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Applied Microbiology and Biotechnology (v.87, #5)

Current advances in Phellinus sensu lato: medicinal species, functions, metabolites and mechanisms by Yu-Cheng Dai; Li-Wei Zhou; Bao-Kai Cui; Yan-Qiu Chen; Cony Decock (pp. 1587-1593).

Twenty-six species of Phellinus sensu lato, reported as medicinal mushrooms, are enumerated in this review. The species’ names were checked and revised according to contemporary taxonomy and the latest version of the International Code of Botanical Nomenclature (Vienna Code); two misapplied names of Phellinus baumii Pilát and Phellinus himalayensis Y.C. Dai in previous reports are also discussed. Of the 20 types of medicinal functions, the most shared functions are antitumor and improving immunity, both of which may be viewed as the basal functions of Phellinus s. l. In addition, alleviating septic shock, anti-inflammatory, and antioxidation are also a routine functions mentioned often. The main medicinal metabolites, including several kinds of polysaccharides and polyphenols, are introduced. Different methods and conditions could purify various polysaccharides with difference in activity level even from the same species, while all polyphenols are hispidin and its derivatives in general. Three aspects of mechanism contribute to antitumor activities of polysaccharides: (1) promoting an immune response, (2) inducing cell apoptosis, and (3) inhibiting metastasis. Other general mechanisms of the metabolites in antioxidant activity, and in treating diabetes, as well as complications are summarized. We also elaborate on potential scientific strategies for obtaining the medicinal metabolites from Phellinus s. l., such as artificial cultivation, the discoveries of more species with medicinal functions, the utilization of species growing quickly, and the optimization of culture conditions and media supplements in fermentation.

Keywords: Phellinus sensu lato; Medicinal mushrooms; Polysaccharides; Polyphenols; Antitumor; Antioxidant

Dioxygen activation responsible for oxidation of aliphatic and aromatic hydrocarbon compounds: current state and variants by Masaaki Morikawa (pp. 1595-1603).

The most significant aspect in microbial metabolisms, especially those of bacteria and archaea, is their marvelously wide acceptability of substrate electron donors and acceptors. This feature makes them to be attractive catalysts for environmental biotechnology in terms of degradation of harmful recalcitrant compounds, including hydrocarbons. Transformation of highly reduced and inert hydrocarbon compounds is with no doubt a challenging biochemical reaction for a single enzyme. However, several multi-component enzyme systems enable microorganisms to utilize hydrocarbons as carbon and energy (electron) sources. Initial biological attack to hydrocarbons is, in most cases, the hydroxylation that requires molecular dioxygen as a co-substrate. Dioxygen also contributes to the ring cleavage reaction of homo- and hetero-cyclic aromatic hydrocarbons. Although the molecular dioxygen is omnipresent and highly soluble in water, activation and splitting this triplet ground-state molecule to wed with difficult hydrocarbons need special devices. Non-heme iron, heme iron, or flavin nucleotide was designated as a major hidden dagger for this purpose.

Keywords: Oxygenase; Rieske dioxygenase; Naphthalene dioxygenase; Alkane monooxygenase; P450; Hydrocarbon

Structural determinants of antimicrobial activity in polymers which mimic host defense peptides by Edmund F. Palermo; Kenichi Kuroda (pp. 1605-1615).

Antimicrobial polymers, designed to mimic the salient structural features of host defense peptides, are an emerging class of materials with potential for applications to combat infectious disease. Because the putative mode of action relies on physiochemical parameters of peptides such as hydrophobicity and cationic charge, rather than specific receptor-mediated interactions, the activity of the polymers can be modulated by tuning key structural parameters. While a wide diversity of chemical structures have been reported as antimicrobial polymers, a precise understanding of the structural factors which control their activity is a subject of current investigations. In this mini-review, we will outline the design principles that have been developed so far to fine tune the activity of these antimicrobial agents. The roles played by specific structural features such as cationic charge, hydrophobicity, and molecular weight will be discussed. Future directions of the field and potential challenges will be proposed.

Keywords: Antimicrobial; Polymer; Peptide mimic; Structure−activity; Membrane disruption; Cell penetrating

Engineering of glycosylation in yeast and other fungi: current state and perspectives by Karen De Pourcq; Kristof De Schutter; Nico Callewaert (pp. 1617-1631).

With the increasing demand for recombinant proteins and glycoproteins, research on hosts for producing these proteins is focusing increasingly on more cost-effective expression systems. Yeasts and other fungi are promising alternatives because they provide easy and cheap systems that can perform eukaryotic post-translational modifications. Unfortunately, yeasts and other fungi modify their glycoproteins with heterogeneous high-mannose glycan structures, which is often detrimental to a therapeutic protein’s pharmacokinetic behavior and can reduce the efficiency of downstream processing. This problem can be solved by engineering the glycosylation pathways to produce homogeneous and, if so desired, human-like glycan structures. In this review, we provide an overview of the most significant recently reported approaches for engineering the glycosylation pathways in yeasts and fungi.

Keywords: N-glycosylation engineering; O-glycosylation engineering; Yeast; Fungi; Biopharmaceuticals; Recombinant proteins

Improved production of A40926 by Nonomuraea sp. through deletion of a pathway-specific acetyltransferase by Margherita Sosio; Augusto Canavesi; Sofia Stinchi; Stefano Donadio (pp. 1633-1638).

Nonomuraea strain ATCC 39727 produces the glycopeptide A40926, used for manufacturing dalbavancin, currently in advanced clinical trials. From the gene cluster involved in A40926 biosynthesis, a strain deleted in dbv23 was constructed. This mutant can produce only the glycopeptides lacking the O-linked acetyl residue at position 6 of the mannose moiety, while, under identical fermentation conditions, the wild-type strain produces mostly glycopeptides carrying an acetylated mannose. Furthermore, the total amount of glycopeptides produced by the mutant strain was found to be approximately twice that of the wild type. The reduced level of glycopeptides observed in the wild-type strain may be due to an inhibitory effect exerted by the acetylated compound on the biosynthesis of A40926. Indeed, spiking production cultures with ≥1 μg/ml of the acetylated glycopeptide inhibited A40926 production in the mutant strain.

Keywords: Actinomycetes; Antibiotics; Glycopeptides

A novel biopesticide production: attagel-mediated precipitation of chitinase from Beauveria bassiana SFB-205 supernatant for thermotolerance by Jae Su Kim; Yeon Ho Je (pp. 1639-1648).

Insect killing fungi have high potential in controlling agriculturally harmful pest, but their slow progress and high variation in killing insect are major impediments to successful industrialization. The present work describes the use of supernatant from the liquid culture of Beauveria bassiana SFB-205 to surmount this problem, particularly efficient production of thermotolerant chitinase, which is one of the major pathogenesis-related enzymes in the supernatant. The chitinase was precipitated using varying mineral precipitants and followed by lyophilization, which was compared with a salting out method using ammonium sulfate in effectiveness. Incorporating of the supernatant fraction of the Beauveria preparation with attagel at 0.5% (w/v) as a precipitant enabled this treatment to show the greatest chitinase-precipitation efficiency (93.4%), followed up with excellent insecticidal activity against cotton aphids when it was mixed with 0.01% (v/v) polyoxyethylene-(3)-isotridecyl ether (TDE-3) as a spreading agent in laboratory conditions. Consequently, lyophilized attagel-mediated precipitation pellet was superior to lyophilized salting out pellet in maintaining chitinase activity against a thermal stress at 50°C. This finding provides that the attagel-mediated precipitation can be exploited to improve the thermotolerance of B. bassiana SFB-205 chitinase as a novel strategy for biopesticide production.

Keywords: Attagel; Beauveria bassiana ; Chitinase; Precipitation; Thermotolerance

Development of a stepwise aeration control strategy for efficient docosahexaenoic acid production by Schizochytrium sp. by Lu-Jing Ren; Xiao-Jun Ji; He Huang; Liang Qu; Yun Feng; Qian-Qian Tong; Ping-Kai Ouyang (pp. 1649-1656).

The effect of aeration on the performance of docosahexaenoic acid (DHA) production by Schizochytrium sp. was investigated in a 1,500-L bioreactor using fed-batch fermentation. Six parameters, including specific growth rate, specific glucose consumption rate, specific lipid accumulation rate, cell yield coefficient, lipid yield coefficient, and DHA yield coefficient, were used to understand the relationship between aeration and the fermentation characteristics. Based on the information obtained from the parameters, a stepwise aeration control strategy was proposed. The aeration rate was controlled at 0.4 volume of air per volume of liquid per minute (vvm) for the first 24 h, then shifted to 0.6 vvm until 96 h, and then switched back to 0.4 vvm until the end of the fermentation. High cell density (71 g/L), high lipid content (35.75 g/L), and high DHA percentage (48.95%) were achieved by using this strategy, and DHA productivity reached 119 mg/L h, which was 11.21% over the best results obtained by constant aeration rate.

Keywords: Docosahexaenoic acid; Schizochytrium sp.; Aeration control strategy; Fed-batch fermentation

Enhancing itaconic acid production by Aspergillus terreus by Gregor Tevž; Mojca Benčina; Matic Legiša (pp. 1657-1664).

Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium.

Keywords: Aspergillus terreus ; Itaconic acid; 6-Phosphofructo-1-kinase; Posttranslational modification; Primary metabolism; Anaplerosis

A comparison of laboratory and pilot-scale fermentations in winemaking conditions by Erick Casalta; Evelyne Aguera; Christian Picou; Juan-Jose Rodriguez-Bencomo; Jean-Michel Salmon; Jean-Marie Sablayrolles (pp. 1665-1673).

We investigated the influence of the fermenter size on alcoholic fermentation. Experiments were carried out at pilot scale, in 100-L fermenters, and at laboratory scale, in stirred and static 1-L fermenters. Two musts, Grenache blanc and Sauvignon, were fermented with and without the addition of solid particles from grape musts. Highly clarified must fermentation kinetics was strongly affected by the scale of the experiment, with slower fermentation occurring in the 100-L fermenter. Alcohol, ester, and thiol synthesis in clarified sauvignon must fermentation was also strongly correlated with the fermentation scale. Addition of solid particles from grape tended to reduce the effects on kinetics associated with increasing the scale of the fermentation, by increasing the maximum rate of CO₂ production, and by shortening the duration of fermentation. The addition of such particles also decreased the effects of scaling up the fermentation on the concentration of some volatile compounds, i.e., isoamyl acetate, ethyl octanoate, but did not decrease this effect for other compounds, such as isobutyl acetate, isobutanol, and 3-mercaptohexanol.

Keywords: Alcoholic fermentation; Yeasts; Wine; Fermenter size

Evaluation of biocathodes in freshwater and brackish sediment microbial fuel cells by Liesje De Schamphelaire; Pascal Boeckx; Willy Verstraete (pp. 1675-1687).

Biofilms on biocathodes can catalyze the cathodic oxygen reduction and accordingly guarantee high cathode redox potentials. The present research assessed the use of biocathodes in full-sediment microbial fuel cells. Carbon felt-based biocathodes were evaluated in freshwater systems, and an extension of their application to brackish systems and/or stainless steel webs as base material was considered. Efficient biocathodes could be developed within days through inoculation with active microorganisms. Carbon felt was found most suited for the biocathodes in freshwater with increased performance at salinities around 80–250 mM. Maximum long-term performance reached 12.3 µW cm⁻² cathode. The relative benefit of stainless steel seemed to increase with increasing salinity. A combination of stainless steel cathodes with biofilms could, however, also result in decreased electrical performance. In an efficiently catalyzing cathodic biofilm, an enrichment with an uncultured Proteobacterium—previously correlated with steel waste—was observed.

Keywords: Biocathode; Carbon; Stainless steel; Sediment microbial fuel cell

Quantitative analysis for the effect of plant oil and fatty acid on Tuber melanosporum culture by uniform design combined with partial least squares regression by Rui-Sang Liu; Ya-Jie Tang (pp. 1689-1697).

Uniform design and partial least squares regression were adopted to quantitatively analyze the effects of plant oil and fatty acid as well as their addition amount and addition time on the performance of Tuber melanosporum submerged fermentation. The regression models showed the optimal scheme was the addition of 1.2 mL soybean oil on day 9, which was validated by experiment. The maximal biomass of 25.89 ± 1.01 g/L and extracellular polysaccharides (EPS) production of 6.51 ± 0.68 g/L were obtained, which were enhanced by 18.5% and 86%, respectively. Palmitic acid was identified to be the key component to stimulate the cell growth and EPS biosynthesis, and stearic acid was beneficial for the production of intracellular polysaccharides. Not only the biomass but also EPS production obtained in this work are the highest reported in the batch fermentation of truffle.

Keywords: Truffle; Tuber melanosporum ; Tuber polysaccharides; Plant oil; Fatty acid; Uniform design; Partial least squares regression

Open circuit versus closed circuit enrichment of anodic biofilms in MFC: effect on performance and anodic communities by Amor Larrosa-Guerrero; Keith Scott; Krishna P. Katuri; Carlos Godinez; Ian M. Head; Thomas Curtis (pp. 1699-1713).

The influence of various carbon anodes; graphite, sponge, paper, cloth, felt, fiber, foam and reticulated vitreous carbon (RVC); on microbial fuel cell (MFC) performance is reported. The feed was brewery wastewater diluted in domestic wastewater. Biofilms were grown at open circuit or under an external load. Microbial diversity was analysed as a function of current and anode material. The bacterial community formed at open circuit was influenced by the anode material. However at closed circuit its role in determining the bacterial consortia formed was less important than the passage of current. The rate and extent of organic matter removal were similar for all materials: over 95% under closed circuit. The biofilm in MFCs working at open circuit and in the control reactors, increased COD removal by up to a factor of nine compared with that for baseline reactors. The average voltage output was 0.6 V at closed circuit, with an external resistor of 300 kΩ and 0.75 V at open circuit for all materials except RVC. The poor performance of this material might be related to the surface area available and concentration polarizations caused by the morphology of the material and the structure of the biofilm. Peak power varied from 1.3 mW m⁻² for RVC to 568 mW m⁻² for graphite with biofilm grown at closed circuit.

Keywords: Microbial fuel cell; Wastewater treatment; Bacteria selection; Anode; Carbon materials

Improving the catalytic activity of hyperthermophilic Pyrococcus prolidases for detoxification of organophosphorus nerve agents over a broad range of temperatures by Casey M. Theriot; Xuelian Du; Sherry R. Tove; Amy M. Grunden (pp. 1715-1726).

Prolidase isolated from the hyperthermophilic archaeon Pyrococcus furiosus has potential for application for decontamination of organophosphorus compounds in certain pesticides and chemical warfare agents under harsh conditions. However, current applications that use an enzyme-based cocktail are limited by poor long-term enzyme stability and low reactivity over a broad range of temperatures. To obtain a better enzyme for OP nerve agent decontamination and to investigate structural factors that influence protein thermostability and thermoactivity, randomly mutated P. furiosus prolidases were prepared by using XL1-red-based mutagenesis and error-prone PCR. An Escherichia coli strain JD1 (λDE3) (auxotrophic for proline [ΔproA] and having deletions in pepQ and pepP dipeptidases with specificity for proline-containing dipeptides) was constructed for screening mutant P. furiosus prolidase expression plasmids. JD1 (λDE3) cells were transformed with mutated prolidase expression plasmids and plated on minimal media supplemented with 50 μM Leu-Pro as the only source of proline. By using this positive selection, Pyrococcus prolidase mutants with improved activity over a broader range of temperatures were isolated. The activities of the mutants over a broad temperature range were measured for both Xaa-Pro dipeptides and OP nerve agents, and the thermoactivity and thermostability of the mutants were determined.

Keywords: Prolidase; Pyrococcus furiosus ; OP nerve agents; Mutagenesis; Directed evolution

Enhancement of hydrogen peroxide stability of a novel Anabaena sp. DyP-type peroxidase by site-directed mutagenesis of methionine residues by Henry Joseph Oduor Ogola; Naoya Hashimoto; Suguru Miyabe; Hiroyuki Ashida; Takahiro Ishikawa; Hitoshi Shibata; Yoshihiro Sawa (pp. 1727-1736).

Previous reports have shown that a unique bacterial dye-decolorizing peroxidase from the cyanobacterium Anabaena sp. strain PCC7120 (AnaPX) efficiently oxidizes both recalcitrant anthraquinone dyes (AQ) and typical aromatic peroxidase substrates. In this study, site-directed mutagenesis to replace five Met residues in AnaPX with high redox residues Ile, Leu, or Phe was performed for the improvement of the enzyme stability toward H₂O₂. The heme cavity mutants M401L, M401I, M401F, and M451I had significantly increased H₂O₂ stabilities of 2.4-, 3.7-, 8.2-, and 5.2-fold, respectively. Surprisingly, the M401F and M451I retained 16% and 5% activity at 100 mM H₂O₂, respectively, in addition to maintaining high dye-decolorization activity toward AQ and azo dyes at 5 mM H₂O₂ and showing a slower rate of heme degradation than the wildtype enzyme. The observed stabilization of AnaPX may be attributed to the replacement of potentially oxidizable Met residues either increasing the local stability of the heme pocket or limiting of the self-inactivation electron transfer pathways due to the above mutations. The increased stability of AnaPX variants coupled with the broad substrate specificity can be potentially useful for the further practical application of these enzymes especially in bioremediation of wastewater contaminated with recalcitrant AQ.

Keywords: DyP-type peroxidase; Oxidative stability; Methionine residue; Site-directed mutagenesis; Bioremediation

Genetic and functional aspects of linoleate isomerase in Lactobacillus acidophilus by Martin Macouzet; Normand Robert; Byong H. Lee (pp. 1737-1742).

While the remarkable health effects of conjugated linoleic acid (CLA) catalyzed from α-linoleic acid by the enzyme linoleate isomerase (LI, EC 5.2.1.5) are well recognized, how widely this biochemical activity is present and the mechanisms of its regulation in lactic acid bacteria are unknown. Although certain strains of Lactobacillus acidophilus can enrich CLA in fermented dairy products, it is unknown if other strains share this capacity. Due to its immense economic importance, this work aimed to investigate genetic aspects of CLA production in L. acidophilus for the first time. The genomic DNA from industrial and type strains of L. acidophilus were subjected to PCR and immunoblot analyses using the putative LI gene of L. reuteri ATCC 55739 as probe. The CLA production ability was estimated by gas chromatography of the biomass extracts. The presumptive LI gene from L. acidophilus ATCC 832 was isolated and sequenced. The resulting sequence shared 71% identity with that of L. reuteri and at least 99% with reported sequences from other L. acidophilus strains. All the strains accumulated detectable levels of CLA and tested positive by PCR and immunoblotting. However, no apparent correlation was observed between the yields and the hybridization patterns. The results suggest that LI activity might be common among L. acidophilus and related species and provide a new tool for screening potential CLA producers.

Keywords: CLA production; Conjugated linoleic acid; Lactobacillus acidophilus ; Linoleate isomerase

Engineering of choline oxidase from Arthrobacter nicotianae for potential use as biological bleach in detergents by Doris Ribitsch; Sonja Winkler; Karl Gruber; Wolfgang Karl; Eva Wehrschütz-Sigl; Inge Eiteljörg; Petra Schratl; Peter Remler; Regina Stehr; Cornelius Bessler; Nina Mußmann; Kerstin Sauter; Karl Heinz Maurer; Helmut Schwab (pp. 1743-1752).

In order to engineer the choline oxidase from Arthrobacter nicotianae (An_CodA) for the potential application as biological bleach in detergents, the specific activity of the enzyme toward the synthetic substrate tris-(2-hydroxyethyl)-methylammonium methylsulfate (MTEA) was improved by methods of directed evolution and rational design. The best mutants (up to 520% wt-activity with MTEA) revealed mutations in the FAD- (A21V, G62D, I69V) and substrate-binding site (S348L, V349L, F351Y). In a separate screening of a library comprising of randomly mutagenised An_CodA, with the natural substrate choline, four mutations were identified, which were further combined in one clone. The constructed clone showed improved activity towards both substrates, MTEA and choline. Mapping these mutation sites onto the structural model of An_CodA revealed that Phe351 is positioned right in the active site of An_CodA and very likely interacts with the bound substrate. Ala21 is part of an α-helix which interacts with the diphosphate moiety of the flavin cofactor and might influence the activity and specificity of the enzyme.

Keywords: Choline oxidase; Arthrobacter nicotianae ; Directed evolution; Biological bleaching; Detergents; Hydrogen peroxide

Hydrolysis of cyclic poly(ethylene terephthalate) trimers by a carboxylesterase from Thermobifida fusca KW3 by Susan Billig; Thorsten Oeser; Claudia Birkemeyer; Wolfgang Zimmermann (pp. 1753-1764).

We have identified a carboxylesterase produced in liquid cultures of the thermophilic actinomycete Thermobifida fusca KW3 that were supplemented with poly(ethylene terephthalate) fibers. The enzyme hydrolyzed highly hydrophobic, synthetic cyclic poly(ethylene terephthalate) trimers with an optimal activity at 60°C and a pH of 6. V _max and K _m values for the hydrolysis were 9.3 µmol⁻¹ min⁻¹ mg⁻¹ and 0.5 mM, respectively. The esterase showed high specificity towards short and middle chain-length fatty acyl esters of p-nitrophenol. The enzyme retained 37% of its activity after 96 h of incubation at 50°C and a pH of 8. Enzyme inhibition studies and analysis of substitution mutants of the carboxylesterase revealed the typical catalytic mechanism of a serine hydrolase with a catalytic triad composed of serine, glutamic acid, and histidine.

Keywords: Thermobifida fusca ; Carboxylesterase; Cutinase; Poly(ethylene terephthalate) (PET); Enzymatic hydrolysis

Functional expression of a thermophilic glucuronoyl esterase from Sporotrichum thermophile: identification of the nucleophilic serine by Evangelos Topakas; Maria Moukouli; Maria Dimarogona; Christina Vafiadi; Paul Christakopoulos (pp. 1765-1772).

A glucuronoyl esterase (GE) from the thermophilic fungus Sporotrichum thermophile, belonging to the carbohydrate esterase family 15 (CE-15), was functionally expressed in the methylotrophic yeast Pichia pastoris. The putative GE gene ge2 from the genomic DNA was successfully cloned in frame with the sequence for the Saccharomyces cerevisiae α-factor secretion signal under the transcriptional control of the alcohol oxidase (AOX1) promoter and integrated in P. pastoris X-33 to confirm that the encoded enzyme StGE2 exhibits esterase activity. The enzyme was active on substrates containing glucuronic acid methyl ester, showing optimal activity at pH 7.0 and 55°C. The esterase displayed broad pH range stability between 4–10 and temperature stability up to 50°C, rendering StGE2 a strong candidate for future biotechnological applications that require robust biocatalysts. ClustalW alignment of StGE2 with characterized GEs and selected homologous sequences, members of CE-15 family, revealed a novel consensus sequence G-C-S-R-X-G that features the characteristic serine residue involved in the generally conserved catalytic mechanism of the esterase family. The putative serine has been mutated, and the corresponding enzyme has been expressed in P. pastoris to prove that the candidate nucleophilic residue is responsible for catalyzing the enzymatic reaction.

Keywords: Glucuronoyl esterase; Nucleophilic serine; Active site; Lignin–carbohydrate complex; Sporotrichum thermophile ; Pichia pastoris

Engineering of a fungal β-galactosidase to remove product inhibition by galactose by Xuejun Hu; Sylvain Robin; Shane O’Connell; Gary Walsh; J. Gerard Wall (pp. 1773-1782).

β-galactosidase is an enzyme administered as a digestive supplement to treat lactose intolerance, a genetic condition prevalent in most world regions. The gene encoding an acid-stable β-galactosidase potentially suited for use as a digestive supplement was cloned from Aspergillus niger van Tiegh, sequenced and expressed in Pichia pastoris. The purified recombinant protein exhibited kinetic properties similar to those of the native enzyme and thus was also competitively inhibited by its product, galactose, at application-relevant concentrations. In order to alleviate this product inhibition, a model of the enzyme structure was generated based on a Penicillium sp. β-galactosidase crystal structure with bound β-galactose. This led to targeted mutagenesis of an Asp²⁵⁸-Ser-Tyr-Pro-Leu-Gly-Phe amino acid motif in the A. niger van Tiegh enzyme and isolation from the resultant library of a mutant β-galactosidase enzyme with reduced sensitivity to inhibition by galactose (K _i of 6.46 mM galactose, compared with 0.76 mM for the wildtype recombinant enzyme). The mutated enzyme also exhibited an increased K _m (3.76 mM compared to 2.21 mM) and reduced V _max (110.8 μmol min⁻¹ mg⁻¹ compared to 172.6 μmol min⁻¹ mg⁻¹) relative to the wild-type enzyme, however, and its stability under simulated fasting gastric conditions was significantly reduced. The study nevertheless demonstrates the potential to rationally engineer the A. niger van Tiegh enzyme to relieve product inhibition and create mutants with improved, application-relevant kinetic properties for treatment of lactose intolerance.

Keywords: β-galactosidase; Lactase; Aspergillus niger van Tiegh; Mutagenesis; Product inhibition; Galactose

Display of both N- and C-terminal target fusion proteins on the Aspergillus oryzae cell surface using a chitin-binding module by Soichiro Tabuchi; Junji Ito; Takashi Adachi; Hiroki Ishida; Yoji Hata; Fumiyoshi Okazaki; Tsutomu Tanaka; Chiaki Ogino; Akihiko Kondo (pp. 1783-1789).

A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein.

Keywords: Cell surface display; Aspergillus oryzae ; Chitin-binding module; Triacylglycerol lipase

Eryngase: a Pleurotus eryngii aminopeptidase exhibiting peptide bond formation activity by Jiro Arima; Masanori Chiba; Tsuyoshi Ichiyanagi; Yukinori Yabuta; Nobuhiro Mori; Tadanori Aimi (pp. 1791-1801).

An aminopeptidase that has peptide bond formation activity was identified in the cell-free extract of carpophore of Pleurotus eryngii. The enzyme, redesignated as eryngase, was purified for homogeneity and characterized. Eryngase had a molecular mass of approximately 79 kDa. It showed somewhat high stability with respect to temperature and pH; it was inhibited by iodoacetate. Among hydrolytic activities toward aminoacyl-p-nitroanilides (aminoacyl-pNAs), eryngase mainly hydrolyzed hydrophobic l-aminoacyl-pNAs and exhibited little activity toward d-Ala-pNA and d-Leu-pNA. In terms of peptide bond formation activity, eryngase used various aminoacyl derivatives as acyl donors and acceptors. The products were all dipeptidyl derivatives. Investigation of time dependence on peptide synthesis revealed that some peptides that are not recognized as substrates for hydrolytic activity of eryngase could become good targets for synthesis. Furthermore, eryngase has produced opioid dipeptides––l-kyotorphin (l-Tyr-l-Arg) and d-kyotorphin (l-Tyr-d-Arg)––using l-Tyr-NH₂ and d- and l-Arg-methyl ester respectively as an acyl donor and acceptor. Yield evaluation of kyotorphin synthesis indicated that the conversion ratio of substrate to kyotorphin was moderate: the value was estimated as greater than 20%.

Keywords: Aminopeptidase; Peptide bond formation; Pleurotus eryngii ; Aminolysis

Establishment of l-arabinose fermentation in glucose/xylose co-fermenting recombinant Saccharomyces cerevisiae 424A(LNH-ST) by genetic engineering by Aloke Kumar Bera; Miroslav Sedlak; Aftab Khan; Nancy W. Y. Ho (pp. 1803-1811).

Cost-effective and efficient ethanol production from lignocellulosic materials requires the fermentation of all sugars recovered from such materials including glucose, xylose, mannose, galactose, and l-arabinose. Wild-type strains of Saccharomyces cerevisiae used in industrial ethanol production cannot ferment d-xylose and l-arabinose. Our genetically engineered recombinant S. cerevisiae yeast 424A(LNH-ST) has been made able to efficiently ferment xylose to ethanol, which was achieved by integrating multiple copies of three xylose-metabolizing genes. This study reports the efficient anaerobic fermentation of l-arabinose by the derivative of 424A(LNH-ST). The new strain was constructed by over-expression of two additional genes from fungi l-arabinose utilization pathways. The resulting new 424A(LNH-ST) strain exhibited production of ethanol from l-arabinose, and the yield was more than 40%. An efficient ethanol production, about 72.5% yield from five-sugar mixtures containing glucose, galactose, mannose, xylose, and arabinose was also achieved. This co-fermentation of five-sugar mixture is important and crucial for application in industrial economical ethanol production using lignocellulosic biomass as the feedstock.

Keywords: l-arabinose; Pentose fermentation; l-arabitol 4-dehydrogenase; l-xylulose reductase; Saccharomyces cerevisiae ; Ethanol

Heterologous co-production of Thermobifida fusca Cel9A with other cellulases in Saccharomyces cerevisiae by Niel van Wyk; Riaan den Haan; Willem H. van Zyl (pp. 1813-1820).

The processive endoglucanase Cel9A of the moderately thermophilic actinomycete Thermobifida fusca was functionally produced in Saccharomyces cerevisiae. Recombinant Cel9A displayed activity on both soluble (carboxymethylcellulose) and insoluble (Avicel) cellulose substrates confirming its processive endoglucanase activity. High-performance anionic exchange chromatography analyses of soluble sugars released from Avicel revealed a cellobiose/glucose ratio of 2.5 ± 0.1. Growth by the recombinant strain on amorphous cellulose was possible due to the sufficient amount of glucose cleaved from the cellulose chain. This is the first confirmed report of S. cerevisiae growing on a cellulosic substrate as sole carbohydrate source while only expressing one recombinant gene. To improve the cellulolytic capability of S. cerevisiae and to investigate the level of synergy among cellulases produced by a recombinant host, the cel9A gene was co-expressed with four cellulase-coding genes of Trichoderma reesei: two endoglucanases cel5A (egII) and cel7B (egI), and two cellobiohydrolases cel6A (cbhII) and cel7A (cbhI). Synergy, especially between the Cel9A and the two cellobiohydrolases, resulted in a higher cellulolytic capability of the recombinant host.

Keywords: Saccharomyces cerevisiae ; Consolidated bioprocessing; Co-expression; Cellulases; Phosphoric acid swollen cellulose

Global transcription engineering of brewer’s yeast enhances the fermentation performance under high-gravity conditions by Cuijuan Gao; Zhikun Wang; Quanfeng Liang; Qingsheng Qi (pp. 1821-1827).

Global transcription engineering was developed as a tool to reprogram gene transcription for eliciting new phenotypes important for technological applications (Science 2006, 314(5805):1565-1568). A recent report indicated that the beneficial growth advantage of yeast cells expressing the SPT15-300 mutation is the result of enhanced uptake and/or improved utilization of leucine and thus was seen only on defined media with low concentrations of leucine (Appl Environ Microbiol 2009, 75(19):6055-6061). Further investigation towards a leucine-prototrophic strain of industrial lager brewer’s yeast indicated that integration one copy of SPT15-300 in SPT15 allele, however, did lead to an increased ethanol tolerance on complex rich medium at high gravity fermentation condition. Under brewing conditions, the SPT15-300 mutant produced 80.78 g/L ethanol from 200 g/L carbohydrates after 384 h, almost twice as much as that of the wild-type strain. The results convinced us that the effect of global regulator modification of yeast is at multi-genes level and is extremely complicated.

Keywords: Brewer’s yeast; gTME; Ethanol; Metabolic engineering; High gravity fermentation

Use of the Aspergillus oryzae actin gene promoter in a novel reporter system for exploring antifungal compounds and their target genes by Junichiro Marui; Akira Yoshimi; Daisuke Hagiwara; Yoshimi Fujii-Watanabe; Ken Oda; Hideaki Koike; Koichi Tamano; Tomoko Ishii; Motoaki Sano; Masayuki Machida; Keietsu Abe (pp. 1829-1840).

Demand for novel antifungal drugs for medical and agricultural uses has been increasing because of the diversity of pathogenic fungi and the emergence of drug-resistant strains. Genomic resources for various living species, including pathogenic fungi, can be utilized to develop novel and effective antifungal compounds. We used Aspergillus oryzae as a model to construct a reporter system for exploring novel antifungal compounds and their target genes. The comprehensive gene expression analysis showed that the actin-encoding actB gene was transcriptionally highly induced by benomyl treatment. We therefore used the actB gene to construct a novel reporter system for monitoring responses to cytoskeletal stress in A. oryzae by introducing the actB promoter::EGFP fusion gene. Distinct fluorescence was observed in the reporter strain with minimum background noise in response to not only benomyl but also compounds inhibiting lipid metabolism that is closely related to cell membrane integrity. The fluorescent responses indicated that the reporter strain can be used to screen for lead compounds affecting fungal microtubule and cell membrane integrity, both of which are attractive antifungal targets. Furthermore, the reporter strain was shown to be technically applicable for identifying novel target genes of antifungal drugs triggering perturbation of fungal microtubules or membrane integrity.

Keywords: Antifungal; Cytoskeleton; Actin; Reporter; Aspergillus oryzae

Construction of a β-glucosidase expression system using the multistress-tolerant yeast Issatchenkia orientalis by Takao Kitagawa; Kenro Tokuhiro; Hidehiko Sugiyama; Katsuhiro Kohda; Naoto Isono; Makoto Hisamatsu; Haruo Takahashi; Takao Imaeda (pp. 1841-1853).

We demonstrate the value of the thermotolerant yeast Issatchenkia orientalis as a candidate microorganism for bioethanol production from lignocellulosic biomass with the goal of consolidated bioprocessing. The I. orientalis MF-121 strain is acid tolerant, ethanol tolerant, and thermotolerant, and is thus a multistress-tolerant yeast. To express heterologous proteins in I. orientalis, we constructed a transformation system for the MF-121 strain and then isolated the promoters of TDH1 and PGK1, two genes that were found to be strongly expressed during ethanol fermentation. As a result, expression of β-glucosidase from Aspergillus aculeatus could be achieved with I. orientalis, demonstrating successful heterologous gene expression in I. orientalis for the first time. The transformant could convert cellobiose to ethanol under acidic conditions and at high temperature. Simultaneous saccharification and fermentation (SSF) was performed with the transformant, which produced 29 g l⁻¹ of ethanol in 72 h at 40°C even without addition of β-glucosidase when SSF was carried out in medium containing 100 g l⁻¹ of microcrystalline cellulose and a commercial cellulase preparation. These results suggest that using a genetically engineered thermotolerant yeast such as I. orientalis in SSF could lead to cost reduction because less saccharification enzymes are required.

Keywords: Thermotolerant yeast; Issatchenkia orientalis ; Microcrystalline cellulose; Ethanol; Beta-glucosidase

Characterization of a 24-kb plasmid pCGR2 newly isolated from Corynebacterium glutamicum by Yoshiki Tsuchida; Sakurako Kimura; Nobuaki Suzuki; Masayuki Inui; Hideaki Yukawa (pp. 1855-1866).

A 24-kb plasmid with 21 open reading frames (ORFs) was newly isolated from Corynebacterium glutamicum ATCC 14997 and named pCGR2. Three of its ORFs were indispensable for stable autonomous replication of pCGR2 in C. glutamicum: in the absence of selective pressure, deletion derivatives of pCGR2 containing the three ORFs showed stability in C. glutamicum for over 50 generations. The first of these ORFs encoded replicase repA whose gene product revealed high amino acid sequence similarity to corresponding gene products of C. glutamicum pCG1-family plasmids in general, and to that of pTET3 plasmid repA in particular. The other two ORFs were located upstream of repA and exhibited high sequence similarity to pTET3 parA and parB, respectively. Interestingly, plasmids based on the pCGR2 were compatible not only with those based on different family plasmids (pBL1, pCASE1) but also with those based on pCG1-family plasmid. Plasmids comprising pCGR2 repA showed a copy number of four in C. glutamicum. The number increased to 240 upon introduction of a mutation within the repA origin of the putative promoter for counter-transcribed RNA. This 60-fold increase in copy number should immensely contribute towards enhanced expression of desired genes in C. glutamicum.

Keywords: Plasmid; pCG1 family; C. glutamicum ; Partitioning genes; Copy number; Shuttle vector

OdhI dephosphorylation kinetics during different glutamate production processes involving Corynebacterium glutamicum by Kenza-Amel Boulahya; Emmanuel Guedon; Stéphane Delaunay; Christian Schultz; Joseph Boudrant; Michael Bott; Jean-Louis Goergen (pp. 1867-1874).

In Corynebacterium glutamicum, the activity of the 2-oxoglutarate dehydrogenase complex was shown to be controlled by the phosphorylation of a 15-kDa protein OdhI by different serine/threonine protein kinases. In this paper, the phosphorylation status and kinetics of OdhI dephosphorylation were assessed during glutamate producing processes triggered by either a biotin limitation or a temperature upshock from 33°C to 39°C. A dephosphorylation of OdhI in C. glutamicum 2262 was observed during the biotin-limited as well as the temperature-induced glutamate-producing process. Deletion of pknG in C. glutamicum 2262 did not affect the phosphorylation status of OdhI during growth and glutamate production phases triggered by a temperature upshock, though a 40% increase in the specific glutamate production rate was measured. These results suggest that, under the conditions analyzed, PknG is not the kinase responsible for the phosphorylation of OdhI in C. glutamicum 2262. The phosphorylation status of OdhI alone is, as expected, not the only parameter that determines the performance of a specific strain, as no clear relation between the specific glutamate production rate and OdhI phosphorylation level was demonstrated.

Keywords: Corynebacterium glutamicum ; Glutamate; OdhI; Biotin limitation; Temperature increase; PknG

Survival of bacteria on metallic copper surfaces in a hospital trial by André Mikolay; Susanne Huggett; Ladji Tikana; Gregor Grass; Jörg Braun; Dietrich H. Nies (pp. 1875-1879).

Basic chemistry of copper is responsible for its Janus-faced feature: on one hand, copper is an essential trace element required to interact efficiently with molecular oxygen. On the other hand, interaction with reactive oxygen species in undesired Fenton-like reactions leads to the production of hydroxyl radicals, which rapidly damage cellular macromolecules. Moreover, copper cations strongly bind to thiol compounds disturbing redox-homeostasis and may also remove cations of other transition metals from their native binding sites in enzymes. Nature has learned during evolution to deal with the dangerous yet important copper cations. Bacterial cells use different efflux systems to detoxify the metal from the cytoplasm or periplasm. Despite this ability, bacteria are rapidly killed on dry metallic copper surfaces. The mode of killing likely involves copper cations being released from the metallic copper and reactive oxygen species. With all this knowledge about the interaction of copper and its cations with cellular macromolecules in mind, experiments were moved to the next level, and the antimicrobial properties of copper-containing alloys in an “everyday” hospital setting were investigated. The alloys tested decreased the number of colony-forming units on metallic copper-containing surfaces by one third compared to control aluminum or plastic surfaces. Moreover, after disinfection, repopulation of the surfaces was delayed on copper alloys. This study bridges a gap between basic research concerning cellular copper homeostasis and application of this knowledge. It demonstrates that the use of copper-containing alloys may limit the spread of multiple drug-resistant bacteria in hospitals.

Keywords: Copper; Copper surfaces; Ciprofloxacin-resistant Staphylococcus (CRS); Methicillin-resistant Staphylococcus aureus (MRSA)

Production and characterization of a group of bioemulsifiers from the marine Bacillus velezensis strain H3 by Xiangyang Liu; Biao Ren; Ming Chen; Haibin Wang; Chandrakant R. Kokare; Xianlong Zhou; Jidong Wang; Huanqin Dai; Fuhang Song; Mei Liu; Jian Wang; Shujin Wang; Lixin Zhang (pp. 1881-1893).

Marine microbes are a rich source of bioactive compounds, such as drugs, enzymes, and biosurfactants. To explore the bioactive compounds from our marine natural product library, an oil emulsification assay was applied to discover biosurfactants and bioemulsifiers. A spore-forming bacterial strain from sea mud was found to produce bioemulsifiers with good biosurfactant activity and a broad spectrum of antimicrobial properties. It was identified as Bacillus velezensis H3 using genomic and phenotypic data analysis. This strain was able to produce biosurfactants with an optimum emulsification activity at pH 6.0 and 2% NaCl by using starch as the carbon source and ammonium sulfate as the nitrogen source. The emulsification-guided isolation and purification procedure led to the discovery of the biosurfactant components, which were mainly composed of nC₁₄-surfactin and anteisoC₁₅-surfactin as determined by NMR and MS spectra. These compounds can reduce the surface tension of phosphate-buffered saline (PBS) from 71.8 to 24.8 mN/m. The critical micelle concentrations (CMCs) of C₁₄-surfactin and C₁₅-surfactin in 0.1 M PBS (pH 8.0) were determined to be 3.06 × 10^-5 and 2.03 × 10^-5 mol/L, respectively. The surface tension values at CMCs for C₁₄-surfactin and C₁₅-surfactin were 25.7 and 27.0 mM/m, respectively. In addition, the H3 biosurfactant exhibited antimicrobial activities against Staphyloccocus aureus, Mycobacterium, Klebsiella peneumoniae, Pseudomonas aeruginosa, and Candida albicans. Thus B. velezensis H3 is an alternative surfactin producer with potential application as an industrial strain for the lipopeptide production.

Keywords: Bacillus velezensis ; Biosurfactant; Lipopeptide; Surfactin; Bioemulsifier

All genomic mutations in the antimicrobial surfactant-resistant mutant, Escherichia coli OW66, are involved in cell resistance to surfactant by Kunihiro Nakata; Myo Myoung Koh; Tetsuaki Tsuchido; Yoshinobu Matsumura (pp. 1895-1905).

The spontaneous antimicrobial surfactant-resistant mutant, Escherichia coli OW66, has been isolated, and its physiological properties have been characterized in our previous paper (Ishikawa et al., J Appl Microbiol 92:261–268, 2002b). This report revealed that strain OW66 had seven mutations in their chromosomal DNA by comparative genomic hybridization microarray, and that their alternative functions were involved in cell resistance to antimicrobial surfactants. These mutations were located in oppB, ydcR, IVR(vacJ-yfdC), rpoN, rpoB, rpoC, and soxR. Furthermore, seven of the single-mutated isogenic strains and seven of the six-mutated isogenic strains were constructed from strains OW6 (NBRC106482) and OW66, respectively, through homologous recombination, and their resistances to an antimicrobial surfactant were measured using the minimum inhibitory concentration method. These results revealed that all six-mutated strains were more sensitive than strain OW66, and that the soxR66 mutation was independently involved in antimicrobial surfactant resistance of E. coli cells. Expression of soxR66 and soxS was increased in both strains OW66 and OW6-soxR66 without the surfactant treatment by the quantitative real time-polymerase chain reaction analysis, compared with strain OW6. Two-dimensional polyacrylamide gel electrophoresis analysis also revealed that some proteins in the soxRS regulon, including Mn-SOD, were overexpressed in both strains OW66 and OW6-soxR66. These results indicate that the soxR66 mutation leads to the constitutive expression of the soxRS regulon, resulting in the acquired resistance of E. coli cells to an antimicrobial surfactant.

Keywords: Spontaneous mutant; Quaternary ammonium compound; Antimicrobial surfactant; Cetyltrimethylammonium bromide; soxRS regulon; Comparative genomic hybridization microarray

Cytochrome P450 monooxygenases involved in anthracene metabolism by the white-rot basidiomycete Phanerochaete chrysosporium by Nomathemba Loice Chigu; Sinji Hirosue; Chie Nakamura; Hiroshi Teramoto; Hirofumi Ichinose; Hiroyuki Wariishi (pp. 1907-1916).

Cytochrome P450 monooxygenases (P450s) involved in anthracene metabolism by the white-rot basidiomycete Phanerochaete chrysosporium were identified by comprehensive screening of both catalytic potentials and transcriptomic profiling. Functional screening of P. chrysosporium P450s (PcCYPs) revealed that 14 PcCYP species catalyze stepwise conversion of anthracene to anthraquinone via intermediate formation of anthrone. Moreover, transcriptomic profiling explored using a complementary DNA microarray system demonstrated that 12 PcCYPs are up-regulated in response to exogenous addition of anthracene. Among the up-regulated PcCYPs, five species showed catalytic activity against anthracene. Based upon both catalytic and transcriptional properties, these five species are most likely to play major roles in anthracene metabolic processes in vivo. Thus, the combination of functional screening and a microarray system may provide a novel strategy for obtaining a thorough understanding of the catalytic functions and biological impacts of PcCYPs.

Keywords: White-rot basidiomycete; Cytochrome P450; Transcriptional regulation; Substrate specificity; Bioremediation

Antibacterial activity of ZnO nanoparticles prepared via non-hydrolytic solution route by Rizwan Wahab; Amrita Mishra; Soon-Il Yun; Young-Soon Kim; Hyung-Shik Shin (pp. 1917-1925).

The antibacterial activity of ZnO nanoparticles has been investigated and presented in this paper. Nanoparticles were prepared via non-hydrolytic solution process using zinc acetate di-hydrate (Zn(CH₃COO)₂·2H₂O) and aniline (C₆H₅NH₂) in 6 h refluxing at ∼65 °C. In the presence of four pathogens such as Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae, the antibacterial study of zinc oxide nanoparticles were observed. The antibacterial activity of ZnO nanoparticles (ZnO-NPs) were studied by spectroscopic method taking different concentrations (5–45 μg/ml) of ZnO-NPs. Our investigation reveals that the lowest concentration of ZnO-NPs solution inhibiting the growth of microbial strain is found to be 5 μg/ml for K. pneumoniae, whereas for E. coli, S. aureus, and S. typhimurium, it was calculated to be 15 μg/ml. The diameter of each ZnO-NPs lies between “20 and 30 nm” as observed from FESEM and transmission electron microscopy images. The composition of synthesized material was analyzed by the Fourier transform infrared spectroscopy, and it shows the band of ZnO at 441 cm⁻¹. Additionally, on the basis of morphological and chemical observations, the chemical reaction mechanism of ZnO-NPs was also proposed.

Keywords: Pathogenic bacteria; X-ray diffraction pattern (XRD); PL spectroscopy; FTIR spectroscopy; ZnO nanoparticles and antibacterial activity

Flotation as a tool for indirect DNA extraction from soil by Nádia Skorupa Parachin; Jenny Schelin; Börje Norling; Peter Rådström; Marie F. Gorwa-Grauslund (pp. 1927-1933).

Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 μg DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences.

Keywords: DNA extraction; Soil; Humic acid and flotation

High efficiency preparation of bioactive human α-defensin 6 in Escherichia coli Origami(DE3)pLysS by soluble fusion expression by Aiping Wang; Yongping Su; Song Wang; Mingqiang Shen; Fang Chen; Mo Chen; Xinze Ran; Tianmin Cheng; Junping Wang (pp. 1935-1942).

Human α-defensin 6 (HD₆), a small cysteine-rich cationic peptide specially expressed in epithelial cells of digestive tract, may play a crucial role in mucosal immunity. This is the first report on efficient production of bioactive HD₆ through a gene-engineering approach in Escherichia coli. The recombinant plasmid pET32a-omHD₆ was primarily constructed by inserting a PCR fragment encoding mature HD₆ peptide (mHD₆) preceded by an enterokinase recognition sequence into the expression vector pET32a(+), in frame with the upstream thioredoxin (TrxA) gene. Under optimized expression conditions, a high percentage (>60%) of soluble TrxA-omHD₆ fusion protein was obtained with a yield of about 1.69 g/l, and the theoretical productivity of recombinant mHD₆ (rmHD₆) reached 0.38 g/l. A feasible three-step purification strategy involving nickel-sepharose chromatography, enterokinase-cleavage and cation exchange chromatography was developed to purify rmHD₆, followed by characteristic identifications by Western blot, mass spectrometry and sequencing. About 102 mg/l of rmHD₆ with its intact N-terminal amino acid sequence was finally achieved. The in vitro experiments showed that rmHD₆ possesses high potency to inhibit herpes simplex virus-2 infection. This work settles substantial foundation for further functional study of HD₆.

Keywords: Human α-defensin 6; Expression; Purification; Escherichia coli ; Antiviral activity

Bioaugmentation treatment for coking wastewater containing pyridine and quinoline in a sequencing batch reactor by Yaohui Bai; Qinghua Sun; Cui Zhao; Donghui Wen; Xiaoyan Tang (pp. 1943-1951).

Two pyridine-degrading bacteria and two quinoline-degrading bacteria were introduced for bioaugmentation to treat the coking wastewater. Sequencing batch reactors (SBRs) were used for a comparative study on the treatment efficiency of pyridine, quinoline, and chemical oxygen demand. Results showed that the treatment efficiency with coking-activated sludge plus a mixture of the four degrading bacteria was much better than that ones with coking-activated sludge only or mixed degrading bacteria only. Moreover, a 52-day continuous operation of the bioaugmented and general SBRs was investigated. The bioaugmented SBR showed better treatment efficiency and stronger capacity to treat high pyridine and quinoline shock loading. The general SBR failed to cope with the shock loading, and the biomass of the activated sludge decreased significantly. In order to monitor the microbial ecological variation during the long-term treatment, the bacterial community in both reactors was monitored by the amplicon length heterogeneity polymerase chain reaction technique. The diversity of the bacterial community decreased in both reactors, but the introduced highly efficient bacteria were dominant in the bioaugmented SBR. Our experiment showed clearly that the use of highly efficient bacteria in SBR process could be a feasible method to treat wastewater containing pyridine or/and quinoline.

Keywords: Bioaugmentation; Pyridine; Quinoline; SBR; Bacterial community

Treatment of Cr(VI)-containing wastewaters with exopolysaccharide-producing cyanobacteria in pilot flow through and batch systems by Giovanni Colica; Pier Cesare Mecarozzi; Roberto De Philippis (pp. 1953-1961).

Seven exopolysaccharide-producing cyanobacteria were tested with regard to their capability to remove Cr(VI) from the wastewater of a plating industry. The cyanobacterium which showed, under lab conditions, the most promising features with regard to both Cr(VI) removal (about 12 mg of Cr(VI) removed per gram of dry biomass) and growth characteristics (highest growth rate and simplest culture medium) was Nostoc PCC7936. Furthermore, in lab experiments, it was also found that a HCl pretreatment is essential to abate the concentration of Cr(VI) in solution and that the viability of the biomass is not necessary. Subsequently, three pilot devices were tested, one batch (a dialysis cell) and two flow-through systems (a filter press and a column filled with quartz grain). The best performances were obtained with the filter press, where it was observed a sharp decrease in the concentration of Cr(VI), partly due to the adsorption of the metal by the biomass (about 50%) and partly due to its reduction to Cr(III). The results are discussed in terms of the role played by the different components (biomass and polysaccharide) of the cyanobacterial cultures in the removal of Cr(VI).

Keywords: Chromium (VI) bioremoval; Cr(VI)-containing wastewaters; Cr-plating industry; Exopolysaccharide-producing cyanobacteria; Confining systems; Biosorption

Quantitative and qualitative transitions of methanogen community structure during the batch anaerobic digestion of cheese-processing wastewater by Changsoo Lee; Jaai Kim; Seung Gu Shin; Vincent O’Flaherty; Seokhwan Hwang (pp. 1963-1973).

Qualitative and quantitative shifts in methanogen community structure, associated with process performance data, were investigated during the batch anaerobic digestion of a cheese-processing wastewater, whey permeate. Denaturing gradient gel electrophoresis (DGGE) and real-time PCR techniques were applied to obtain qualitative and quantitative microbial data sets, respectively, based on methanogen 16S rRNA genes. Throughout the operation, dynamic variations in both qualitative and quantitative community structures were observed, with repeated shifts in dominance between the aceticlastic Methanosarcinaceae (suggested mainly by the detection of a Methanosarcina-like population) and the hydrogenotrophic Methanomicrobiales (suggested mainly by the detection of a Methanofollis-like population). This trend corresponded well to the diauxic utilization of acetate and longer-chain fatty acids (C₃–C₆), mainly propionate. Joint-plot non-metric multidimensional scaling (NMS) analysis demonstrated that the qualitative and quantitative community shifts had significant correlations with the composition of residual organic acids and the methane production rate, respectively. This suggests the potential use of microbial community shift analysis as an indicative tool for diagnosing anaerobic digestion processes. The results suggest that more attention should be directed to quantitative, as well as qualitative, approaches for a better understanding of anaerobic digestion, particularly in terms of biogas production efficiency, under dynamic and transitional conditions.

Keywords: Anaerobic digestion; Denaturing gradient gel electrophoresis (DGGE); Microbial community structure; Non-metric multidimensional scaling (NMS); Real-time PCR

Co-fermentation of cellobiose and xylose using beta-glucosidase displaying diploid industrial yeast strain OC-2 by Satoshi Saitoh; Tomohisa Hasunuma; Tsutomu Tanaka; Akihiko Kondo (pp. 1975-1982).

The co-utilization of sugars, particularly xylose and glucose, during industrial fermentation is essential for economically feasible processes with high ethanol productivity. However, the major problem encountered during xylose/glucose co-fermentation is the lower consumption rate of xylose compared with that of glucose fermentation. Here, we therefore attempted to construct high xylose assimilation yeast by using industrial yeast strain with high β-glucosidase activity on the cell surface. We first constructed the triple auxotrophic industrial strain OC2-HUT and introduced four copies of the cell-surface-displaying β-glucosidase (BGL) gene and two copies of a xylose-assimilating gene into its genome to generate strain OC2-ABGL4Xyl2. It was confirmed that the introduction of multiple copies of the BGL gene increased the cell-surface BGL activity, which was also correlated to the observed increase in xylose-assimilating ability. The strain OC2-ABGL4Xyl2 was able to consume xylose during cellobiose/xylose co-fermentation (0.38 g/h/g-DW) more rapidly than during glucose/xylose co-fermentation (0.18 g/h/g-DW). After 48 h, 5.77% of the xylose was consumed despite the co-fermentation conditions, and the observed ethanol yield was 0.39 g-ethanol/g-total sugar. Our results demonstrate that a BGL-displaying and xylose-assimilating industrial yeast strain is capable of efficient xylose consumption during the co-fermentation with cellobiose. Due to its high performance for fermentation of mixtures of cellobiose and xylose, OC2-ABGL4Xyl2 does not require the addition of β-glucosidase and is therefore a promising yeast strain for cost-effective ethanol production from lignocellulosic biomass.

Keywords: Saccharomyces cerevisiae ; Co-fermentation; β-Glucosidase; Xylose fermentation

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Applied Microbiology and Biotechnology (v.87, #5)