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Applied Microbiology and Biotechnology (v.87, #2)

Saccharomyces cerevisiae and DNA microarray analyses: what did we learn from it for a better understanding and exploitation of yeast biotechnology? by Takashi Hirasawa; Chikara Furusawa; Hiroshi Shimizu (pp. 391-400).

Saccharomyces cerevisiae has been widely used in industrial fields such as in the production of alcoholic beverages and useful chemicals and in bakery. Since S. cerevisiae was the first organism whose genome sequence was determined in eukaryotes, genome-wide analysis systems such as DNA microarrays also developed early for this organism. Many researches related to the analysis of transcriptional profiles during the processes and transcriptional responses to the environmental stresses that are encountered during production processes using DNA microarray were reported in the literature. In addition, DNA microarrays can be used in detecting transcription factor binding sites and single nucleotide polymorphisms. In this paper, we review transcriptome analysis toward industrial production processes involving yeast, as in the case of wine, beer, and sake. Moreover, identification of the target genes for genetic manipulation to confer useful phenotypes, such as stress tolerance and high fermentation activity, and to improve production of target product in useful chemicals production using DNA microarray analysis is described. Finally, recent advances of DNA microarray analysis are briefly discussed.

Keywords: Saccharomyces cerevisiae ; DNA microarray; Transcriptome; Biotechnology; Metabolic engineering

Recombinant immunotherapeutics: current state and perspectives regarding the feasibility and market by Chung-Jr Huang; Adam J. Lowe; Carl A. Batt (pp. 401-410).

Recombinant immunotherapeutics are important biologics for the treatment and prevention of various diseases. Immunotherapy can be divided into two categories, passive and active. For passive immunotherapy, the successes of antibody and cytokine therapeutics represent a promising future and opportunities for improvements. Efforts, such as cell engineering, antibody engineering, human-like glycosylation in yeast, and Fab fragment development, have led the way to improve antibody efficacy while decreasing its high manufacturing costs. Both new cytokines and currently used cytokines have demonstrated therapeutic effects for different indications. As for active immunotherapy, recently approved HPV vaccines have encouraged the development of preventative vaccines for other infectious diseases. Immunogenic antigens of pathogenic bacteria can now be identified by genomic means (reverse vaccinology). Due to the recent outbreaks of pandemic H1N1 influenza virus, recombinant influenza vaccines using virus-like particles and other antigens have also been engineered in several different recombinant systems. However, limitations are found in existing immunotherapeutics for cancer treatment, and recent development of therapeutic cancer vaccines such as MAGE-A3 and NY-ESO-1 may provide alternative therapeutic strategy.

Keywords: Recombinant immunotherapeutics; Immunotherapy; Antibodies; Cytokines; Preventative recombinant vaccines; Therapeutic cancer vaccine

Integrated production of xylitol and ethanol using corncob by Ke-Ke Cheng; Jian-An Zhang; Erik Chavez; Jin-Ping Li (pp. 411-417).

Xylitol production from corncob hemicellulose is a popular process in China. Microbial conversion of xylose to xylitol, as a biological process with many advantages, has drawn increasing attention. As a by-product from the manufacturing of xylitol, corncob cellulosic residues are produced in very large amounts and represent an environmental problem. As a result, considering the large amount of xylitol production in China, the conversion of corncob cellulosic residues has become a widespread issue having to be tackled. After the hemicellulose in corncob has been hydrolyzed for xylitol production, the corncob cellulosic residue is porous and can easily be hydrolyzed by cellulases into glucose and further converted to ethanol, another high-added-value chemical. Based on the latest technology advancements in xylitol, cellulase, and ethanol production, the integrated production of ethanol from corncob cellulosic residues appears as a promising way to improve the profit of the whole xylitol production process.

Keywords: Xylitol; Ethanol; Corncob; Fermentation; Cellulase

Comparison of hybridization methods and real-time PCR: their value in animal cell line characterization by Helga Böhm-Hofstätter; Marion Tschernutter; Renate Kunert (pp. 419-425).

For biotechnological protein production, the International Conference of Harmonization (ICH) recommends appropriate testing of expression constructs and characterization of producer cell lines in order to assure uniform specifications, improve safety, and confirm stable productivity. Commonly, hybridization analyses are used for the evaluation of genetic stability, gene and transcript copy numbers as well as the integrity of the coding sequence. However, improvements in polymerase chain reaction (PCR) techniques have accelerated the analysis of genetic parameters and have nowadays become the method of choice for the evaluation of gene copy numbers and transcript levels. Nevertheless, Southern and Northern blot analyses are still valuable tools that deliver additional information to PCR results during cell clone characterization studies. In this study, we discuss advantages and drawbacks of hybridization and PCR methods in regard to their applicability and efficiency during the development of recombinant mammalian cell lines. A comparative review of the literature as well as an overview of findings from our own group will be given.

Keywords: Southern blot; Northern blot; Real-time PCR; Gene copy number; mRNA

Microbial biosurfactants production, applications and future potential by Ibrahim M. Banat; Andrea Franzetti; Isabella Gandolfi; Giuseppina Bestetti; Maria G. Martinotti; Letizia Fracchia; Thomas J. Smyth; Roger Marchant (pp. 427-444).

Microorganisms synthesise a wide range of surface-active compounds (SAC), generally called biosurfactants. These compounds are mainly classified according to their molecular weight, physico-chemical properties and mode of action. The low-molecular-weight SACs or biosurfactants reduce the surface tension at the air/water interfaces and the interfacial tension at oil/water interfaces, whereas the high-molecular-weight SACs, also called bioemulsifiers, are more effective in stabilising oil-in-water emulsions. Biosurfactants are attracting much interest due to their potential advantages over their synthetic counterparts in many fields spanning environmental, food, biomedical, and other industrial applications. Their large-scale application and production, however, are currently limited by the high cost of production and by limited understanding of their interactions with cells and with the abiotic environment. In this paper, we review the current knowledge and the latest advances in biosurfactant applications and the biotechnological strategies being developed for improving production processes and future potential.

Keywords: Biosurfactants; Bioemulsifiers; Surfactants; Emulsifiers

Advances in biofilm reactors for production of value-added products by Kuan-Chen Cheng; Ali Demirci; Jeffrey M. Catchmark (pp. 445-456).

Biofilms are defined as microbial cell layers, which are irreversibly or reversibly attached on solid surfaces. These attached cells are embedded in a self-produced exopolysaccharide matrix, and exhibit different growth and bioactivity compared with suspended cells. With their high biomass density, stability, and potential for long-term fermentation, biofilm reactors are employed for the fermentation and bioconversion, which need large amount of biomass. During the past decade, biofilm reactors have been successfully applied for production of many value-added products. This review article summarizes the applications of biofilm reactors with different novel designs. Advantages and concerns using biofilm reactors, potential uses for industrial-scale production, and further investigation needs are discussed.

Keywords: Biofilm; Biofilm reactors; Plastic-composite supports; Cell immobilization; Fermentation

Biotechnological production and application of ganoderic acids by Jun-Wei Xu; Wei Zhao; Jian-Jiang Zhong (pp. 457-466).

Ganoderic acids (GAs), a kind of highly oxygenated lanostane-type triterpenoids, are important bioactive constituents of the famous medicinal mushroom Ganoderma lucidum. They have received wide attention in recent years due to extraordinarily pharmacological functions. Submerged fermentation of G. lucidum is viewed as a promising technology for production of GAs, and substantial efforts have been devoted to process development for enhancing GA production in the last decade. This article reviews recent publication about fermentative production of GAs and their potential applications, especially the progresses toward manipulation of fermentation conditions and bioprocessing strategies are summarized. The biosynthetic pathway of GAs is also outlined.

Keywords: Ganoderic acids; Ganoderma lucidum ; Medicinal mushroom fermentation; Biosynthesis; Bioactivities; Application

Bacteria causing important diseases of citrus utilise distinct modes of pathogenesis to attack a common host by Adrián Alberto Vojnov; Alexandre Morais do Amaral; John Maxwell Dow; Atilio Pedro Castagnaro; Marìa Rosa Marano (pp. 467-477).

In this review, we summarise the current knowledge on three pathogens that exhibit distinct tissue specificity and modes of pathogenesis in citrus plants. Xanthomonas axonopodis pv. citri causes canker disease and invades the host leaf mesophyll tissue through natural openings and can also survive as an epiphyte. Xylella fastidiosa and Candidatus Liberibacter are vectored by insects and proliferate in the vascular system of the host, either in the phloem (Candidatus Liberibacter) or xylem (X. fastidiosa) causing variegated chlorosis and huanglongbing diseases, respectively. Candidatus Liberibacter can be found within host cells and is thus unique as an intracellular phytopathogenic bacterium. Genome sequence comparisons have identified groups of species-specific genes that may be associated with the particular lifestyle, mode of transmission or symptoms produced by each phytopathogen. In addition, components that are conserved amongst bacteria may have diverse regulatory actions underpinning the different bacterial lifestyles; one example is the divergent role of the Rpf/DSF cell–cell signalling system in X. citri and X. fastidiosa. Biofilm plays a key role in epiphytic fitness and canker development in X. citri and in the symptoms produced by X. fastidiosa. Bacterial aggregation may be associated with vascular occlusion of the xylem vessels and symptomatology of variegated chlorosis.

Keywords: Citrus

Inteins, valuable genetic elements in molecular biology and biotechnology by Skander Elleuche; Stefanie Pöggeler (pp. 479-489).

Inteins are internal protein elements that self-excise from their host protein and catalyze ligation of the flanking sequences (exteins) with a peptide bond. They are found in organisms in all three domains of life, and in viral proteins. Intein excision is a posttranslational process that does not require auxiliary enzymes or cofactors. This self-excision process is called protein splicing, by analogy to the splicing of RNA introns from pre-mRNA. Protein splicing involves only four intramolecular reactions, and a small number of key catalytic residues in the intein and exteins. Protein-splicing can also occur in trans. In this case, the intein is separated into N- and C-terminal domains, which are synthesized as separate components, each joined to an extein. The intein domains reassemble and link the joined exteins into a single functional protein. Understanding the cis- and trans-protein splicing mechanisms led to the development of intein-mediated protein-engineering applications, such as protein purification, ligation, cyclization, and selenoprotein production. This review summarizes the catalytic activities and structures of inteins, and focuses on the advantages of some recent intein applications in molecular biology and biotechnology.

Keywords: Intein; Protein splicing; Protein purification; Cyclic proteins; Selenoproteins

Possible incorporation of petroleum-based carbons in biochemicals produced by bioprocess by Masao Kunioka (pp. 491-497).

The biomass carbon ratios of biochemicals related to biomass have been reviewed. Commercial products from biomass were explained. The biomass carbon ratios of biochemical compounds were measured by accelerator mass spectrometry (AMS) based on the ¹⁴C concentration of carbons in the compounds. This measuring method uses the mechanism that biomass carbons include a very low level of ¹⁴C and petroleum carbons do not include ¹⁴C similar to the carbon dating measuring method. It was confirmed that there were some biochemicals synthesized from petroleum-based carbons. This AMS method has a high accuracy with a small standard deviation and can be applied to plastic products.

Keywords: Biomass based; Petroleum based; Accelerator mass spectrometry; Biomass carbon ratio; Biobased content; ASTM D6866; Bioplastics; Biomass-based plastics

Production of surfactin and fengycin by Bacillus subtilis in a bubbleless membrane bioreactor by François Coutte; Didier Lecouturier; Saliha Ait Yahia; Valérie Leclère; Max Béchet; Philippe Jacques; Pascal Dhulster (pp. 499-507).

Surfactin and fengycin are lipopeptide biosurfactants produced by Bacillus subtilis. This work describes for the first time the use of bubbleless bioreactors for the production of these lipopeptides by B. subtilis ATCC 21332 with aeration by a hollow fiber membrane air–liquid contactor to prevent foam formation. Three different configurations were tested: external aeration module made from either polyethersulfone (reactor BB1) or polypropylene (reactor BB2) and a submerged module in polypropylene (reactor BB3). Bacterial growth, glucose consumption, lipopeptide production, and oxygen uptake rate were monitored during the culture in the bioreactors. For all the tested membranes, the bioreactors were of satisfactory bacterial growth and lipopeptide production. In the three configurations, surfactin production related to the culture volume was in the same range: 242, 230, and 188 mg l⁻¹ for BB1, BB2, and BB3, respectively. Interestingly, high differences were observed for fengycin production: 47 mg l⁻¹ for BB1, 207 mg l⁻¹ for BB2, and 393 mg l⁻¹ for BB3. A significant proportion of surfactin was adsorbed on the membranes and reduced the volumetric oxygen mass transfer coefficient. The degree of adsorption depended on both the material and the structure of the membrane and was higher with the submerged polypropylene membrane.

Keywords: Bacillus subtilis ; Surfactin; Fengycin; Bubbleless membrane bioreactor; Oxygen transfer; Adsorption

A novel nitric oxide producing probiotic patch and its antimicrobial efficacy: preparation and in vitro analysis by Mitchell Lawrence Jones; Jorge G. Ganopolsky; Alain Labbé; Satya Prakash (pp. 509-516).

Microbial and fungal infections are a significant consideration in the etiology of all wounds. Numerous antimicrobial and antifungal formulations have been developed with varying degrees of efficacy and stability. Here, we report a nitric oxide producing probiotic adhesive patch device and investigate its antimicrobial and antifungal efficacy in vitro. This probiotic patch utilizes the metabolic activity of immobilized lactic acid bacteria, glucose, and nitrite salts for the production of gaseous nitric oxide (gNO), which is used as an antimicrobial agent against bacterial and fungal pathogens. Results show that application of gNO-producing probiotic patches to cultures of E. coli, S. aureus, P. aeruginosa, MRSA, T. mentagrophytes, and T. rubrum resulted in complete cell death at between 4 and 8 h, and application to cultures of A. baumannii, resulted in fewer than ten colonies detected per milliliter at 6 h. These results demonstrate that a gNO-producing probiotic patch device containing bacteria, glucose, and nitrite salts can produce sufficient levels of gNO over a therapeutically relevant duration to kill common bacterial and fungal wound pathogens in humans.

Keywords: Nitric oxide; Antimicrobial; Bacteria; Infection; Wound healing; Diabetic ulcer; Venous stasis ulcer; Artificial cell; Microcapsule; Patch; Dressing

High-level production of heterologous proteins using untreated cane molasses and corn steep liquor in Escherichia coli medium by Qi Ye; Ximu Li; Ming Yan; Hou Cao; Lin Xu; Yueyuan Zhang; Yong Chen; Jian Xiong; Pingkai Ouyang; Hanjie Ying (pp. 517-525).

To develop an economical industrial medium, untreated cane molasses (UCM) was tested as a carbon source for fermentation culturing of Escherichia coli. To test the industrial application of this medium, we chose a strain co-expressing a carbonyl reductase (PsCR) and a glucose dehydrogenase (BmGDH). Although corn steep liquor (CSL) could be used as an inexpensive nitrogen source to replace peptone, yeast extract could not be replaced in E. coli media. In a volume of 40 ml per 1-l flask, a cell concentration of optical density (OD₆₀₀) 15.1 and enzyme activities of 6.51 U/ml PsCR and 3.32 U/ml BmGDH were obtained in an optimized medium containing 25.66 g/l yeast extract, 3.88 g/l UCM, and 7.1% (v/v) CSL. When 3.88 g/l UCM was added to the medium at 6 h in a fed-batch process, the E. coli concentration increased to OD₆₀₀ of 24, and expression of both PsCR and BmGDH were twofold higher than that of a batch process. Recombinant cells from batch or fed-batch cultures were assayed for recombinant enzyme activity by testing the reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE). Compared to cells from batch cultures, fed-batch cultured cells showed higher recombinant enzyme expression, producing 560 mM CHBE in the organic phase with a molar yield of 92% and an optical purity of the (S)-isomer of >99% enantiomeric excess.

Keywords: Escherichia coli Rosetta; Untreated cane molasses; Corn steep liquor; Fed-batch; Ethyl (S)-4-chloro-3-hydroxybutanoate; Ethyl 4-chloro-3-oxobutanoate

Investigation of factors influencing spore germination of Paenibacillus polymyxa ACCC10252 and SQR-21 by Zhenhua Huo; Xingming Yang; Waseem Raza; Qiwei Huang; Yangchun Xu; Qirong Shen (pp. 527-536).

Bioorganic fertilizer containing Paenibacillus polymyxa SQR-21 showed very good antagonistic activity against Fusarium oxysporum. To optimize the role of P. polymyxa SQR-21 in bioorganic fertilizer, we conducted a study of spore germination under various conditions. In this study, l-asparagine, glucose, fructose and K⁺ (AGFK), and sugars (glucose, fructose, sucrose, and lactose) plus l-alanine were evaluated to determine their ability to induce spore germination of two strains; P. polymyxa ACCC10252 and SQR-21. Spore germination was measured as a decrease in optical density at 600 nm. The effect of heat activation and germination temperature were important for germination of spores of both strains on AGFK in Tris–HCl. l-Alanine alone showed a slight increase in spore germination; however, fructose plus l-alanine significantly induced spore germination, and the maximum spore germination rate was observed with 10 mmol l⁻¹ l-alanine in the presence of 1 mmol l⁻¹ fructose in phosphate-buffered saline (PBS). In contrast, fructose plus l-alanine hardly induced spore germination in Tris–HCl; however, in addition of 10 mmol l⁻¹ NaCl into Tris–HCl, the percentages of OD₆₀₀ fall were increased by 19.6% and 24.3% for ACCC10252 and SQR-21, respectively. AGFK-induced spore germination was much more strict to germination temperature than that induced by fructose plus l-alanine. For both strains, fructose plus l-alanine-induced spore germination was not sensitive to pH. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination diversity, which will be beneficial for bioorganic fertilizer storage and transportation to improve the P. polymyxa efficacy as biological control agent.

Keywords: Paenibacillus polymyxa ; AGFK; Fructose plus l-alanine; Germination temperature; Germination buffer; pH

Surfactant-modified yeast whole-cell biocatalyst displaying lipase on cell surface for enzymatic production of structured lipids in organic media by Shinji Hama; Ayumi Yoshida; Kazunori Nakashima; Hideo Noda; Hideki Fukuda; Akihiko Kondo (pp. 537-543).

The cell surface engineering system, in which functional proteins are genetically displayed on microbial cell surfaces, has recently become a powerful tool for applied biotechnology. Here, we report on the surfactant modification of surface-displayed lipase to improve its performance for enzymatic synthesis reactions. The lipase activities of the surfactant-modified yeast displaying Rhizopus oryzae lipase (ROL) were evaluated in both aqueous and nonaqueous systems. Despite the similar lipase activities of control and surfactant-modified cells in aqueous media, the treatment with nonionic surfactants increased the specific lipase activity of the ROL-displaying yeast in n-hexane. In particular, the Tween 20-modified cells increased the cell surface hydrophobicity significantly among a series of Tween surfactants tested, resulting in 8–30 times higher specific activity in organic solvents with relatively high log P values. The developed cells were successfully used for the enzymatic synthesis of phospholipids and fatty acid methyl esters in n-hexane, whereas the nontreated cells produced a significantly low yield. Our results thus indicate that surfactant modification of the cell surface can enhance the potential of the surface-displayed lipase for bioconversion.

Keywords: Surfactant; Saccharomyces cerevisiae ; Cell surface display; Lipase; Transesterification; Organic solvent

Cellulase production by Aspergillus niger in biofilm, solid-state, and submerged fermentations by Norma N. Gamarra; Gretty K. Villena; Marcel Gutiérrez-Correa (pp. 545-551).

Cellulase production by Aspergillus niger was compared in three different culture systems: biofilm, solid-state, and submerged fermentation. Biofilm and solid-state fermentations were carried out on perlite as inert support, and lactose was used as a carbon source in the three culture systems. In cryo-scanning electron microscopy, biofilm and solid-state cultures gave similar morphological patterns and confirmed that both spore first attachment and hyphal adhered growth are helped by the production of an adhesive extracellular matrix. Biofilm cultures produced higher cellulase activities than those in submerged and solid-state cultures (1,768, 1,165, and 1,174 U l⁻¹, respectively). Although biofilm cultures grew less than the other cultures, they produced significantly higher cellulase yields (370, 212, and 217 U g⁻¹ lactose, respectively) and volumetric productivities (24, 16, and 16 U l⁻¹ h⁻¹, respectively). Likewise, endoglucanase and xylanase activities were higher in biofilm cultures. Under the conditions tested, it seems that fungal attached growth on perlite may favor better enzyme production. Biofilms are efficient systems for cellulase production and may replace solid-state fermentation. Biofilm fermentation holds promise for further optimization and development. The results of this work reveal that fungal biofilms may be used for the commercial production of cellulase employing the technology developed for submerged fermentation at high cell densities.

Keywords: Cellulase; Aspergillus niger ; Biofilm; Solid-state fermentation; Submerged fermentation

Effects of pH and corn steep liquor variability on mannitol production by Lactobacillus intermedius NRRL B-3693 by Badal C. Saha; F. Michael Racine (pp. 553-560).

Lactobacillus intermedius NRRL B-3693 produced mannitol, lactic acid, and acetic acid when grown on fructose at 37°C. The optimal pH for mannitol production from fructose by the heterofermentative lactic acid bacterium (LAB) in pH-controlled fermentation was at pH 5.0. It produced 160.7 ± 1.1 g mannitol in 40 h with a volumetric productivity of 4.0 g l⁻¹ h⁻¹ in a simplified medium containing 250 g fructose, 50 g corn steep liquor (CSL), and 33 mg MnSO₄ per liter. However, the mannitol production by the LAB was severely affected by the variability of CSL. The supplementation of CSL with soy peptone (5 g/l), tryptophan (50 mg/l), tryptophan (50 mg/l) plus tyrosine (50 mg/l), or commercial protease preparation (2 ml/100 g of CSL) enhanced the performance of the inferior CSL and thus helped to overcome the nutrient limitations.

Keywords: Mannitol production; Fructose fermentation; pH-controlled fermentation; Lactobacillus intermedius ; Lactic acid bacterium; Corn steep liquor

Mercury resistance and accumulation in Escherichia coli with cell surface expression of fish metallothionein by Kuo-Hsing Lin; Mei-Fang Chien; Ju-Liang Hsieh; Chieh-Chen Huang (pp. 561-569).

Recombinant tilapia (Oreochromis mossambicus) fish metallothionein (MT) was used as a surface biosorbent for mercury removal in Escherichia coli. Fish MT conferred better resistance than did mouse or human MT. When tilapia MT (tMT) was fused with an outer-membrane protein, outer membrane protein C (OmpC), the membrane-targeted fusion protein, OmpC–tMT, gave enhanced resistance compared with cytoplasmic tMT expressed in the same host cell. The cytoplasmically expressed tMT showed high mercury adsorption (4.3 ± 0.4 mg/g cell dry weight). The cell surface that expressed E. coli showed about 25% higher adsorption ability (5.6 ± 0.4 mg/g) than the cells expressing cytoplasmic MT, attaining almost twice the level of adsorption of the control plasmid (3.0 ± 0.4 mg/g). As MTs are also known for their ability to scavenge hydroxyl-free radicals, it was also shown that tMT exhibited better radical-scavenging activities than glutathione. These results suggest that fish MT has potential for the development of a bioremediation system for mercury removal that protects the harboring E. coli host by free-radical scavenging.

Keywords: Metallothionein; Mercury; Bioadsorption; OmpC; Tilapia

Cloning and characterization of a thermostable xylitol dehydrogenase from Rhizobium etli CFN42 by Manish Kumar Tiwari; Hee-Jung Moon; Marimuthu Jeya; Jung-Kul Lee (pp. 571-581).

An NAD⁺-dependent xylitol dehydrogenase from Rhizobium etli CFN42 (ReXDH) was cloned and overexpressed in Escherichia coli. The DNA sequence analysis revealed an open reading frame of 1,044 bp, capable of encoding a polypeptide of 347 amino acid residues with a calculated molecular mass of 35,858 Da. The ReXDH protein was purified as an active soluble form using GST affinity chromatography. The molecular mass of the purified enzyme was estimated to be ∼34 kDa by sodium dodecyl sulfate–polyacrylamide gel and ∼135 kDa with gel filtration chromatography, suggesting that the enzyme is a homotetramer. Among various polyols, xylitol was the preferred substrate of ReXDH with a K _m = 17.9 mM and k_cat /K _m = 0.5 mM⁻¹ s⁻¹ for xylitol. The enzyme had an optimal pH and temperature of 9.5 and 70 °C, respectively. Heat inactivation studies revealed a half life of the ReXDH at 40 °C of 120 min and a half denaturation temperature (T _1/2) of 53.1 °C. ReXDH showed the highest optimum temperature and thermal stability among the known XDHs. Homology modeling and sequence analysis of ReXDH shed light on the factors contributing to the high thermostability of ReXDH. Although XDHs have been characterized from several other sources, ReXDH is distinguished from other XDHs by its high thermostability.

Keywords: Characterization; Homology modeling; Rhizobium etli ; Thermostability; Xylitol dehydrogenase

Polyphosphate/ATP-dependent NAD kinase of Corynebacterium glutamicum: biochemical properties and impact of ppnK overexpression on lysine production by Steffen N. Lindner; Henrike Niederholtmeyer; Katja Schmitz; Siegfried M. Schoberth; Volker F. Wendisch (pp. 583-593).

Nicotinamide adenine dinucleotide phosphate (NADP) is synthesized by phosphorylation of either oxidized or reduced nicotinamide adenine dinucleotide (NAD/NADH). Here, the cg1601/ppnK gene product from Corynebacterium glutamicum genome was purified from recombinant Escherichia coli and enzymatic characterization revealed its activity as a polyphosphate (PolyP)/ATP-dependent NAD kinase (PPNK). PPNK from C. glutamicum was shown to be active as homotetramer accepting PolyP, ATP, and even ADP for phosphorylation of NAD. The catalytic efficiency with ATP as phosphate donor for phosphorylation of NAD was higher than with PolyP. With respect to the chain length of PolyP, PPNK was active with short-chain PolyPs. PPNK activity was independent of bivalent cations when using ATP, but was enhanced by manganese and in particular by magnesium ions. When using PolyP, PPNK required bivalent cations, preferably manganese ions, for activity. PPNK was inhibited by NADP and NADH at concentrations below millimolar. Overexpression of ppnK in C. glutamicum wild type slightly reduced growth and ppnK overexpression in the lysine producing strain DM1729 resulted in a lysine product yield on glucose of 0.136 ± 0.006 mol lysine (mol glucose)⁻¹, which was 12% higher than that of the empty vector control strain.

Keywords: Polyphosphate; NAD kinase; Corynebacterium; Lysine production

Characterization of the versatile monooxygenase CYP109B1 from Bacillus subtilis by Marco Girhard; Tobias Klaus; Yogan Khatri; Rita Bernhardt; Vlada B. Urlacher (pp. 595-607).

The oxidizing activity of CYP109B1 from Bacillus subtilis was reconstituted in vitro with various artificial redox proteins including putidaredoxin reductase and putidaredoxin from Pseudomonas putida, truncated bovine adrenodoxin reductase and adrenodoxin, flavodoxin reductase and flavodoxin from Escherichia coli, and two flavodoxins from B. subtilis (YkuN and YkuP). Binding and oxidation of a broad range of chemically different substrates (fatty acids, n-alkanes, primary n-alcohols, terpenoids like (+)-valencene, α- and β-ionone, and the steroid testosterone) were investigated. CYP109B1was found to oxidize saturated fatty acids (conversion up to 99%) and their methyl and ethyl esters (conversion up to 80%) at subterminal positions with a preference for the carbon atoms C11 and C12 counted from the carboxyl group. For the hydroxylation of primary n-alcohols, the ω₋₂ position was preferred. n-Alkanes were not accepted as substrates by CYP109B1. Regioselective hydroxylation of terpenoids α-ionone (∼70% conversion) and β-ionone (∼ 91% conversion) yielded the allylic alcohols 3-hydroxy-α-ionone and 4-hydroxy-β-ionone, respectively. Furthermore, indole was demonstrated to inhibit fatty acid oxidation.

Keywords: Bacillus subtilis ; P450 monooxygenase; Fatty acid; Terpene; Ionone; Activity reconstitution

Soluble expression and purification of the anthrax protective antigen in E. coli and identification of a novel dominant-negative mutant N435C by Gaobing Wu; Chunfang Feng; Yuzhi Hong; Aizhen Guo; Sha Cao; Junli Dong; Ling Lin; Ziduo Liu (pp. 609-616).

The anthrax toxin is an AB-type bacterium toxin composed of the protective antigen (PA) as the cell-binding B component, and the lethal factor (LF) and edema toxin (EF) as the catalytic A components. The PA component is a key factor in anthrax-related research and recombinant PA can be produced in general in Escherichia coli. However, such recombinant PA always forms inclusion bodies in the cytoplasm of E. coli, making difficult the procedure of its purification. In this study, we found that the solubility of recombinant PA was dramatically enhanced by fusion with glutathione S-transferase (GST) and an induction of its expression at 28°C. The PA was purified to high homogeneity and a yield of 3 mg protein was obtained from 1 l culture by an affinity-chromatography approach. Moreover, we expressed and purified three PA mutants, I394C, A396C, and N435C, which were impaired in expression in previous study. Among them, a novel mutant N435C which conferred dominant-negative inhibitory activity on PA was identified. This new mutant may be useful in designing new antitoxin for anthrax prophylaxis and therapy.

Keywords: Bacillus anthracis ; Protective antigen; Soluble expression; Dominant-negative mutant

The antifungal protein AFP from Aspergillus giganteus prevents secondary growth of different Fusarium species on barley by Hassan Barakat; Anja Spielvogel; Mahmoud Hassan; Ahmed El-Desouky; Hamdy El-Mansy; Frank Rath; Vera Meyer; Ulf Stahl (pp. 617-624).

Secondary growth is a common post-harvest problem when pre-infected crops are attacked by filamentous fungi during storage or processing. Several antifungal approaches are thus pursued based on chemical, physical, or bio-control treatments; however, many of these methods are inefficient, affect product quality, or cause severe side effects on the environment. A protein that can potentially overcome these limitations is the antifungal protein AFP, an abundantly secreted peptide of the filamentous fungus Aspergillus giganteus. This protein specifically and at low concentrations disturbs the integrity of fungal cell walls and plasma membranes but does not interfere with the viability of other pro- and eukaryotic systems. We thus studied in this work the applicability of AFP to efficiently prevent secondary growth of filamentous fungi on food stuff and chose, as a case study, the malting process where naturally infested raw barley is often to be used as starting material. Malting was performed under lab scale conditions as well as in a pilot plant, and AFP was applied at different steps during the process. AFP appeared to be very efficient against the main fungal contaminants, mainly belonging to the genus Fusarium. Fungal growth was completely blocked after the addition of AFP, a result that was not observed for traditional disinfectants such as ozone, hydrogen peroxide, and chlorine dioxide. We furthermore detected reduced levels of the mycotoxin deoxynivalenol after AFP treatment, further supporting the fungicidal activity of the protein. As AFP treatments did not compromise any properties and qualities of the final products malt and wort, we consider the protein as an excellent biological alternative to combat secondary growth of filamentous fungi on food stuff.

Keywords: Antifungal protein AFP; Barley; Malting; Fusarium ; Mycotoxin; Bio-control

Purification and characterization of extreme alkaline, thermostable keratinase, and keratin disulfide reductase produced by Bacillus halodurans PPKS-2 by Pathange Prakash; Senigala K. Jayalakshmi; Kuruba Sreeramulu (pp. 625-633).

Two alkaline keratinases-I and II secreted by Bacillus halodurans PPKS-2 were purified and characterized. Both the keratinases were purified using ammonium sulfate, DEAE-Sephadex followed by Sephadex G-200 column chromatography. The purification was 21.5-fold and 11.17% yield for keratinase-I and 23.7-fold with yield 18.46 for keratinase-II and its molecular weights 30 and 66 kDa. Both purified enzymes were relatively stable over a broad pH range 7.0–13.0 and optimally active at pH 11.0 and 60–70 °C. Keratinase-II was found to be more stable at 70 °C for 3 h and retained 100% of its activity, whereas keratinase-I lost 10% activity. Keratinase-I had high keratin disulfide reductase activity with low keratinase activity whereas keratinase-II had high keratinase activity with low keratin disulfide reductase activity. Keratinase activities of both the enzymes were completely inhibited by PMSF at 1 mM, whereas keratin disulfide reductase activity of keratinase-I was not affected. Enzymes were active and stable in the presence of the surfactants, bleaching agents (20% H₂O₂), commercial detergents (1%), and SDS (20%). Both the enzymes were partially sequenced and found that keratinase-I and II had a homology with disulfide reductases and serine type of proteases, respectively.

Keywords: Keratinases; Keratin; Bleach stable; Disulfide reductase and glutathione

Reconstructing the clostridial n-butanol metabolic pathway in Lactobacillus brevis by Oksana V. Berezina; Natalia V. Zakharova; Agnieszka Brandt; Sergey V. Yarotsky; Wolfgang H. Schwarz; Vladimir V. Zverlov (pp. 635-646).

A Lactobacillus brevis strain with the ability to synthesize butanol from glucose was constructed by metabolic engineering. The genes crt, bcd, etfB, etfA, and hbd, composing the bcs-operon, and the thl gene encode the enzymes of the lower part of the clostridial butanol pathway (crotonase, butyryl-CoA-dehydrogenase, two subunits of the electron transfer flavoprotein, 3-hydroxybutyryl-CoA dehydrogenase, and thiolase) of Clostridium acetobutylicum. They were cloned into the Gram-positive/Gram-negative shuttle plasmid vector pHYc. The two resulting plasmids pHYc-thl-bcs and pHYc-bcs (respectively, with and without the clostridial thl gene) were transferred to Escherichia coli and L. brevis. The recombinant L. brevis strains were able to synthesize up to 300 mg l⁻¹ or 4.1 mM of butanol on a glucose-containing medium. A L. brevis strain carrying the clostridial bcs-operon has the ability to synthesize butanol with participation of its own thiolase, aldehyde dehydrogenase, and alcohol dehydrogenase. The particular role of the enzymes involved in butanol production and the suitability of L. brevis as an n-butanol producer are discussed.

Keywords: Lactobacillus brevis ; Clostridium acetobutylicum ; Biofuels; Butanol; Metabolic engineering

Systematic screening of Escherichia coli single-gene knockout mutants for improving recombinant whole-cell biocatalysts by Ying Zhou; Takeshi Minami; Kohsuke Honda; Takeshi Omasa; Hisao Ohtake (pp. 647-655).

Systematic screening of single-gene knockout collection of Escherichia coli BW25113 (the Keio collection) was performed to select mutants that could enhance the deethylation of 7-ethoxycoumarin catalyzed by CYP154A1. After 96-well plate high-throughput screening followed by test tube assays, four mutants (ΔcpxA, ΔgcvR, ΔglnL, and an unknown-gene-deleted one (Δuk)) were able to increase the CYP154A1 activity by approximately 1.4–1.7 times compared with that of the control strain. When new mutants were constructed by disrupting individually the cpxA, gcvR, glnL, and uk genes in E. coli BW25113, three of them (ΔcpxA, ΔgcvR, and ΔglnL) showed high levels of CYP154A1 activity. However, the uk-disruptant failed to enhance the CYP154A1 activity, suggesting that the high CYP154A1 activity of the Δuk mutant in the Keio collection was due to a spontaneous mutation in the chromosome. In-frame deletion mutants of ΔcpxA, ΔgcvR, and ΔglnL also exhibited high enzyme activity, and complementation of these mutations could decrease CYP154A1 activity. These results indicated that the enhancement of the enzyme activity was not caused by polar effects on their neighbor genes. To our knowledge, this is the first report on a genome-wide screening of the genes for deletion to improve the activity of a recombinant whole-cell biocatalyst.

Keywords: Systematic screening; Keio collection; Whole-cell biocatalysis; Cytochrome P450 monooxygenase

Characterization and separate activities of the two promoters of the Lactobacillus brevis S-layer protein gene by Ulla Hynönen; Silja Åvall-Jääskeläinen; Airi Palva (pp. 657-668).

Lactobacillus brevis ATCC 8287 possesses a surface (S)-layer protein SlpA, the gene of which is very efficiently expressed. To study the expression signals of the slpA gene, several different reporter plasmids, based on the low-copy-number vector pKTH2121 derived from pGK12, were constructed. In the reporter plasmids, only one of the two consecutive slpA promoters (P1, P2) was placed upstream of the Lactobacillus helveticus proline iminopeptidase (pepI) gene, and defined parts of the sequences upstream of the promoter were deleted. As indicated by reporter enzyme activities, both promoters were efficiently recognized at different growth stages in L. brevis. An upstream region important for the full activity of P1 was identified. The quantification of pepI-specific mRNA in L. brevis and SDS-PAGE indicated that slpA expression is not regulated at the post-transcriptional level and revealed no regulation of slpA promoters under the conditions tested. The high expression levels of both slpA and the reporter gene in L. brevis were found to remain at a high level after the addition of bile or pancreatin in the growth medium or after a change of the carbon source, which is advantageous for the potential use of SlpA as a carrier in live oral vaccines.

Keywords: S-layer; Promoter; Gene expression; Lactobacillus

Surface display of acid protease on the cells of Yarrowia lipolytica for milk clotting by Xin-Jun Yu; Catherine Madzak; Hui-Juan Li; Zhen-Ming Chi; Jing Li (pp. 669-677).

The acid protease structural gene was amplified from the genomic DNA of Saccharomycopsis fibuligera A11. When the gene was cloned into the multiple cloning site of the surface display vector pINA1317-YlCWP110 and expressed in the cells of Yarrowia lipolytica, the cells displaying the acid protease could form clear zone on the plate-containing milk indicating that they had extracellular acid protease activity. The cells displaying the acid protease can be used to effectively clot skimmed milk. The highest clotting milk activity (1,142.9 U/ml) was observed under the conditions of pH 3.0, 40 °C, 20 mM of CaCl₂, and 10% skimmed milk powder. We found that the acid protease displayed on the cells of Y. lipolytica which has generally regarded as safe status could be easily isolated and concentrated compared to the free acid protease. Therefore, the displayed acid protease may have many potential applications in food and cheese industries. This is the first report that the yeast cells displaying the acid protease were used to clot milk.

Keywords: Acid protease gene; Milk clotting; Saccharomycopsis fibuligera ; Yeast surface display; Yarrowia lipolytica

Comparative transcriptomics and proteomics of p-hydroxybenzoate producing Pseudomonas putida S12: novel responses and implications for strain improvement by Suzanne Verhoef; Hendrik Ballerstedt; Rita J. M. Volkers; Johannes H. de Winde; Harald J. Ruijssenaars (pp. 679-690).

A transcriptomics and proteomics approach was employed to study the expression changes associated with p-hydroxybenzoate production by the engineered Pseudomonas putida strain S12palB1. To establish p-hydroxybenzoate production, phenylalanine-tyrosine ammonia lyase (pal/tal) was introduced to connect the tyrosine biosynthetic and p-coumarate degradation pathways. In agreement with the efficient p-hydroxybenzoate production, the tyrosine biosynthetic and p-coumarate catabolic pathways were upregulated. Also many transporters were differentially expressed, one of which—a previously uncharacterized multidrug efflux transporter with locus tags PP1271-PP1273—was found to be associated with p-hydroxybenzoate export. In addition to tyrosine biosynthesis, also tyrosine degradative pathways were upregulated. Eliminating the most prominent of these resulted in a 22% p-hydroxybenzoate yield improvement. Remarkably, the upregulation of genes contributing to p-hydroxybenzoate formation was much higher in glucose than in glycerol-cultured cells.

Keywords: Pseudomonas putida S12; p-hydroxybenzoate; Transcriptomics; Proteomics; Metabolic engineering

Role of lactose on the production of d-arabitol by Kluyveromyces lactis grown on lactose by Tomoyuki Toyoda; Kazuhisa Ohtaguchi (pp. 691-701).

There are remarkably few reports on d-arabitol production from lactose. Previous studies in our laboratory have shown that the osmophilic yeast Kluyveromyces lactis NBRC 1903 convert lactose to extracellular d-arabitol. The present study was undertaken to determine the participation of osmotic stress caused by lactose on d-arabitol production by K. lactis NBRC 1903 and to provide the information on the kinetics of d-arabitol production from lactose by K. lactis NBRC 1903. It was confirmed that d-arabitol production was triggered when an initial lactose concentration was above 278 mmol L⁻¹. d-Arabitol yield increased with an increase in initial lactose concentration. The highest d-arabitol concentration of 79.5 mmol L⁻¹ was achieved in the cultivation of K. lactis NBRC 1903 in a medium containing 555 mmol L⁻¹ lactose and 40 g L⁻¹ yeast extract. Lactose was found to play two important roles in d-arabitol production by K. lactis NBRC 1903 grown on lactose. First, lactose was assimilated as the substrate both for cell growth and d-arabitol production. Second, a high lactose concentration induced cellular response to high osmotic stress and up-regulated the flow from d-glucose-6-phosphate to d-arabitol. The arrest of cell growth triggered d-arabitol production.

Keywords: Kluyveromyces lactis ; d-Arabitol; Lactose; Osmotic stress; Metabolic changes

Cg2091 encodes a polyphosphate/ATP-dependent glucokinase of Corynebacterium glutamicum by Steffen N. Lindner; Sandra Knebel; Srinivas R. Pallerla; Siegfried M. Schoberth; Volker F. Wendisch (pp. 703-713).

The Corynebacterium glutamicum gene cg2091 is encoding a polyphosphate (PolyP)/ATP-dependent glucokinase (PPGK). Previous work demonstrated the association of PPGK to PolyP granules. The deduced amino acid sequence of PPGK shows 45% sequence identity to PolyP/ATP glucomannokinase of Arthrobacter sp. strain KM and 50% sequence identity to PolyP glucokinase of Mycobacterium tuberculosis H37Rv. PPGK from C. glutamicum was purified from recombinant Escherichia coli. PolyP was highly preferred over ATP and other NTPs as substrate and with respect to the tested PolyPs differing in chain length; the protein was most active with PolyP₇₅. Gel filtration analysis revealed that PolyP supported the formation of homodimers of PPGK and that PPGK was active as a homodimer. A ppgK deletion mutant (ΔppgK) showed slowed growth in minimal medium with maltose as sole carbon source. Moreover, in minimal medium containing 2 to 4% (w/v) glucose as carbon source, ΔppgK grew to lower final biomass concentrations than the wild type. Under phosphate starvation conditions, growth of ΔppgK was reduced, and growth of a ppgK overexpressing strain was increased as compared to wild type and empty vector control, respectively. Thus, under conditions of glucose excess, the presence of PPGK entailed a growth advantage.

Keywords: Corynebacterium ; Glucokinase; Polyphosphate; Poly P; Maltose; Phosphate starvation

The gld1 ⁺ gene encoding glycerol dehydrogenase is required for glycerol metabolism in Schizosaccharomyces pombe by Tomohiko Matsuzawa; Takao Ohashi; Akira Hosomi; Naotaka Tanaka; Hideki Tohda; Kaoru Takegawa (pp. 715-727).

The budding yeast Saccharomyces cerevisiae is able to utilize glycerol as the sole carbon source via two pathways (glycerol 3-phosphate pathway and dihydroxyacetone [DHA] pathway). In contrast, the fission yeast Schizosaccharomyces pombe does not grow on media containing glycerol as the sole carbon source. However, in the presence of other carbon sources such as galactose and ethanol, S. pombe could assimilate glycerol and glycerol was preferentially utilized over ethanol and galactose. No equivalent of S. cerevisiae Gcy1/glycerol dehydrogenase has been identified in S. pombe. However, we identified a gene in S. pombe, SPAC13F5.03c (gld1 ⁺), that is homologous to bacterial glycerol dehydrogenase. Deletion of gld1 caused a reduction in glycerol dehydrogenase activity and prevented glycerol assimilation. The gld1Δ cells grew on 50 mM DHA as the sole carbon source, indicating that the glycerol dehydrogenase encoded by gld1 ⁺ is essential for glycerol assimilation in S. pombe. Strains of S. pombe deleted for dak1 ⁺ and dak2 ⁺ encoding DHA kinases could not grow on glycerol and showed sensitivity to a higher concentration of DHA. The dak1Δ strain showed a more severe reduction of growth on glycerol and DHA than the dak2Δ strain because the expression of dak1 ⁺ mRNA was higher than that of dak2 ⁺. In wild-type S. pombe, expression of the gld1 ⁺, dak1 ⁺, and dak2 ⁺ genes was repressed at a high concentration of glucose and was derepressed during glucose starvation. We found that gld1 ⁺ was regulated by glucose repression and that it was derepressed in scr1Δ and tup12Δ strains.

Keywords: Glycerol; Glycerol dehydrogenase; Glucose repression; Fission yeast

Control of biofilm formation by poly-ethylene-co-vinyl acetate films incorporating nisin by Antonia Nostro; Roberto Scaffaro; Giovanna Ginestra; Manuela D’Arrigo; Luigi Botta; Andreana Marino; Giuseppe Bisignano (pp. 729-737).

The aim of this study was to evaluate the effect of poly-ethylene-co-vinyl acetate (EVA) films incorporating different concentrations (0.1%, 0.5% and 1%) of nisin on the biofilm-forming ability of Listeria monocytogenes ATCC 7644, Staphylococcus aureus 815 and Staphylococcus epidermidis ATCC 35984. Nisin was incorporated into two grades of EVA (EVA14 and EVA28) in the melt during a common film-blowing operation. The efficacy of EVA/nisin films was evaluated by biofilm biomass measurements and Live/Dead staining in combination with fluorescence microscopy. In order to evaluate whether the nisin incorporation could modify the film surface properties, contact angle measurements and scanning electron microscopy were performed. The results revealed the efficacy of EVA14/nisin films in reducing biofilm formation on their surfaces with more evident effect for S. epidermidis than L. monocytogenes and S. aureus strains. In contrast, EVA28/nisin films showed unsatisfactory activity. Fluorescence microscopy confirmed poor biofilm formation on EVA14/nisin films, also characterised by the presence of dead cells. The data presented in this study offer new potential applications for developing strategies aimed to improve the effect of antimicrobial agents.

Keywords: Nisin; Polymeric film; Biofilm; Listeria monocytogenes ; Staphylococcus aureus ; Staphylococcus epidermidis

A gyrB-targeted PCR for rapid identification of Paenibacillus mucilaginosus by Jin-Guang Wu; Jian-Feng Wang; Xing-Hai Zhang; Shan-Shan Zhang; Xiu-Fang Hu; Ji-Shuang Chen (pp. 739-747).

Paenibacillus mucilaginosus, one of the typical silicate bacteria, has long been used as a biofertilizer in agriculture and has recently shown potential in bioleaching and wastewater engineering. There has been considerable research involving the isolation of P. mucilaginosus for various utilizations; therefore, rapid identification of this species is of great interest. Herein, we describe a specific polymerase chain reaction (PCR) method developed for a rapid identification of P. mucilaginosus, which might provide potential utilization in the investigation of populations, detection of biofertilizers, and identification of novel isolates on a large scale. A gyrB-targeted species-specific primer pair, F2 (5′-ACG GAT ATC TCC CAG ACG TTC AT-3′) and R5 (5′-ACG GGC ACG CTG CGC CTG TAC G-3′), was successfully designed to selectively amplify a 519-bp amplicon from P. mucilaginosus. Good specificity was demonstrated by both reference strains and total soil deoxyribonucleic acid, from which only the gyrB gene of P. mucilaginosus was amplified. The detection limit was 4–10 cells per assay. Using the culture-PCR method, 20 of 26 soil isolates on a nitrogen-free medium were rapidly identified as P. mucilaginosus, which was confirmed by sequencing of the gyrB gene.

Keywords: Paenibacillus mucilaginosus ; gyrB; Culture-PCR; Rapid identification

A new coupon design for simultaneous analysis of in situ microbial biofilm formation and community structure in drinking water distribution systems by Peter Deines; Raju Sekar; P. Stewart Husband; Joby B. Boxall; A. Mark Osborn; Catherine A. Biggs (pp. 749-756).

This study presents a new coupon sampling device that can be inserted directly into the pipes within water distribution systems (WDS), maintaining representative near wall pipe flow conditions and enabling simultaneous microscopy and DNA-based analysis of biofilms formed in situ. To evaluate this sampling device, fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analyses were used to investigate changes in biofilms on replicate coupons within a non-sterile pilot-scale WDS. FISH analysis demonstrated increases in bacterial biofilm coverage of the coupon surface over time, while the DGGE analysis showed the development of increasingly complex biofilm communities, with time-specific clustering of these communities. This coupon design offers improvements over existing biofilm sampling devices in that it enables simultaneous quantitative and qualitative compositional characterization of biofilm assemblages formed within a WDS, while importantly maintaining fully representative near wall pipe flow conditions. Hence, it provides a practical approach that can be used to capture the interactions between biofilm formation and changing abiotic conditions, boundary shear stress, and turbulent driven exchange within WDS.

Keywords: Biofilms; CARD-FISH; DGGE; Drinking water distribution systems; Sampling coupon

Efficient amplification of genes involved in microbial secondary metabolism by an improved genome walking method by Jing Deng; Maochen Wei; Boyang Yu; Yijun Chen (pp. 757-764).

Genome walking is a commonly used technique for the identification of DNA sequences adjacent to known regions. Despite the development of various genome walking methods, nonspecific products are often produced in certain circumstances, especially when GC-rich DNA sequences are dealt with. To effectively resolve such technical issues, a simple nested polymerase chain reaction-based genome walking method has been developed by implementing a progressively decreased annealing temperature from 70°C to 47.5°C in the first round of amplification and a high annealing temperature of 65°C in the second round of amplification. During the entire process, a lower ramp rate of 1.5°C s⁻¹ and cooling rate of 2.5°C s⁻¹ are performed to reach the annealing temperature. Using this method, we successfully obtained the upstream and downstream sequences of three GC-rich genes involved in the biosynthetic pathways of secondary metabolites from two bacterial genomes. The efficient amplification of DNA target longer than 1.5 Kb with GC content up to 75.0% indicates that the present technique could be a valuable tool for the investigation of biosynthetic pathways of various secondary metabolites.

Keywords: Genome walking; Flanking sequence amplification; Nested PCR; GC-rich DNA

Effect of microwave irradiation on cellular disintegration of Gram positive and negative cells by Bi Wen Zhou; Seung Gu Shin; KwangHyun Hwang; Johng-Hwa Ahn; Seokhwan Hwang (pp. 765-770).

This research investigated the effect of microwave irradiation (MWI) on cell disintegration in municipal secondary sludge (MSS). A representative MSS Gram-positive bacterium (Bacillus subtilis) and Gram-negative bacteria (Acinetobacter calcoaceticus and Pseudomonas aeruginosa) were pure cultured separately and treated using MWI. Compared to untreated controls, MWI significantly increased the soluble chemical oxygen demand (COD) (1.8–4.0-fold), soluble protein concentration (1.1–1.8-fold), and soluble carbohydrate concentration (3.2–14.1-fold), with greater increase in the Gram-negative bacteria. After MSS was MWI-treated with different irradiation times, from 0 to 9 min, soluble COD increased gradually from 0.14 to 2.38 g/L (i.e., 72-fold). Effective disintegration of Gram-negative cell walls and of MSS by MWI was confirmed by scanning electron microscopy. These findings suggest that MWI could be an effective pretreatment method for MSS that is dominated by Gram-negative microorganisms.

Keywords: Acinetobacter calcoaceticus ; Bacillus subtilis ; Cell disintegration; Microwave irradiation; Municipal secondary sludge; Pseudomonas aeruginosa

Diversity of polyester-degrading bacteria in compost and molecular analysis of a thermoactive esterase from Thermobifida alba AHK119 by Xiaoping Hu; Uschara Thumarat; Xian Zhang; Ming Tang; Fusako Kawai (pp. 771-779).

More than 100 bacterial strains were isolated from composted polyester films and categorized into two groups, Actinomycetes (four genera) and Bacillus (three genera). Of these isolates, Thermobifida alba strain AHK119 (AB298783) was shown to possess the ability to significantly degrade aliphatic-aromatic copolyester film as well as decreasing the polymer particle sizes when grown at 50°C on LB medium supplemented with polymer particles, yielding terephthalic acid. The esterase gene (est119, 903 bp, encoding a signal peptide and a mature protein of 34 and 266 amino acids, respectively) was cloned from AHK119. The Est119 sequence contains a conserved lipase box (–G-X-S-X-G-) and a catalytic triad (Ser129, His207, and Asp175). Furthermore, Tyr59 and Met130 likely form an oxyanion hole. The recombinant enzyme was purified from cell-free extracts of Escherichia coli Rosetta-gami B (DE3) harboring pQE80L-est119. The enzyme is a monomeric protein of ca. 30 kDa, which is active from 20°C to 75°C (with an optimal range of 45 to 55°C) and in a pH range of 5.5 to 7.0 (with an optimal pH of 6.0). Its preferred substrate among the p-nitrophenyl acyl esters (C₂ to C₈) is p-nitrophenyl hexanoate (C₆), indicating that the enzyme is an esterase rather than a lipase.

Keywords: Thermophilic bacteria in compost; Aliphatic-aromatic copolyester degraders; Thermoactive esterase; Serine hydrolase; Thermobifida alba strain AHK119

Development of a transgenic tobacco plant for phytoremediation of methylmercury pollution by Takeshi Nagata; Hirofumi Morita; Toshifumi Akizawa; Hidemitsu Pan-Hou (pp. 781-786).

To develop the potential of plant for phytoremediation of methylmercury pollution, a genetically engineered tobacco plant that coexpresses organomercurial lyase (MerB) with the ppk-specified polyphosphate (polyP) and merT-encoding mercury transporter was constructed by integrating a bacterial merB gene into ppk/merT-transgenic tobacco. A large number of independent transgenic tobaccos was obtained, in some of which the merB gene was stably integrated in the plant genome and substantially translated to the expected MerB enzyme in the transgenic tobacco. The ppk/merT/merB-transgenic tobacco callus showed more resistance to methylmercury (CH₃Hg⁺) and accumulated more mercury from CH₃Hg⁺-containing medium than the ppk/merT-transgenic and wild-type progenitors. These results suggest that the MerB enzyme encoded by merB degraded the incorporated CH₃Hg⁺ to Hg²⁺, which then accumulated as a less toxic Hg-polyP complex in the tobacco cells. Phytoremediation of CH₃Hg⁺ and Hg²⁺ in the environment with this engineered ppk/merT/merB-transgenic plant, which prevents the release mercury vapor (Hg⁰) into the atmosphere in addition to generating potentially recyclable mercury-rich plant residues, is believed to be more acceptable to the public than other competing technologies, including phytovolatilization.

Keywords: Phytoremediation; Methylmercury pollution; Transgenic tobacco; Polyphosphate; merB gene; Mercury accumulation

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Applied Microbiology and Biotechnology (v.87, #2)

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Applied Microbiology and Biotechnology (v.87, #2)