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Applied Microbiology and Biotechnology (v.77, #5)

Biologically active components and nutraceuticals in the Monascus-fermented rice: a review by Yii-Lih Lin; Teng-Hsu Wang; Min-Hsiung Lee; Nan-Wei Su (pp. 965-973).

Monascus-fermented rice has traditionally been used as a natural food colorant and food preservative of meat and fish for centuries. It has recently become a popular dietary supplement because of many of its bioactive constituents being discovered, including a series of active drug compounds, monacolins, indicated as the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors for reducing serum cholesterol level. The controversy of its safety has been provoked because a mycotoxin, citrinin, is also produced along with the Monascus secondary metabolites by certain strains or under certain cultivation conditions. This review introduces the basic production process and addresses on the compounds with bioactive functions. Current advances in avoiding the harmful ingredient citrinin are also discussed.

Keywords: Koji; Metabolites; Monascus; Nutraceuticals

Characterization of a family 54 α-l-arabinofuranosidase from Aureobasidium pullulans by Barend J. M. de Wet; Mark K. A. Matthew; Karl-Heinz Storbeck; Willem H. van Zyl; Bernard A. Prior (pp. 975-983).

A glycosyl hydrolase family 54 (GH54) α-l-arabinofuranosidase gene (abfA) of Aureobasidium pullulans was amplified by polymerase chain reaction from genomic DNA and a 498-amino-acid open reading frame deduced from the DNA sequence. Modeling of the highly conserved A. pullulans AbfA protein sequence on the crystal structure of Aspergillus kawachii AkabfB showed that the catalytic amino acid arrangement and overall structure were highly similar including the N-terminal catalytic and C-terminal arabinose binding domains. The abfA gene was expressed in Saccharomyces cerevisiae, and the heterologous enzyme was purified. The protein was monomeric, migrating at 49 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and eluting at 36 kDa upon gel filtration. AbfA showed maximal activity at 55°C and between pH 3.5 and pH 4. The enzyme had a K _m value for p-nitrophenyl-α-l-arabinofuranoside of 3.7 mM and a V _max of 34.8 μmol min⁻¹ mg protein⁻¹. Arabinose acted as a noncompetitive inhibitor with a K _i of 38.4 mM. The enzyme released arabinose from maize fiber, oat spelt arabinoxylan, and wheat arabinoxylan, but not from larch wood arabinogalactan or α-1,5-debranched arabinan. AbfA displayed low activity against α-1,5-l-arabino-oligosaccharides. The enzyme acted synergistically with endo-β-1,4-xylanase in the breakdown of wheat arabinoxylan. Binding of AbfA to xylan from several sources confirmed the presence of a functional carbohydrate-binding module.

Isolation, identification and characterization of Bacillus subtilis ZJB-063, a versatile nitrile-converting bacterium by Yu-Guo Zheng; Jing Chen; Zhi-Qiang Liu; Ming-Huo Wu; Liang-Ying Xing; Yin-Chu Shen (pp. 985-993).

Strain ZJB-063, a versatile nitrile-amide-degrading strain, was newly isolated from soil in this study. Based on morphology, physiological tests, Biolog and the 16S rDNA sequence, strain ZJB-063 was identified as Bacillus subtilis. ZJB-063 exhibited nitrilase activity without addition of inducers, indicating that the nitrilase in B. subtilis ZJB-063 is constitutive. Interestingly, the strain exhibited nitrile hydratase and amidase activity with the addition of ɛ-caprolactam. Moreover, the substrate spectrum altered with the alteration of enzyme systems due to the addition of ɛ-caprolactam. The constitutive nitrilase was highly specific for arylacetonitriles, while the nitrile hydratase/amidase in B. subtilis ZJB-063 could not only hydrolyze arylacetonitriles but also other nitriles including some aliphatic nitriles and heterocyclic nitriles. Despite comparatively low activity, the amidase of hydratase/amidase system was effective in converting amides to acids. The versatility of this strain in the hydrolysis of various nitriles and amides makes it a potential biocatalyst in organic synthesis.

Keywords: Bacillus subtilis ; Isolation; Identification; Nitrilase; Nitrile hydratase; Substrate spectrum

Purification and characterization of alcohol oxidase from Paecilomyces variotii isolated as a formaldehyde-resistant fungus by Tetsuya Kondo; Yutaka Morikawa; Naohiro Hayashi (pp. 995-1002).

Paecilomyces variotii IRI017 was isolated as a formaldehyde-resistant fungus from wastewater containing formaldehyde. The fungus grew in a medium containing 0.5% formaldehyde and had consumed formaldehyde completely after 5 days. Alcohol oxidase was purified from the fungus grown on methanol. A 20-fold purification was achieved with a yield of 44%. The molecular mass of the purified enzyme was estimated to be 73 and 450 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively, suggesting that the enzyme consists of six identical subunits. The N-terminal amino acid sequence of the subunit was TIPDEVDIII. The enzyme showed an absorption spectrum typical of a flavoprotein and had a noncovalently bound flavin different from FAD, FMN, and riboflavin. The pH optimum of the enzyme activity was pH 6–10. The enzyme was stable in the pH range of pH 5–10. The enzyme retained full activity after incubation at 50°C for 30 min. The enzyme oxidized not only methanol but also lower primary alcohols and formaldehyde. The K _m values for methanol, ethanol, and formaldehyde were 1.9, 3.8, and 4.9 mmol l⁻¹, respectively.

Keywords: Alcohol oxidase; Enzyme purification; Formaldehyde; Methanol; Paecilomyces variotii

Archaeal alcohol dehydrogenase active at increased temperatures and in the presence of organic solvents by Matthias Hess; Garabed Antranikian (pp. 1003-1013).

The adhA gene of the extreme thermoacidophilic Archaeon Picrophilus torridus was identified by the means of genome analysis and was subsequently cloned in Escherichia coli. PTO 0846, encoding AdhA, consists of 954 bp corresponding to 317 aa. Sequence comparison revealed that the novel biocatalyst has a low sequence identity (<26%) to previously characterized enzymes. The recombinant alcohol dehydrogenase was purified using hydroxyapatite, and alcohol oxidative activity of the purified AdhA was measured over a wide pH and temperature range with maximal activity at 83°C and pH 7.8. Detailed analysis suggests that the active AdhA is a multimer, consisting of 12 identical subunits, with a molecular mass of 35 kDa each. AdhA represents the first dodecameric alcohol dehydrogenase characterized until to date. AdhA is able to oxidize primary and secondary alcohols with ethanol and 1-phenylalcohol as preferred substrates and NAD⁺ as preferred cofactor. In addition, isopropanol, which has been used successfully as cosubstrate in cofactor regeneration, is oxidized as well by AdhA. Besides being thermostable (t _1/2 = 42 min at 70°C), AdhA is also active in the presence of increased concentrations of urea (up to 5 M) and in the presence of organic solvents [up to 50% (v/v)] commonly used for organic synthesis.

Keywords: Alcohol dehydrogenase; Picrophilus torridus ; Thermoactive enzyme

High-level expression and purification of Tat-haFGF_19-154 by Yadong Huang; Yulan Rao; Chengli Feng; Yanmei Li; Xiaoping Wu; Zhijian Su; Jian Xiao; Yechen Xiao; Wenke Feng; Xiaokun Li (pp. 1015-1022).

Human acidic fibroblast growth factor (haFGF) stimulates repair and regeneration of central and peripheral nerves after various injuries. However, it is unable to cross the blood–brain barrier (BBB). To produce a therapeutic haFGF with cell-permeable activity, we fused the haFGF_19-154 gene with Tat-PTD. After its construction by a single-step insertion of a polymerase chain reaction (PCR)–amplified coding sequence, the vector pTat-haFGF_19-154-His was expressed in Escherichia coli BL21 (DE3) cells. The optimal expression level of the soluble fusion protein was up to 36.7% of the total cellular protein. The recombinant Tat-haFGF_19-154-His was purified by a combination of Ni–NTA affinity, Sephadex G-25, and heparin affinity chromatography to 95% as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The final yield was 171 mg/l culture. Purified Tat-haFGF_19-154-His had distinct mitogenic activity in Balb/c 3T3 cells, as measured by methylthiazoletetrazolium (MTT) assay and its ED₅₀ was 3.931 × 10⁻⁴ µmol/l. Tat-haFGF_19-154-His protein intravenously injected at the dose of 10 mg/kg could be detected in the pallium and hippocampi.

Keywords: Acidic fibroblast growth factor; Tat-PTD; Expression; Purification; Mitogenic activity

Production of surfactant and detergent-stable, halophilic, and alkalitolerant alpha-amylase by a moderately halophilic Bacillus sp. Strain TSCVKK by Kondepudi Kanthi Kiran; T. S. Chandra (pp. 1023-1031).

A moderately halophilic alkalitolerant Bacillus sp. Strain TSCVKK, with an ability to produce extracellular halophilic, alkalitolerant, surfactant, and detergent-stable alpha-amylase was isolated from soil samples obtained from a salt-manufacturing industry in Chennai. The culture conditions for higher amylase production were optimized with respect to NaCl, substrate, pH, and temperature. Maximum amylase production of 592 mU/ml was achieved in the medium at 48 h with 10% NaCl, 1% dextrin, 0.4% yeast extract, 0.2% tryptone, and 0.2% CaCl₂ at pH 8.0 at 30 °C. The enzyme activity in the culture supernatant was highest with 10% NaCl at pH 7.5 and 55 °C. The amylase that was partially purified by acetone precipitation was highly stable in various surfactants and detergents. Glucose, maltose, and maltooligosaccharides were the main end products of starch hydrolysis indicating that it is an alpha-amylase.

Keywords: Moderate halophile; Bacillus sp. Strain TSCVKK ; Alpha-amylase; Alkalitolerant; Surfactant stable; Detergent stable

Cumulative improvements of thermostability and pH-activity profile of Aspergillus niger PhyA phytase by site-directed mutagenesis by Wanming Zhang; Xin Gen Lei (pp. 1033-1040).

Aspergillus niger phytase (PhyA) has been used as a feed supplement to reduce manure phosphorus excretion of swine and poultry but lacks sufficient thermostability for feed pelleting and appropriate pH-activity profile for phytate hydrolysis in the stomach of animals. Previously, a thermostable mutant PhyA18 and two pH-activity profile-improved mutants E228K and K300E were developed. In this study, the mutations were combined to determine if both improvements were cumulative. Four substitutions (S149P, F131L, K112R, and K195R) identified from random mutagenesis were added sequentially to the combined mutants to further improve their thermostability. Mutant E228K shifted the optimum pH of the parent one from 5.5 to 4.0 and increased (P < 0.05) its specific activity at pH 3.5, whereas mutant K300E eliminated the activity dip at pH 3.5 shown in the wild type. Mutant S149P further improved thermostability over PhyA18. Our results illustrate the feasibility and structural basis to improve thermostability and pH-activity profile of PhyA phytase by assembling mutations derived from rational design and random mutagenesis.

Discovery of three novel lipase (lipA 1, lipA 2, and lipA 3) and lipase-specific chaperone (lipB) genes present in Acinetobacter sp. DYL129 by Sun-Hee Kim; In-Hye Park; Sang-Cheol Lee; Yong-Seok Lee; Zhou-Yi; Cheol-Min Kim; Soon-Cheol Ahn; Yong-Lark Choi (pp. 1041-1051).

A microbe isolated from a soil sample obtained on Deog-yu Mountain in Korea, was found to produce an extracellular lipase. This microbe, which was designated as DYL129, was identified as an Acinetobacter sp. based on phylogenetic analysis of its 16S rDNA. A genomic library was constructed by using DYL129 fragment digested with HindIII and a recombinant plasmid, pLip-1, was selected for further analysis by colony polymerase chain reaction (PCR). Sequencing of a 3.8-kb insert in the pLip-1 clone revealed the presence of one incomplete and three complete open reading frames (ORFs). The ORFs were predicted to encode a partial lipase, two putative lipases and a 50S ribosomal protein. Genome-walking PCR also identified transcripts encoding a complete lipase chaperone and three lipaseA proteins. The lipase structural gene present in Acinetobacter sp. DYL129 was similar to the lipase structural gene found in Acinetobacter calcoaceticus BD413 (lipBA). However, the three lipase genes were located downstream of the chaperone gene in Acinetobacter sp. DYL129 (lipBA 1 A 2 A 3), which differs from the location of these genes in A. calcoaceticus BD413. Although the amino acid sequences of these lipases (LipA₁, LipA₂, and LipA₃) differed, both strains had a common “GHSHG” consensus motif, which is the conserved active-site pentapeptide of lipaseA. Moreover, all three lipases were found to share a conserved domain, the so-called α/β hydrolase fold.

Keywords: Acinetobacter ; lipA 1 ; lipA 2 ; lipA 3

Engineering of an l-arabinose metabolic pathway in Corynebacterium glutamicum by Hideo Kawaguchi; Miho Sasaki; Alain A. Vertès; Masayuki Inui; Hideaki Yukawa (pp. 1053-1062).

Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar l-arabinose, a product of the degradation of lignocellulosic biomass. The resultant CRA1 recombinant strain expressed the Escherichia coli genes araA, araB, and araD encoding l-arabinose isomerase, l-ribulokinase, and l-ribulose-5-phosphate 4-epimerase, respectively, under the control of a constitutive promoter. Unlike the wild-type strain, CRA1 was able to grow on mineral salts medium containing l-arabinose as the sole carbon and energy source. The three cloned genes were expressed to the same levels whether cells were cultured in the presence of d-glucose or l-arabinose. Under oxygen deprivation and with l-arabinose as the sole carbon and energy source, strain CRA1 carbon flow was redirected to produce up to 40, 37, and 11%, respectively, of the theoretical yields of succinic, lactic, and acetic acids. Using a sugar mixture containing 5% d-glucose and 1% l-arabinose under oxygen deprivation, CRA1 cells metabolized l-arabinose at a constant rate, resulting in combined organic acids yield based on the amount of sugar mixture consumed after d-glucose depletion (83%) that was comparable to that before d-glucose depletion (89%). Strain CRA1 is, therefore, able to utilize l-arabinose as a substrate for organic acid production even in the presence of d-glucose.

Keywords: Corynebacterium glutamicum ; l-arabinose; Lignocellulosic biomass

Construction and characterization of a clostripain-like protease-deficient mutant of Clostridium perfringens as a strain for clostridial gene expression by Hiroaki Tanaka; Eiji Tamai; Shigeru Miyata; Yuki Taniguchi; Hirofumi Nariya; Naoya Hatano; Hitoshi Houchi; Akinobu Okabe (pp. 1063-1071).

The inherent difficulty of expressing clostridial AT-rich genes in a heterologous host has limited their biotechnological application. We previously reported a plasmid for high-level expression of clostridial genes in Clostridium perfringens (Takamizawa et al., Protein Expr Purif 36:70–75, 2004). In this study, we examined the extracellular proteases of C. perfringens strain 13. Zymographic analysis and caseinase assaying of a culture supernatant showed that it contained a protease activated by dithiothreitol and Ca²⁺, suggesting that clostripain-like protease (Clp) is the most likely candidate for the major extracellular protease. Disruption of the clp gene by homologous recombination markedly decreased the level of caseinase activity in the culture supernatant. Analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the Clp⁻ mutant but not the wild type strain increased the levels of many polypeptides in the culture supernatant after the late exponential growth phase. Such polypeptides included both cytoplasmic and secretory proteins, suggesting proteins secreted or released into the medium were degraded by Clp. To assess the effects of Clp on the productivity and stability of recombinant proteins, 74-kDa NanI sialidase was expressed in the two strains. The mutant strain produced a higher level of NanI activity than the wild type strain. Furthermore, under the conditions where Clp was activated, NanI was degraded easily in the latter culture but not in the former one. These results indicate that the Clp⁻ mutant could serve as a useful strain for efficiently expressing and preparing protease-free clostridial proteins.

Keywords: Clostripain; Thiol-protease; Clostridium perfringens ; Protease-deficient mutant; Expression system

Loss of heterozygosity is induced in Candida albicans by ultraviolet irradiation by Yukinobu Takagi; Rinji Akada; Hidehiko Kumagai; Kenji Yamamoto; Hisanori Tamaki (pp. 1073-1082).

Candida albicans is a human fungal pathogen and has been extensively studied because of its clinical importance. Comprehensive gene analyses have, however, made little progress. This is because of the diploid and asexual characteristics of the fungus that hamper gene disruptions. In this study, we found that ultraviolet (UV) irradiation, as well as mutagen treatment, strongly stimulated loss of heterozygosity (LOH) in strains harboring artificially constructed heterozygosity. UV-induced LOH occurred more frequently in cells within the logarithmic phase of growth compared to those within the stationary phase of growth. This was observed at all loci tested on chromosome 7, except for a locus neighboring the centromere. C. albicans RAD52, whose orthologue in Saccharomyces cerevisiae was reported to be involved in DNA repair by homologous recombination, was shown to be required for UV-induced LOH. These results suggest that high efficiency LOH caused by UV irradiation could be a prominent tool for gene analyses in C. albicans.

Keywords: Candida albicans ; Loss of heterozygosity; UV; RAD52

Correlation between glucose/fructose discrepancy and hexokinase kinetic properties in different Saccharomyces cerevisiae wine yeast strains by Nele J. Berthels; Ricardo R. Cordero Otero; Florian F. Bauer; Isak S. Pretorius; Johan M. Thevelein (pp. 1083-1091).

Grape juice contains about equal amounts of glucose and fructose, but wine strains of Saccharomyces cerevisiae ferment glucose slightly faster than fructose, leading to fructose concentrations that exceed glucose concentrations in the fermenting must. A high fructose/glucose ratio may contribute to sluggish and stuck fermentations, a major problem in the global wine industry. We evaluated wine yeast strains with different glucose and fructose consumption rates to show that a lower glucose preference correlates with a higher fructose/glucose phosphorylation ratio in cell extracts and a lower K _m for both sugars. Hxk1 has a threefold higher V _max with fructose than with glucose, whereas Hxk2 has only a slightly higher V _max with glucose than with fructose. Overexpression of HXK1 in a laboratory strain of S. cerevisiae (W303–1A) accelerated fructose consumption more than glucose consumption, but overexpression in a wine yeast strain (VIN13) reduced fructose consumption less than glucose consumption. Results with laboratory strains expressing a single kinase showed that total hexokinase activity is inversely correlated with the glucose/fructose (G/F) discrepancy. The latter has been defined as the difference between the rate of glucose and fructose fermentation. We conclude that the G/F discrepancy in wine yeast strains correlates with the kinetic properties of hexokinase-mediated sugar phosphorylation. A higher fructose/glucose phosphorylation ratio and a lower K _m might serve as markers in selection and breeding of wine yeast strains with a lower tendency for sluggish fructose fermentation.

Keywords: Wine yeast; Sugar preference; Sugar phosphorylation; Fermentation

Acetaldehyde addition throughout the growth phase alleviates the phenotypic effect of zinc deficiency in Saccharomyces cerevisiae by Naoufel Cheraiti; François-Xavier Sauvage; Jean-Michel Salmon (pp. 1093-1109).

During experiments to determine the effects of exogenously added acetaldehyde on pure cultures of various yeast strains, we discovered that an early acetaldehyde perfusion during the growth phase allowed several yeasts to partially overcome the phenotypic effects of zinc depletion during alcoholic fermentation. We, therefore, performed genome-wide expression and proteomic analysis on an industrial Saccharomyces cerevisiae yeast strain (VL1) growing in zinc-replete or zinc-depleted conditions in the presence of perfused acetaldehyde to identify molecular markers of this effect. Zinc depletion severely affects ethanol production and therefore nicotinamide adenine dinucleotide (NAD) regeneration, although we observed partial compensation by the upregulation of the poorly efficient Fe-dependent Adh4p in our conditions. A coordinate metabolic response was indeed observed in response to the early acetaldehyde perfusion, and particularly of the lower part of glycolysis, leading to the cellular replenishment of NAD cofactor. These various findings suggest that acetaldehyde exchange between strains may inhibit the growth of some yeast strains while encouraging the growth of others. This phenomenon could be particularly important for understanding the ecology of colonization of complex fermentation media by S. cerevisiae after elimination of non-Saccharomyces yeasts.

Keywords: Yeast; Zinc depletion; Acetaldehyde; Anaerobiosis; Alcoholic fermentation

Reaction and strain engineering for improved stereo-selective whole-cell reduction of a bicyclic diketone by Ted Johanson; Magnus Carlquist; Cecilia Olsson; Andreas Rudolf; Torbjörn Frejd; Marie F. Gorwa-Grauslund (pp. 1111-1118).

Reduction of bicyclo[2.2.2]octane-2,6-dione to (1R, 4S, 6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one by whole cells of Saccharomyces cerevisiae was improved using an engineered recombinant strain and process design. The substrate inhibition followed a Han-Levenspiel model showing an effective concentration window between 12 and 22 g/l, in which the activity was kept above 95%. Yeast growth stage, substrate concentration and a stable pH were shown to be important parameters for effective conversion. The over-expression of the reductase gene YDR368w significantly improved diastereoselectivity compared to previously reported results. Using strain TMB4110 expressing YDR368w in batch reduction with pH control, complete conversion of 40 g/l (290 mM) substrate was achieved with 97% diastereomeric excess (de) and >99 enantiomeric excess (ee), allowing isolation of the optically pure ketoalcohol in 84% yield.

Keywords: Whole-cell; Bioreduction; Reductase; Yeast; Dicarbonyl; Process optimisation; Toxicity; Substrate inhibition; Diastereoselectivity

Metabolites produced by Pseudomonas sp. enable a Gram-positive bacterium to achieve extracellular electron transfer by The Hai Pham; Nico Boon; Peter Aelterman; Peter Clauwaert; Liesje De Schamphelaire; Lynn Vanhaecke; Katrien De Maeyer; Monica Höfte; Willy Verstraete; Korneel Rabaey (pp. 1119-1129).

Previous studies revealed the abundance of Pseudomonas sp. in the microbial community of a microbial fuel cell (MFC). These bacteria can transfer electrons to the electrode via self-produced phenazine-based mediators. A MFC fed with acetate where several Pseudomonas sp. were present was found to be rich in a Gram-positive bacterium, identified as Brevibacillus sp. PTH1. Remarkably, MFCs operated with only the Brevibacillus strain in their anodes had poor electricity generation. Upon replacement of the anodic aqueous part of Brevibacillus containing MFCs with the cell-free anodic supernatants of MFCs operated with Pseudomonas sp. CMR12a, a strain producing considerable amounts of phenazine-1-carboxamide (PCN) and biosurfactants, the electricity generation was improved significantly. Supernatants of Pseudomonas sp. CMR12a_Reg, a regulatory mutant lacking the ability to produce PCN, had no similar improvement effect. Purified PCN, together with rhamnolipids as biosurfactants (1 mg L⁻¹), could clearly improve electricity generation by Brevibacillus sp. PTH1, as well as enable this bacterium to oxidize acetate with concomitant reduction of ferric iron, supplied as goethite (FeOOH). When added alone, PCN had no observable effects on Brevibacillus’ electron transfer. This work demonstrates that metabolites produced by Pseudomonas sp. enable Gram-positive bacteria to achieve extracellular electron transfer. Possibly, this bacterial interaction is a key process in the anodic electron transfer of a MFC, enabling Brevibacillus sp. PTH1 to achieve its dominance.

Keywords: Electrochemically active bacteria; Microbial interaction; Electron shuttle; Phenazines; Biosurfactant; Synergistic effect

Obtaining and selection of hexokinases-less strains of Saccharomyces cerevisiae for production of ethanol and fructose from sucrose by Rodrigo Setem Carvalho; Luiz Humberto Gomes; Luiz Gonzaga do P. Filho; Flávio C. A. Tavares (pp. 1131-1137).

Saccharomyces cerevisiae hexokinase-less strains were produced to study the production of ethanol and fructose from sucrose. These strains do not have the hexokinases A and B. Twenty-three double-mutant strains were produced, and then, three were selected for presenting a smaller growth in yeast extract–peptone–fructose. In fermentations with a medium containing sucrose (180.3 g L⁻¹) and with cell recycles, simulating industrial conditions, the capacity of these mutant yeasts in inverting sucrose and fermenting only glucose was well characterized. Besides that, we could also see their great tolerance to the stresses of fermentative recycles, where fructose production (until 90 g L⁻¹) and ethanol production (until 42.3 g L⁻¹) occurred in cycles of 12 h, in which hexokinase-less yeasts performed high growth (51.2% of wet biomass) and viability rates (77% of viable cells) after nine consecutive cycles.

Keywords: Saccharomyces cerevisiae ; Hexokinase; Fermentation; Fructose; Ethanol

Azoreductase and dye detoxification activities of Bacillus velezensis strain AB by Amit Bafana; Tapan Chakrabarti; Sivanesan Saravana Devi (pp. 1139-1144).

Azo dyes are known to be a very important and widely used class of toxic and carcinogenic compounds. Although lot of research has been carried out for their removal from industrial effluents, very little attention is given to changes in their toxicity and mutagenicity during the treatment processes. Present investigation describes isolation of a Bacillus velezensis culture capable of degrading azo dye Direct Red 28 (DR28). Azoreductase enzyme was isolated from it, and its molecular weight was found to be 60 kDa. The enzyme required NADH as cofactor and was oxygen-insensitive. Toxicity and mutagenicity of the dye during biodegradation was monitored by using a battery of carefully selected in vitro tests. The culture was found to degrade DR28 to benzidine and 4-aminobiphenyl, both of which are potent mutagens. However, on longer incubation, both the compounds were degraded further, resulting in reduction in toxicity and mutagenicity of the dye. Thus, the culture seems to be a suitable candidate for further study for both decolourization and detoxification of azo dyes, resulting in their safe disposal.

Keywords: Direct red 28; Azoreductase; Zymogram; Ames test; Biodegradation

Copper resistance of biofilm cells of the plant pathogen Xylella fastidiosa by Carolina M. Rodrigues; Marco A. Takita; Helvécio D. Coletta-Filho; Jacqueline C. Olivato; Raquel Caserta; Marcos A. Machado; Alessandra A. de Souza (pp. 1145-1157).

Xylella fastidiosa is a phytopathogen that causes diseases in different plant species. The development of disease symptoms is associated to the blockage of the xylem vessels caused by biofilm formation. In this study, we evaluated the sensitivity of biofilm and planktonic cells to copper, one of the most important antimicrobial agents used in agriculture. We measured the exopolysaccharides (EPS) content in biofilm and planktonic cells and used real-time reverse transcription polymerase chain reaction to evaluate the expression of the genes encoding proteins involved in cation/multidrug extrusion (acrA/B, mexE/czcA, and metI) and others associated with different copper resistance mechanisms (copB, cutA1, cutA2, and cutC) in the X. fastidiosa biofilm formed in two different media. We confirmed that biofilms are less susceptible to copper than planktonic cells. The amount of EPS seems to be directly related to the resistance and it varies according to the media where the cells are grown. The same was observed for gene expression. Nevertheless, some genes seem to have a greater importance in biofilm cells resistance to copper. Our results suggest a synergistic effect between diffusion barriers and other mechanisms associated with bacterial resistance in this phytopathogen. These mechanisms are important for a bacterium that is constantly under stress conditions in the host.

Keywords: metal; EPS; real-time RT-PCR

Enrichment of anammox bacteria from marine environment for the construction of a bioremediation reactor by Jun Nakajima; Makiko Sakka; Tetsuya Kimura; Kenji Furukawa; Kazuo Sakka (pp. 1159-1166).

In the global ocean nitrogen cycle, the anaerobic ammonium-oxidizing (anammox) process is recognized as important. In this study, we established an enrichment culture of marine anammox bacteria (MAB) in a column-type reactor. The reactor, which included a porous polyester non-woven fabric that had been placed at the sea floor in advance for enrichment, was continuously fed with NH₄Cl and NaNO₂ for more than 1 year. Anammox activity in the MAB reactor was confirmed by ¹⁵N tracer analysis using ¹⁵NH₄Cl and Na¹⁴NO₂. We identified two 16S rRNA genes in the amplified DNA fragments derived from MAB, which were highly homologous with those from Candidatus “Scalindua wagneri” and an uncultured planctomycete clone. Fluorescence in situ hybridization analysis using an anammox-specific probe also confirmed that MAB predominated in the reactor. To our knowledge, this is the first report on the establishment of an enrichment culture of anammox bacteria from the marine environment using a continuous culture system.

Keywords: Anammox; Denitrification; Marine; Candidatus “Scalindua wagneri”; FISH

Cloning, stable expression of human phosphodiesterase 7A and development of an assay for screening of PDE7 selective inhibitors by Renu Malik; Roop Singh Bora; Dikshi Gupta; Pratibha Sharma; Ranjana Arya; Shiwani Chaudhary; Kulvinder Singh Saini (pp. 1167-1173).

Phosphodiesterases (PDEs) constitute a superfamily of enzymes that plays an important role in signal transduction by catalysing the hydrolysis of cAMP and cGMP. cDNA encoding PDE7A1 subtype was cloned and a stable recombinant HEK 293 cell line expressing high levels of PDE7A1 was generated. Transient transfection of pCRE-Luc plasmid, harboring luciferase reporter gene into the stable recombinant cell line and subsequent treatment with PDE7 inhibitor, resulted in a dose-dependent increase in luciferase activity. This method provides a simple and sensitive cell-based assay for screening of PDE7 selective inhibitors for the treatment of T cell mediated diseases.

Purification of green fluorescent protein using a two-intein system by Zhonglin Zhao; Wei Lu; Baoqing Dun; Dan Jin; Shuzhen Ping; Wei Zhang; Ming Chen; Ming-Qun Xu; Min Lin (pp. 1175-1180).

A two-intein purification system was developed for the affinity purification of GFPmut3*, a mutant of green fluorescent protein. The GFPmut3* was sandwiched between two self-cleaving inteins. This approach avoided the loss of the target protein which may result from in vivo cleavage of a single intein tag. The presence of N- and C-terminal chitin-binding domains allowed the affinity purification by a single-affinity chitin column. After the fusion protein was expressed and immobilized on the affinity column, self-cleavage of the inteins was sequentially induced to release the GFPmut3*. The yield was 2.41 mg from 1 l of bacterial culture. Assays revealed that the purity was up to 98% of the total protein. The fluorescence and circular dichroism spectrum of GFPmut3* demonstrated that the purified protein retains the correctly folded structure and function.

Keywords: Intein; Protein splicing; Green fluorescent protein; Protein purification

Electrical DNA-chip-based identification of different species of the genus Kitasatospora by Robert Möller; Thomas Schüler; Sebastian Günther; Marc René Carlsohn; Thomas Munder; Wolfgang Fritzsche (pp. 1181-1188).

The identification of different Kitasatospora strains has been shown with a DNA-chip based on an electrical readout scheme. The 16S-23S rDNA internal transcribed spacer region of these Actinomycetes was used for identification. Two different capture probes per strain were immobilized on the chip. The capture probes were spotted on a DNA-chip with electrode structures for an electrical DNA detection. A biotinylated PCR product of the 16S-23S rDNA region was incubated on the chips and bound to its complementary capture sequences. Followed by a gold nanoparticle or enzyme labeling and a deposition of silver, the binding of the PCR product was detected by an increase of the measured conductivity on the chip. To show the applicability of this detection system, four strains of Kitasatospora were chosen for an identification using the DNA-chip with electrical detection. Each strain was clearly identified using the system. Concentrations of the polymerase chain reaction (PCR) products within the range of 1 ng/ml to 1 μg/ml were detected and identified. These tests are the first application of this novel electrical detection scheme for the identification and classification of microorganisms. The presented results show that the DNA-chip with electrical detection can be used for a robust and cost-efficient DNA analysis.

Keywords: DNA-chip; Electrical readout; Kitasatospora ; Gold nanoparticles; 16S-23S rDNA

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Applied Microbiology and Biotechnology (v.77, #5)

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Applied Microbiology and Biotechnology (v.77, #5)