, engineering of flavonoid biosynthesis in plants or in microorganisms has significant scientific and economical importance. Construction of biosynthetic pathways in heterologous systems offers promising results for large-scale flavonoid production by fermentation or bioconversion. Genomics and metabolomics now offer unprecedented tools for detailed understanding of the engineered transgenic organism and for developing novel technologies to further increase flavonoid production yields. We summarize some of the recent metabolic engineering strategies in plants and microorganisms, with a focus on applications of metabolic flux analysis. We are confident that these engineering approaches will lead to successful industrial flavonoid production in the near future.

Keywords: Flavonoids; Resveratrol; Metabolic engineering; Metabolic flux

Sulfhydryl oxidases: sources, properties, production and applications by Greta Faccio; Outi Nivala; Kristiina Kruus; Johanna Buchert; Markku Saloheimo (pp. 957-966).

The formation of disulfide bonds in proteins and small molecules can greatly affect their functionality. Sulfhydryl oxidases (SOXs) are enzymes capable of oxidising the free sulfhydryl groups in proteins and thiol-containing small molecules by using molecular oxygen as an electron acceptor. SOXs have been isolated from the intracellular compartments of many organisms, but also secreted SOXs are known. These latter enzymes are generally active on small compounds and their physiological role is unknown, whereas the intracellular enzymes prefer proteins as substrates and are involved in protein folding. An increasing number of scientific publications and patent applications on SOXs have been published in recent years. The present mini-review provides an up-to-date summary of SOXs from various families, their production and their actual or suggested applications. The sequence features and domain organisation of the characterised SOXs are reviewed, and special attention is paid to the physicochemical features of the enzymes. A review of patents and patent applications regarding this class of enzymes is also provided.

Keywords: Application; Disulfide bond; Glutathione; Sulfhydryl oxidase; Sulphydryl oxidase; Thiol oxidase

Enhanced production of GDP-l-fucose by overexpression of NADPH regenerator in recombinant Escherichia coli by Won-Heong Lee; Young-Wook Chin; Nam Soo Han; Myoung-Dong Kim; Jin-Ho Seo (pp. 967-976).

Biosynthesis of guanosine 5′-diphosphate-l-fucose (GDP-l-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP⁺-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-l-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-l-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-l-fucose production. However, GDP-l-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-l-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-l-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-l-fucose concentration of 235.2 ± 3.3 mg l⁻¹, corresponding to a 21% enhancement in the GDP-l-fucose production compared with the control strain overexpressing GDP-l-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-l-fucose production in recombinant E. coli.

Keywords: Recombinant Escherichia coli ; GDP-l-fucose; NADPH; Glucose 6-phosphate dehydrogenase; pH-stat fed-batch fermentation

Root cultures of Hypericum perforatum subsp. angustifolium elicited with chitosan and production of xanthone-rich extracts with antifungal activity by Noemi Tocci; Giovanna Simonetti; Felicia Diodata D’Auria; Simona Panella; Anna Teresa Palamara; Alessio Valletta; Gabriella Pasqua (pp. 977-987).

Hypericum perforatum is a well-known medicinal plant which contains a wide variety of metabolites, including xanthones, which have a wide range of biological properties, including antifungal activity. In the present study, we evaluated the capability of roots regenerated from calli of H. perforatum subsp. angustifolium to produce xanthones. Root biomass was positively correlated with the indole-3-butyric acid concentration, whereas a concentration of 1 mg l⁻¹ was the most suitable for the development of roots. High auxin concentrations also inhibited xanthone accumulation. Xanthones were produced in large amounts, with a very stable trend throughout the culture period. When the roots were treated with chitosan, the xanthone content dramatically increased, peaking after 7 days. Chitosan also induced a release of these metabolites into the culture. The maximum accumulation (14.26 ± 0.62 mg g⁻¹ dry weight [DW]) and release (2.64 ± 0.13 mg g⁻¹ DW) of xanthones were recorded 7 days after treatment. The most represented xanthones were isolated, purified, and spectroscopically characterized. Antifungal activity of the total root extracts was tested against a broad panel of human fungal pathogen strains (30 Candida species, 12 Cryptococcus neoformans, and 16 dermatophytes); this activity significantly increased when using chitosan. Extracts obtained after 7 days of chitosan treatment showed high antifungal activity (mean minimum inhibitory concentration of 83.4, 39.1, and 114 μg ml⁻¹ against Candida spp., C. neoformans, and dermatophytes, respectively). Our results suggest that root cultures can be considered as a potential tool for large-scale production of extracts with stable quantities of xanthones.

Keywords: Hypericum perforatum ; Root cultures; Xanthones; Candida spp.; Cryptococcus neoformans ; Dermatophytes

First steps towards a Zn/Co(III)sep-driven P450 BM3 reactor by Liqing Zhao; Güray Güven; Yin Li; Ulrich Schwaneberg (pp. 989-999).

Cytochrome P450s are synthetically attractive hydroxylation catalysts. For cell-free applications, a constant supply of NAD(P)H can be very costly. Mediators such as Zn/Co(III)sep can be an alternative cofactor system to NAD(P)H. Several mutants of cytochrome P450 BM3 with improved electron transfer rate to Zn/Co(III)sep have been obtained in our group. P450 BM3 M7 (F87A V281G M354S R471C A1011T S1016G Q1022R) was immobilized on DEAE-650S, further entrapped with k-carrageenan together with zinc dust which function as electron source and catalase which removes produced hydrogen peroxide instantly. Immobilized P450 BM3 M7 were treated with 0.05% (v/v) glutaraldehyde to enhance operational stability. P450 BM3 M7 retained around 76% of its activity and conversions stayed above 80% in 10 batch cycles, indicating a high stability of immobilized P450 BM3 M7. To explore the synthetic potential, a small-scale bioreactor was developed to investigate the stability and efficiencies of P450 BM3 M9 (R47F F87A M238K V281G M354S D363H W575C A595T). P450 BM3 M9 was used for the continuous conversion of 3-phenoxytoluene in a plug flow reactor (PFR) since P450 BM3 M9 has a 3-fold higher activity for 3-phenoxytoluene compared to P450 BM3 M7 which was used for optimizing immobilization conditions with the highest activity for 12-pNCA assay. The reactor could be operated for 5 days with total turnover numbers (TTNs) over 2,000.

Keywords: P450 BM3; Enzyme co-immobilization; Plug flow reactor (PFR); Alternative cofactor; Resin DEAE-650S; k-carrageenan

Enzymatic glutathione production using metabolically engineered Saccharomyces cerevisiae as a whole-cell biocatalyst by Hideyo Yoshida; Kiyotaka Y. Hara; Kentaro Kiriyama; Hideki Nakayama; Fumiyoshi Okazaki; Fumio Matsuda; Chiaki Ogino; Hideki Fukuda; Akihiko Kondo (pp. 1001-1006).

We developed a novel enzymatic glutathione (GSH) production system using Saccharomyces cerevisiae as a whole-cell biocatalyst, and improved its GSH productivity by metabolic engineering. We demonstrated that the metabolic engineering of GSH pathway and ATP regeneration can significantly improve GSH productivity by up to 1.7-fold higher compared with the parental strain, respectively. Furthermore, the combination of both improvements in GSH pathway and ATP regeneration is more effective (2.6-fold) than either improvement individually for GSH enzymatic production using yeast. The improved whole-cell biocatalyst indicates its great potential for applications to other kinds of ATP-dependent bioproduction.

Keywords: Glutathione; ATP; Yeast; Permeated cell; Biocatalyst

Enhanced degradation of caffeine and caffeine demethylase production by Pseudomonas sp. in bioreactors under fed-batch mode by Sathyanarayana N. Gummadi; B. Bhavya (pp. 1007-1017).

The growth of Pseudomonas sp. was studied in fed-batch process with an aim to improve the caffeine degradation rate and caffeine demethylase activity. The effects of varying initial caffeine concentrations in the batch mode, increase in the number of feeds, varying feed flow rates, and added nutrients to the feed on the fed-batch process were investigated. A maximum caffeine degradation rate of 0.82 g/L h and maximum caffeine demethylase activity of 2.6 U/mg were achieved using manual intermittent pulse feeds of caffeine with substrate concentration as feedback parameter for the fed batch started with an initial caffeine concentration of 3 g/L. A slight increase in the caffeine degradation rate (0.85 g/L h) and caffeine demethylase activity (3.4 U/mg) was observed when the additional nutrients were added along with caffeine in the feed. This is the first report showing complete degradation of large magnitudes of caffeine amounting to 237 g in 75 h. These results show that the fed-batch conditions achieved in this study using Pseudomonas sp. facilitate the development of a sustainable bioprocess to degrade the high concentrations of caffeine in industrial effluents.

Keywords: Pseudomonas sp.; Caffeine degradation; Caffeine demethylase; Bioreactor; Fed-batch

Role of extracellular protease in nitrogen substrate management during antibiotic fermentation: a process model and experimental validation by Prashant M. Bapat; Avinash Sinha; Pramod P. Wangikar (pp. 1019-1028).

Kinetics of extracellular protease (ECP) production has typically been studied for processes that involve protease as a product. We argue that ECP is equally important in fermentations where protease is not a product of interest. Industrial fermentations typically use complex nitrogen substrates, which are proteolytically hydrolyzed to amino acids (AA) by ECP before assimilation. However, high AA concentrations may lead to nitrogen catabolite repression (NCR) of the products such as antibiotics. Thus, ECP plays a crucial role in managing the nitrogen substrate supply thereby affecting the antibiotic productivity. Here, we have studied the induction of ECP and its effect on the antibiotic productivity for a rifamycin B overproducer strain Amycolatopsis meditterranei S699. This organism produces ECP at the level of 14 U mL⁻¹ in complex media, which is sufficient for hydrolysis of proteins in the media but low compared to other ECP overproducers. We find ECP secretion to be repressed by ammonia, AA, and under conditions that support high growth rate. We propose a structured kinetic model which accounts for the kinetics of ECP secretion, amino acid availability, growth, and antibiotic production. In addition to the quantity, the timing of ECP induction was critical in achieving higher rifamycin productivity. We artificially created conditions that led to delayed protease secretion, which in turn led to premature termination of batch and lower productivity. The predictive value of the model can be useful in better management of the available nitrogen supply, minimization of NCR, and in the monitoring of fermentation batches.

Keywords: Structured model; Complex media; Nitrogen catabolite repression; Preculture conditions; Protease regulation

Biochemical characterization of the carotenoid 1,2-hydratases (CrtC) from Rubrivivax gelatinosus and Thiocapsa roseopersicina by Aida Hiseni; Isabel W. C. E. Arends; Linda G. Otten (pp. 1029-1036).

Two carotenoid 1,2-hydratase (CrtC) genes from the photosynthetic bacteria Rubrivivax gelatinosus and Thiocapsa roseopersicina were cloned and expressed in Escherichia coli in an active form and purified by affinity chromatography. The biochemical properties of the recombinant enzymes and their substrate specificities were studied. The purified CrtCs catalyze cofactor independently the conversion of lycopene to 1-HO- and 1,1′-(HO)₂-lycopene. The optimal pH and temperature for hydratase activity was 8.0 and 30°C, respectively. The apparent K _m and V _max values obtained for the hydration of lycopene were 24 μM and 0.31 nmol h⁻¹ mg⁻¹ for RgCrtC and 9.5 μM and 0.15 nmol h⁻¹ mg⁻¹ for TrCrtC, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed two protein bands of 44 and 38 kDa for TrCrtC, which indicate protein processing. Both hydratases are also able to convert the unnatural substrate geranylgeraniol (C20 substrate), which functionally resembles the natural substrate lycopene.

Keywords: Enzyme kinetics; Biocatalysis; Carotenoid hydratase; CrtC; Recombinant expression; Hydro-lyase

Application of polyhydroxyalkanoate binding protein PhaP as a bio-surfactant by Dai-Xu Wei; Chong-Bo Chen; Guo Fang; Shi-Yan Li; Guo-Qiang Chen (pp. 1037-1047).

PhaP or phasin is an amphiphilic protein located on surfaces of microbial storage polyhydroxyalkanoates granules. This study aimed to explore amphiphilic properties of PhaP for possible application as a protein surfactant. Following agents were used to conduct this study as controls including bovine serum albumin, sodium dodecyl sulfate (SDS), Tween 20, sodium oleate, a commercial liquefied detergent together with the same amount of PhaP. Among all these tested control surfactants, PhaP showed the strongest effect to form emulsions with lubricating oil, diesel, and soybean oil, respectively. PhaP emulsion stability study compared with SDS revealed that PhaP had a stronger capability to maintain a very stable emulsion layer after 30 days while SDS lost half and two-thirds of its capacity after 2 and 30 days, respectively. When PhaP was more than 200 μg/ml in the water, all liquids started to exhibit stable emulsion layers. Similar to SDS, PhaP significantly reduced the water contact angles of water on a hydrophobic film of biaxially oriented polypropylene. PhaP was thermally very stable, it showed ability to form emulsion and to bind to the surface of polyhydroxybutyrate nanoparticles after a 60- min heating process at 95 °C. It is therefore concluded that PhaP is a protein with thermally stable property for application as natural and environmentally friendly surfactant for food, cosmetic, and pharmaceutical usages.

Keywords: PHB; Polyhydroxyalkanoates; PhaP; Phasin; Amphiphilic protein; Emulsion; Surfactant

Cloning, functional expression, biochemical characterization, and structural analysis of a haloalkane dehalogenase from Plesiocystis pacifica SIR-1 by Martin Hesseler; Xenia Bogdanović; Aurelio Hidalgo; Jose Berenguer; Gottfried J. Palm; Winfried Hinrichs; Uwe T. Bornscheuer (pp. 1049-1060).

A haloalkane dehalogenase (DppA) from Plesiocystis pacifica SIR-1 was identified by sequence comparison in the NCBI database, cloned, functionally expressed in Escherichia coli, purified, and biochemically characterized. The three-dimensional (3D) structure was determined by X-ray crystallography and has been refined at 1.95 Å resolution to an R-factor of 21.93%. The enzyme is composed of an α/β-hydrolase fold and a cap domain and the overall fold is similar to other known haloalkane dehalogenases. Active site residues were identified as Asp123, His278, and Asp249 and Trp124 and Trp163 as halide-stabilizing residues. DppA, like DhlA from Xanthobacter autotrophicus GJ10, is a member of the haloalkane dehalogenase subfamily HLD-I. As a consequence, these enzymes have in common the relative position of their catalytic residues within the structure and also show some similarities in the substrate specificity. The enzyme shows high preference for 1-bromobutane and does not accept chlorinated alkanes, halo acids, or halo alcohols. It is a monomeric protein with a molecular mass of 32.6 kDa and exhibits maximum activity between 33 and 37°C with a pH optimum between pH 8 and 9. The K_m and k_cat values for 1-bromobutane were 24.0 mM and 8.08 s⁻¹. Furthermore, from the 3D-structure of DppA, it was found that the enzyme possesses a large and open active site pocket. Docking experiments were performed to explain the experimentally determined substrate preferences.

Keywords: Haloalkane dehalogenase; Plesiocystis pacifica ; Biochemical characterization; Structure

The crystal structure of an esterase from the hyperthermophilic microorganism Pyrobaculum calidifontis VA1 explains its enantioselectivity by Gottfried J. Palm; Elena Fernández-Álvaro; Xenia Bogdanović; Sebastian Bartsch; Jaroslaw Sczodrok; Rajesh K. Singh; Dominique Böttcher; Haruyuki Atomi; Uwe T. Bornscheuer; Winfried Hinrichs (pp. 1061-1072).

The highly thermostable esterase from the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 (PestE) shows high enantioselectivity (E > 100) in the kinetic resolution of racemic chiral carboxylic acids, but little selectivity towards acetates of tertiary alcohols (E = 2–4). To explain these unique properties, its crystal structure has been determined at 2.0 Å resolution. The enzyme is a member of the hormone-sensitive lipase group (group H) of the esterase/lipase superfamily on the basis of the amino acid sequence identity. The PestE structure shows a canonical α/β-hydrolase fold as core domain with a cap structure at the C-terminal end of the β-sheet. A tetramer in the crystal packing is formed of two dimers; the dimeric form is observed in solution. Conserved dimers and even tetramers are found in other group H proteins. The amino acid residues Ser157, His284, and Asp254 form the catalytic triad, which is typically found in α/β-hydrolases. The oxyanion hole is composed of Gly85 and Gly86 within the conserved sequence motif HGGG(M,F,W) (amino acid residues 83–87) and Ala158. With the elucidated structure, experimental results about enantioselectivity towards the two model substrate classes (as exemplified for 3-phenylbutanoic acid ethyl ester and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate) could be explained by molecular modeling. For both enantiomers of the tertiary alcohol, orientations in two binding pockets were obtained without significant energy differences corresponding to the observed low enantioselectivity due to missing steric repulsions. In contrast, for the carboxylic acid ester, two different orientations with significant energy differences for each enantiomer were found matching the high E values.

Keywords: Hyperthermophilic esterase; α/β-Hydrolase fold; Crystal structure; Enantioselectivity

Biochemical characterization of Magnaporthe oryzae β-glucosidases for efficient β-glucan hydrolysis by Machiko Takahashi; Teruko Konishi; Takumi Takeda (pp. 1073-1082).

β-Glucosidases designated MoCel3A and MoCel3B were successfully overexpressed in Magnaporthe oryzae. MoCel3A and MoCel3B showed optimal activity at 50 °C and pH 5.0–5.5. MoCel3A exhibited higher activity on higher degree of polymerization (DP) oligosaccharides and on β-1,3-linked oligosaccharides than on β-1,4-linked oligosaccharides. Furthermore, MoCel3A could liberate glucose from polysaccharides such as laminarin, 1,3-1,4-β-glucan, phosphoric acid-swollen cellulose, and pustulan, of which laminarin was the most suitable substrate. Conversely, MoCel3B preferentially hydrolyzed lower DP oligosaccharides such as cellobiose, cellotriose, and laminaribiose. Furthermore, the synergistic effects of combining enzymes including MoCel3A and MoCel3B were investigated. Depolymerization of 1,3-1,4-β-glucan by M. oryzae cellobiohydrolase (MoCel6A) enhanced the production of glucose by the actions of MoCel3A and MoCel3B. In these reactions, MoCel3A hydrolyzed higher DP oligosaccharides, resulting in the release of glucose and cellobiose, and MoCel3B preferentially hydrolyzed lower DP oligosaccharides including cellobiose. On the other hand, MoCel3A alone produced glucose from laminarin at levels equivalent to 80% of maximal hydrolysis obtained by the combined action of MoCel3A, MoCel3B, and endo-1,3-β-glucanase. Therefore, MoCel3A and MoCel3B activities yield glucose from not only cellulosic materials but also hemicellulosic polysaccharides.

Keywords: β-Glucosidase; GH family 3; Homologous overexpression; β-Glucan hydrolysis; Glucose production

Biosurfactant-producing Bacillus are present in produced brines from Oklahoma oil reservoirs with a wide range of salinities by D. Randall Simpson; Nisha Ravi Natraj; Michael J. McInerney; Kathleen E. Duncan (pp. 1083-1093).

Nine wells producing from six different reservoirs with salinities ranging from 2.1% to 15.9% were surveyed for presence of surface-active compounds and biosurfactant-producing microbes. Degenerate primers were designed to detect the presence of the surfactin/lichenysin (srfA3/licA3) gene involved in lipopeptide biosurfactant production in members of Bacillus subtilis/licheniformis group and the rhlR gene involved in regulation of rhamnolipid production in pseudomonads. Polymerase chain reaction amplification, cloning, and sequencing confirmed the presence of the srfA3/licA3 genes in brines collected from all nine wells. The presence of B. subtilis/licheniformis strains was confirmed by sequencing two other genes commonly used for taxonomic identification of bacteria, gyrA (gyrase A) and the 16S rRNA gene. Neither rhlR nor 16S rRNA gene related to pseudomonads was detected in any of the brines. Intrinsic levels of surface-active compounds in brines were low or not detected, but biosurfactant production could be stimulated by nutrient addition. Supplementation with a known biosurfactant-producing Bacillus strain together with nutrients increased biosurfactant production. The genetic potential to produce lipopeptide biosurfactants (e.g., srfA3/licA3 gene) is prevalent, and nutrient addition stimulated biosurfactant production in brines from diverse reservoirs, suggesting that a biostimulation approach for biosurfactant-mediated oil recovery may be technically feasible.

Keywords: Biosurfactant; MEOR; Surfactin; Lichenysin; Rhamnolipid; Biostimulation; Bioaugmentation; Oil reservoir

Soybean transcription factor GmMYBZ2 represses catharanthine biosynthesis in hairy roots of Catharanthus roseus by Mei-Liang Zhou; Hong-Li Hou; Xue-Mei Zhu; Ji-Rong Shao; Yan-Min Wu; Yi-Xiong Tang (pp. 1095-1105).

Catharanthus roseus (L.) G. Don is a plant species known for its production of a variety of terpenoid indole alkaloids, many of which have pharmacological activities. Production of catharanthine in cell cultures or in hairy roots established by transformation with Agrobacterium rhizogenes is of interest because catharanthine can be chemically coupled to the abundant leaf alkaloid vindoline to form the valuable anticancer drug vinblastine. Here, we observed a high amount of catharanthine in hairy roots of C. roseus, established by infecting leaf explants with the A. rhizogenes >agropine-type A4 strain carrying plasmid pRi. T-DNA transfer from plasmid pRi into hairy roots was confirmed by PCR for the essential T-DNA genes rolA and rolB and the agropine synthesis gene ags. The results suggest that integration of T-DNA into the plant DNA plays a positive role on the catharanthine pathway in C. roseus hairy roots. Furthermore, co-transformation with the soybean transcription factor GmMYBZ2 indicated that GmMYBZ2 reduces the catharanthine production by alteration of expression of a number of genes linked to the pathway. Transcription levels of the zinc-finger transcription factor 1 gene ZCT1 were high, and the transcription levels of the anthranilate synthase gene ASα, the strictosidine synthase gene STR, and the key transcription factor gene octadecanoid-responsive Catharanthus APETALA2/ethylene response factor were low. In addition, GmMYBZ2 had a negative effect on the gene expression levels of A-type cyclin CYSA and B-type cyclin CYSB, which was correlated with a reduced growth rate of the hairy roots.

Keywords: Catharanthine; Growth index; Hairy roots; Metabolic engineering; MYB transcription factor; Transcript profiling

PdCYP51B, a new putative sterol 14α-demethylase gene of Penicillium digitatum involved in resistance to imazalil and other fungicides inhibiting ergosterol synthesis by Xuepeng Sun; Jiye Wang; Dan Feng; Zhonghua Ma; Hongye Li (pp. 1107-1119).

Penicillium digitatum, causing green mold decay, is the most destructive postharvest pathogen of citrus fruits worldwide. The phenotypes and genotypes of 403 isolates of P. digitatum, collected from packing houses and supermarkets in Zhejiang, China, during 2000 to 2010, were characterized in terms of their imazalil sensitivity. The frequency of detected imazalil-resistant (IMZ-R) isolates increased from 2.1% in 2000 to 60–84% during 2005–2010. Only 6.5% and 4.5% of the collected IMZ-R isolates belong to the previously described IMZ-R1 and IMZ-R2 genotypes, respectively. To determine the resistance mechanism of the predominant and novel IMZ-R isolates of P. digitatum (termed IMZ-R3), genes PdCYP51B and PdCYP51C, homologous to the sterol 14α-demethylase encoded gene PdCYP51, were cloned from six IMZ-R3 and eight imazalil-sensitive (IMZ-S) isolates of P. digitatum. A unique 199-bp insertion was observed in the promoter region of PdCYP51B in all IMZ-R3 isolates examined but in none of the tested IMZ-S isolates. Further analysis by PCR confirmed that this insertion was present in all IMZ-R3 isolates but absent in IMZ-S, IMZ-R1, and IMZ-R2 isolates. Transcription levels of PdCYP51B in three IMZ-R3 isolates were found to be 7.5- to 13.6-fold higher than that in two IMZ-S isolates of P. digitatum. Introduction of another copy of PdCYP51B s (from IMZ-S) into an IMZ-S isolate decreased the sensitivity of P. digitatum to 14α-demethylation inhibitors (DMIs) only to a small extent, but introduction of a copy of PdCYP51B R (from IMZ-R3) dramatically increased the resistance level of P. digitatum to DMIs. Regarding PdCYP51C, no consistent changes in either nucleotide sequence or expression level were correlated with imazalil resistance among IMZ-R and IMZ-S isolates. Based on these results, we concluded that (1) the CYP51 family of P. digitatum contains the PdCYP51B and PdCYP51C genes, in addition to the known gene PdCYP51A (previously PdCYP51); (2) PdCYP51B is involved in DMI fungicide resistance; and (3) overexpression of PdCYP51B resulting from a 199-bp insertion mutation in the promoter region of PdCYP51B is responsible for the IMZ-R3 type of DMI resistance in P. digitatum.

Keywords: Penicillium digitatum ; CYP51 family; Gene mutation; Gene expression; Fungicide resistance; Demethylation inhibitors (DMIs)

Identification of a cyclooxygenase gene from the red alga Gracilaria vermiculophylla and bioconversion of arachidonic acid to PGF_2α in engineered Escherichia coli by Hirosuke Kanamoto; Miho Takemura; Kanji Ohyama (pp. 1121-1129).

Prostaglandins (PGs) are important local messenger molecules in many tissues and organs of animals including human. For applications in medicine and animal care, PGs are mostly purified from animal tissues or chemically synthesized. To generate a clean, reliable, and inexpensive source for PGs, we have now engineered expression of a suitable cyclooxygenase gene in Escherichia coli and achieved production levels of up to 2.7 mg l⁻¹ PGF_2α. The cyclooxygenase gene cloned from the red alga Gracilaria vermiculophylla appears to be fully functional without any eukaryotic modifications in E. coli. A crude extract of the recombinant E. coli cells is able to convert in vitro the substrate arachidonic acid (AA) to PGF_2α. Furthermore, these E. coli cells produced PGF_2α in a medium supplemented with AA and secreted the PGF_2α product. To our knowledge, this is the first report of the functional expression of a cyclooxygenase gene and concomitant production of PGF_2α in E. coli. The successful microbial synthesis of PGs with reliable yields promises a novel pharmaceutical tool to produce PGF_2α at significantly reduced prices and greater purity.

Keywords: Prostaglandin; Cyclooxygenase; Gracilaria vermiculophylla ; Arachidonic acid; E. coli

Involvement of ligninolytic enzymes and Fenton-like reaction in humic acid degradation by Trametes sp. by Tzafrir Grinhut; Tomer Meir Salame; Yona Chen; Yitzhak Hadar (pp. 1131-1140).

Trametes sp. M23, isolated from biosolids compost was found to decompose humic acids (HA). A low N (LN) medium (C/N, 53) provided suitable conditions for HA degradation, whereas in a high N (HN) medium (C/N, 10), HA was not degraded. In the absence of Mn²⁺, HA degradation was similar to that in Mn²⁺-containing medium. In contrast, MnP activity was significantly affected by Mn²⁺. Laccase activity exhibited a negative correlation to HA degradation, while LiP activity was not detected. Thus, ligninolytic enzymes activity could provide only a partial explanation for the HA-degradation mechanism. The decolorization of two dyes, Orange II and Brilliant Blue R250, was also determined. Similar to HA degradation, under LN conditions, decolorization occurred independently of the presence of Mn²⁺. We investigated the possible involvement of a Fenton-like reaction in HA degradation. The addition of DMSO, an OH-radical scavenger, to LN media resulted in a significant decrease in HA bleaching. The rate of extracellular Fe³⁺ reduction was much higher in the LN vs. HN medium. In addition, the rate of reduction was even higher in the presence of HA in the medium. In vitro HA bleaching in non-inoculated media was observed with H₂O₂ amendment to a final concentration of 200 mM (obtained by 50 mM amendments for 4 days) and Fe²⁺ (36 mM). After 4 days of incubation, HA decolorization was similar to the biological treatment. These results support our hypothesis that a Fenton-like reaction is involved in HA degradation by Trametes sp. M23.

Keywords: Ligninolytic enzymes; Humic acid degradation; Trametes sp.; Humic substances

Basidiomycete Clitocybe nebularis is rich in lectins with insecticidal activities by Jure Pohleven; Jože Brzin; Lara Vrabec; Adrijana Leonardi; Andrej Čokl; Borut Štrukelj; Janko Kos; Jerica Sabotič (pp. 1141-1148).

Basidiomycete mushrooms are a rich source of unique substances, including lectins, that could potentially be useful in biotechnology or biomedical applications. Lectins are a group of carbohydrate-binding proteins with diverse biological activities and functions. Here, we demonstrate the presence of a number of lectins in the basidiomycete mushroom Clitocybe nebularis. Glucose-, galactose-, sucrose-, lactose-, and Sepharose-binding lectins were isolated from fruiting bodies using affinity chromatography on Sepharose-immobilized sugars or on Sepharose. The lectins were characterized biochemically and their binding specificities examined by agglutination and agglutination inhibition assays. In addition, insecticidal and anti-nutritional properties of the lectins were studied against a model organism, fruit fly (Drosophila melanogaster), and Colorado potato beetle (Leptinotarsa decemlineata). Of the several basidiomycete mushrooms screened, C. nebularis extract showed the most potent insecticidal activity. Sucrose-binding lectin showed the strongest activity against D. melanogaster, followed by lactose- and galactose-binding lectins. Feeding bioassays with Colorado potato beetle revealed that C. nebularis extract exhibited high anti-nutritional activity against the insect; and of those tested, only lactose-binding lectin, named CNL showed the effect. Mushroom C. nebularis is shown to be rich in a variety of lectins with versatile biological activities, including insecticidal and anti-nutritional effects. C. nebularis lectins could thus have potential for use as natural insecticides.

Keywords: Basidiomycete; Clitocybe nebularis ; Lectins; Insecticidal activity; Drosophila melanogaster ; Leptinotarsa decemlineata

The influence of nanoscopically thin silver films on bacterial viability and attachment by Elena P. Ivanova; Jafar Hasan; Vi Khanh Truong; James Y. Wang; Massimo Raveggi; Christopher Fluke; Russell J. Crawford (pp. 1149-1157).

The physicochemical and bactericidal properties of thin silver films have been analysed. Silver films of 3 and 150 nm thicknesses were fabricated using a magnetron sputtering thin-film deposition system. X-ray photoelectron and energy dispersive X-ray spectroscopy and atomic force microscopy analyses confirmed that the resulting surfaces were homogeneous, and that silver was the most abundant element present on both surfaces, being 45 and 53 at.% on the 3- and 150-nm films, respectively. Inductively coupled plasma time of flight mass spectroscopy (ICP-TOF-MS) was used to measure the concentration of silver ions released from these films. Concentrations of 0.9 and 5.2 ppb were detected for the 3- and 150-nm films, respectively. The surface wettability of the films remained nearly identical for both film thicknesses, displaying a static water contact angle of 95°, while the surface free energy of the 150-nm film was found to be slightly greater than that of the 3-nm film, being 28.8 and 23.9 mN m⁻¹, respectively. The two silver film thicknesses exhibited statistically significant differences in surface topographic profiles on the nanoscopic scale, with R _a, R _q and R _max values of 1.4, 1.8 and 15.4 nm for the 3-nm film and 0.8, 1.2 and 10.7 nm for the 150-nm film over a 5 × 5 μm scanning area. Confocal scanning laser microscopy and scanning electron microscopy revealed that the bactericidal activity of the 3-nm silver film was not significant, whereas the nanoscopically smoother 150-nm silver film exhibited appreciable bactericidal activity towards Pseudomonas aeruginosa ATCC 9027 cells and Staphylococcus aureus CIP 65.8 cells, obtaining up to 75% and 27% sterilisation effect, respectively.

Keywords: Nanoscopically thin silver coating; Film; Bactericidal activity; S. aureus ; P. aeruginosa

Identification of novel genes responsible for ethanol and/or thermotolerance by transposon mutagenesis in Saccharomyces cerevisiae by Hyun-Soo Kim; Na-Rae Kim; Jungwoo Yang; Wonja Choi (pp. 1159-1172).

Saccharomyces cerevisiae strains tolerant to ethanol and heat stresses are important for industrial ethanol production. In this study, five strains (Tn 1–5) tolerant to up to 15% ethanol were isolated by screening a transposon-mediated mutant library. Two of them displayed tolerance to heat (42 °C). The determination of transposon insertion sites and Northern blot analysis identified seven putative genes (CMP2, IMD4, SSK2, PPG1, DLD3, PAM1, and MSN2) and revealed simultaneous down-regulations of CMP2 and IMD4, and SSK2 and PPG1, down-regulation of DLD3, and disruptions of the open reading frame of PAM1 and MSN2, indicating that ethanol and/or heat tolerance can be conferred. Knockout mutants of these seven individual genes were ethanol tolerant and three of them (SSK2, PPG1, and PAM1) were tolerant to heat. Such tolerant phenotypes reverted to sensitive phenotypes by the autologous or overexpression of each gene. Five transposon mutants showed higher ethanol production and grew faster than the control strain when cultured in rich media containing 30% glucose and initial 6% ethanol at 30 °C. Of those, two thermotolerant transposon mutants (Tn 2 and Tn 3) exhibited significantly enhanced growth and ethanol production compared to the control at 42 °C. The genes identified in this study may provide a basis for the application in developing industrial yeast strains.

Keywords: Bioethanol; Transposon; Ethanol tolerance; Thermotolerance

Stereospecific microbial production of isoflavanones from isoflavones and isoflavone glucosides by Hye-Yeon Park; Mihyang Kim; Jaehong Han (pp. 1173-1181).

A Gram-negative anaerobic microorganism, MRG-1, isolated from human intestine showed high activities of deglycosylation and reduction of daidzin, based on rapid TLC analysis. A rod-shaped strain MRG-1was identified as a new species showing 91.0% homology to Coprobacillus species, based on 16S rRNA sequence analysis. The strain MRG-1 showed β-glucosidase activity toward daidzin and genistin, and daidzein and genistein were produced, respectively. However, the strain MRG-1 did not react with flavone glycosides, flavanone glycosides, and isoflavone C-glucoside. Besides, MRG-1 showed stereoselective reductase activity to isoflavone, daidzein, genistein, 7-hydroxyisoflavone, and formononetin, resulting in the formation of corresponding R-isoflavanone enantiomers. The new isoflavanones of 7-hydroxyisoflavanone and dihydroformononetin were characterized by NMR, and the absolute configurations of the enantiomers were determined with CD spectroscopy. The kinetic study of the anaerobic biotransformation showed both activities were exceptionally fast compared to the reported conversion by other anaerobic bacteria.

Keywords: Biotransformation; Daidzein; Equol; Isoflavonoids; Stereospecificity

Development of a simple cultivation method for isolating hitherto-uncultured cellulase-producing microbes by Katsuhiko Fujii; Anna Kuwahara; Kanako Nakamura; Yuki Yamashita (pp. 1183-1192).

Although enrichment culture is typically employed to isolate cellulolytic microbes, this approach tends to favor fast-growing species and discriminates against all others. Therefore, efforts to prevent the overgrowth of fast-growing species are necessary to isolate novel cellulase-producing strains. In this study, we developed a simple culture method for isolating hitherto-uncultured microbes that possess cellulase activity, particularly exocellulase. In this method, the microbial source (a forest soil) was suspended in sterilized water and inoculated onto a mineral salts agar medium, which was then overlaid with filter paper to sandwich the microbial suspension between the agar surface and paper. The filter paper fibers served to immobilize the microbial cells and were the dominant carbon source. Following cultivation at 30°C for 2 weeks, emerging colonies were isolated based on their morphology and were then subjected to phylogenetic and enzyme analyses. Using this method, 2,150 CFUs/g dry soil were obtained, and the ratio of fungal to bacterial isolates was approximately 4:1. Phylogenetic analyses revealed that most fungal and bacterial isolates belong to ten and two genera, respectively. Notably, all isolates possessed exocellulase activity, and several strains showed strong activity that was comparable to Trichoderma cellulase. Many isolates also exhibited cellulase and xylanase activity, and several strains possessed laccase activity. It is expected that the culture method described here will be useful for the isolation of hitherto-uncultured cellulolytic microbes and the identification of novel cellulases.

Keywords: Cellulolytic microbe; Hitherto-uncultured strain; Exocellulase; Endocellulase; Xylanase; Laccase

Use of primer selection and restriction enzymes to assess bacterial community diversity in an agricultural soil used for potato production via terminal restriction fragment length polymorphism by Ann-Marie Fortuna; Terence L. Marsh; C. Wayne Honeycutt; William A. Halteman (pp. 1193-1202).

Terminal restriction fragment length polymorphism (T-RFLP) can be used to assess how land use management changes the dominant members of bacterial communities. We compared T-RFLP profiles obtained via amplification with forward primers (27, 63F) each coupled with the fluorescently labeled reverse primer (1392R) and multiple restriction enzymes to determine the best combination for interrogating soil bacterial populations in an agricultural soil used for potato production. Both primer pairs provide nearly universal recognition of a 1,400-bp sequence of the bacterial domain in the V₁–V₃ region of the 16S ribosomal RNA (rRNA) gene relative to known sequences. Labeling the reverse primer allowed for direct comparison of each forward primer and the terminal restriction fragments’ relative migration units obtained with each primer pair and restriction enzyme. Redundancy analysis (RDA) and nested multivariate analysis of variance (MANOVA) were used to assess the effects of primer pair and choice of restriction enzyme on the measured relative migration units. Our research indicates that the 63F–1392R amplimer pair provides a more complete description with respect to the bacterial communities present in this potato (Solanum tuberosum L.)–barley (Hordeum vulgare L.) rotation over seeded to crimson clover (Trifolium praense L.). Domain-specific 16S rRNA gene primers are rigorously tested to determine their ability to amplify across a target region of the gene. Yet, variability within or between T-RFLP profiles can result from factors independent of the primer pair. Therefore, researchers should use RDA and MANOVA analyses to evaluate the effects that additional laboratory and environmental variables have on bacterial diversity.

Keywords: Bacterial community diversity; T-RFLP; 63F; 8-27F

Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems by Zhaopeng Li; Wolfgang Kessler; Joop van den Heuvel; Ursula Rinas (pp. 1203-1213).

Protein production under the control of lac operon regulatory elements using autoinduction is based on diauxic growth of Escherichia coli on lactose after consumption of more preferred carbon substrates. A novel simple and cost-effective defined autoinduction medium using a mixture of glucose, glycerol, and lactose as carbon substrate and NH ₄ ⁺ as sole nitrogen source without any supplementation of amino acids and vitamins was developed for T7-based E. coli expression systems. This medium was successfully employed in 96-well microtiter plates, test tubes, shake flasks, and 15-L bioreactor cultivations for production of different types of proteins achieving an average yield of 500 mg L⁻¹ product. Cell-specific protein concentrations and solubility were similar as during conventional isopropyl β-d-1-thiogalactopyranoside induction using Luria-Bertani broth. However, the final yield of target proteins was about four times higher, as a higher final biomass was achieved using this novel defined autoinduction broth.

Keywords: Escherichia coli ; Recombinant protein production; Autoinduction; Defined medium

Ammonia-oxidizing archaea and ammonia-oxidizing bacteria in six full-scale wastewater treatment bioreactors by Tong Zhang; Lin Ye; Amy Hin Yan Tong; Ming-Fei Shao; Si Lok (pp. 1215-1225).

In this study, dideoxy sequencing and 454 high-throughput sequencing were used to analyze diversities of the ammonia monooxygenase (amoA) genes and the 16S rRNA genes of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in six municipal wastewater treatment plants. The results showed that AOB amoA genes were quite diverse in different wastewater treatment plants while the 16S rRNA genes were relatively conserved. Based on the observed complexity of amoA and 16S rRNA genes, most of the AOB can be assigned to the Nitrosomonas genus, with Nitrosomonas ureae, Nitrosomonas oligotropha, Nitrosomonas marina, and Nitrosomonas aestuarii being the four most dominant species. From the sequences of the AOA amoA genes, most AOA observed in this study belong to the CGI.1b group, i.e., the soil lineage. The AOB amoA and 16S rRNA genes were quantified by quantitative PCR and 454 high-throughput pyrosequencing, respectively. Although the results from the two approaches show some disconcordance, they both indicated that the abundance of AOB in activated sludge was very low.

Keywords: Ammonia-oxidizing archaea; Ammonia-oxidizing bacteria; Municipal wastewater treatment plants; 454 Pyrosequencing

Isolation and characterization of four novel Gram-positive bacteria associated with the rhizosphere of two endemorelict plants capable of degrading a broad range of aromatic substrates by Lidija Djokic; Tanja Narancic; Jasmina Nikodinovic-Runic; Miloje Savic; Branka Vasiljevic (pp. 1227-1238).

Four new Gram-positive, phenol-degrading strains were isolated from the rhizospheres of endemorelict plants Ramonda serbica and Ramonda nathaliae known to exude high amounts of phenolics in the soil. Isolates were designated Bacillus sp. PS1, Bacillus sp. PS11, Streptomyces sp. PS12, and Streptomyces sp. PN1 based on 16S rDNA sequence and biochemical analysis. In addition to their ability to tolerate and utilize high amounts of phenol of either up to 800 or up to 1,400 mg l⁻¹ without apparent inhibition in growth, all four strains were also able to degrade a broad range of aromatic substrates including benzene, toluene, ethylbenzene, xylenes, styrene, halogenated benzenes, and naphthalene. Isolates were able to grow in pure culture and in defined mixed culture on phenol and on the mixture of BTEX (benzene, toluene, ethylbenzene, and xylenes) compounds as a sole source of carbon and energy. Pure culture of Bacillus sp. PS11 yielded 1.5-fold higher biomass amounts in comparison to mixed culture, under all conditions. Strains successfully degraded phenol in the soil model system (2 g kg⁻¹) within 6 days. Activities of phenol hydroxylase, catechol 1,2-dioxygenase, and catechol 2,3-dioxygenase were detected and analyzed from the crude cell extract of the isolates. While all four strains use ortho degradation pathway, enzyme indicative of meta degradation pathway (catechol 2,3-dioxygenase) was also detected in Bacillus sp. PS11 and Streptomyces sp. PN1. Phenol degradation activities were induced 2 h after supplementation by phenol, but not by catechol. Catechol slightly inhibited activity of catechol 2,3-dioxygenase in strains PS11 and PN1.

Keywords: Biodegradation; Phenol; BTEX; Bacillus ; Streptomyces ; Soil model experiments

Improving ethanol fermentation performance of Saccharomyces cerevisiae in very high-gravity fermentation through chemical mutagenesis and meiotic recombination by Jing-Jing Liu; Wen-Tao Ding; Guo-Chang Zhang; Jing-Yu Wang (pp. 1239-1246).

Genome shuffling is an efficient way to improve complex phenotypes under the control of multiple genes. For the improvement of strain’s performance in very high-gravity (VHG) fermentation, we developed a new method of genome shuffling. A diploid ste2/ste2 strain was subjected to EMS (ethyl methanesulfonate) mutagenesis followed by meiotic recombination-mediated genome shuffling. The resulting haploid progenies were intrapopulation sterile and therefore haploid recombinant cells with improved phenotypes were directly selected under selection condition. In VHG fermentation, strain WS1D and WS5D obtained by this approach exhibited remarkably enhanced tolerance to ethanol and osmolarity, increased metabolic rate, and 15.12% and 15.59% increased ethanol yield compared to the starting strain W303D, respectively. These results verified the feasibility of the strain improvement strategy and suggested that it is a powerful and high throughput method for development of Saccharomyces cerevisiae strains with desired phenotypes that is complex and cannot be addressed with rational approaches.

Keywords: Saccharomyces cerevisiae ; Ethanol fermentation; Mutagenesis; Meiotic recombination; STE2

Sections

Personal tools

Applied Microbiology and Biotechnology (v.91, #4)

Sections

Personal tools

Local resources

Applied Microbiology and Biotechnology (v.91, #4)