Check out our New Publishers' Select for Free Articles

Applied Microbiology and Biotechnology (v.78, #1)

Properties and applications of microbial D-amino acid oxidases: current state and perspectives by Loredano Pollegioni; Gianluca Molla; Silvia Sacchi; Elena Rosini; Roberto Verga; Mirella S. Pilone (pp. 1-16).

D-Amino acid oxidase (DAAO) is a biotechnologically relevant enzyme that is used in a variety of applications. DAAO is a flavine adenine dinucleotide-containing flavoenzyme that catalyzes the oxidative deamination of D-isomer of uncharged aliphatic, aromatic, and polar amino acids yielding the corresponding imino acid (which hydrolyzes spontaneously to the α-keto acid and ammonia) and hydrogen peroxide. This enzymatic activity is produced by few bacteria and by most eukaryotic organisms. In the past few years, DAAO from mammals has been the subject of a large number of investigations, becoming a model for the dehydrogenase-oxidase class of flavoproteins. However, DAAO from microorganisms show properties that render them more suitable for the biotechnological applications, such as a high level of protein expression (as native and recombinant protein), a high turnover number, and a tight binding of the coenzyme. Some important DAAO-producing microorganisms include Trigonopsis variabilis, Rhodotorula gracilis, and Fusarium solani. The aim of this paper is to provide an overview of the main biotechnological applications of DAAO (ranging from biocatalysis to convert cephalosporin C into 7-amino cephalosporanic acid to gene therapy for tumor treatment) and to illustrate the advantages of using the microbial DAAOs, employing both the native and the improved DAAO variants obtained by enzyme engineering.

Keywords: D-amino acid; Cephalosporin C; Oxidative stress; Flavoprotein; Bioconversion; D-amino acid oxidase (DAAO, EC 1.4.3.3); D-aspartate oxidase (DASPO, EC 1.4.3.1); D-amino acid dehydrogenase (DAAdH, EC 1.4.99.1); alkaline phosphatase (AP, EC 3.1.3.1)

A small protein that fights fungi: AFP as a new promising antifungal agent of biotechnological value by Vera Meyer (pp. 17-28).

As fungal infections are becoming more prevalent in the medical or agricultural fields, novel and more efficient antifungal agents are badly needed. Within the scope of developing new strategies for the management of fungal infections, antifungal compounds that target essential fungal cell wall components are highly preferable. Ideally, newly developed antimycotics should also combine major aspects such as sustainability, high efficacy, limited toxicity and low costs of production. A naturally derived molecule that possesses all the desired characteristics is the antifungal protein (AFP) secreted by the filamentous ascomycete Aspergillus giganteus. AFP is a small, basic and cysteine-rich peptide that exerts extremely potent antifungal activity against human- and plant-pathogenic fungi without affecting the viability of bacteria, yeast, plant and mammalian cells. This review summarises the current knowledge of the structure, mode of action and expression of AFP, and highlights similarities and differences concerning these issues between AFP and its related proteins from other Ascomycetes. Furthermore, the potential use of AFP in the combat against fungal contaminations and infections will be discussed.

Keywords: Antifungal protein; Aspergillus giganteus ; Pathogenic fungi; Cell wall integrity; Chitin biosynthesis; Antifungal treatment

High-density fermentation of microalga Chlorella protothecoides in bioreactor for microbio-diesel production by Wei Xiong; Xiufeng Li; Jinyi Xiang; Qingyu Wu (pp. 29-36).

Agal-fermentation-based microbio-diesel production was realized through high-cell-density fermentation of Chlorella protothecoides and efficient transesterification process. Cell density achieved was 16.8 g l⁻¹ in 184 h and 51.2 g l⁻¹ in 167 h in a 5-l bioreactor by performing preliminary and improved fed-batch culture strategy, respectively. The lipid content was 57.8, 55.2, and 50.3% of cell dry weight from batch, primary, and improved fed-batch culture in 5-l bioreactor. Transesterification was catalyzed by immobilized lipase, and the conversion rate reached up to 98%. The properties of biodiesel from Chlorella were comparable to conventional diesel fuel and comply with US standard for Biodiesel. In a word, the approach including high-density fermentation of Chlorella and enzymatic transesterification process were set up and proved to be a promising alternative for biodiesel production.

Keywords: Agal-fermentation-based microbio-diesel (AFMD); Chlorella protothecoides ; Fed-batch; High-density fermentation; Immobilized lipase

Efficient production of mannosylerythritol lipids with high hydrophilicity by Pseudozyma hubeiensis KM-59 by Masaaki Konishi; Tomotake Morita; Tokuma Fukuoka; Tomohiro Imura; Koji Kakugawa; Dai Kitamoto (pp. 37-46).

Mannosylerythritol lipids (MELs) are one of the most promising biosurfactants known because of their multifunctionality and biocompatibility. A previously isolated yeast strain, Pseudozyma sp. KM-59, mainly produced a hydrophilic MEL, namely MEL-C (4-O-[4′-O-acetyl-2′,3′-di-O-alka(e)noyl-β-d-mannopyranosyl]-d-erythritol). In this study, we taxonomically characterize the strain in detail and investigate the culture conditions. The genetic, morphological, and physiological characteristics of the strain coincided well with those of Pseudozyma hubeiensis. On batch culture for 4 days under optimal conditions, the yield of all MELs was 21.8 g/l; MEL-C comprised approximately 65% of the all MELs. Consequently, on fed-batch culture for 16 days, the yield reached 76.3 g/l; the volumetric productivity was approximately 4.8 g l⁻¹ day⁻¹. We further examined the surface-active and self-assembling properties of the hydrophilic MELs produced by the yeast strain. They showed higher emulsifying activities against soybean oil and a mixture of hydrocarbons (2-methylnaphtarene and hexadecane, 1:1) than the synthetic surfactants tested. On water penetration scans, they efficiently formed lyotropic liquid crystalline phases such as myelines and lamella ( $$ { ext{L}}_{alpha } $$ ) in a broad range of their concentrations, indicating higher hydrophilicity than conventional MELs. More interestingly, there was little difference in the liquid crystal formation between the crude product and purified MEL-C. The present glycolipids with high hydrophilicity are thus very likely to have practical potential without further purification and to expand the application of MELs especially their use in washing detergents and oil-in-water-type emulsifiers.

Keywords: Biosurfactant; Mannosylerythritol lipid; Pseudozyma hubeiensis ; Lyotropic liquid crystal; Hydrophilicity; Fed-batch cultivation

Complete hydrolysis of myo-inositol hexakisphosphate by a novel phytase from Debaryomyces castellii CBS 2923 by Mélanie Ragon; André Aumelas; Patrick Chemardin; Santiago Galvez; Guy Moulin; Hélène Boze (pp. 47-53).

Debaryomyces castellii phytase was purified to homogeneity in a single step by hydrophobic interaction chromatography. Its molecular mass is 74 kDa with 28.8% glycosylation. Its activity was optimal at 60°C and pH 4.0. The K _m value for sodium phytate was 0.532 mM. The enzyme exhibited a low specificity and hydrolyzed many phosphate esters. The phytase fully hydrolyzed myo-inositol hexakisphosphate (or phytic acid, Ins P₆) to inositol and inorganic phosphate. The sequence of Ins P₆ hydrolysis was determined by combining results from high-performance ionic chromatography and nuclear magnetic resonance. D. castellii phytase is a 3-phytase that sequentially releases phosphate groups through Ins (1,2,4,5,6) P₅, Ins (1,2,5,6) P₄, Ins (1,2,6) P₃, Ins (1,2) P₂, Ins (1 or 2) P₁, and inositol (notation 3/4/5/6/1 or 2).

Keywords: Debaryomyces castellii ; Yeast; Phytase; Enzyme characterization; Inositol phosphates; Phytate degradation; NMR; HPIC

Improvement of P450_BM-3 whole-cell biocatalysis by integrating heterologous cofactor regeneration combining glucose facilitator and dehydrogenase in E. coli by Hendrik Schewe; Bjoern-Arne Kaup; Jens Schrader (pp. 55-65).

Escherichia coli BL21, expressing a quintuple mutant of P450_BM-3, oxyfunctionalizes α-pinene in an NADPH-dependent reaction to α-pinene oxide, verbenol, and myrtenol. We optimized the whole-cell biocatalyst by integrating a recombinant intracellular NADPH regeneration system through co-expression of a glucose facilitator from Zymomonas mobilis for uptake of unphosphorylated glucose and a NADP⁺-dependent glucose dehydrogenase from Bacillus megaterium that oxidizes glucose to gluconolactone. The engineered strain showed a nine times higher initial α-pinene oxide formation rate corresponding to a sixfold higher yield of 20 mg g⁻¹ cell dry weight after 1.5 h. The initial total product formation rate was 1,000 μmol h⁻¹ μmol⁻¹ P450 leading to a total of 32 mg oxidized products per gram cell of dry weight after 1.5 h. The physiological functioning of the heterologous cofactor regeneration system was illustrated by a sevenfold increased α-pinene oxide yield in the presence of glucose compared to glucose-free conditions.

Production of Chryseobacterium proteolyticum protein-glutaminase using the twin-arginine translocation pathway in Corynebacterium glutamicum by Yoshimi Kikuchi; Hiroshi Itaya; Masayo Date; Kazuhiko Matsui; Long-Fei Wu (pp. 67-74).

The protein glutaminase (PG) secreted by the Gram-negative bacterium Chryseobacterium proteolyticum can deamidate glutaminyl residues in several substrate proteins, including insoluble wheat glutens. This enzyme therefore has potential application in the food industry. We assessed the possibility to produce PG containing a pro-domain in Corynebacterium glutamicum which we have successfully used for production of several kinds of proteins at industrial-scale. When it was targeted to the general protein secretion pathway (Sec) via its own signal sequence, the protein glutaminase was not secreted in this strain. In contrast, we showed that pro-PG could be efficiently produced using the recently discovered twin-arginine translocation (Tat) pathway when the typical Sec-dependent signal peptide was replaced by a Tat-dependent signal sequence from various bacteria. The accumulation of pro-PG in C. glutamicum ATCC13869 reached 183 mg/l, and the pro-PG was converted to an active form as the native one by SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus. The successful secretion of PG via this approach confirms that the Tat pathway of C. glutamicum is an efficient alternative for the industrial-scale production of proteins that are not efficiently secreted by other systems.

Keywords: Corynebacterium glutamicum ; Protein secretion; Twin-arginine; Translocation pathway; Protein glutaminase

Genetic and biochemical characterization of a 4-hydroxybenzoate hydroxylase from Corynebacterium glutamicum by Yan Huang; Ke-xin Zhao; Xi-Hui Shen; Chen-Ying Jiang; Shuang-Jiang Liu (pp. 75-83).

Corynebacterium glutamicum uses 4-hydroxybenzoic acid (4HBA) as sole carbon source for growth. Previous studies showed that 4HBA was taken up into cells via PcaK, and the aromatic ring was cleaved via protocatechuate 3,4-dioxygenase. In this study, the gene pobA Cg (ncgl1032) involved in the conversion of 4HBA into 3,4-dihydroxybenzoate (protocatechuate) was identified, and the gene product PobA _Cg was characterized as a 4HBA 3-hydroxylase, which is a homodimer of PobA_Cg. The pobA _Cg is physically associated with pcaK and formed a putative operon, but the two genes were located distantly to the pca cluster, which encode other enzymes for 4HBA/protocatechuate degradation. This new 4HBA 3-hydroxylase is unique in that it prefers NADPH to NADH as a cosubstrate, although its sequence is similar to other 4HBA 3-hydroxylases that prefer NADH as a cosubstrate. Sited-directed mutagenesis on putative NADPH-binding sites, D38 and T42, further improved its affinity to NADPH as well as its catalytic efficiency.

Keywords: 4-Hydroxybenzoate hydroxylase; Corynebacterium glutamicum ; 4-Hydroxybenzoate; Protochatechuate

Characterisation of mutagenised acid-resistant alpha-amylase expressed in Bacillus subtilis WB600 by Yi-han Liu; Fu-ping Lu; Yu Li; Xiang-bin Yin; Yi Wang; Chen Gao (pp. 85-94).

Based on the original thermostable alpha-amylase gene from Bacillus licheniformis, two amino acids were site-directed mutagenised by polymerase chain reaction to obtain a new gene. This gene, with Leu134→Arg and Ser320→Ala, was substituted for acid-resistant capability previously. To favor purification of the product, high-level expression and secretion of mature, authentic and stable recombinant mutagenised alpha-amylase were achieved with protease-deficient strain Bacillus subtilis WB600 as the host. The recombinant mutagenised alpha-amylase with the activity of 4,700 U/mL was then purified by ammonium sulphate fractionation, anion exchange and gel filtration, consecutively. By multi-step purification, the specific activity of the recombinant protein was up to 916.7 U/mg with a 187.1-fold purification. The mutagenised protein was found to be more acid resistant than the native protein. The optimum pH and stable range of pH with the mutagenised protein was 4.5 and 4.0 to 6.5, respectively, compared with pH 6.5 and 5.5 to 7.0 as the favorite pH and pH stability range of the native protein.

Keywords: Alpha-amylase; Acid resistant; Characterisation; Mutagenised gene; B. subtilis

High-level expression of a truncated 1,3-1,4-β-d-glucanase from Fibrobacter succinogenes in Pichia pastoris by optimization of codons and fermentation by Huoqing Huang; Peilong Yang; Huiying Luo; Huigui Tang; Na Shao; Tiezheng Yuan; Yaru Wang; Yingguo Bai; Bin Yao (pp. 95-103).

1,3-1,4-β-d-glucanase is an important endoglycosidase in the brewing and animal feed industries. To achieve high-level expression of recombinant glucanase in Pichia pastoris, we designed sequences encoding the α-factor signal peptide from Saccharomyces cerevisiae and the truncated 1,3-1,4-β-d-glucanase from Fibrobacter succinogenes as a whole. The codons encoding the 52 amino acids of the signal peptide and 106 residues of the glucanase protein were optimized for expression in P. pastoris; 189 nucleotides were changed. The G + C content was adjusted to 48–49%, and AT-rich stretches were eliminated to avoid premature termination. The messenger ribonucleic acid secondary structure near the AUG start codon was also adjusted to ensure efficient translation; the resulting glucanase production was twofold higher compared with that achieved with gene structure optimization alone. We also propose a new fermentation strategy for the induction phase, in which 5/95% glycerol/methanol mixed feed was used in days 1–3 and 100% methanol was used on days 4–6. By comparison with methanol feed and glycerol/methanol-mixed feed alone, the yield of recombinant glucanase increased by 38.5 and 16.5%, respectively. The expressed optimized recombinant 1,3-1,4-β-d-glucanase constituted ~90% of the total secreted protein, reaching up to 3 g l⁻¹ in the medium.

Keywords: 1,3-1,4-β-d-Glucanase; Codon optimization; Fibrobacter succinogenes ; Mixed feed; Pichia pastoris

A thermoactive glucoamylase with biotechnological relevance from the thermoacidophilic Euryarchaeon Thermoplasma acidophilum by Christiane Dock; Matthias Hess; Garabed Antranikian (pp. 105-114).

A gene encoding an intracellular glucoamylase was identified in the genome of the extreme thermoacidophilic Archaeon Thermoplasma acidophilum. The gene taGA, consisting of 1,911 bp, was cloned and successfully expressed in Escherichia coli. The recombinant protein was purified 22-fold to homogeneity using heat treatment, anion-exchange chromatography, and gel filtration. Detailed analysis shows that the glucoamylase, with a molecular weight of 66 kDa per subunit, is a homodimer in its active state. Amylolytic activity was measured over a wide range of temperature (40–90°C) and pH (pH 3.5–7) and was maximal at 75°C and at acidic condition (pH 5). The recombinant archaeal glucoamylase uses a variety of polysaccharides as substrate, including glycogen and amylose. Maximal activity was measured towards amylopectin with a specific activity of 4.2 U/mg and increased almost threefold in the presence of manganese. Calcium ions have a pronounced effect on enzyme stability; in the presence of 5 mM CaCl₂, the half-life increased from 15 min to 2 h at 80°C.

Keywords: Glucoamylase; Thermoplasma acidophilum; Archaeon; Starch processing; High fructose corn syrup; HPLC

Purification and characterization of an extracellular α-l-arabinosidase from a novel isolate Bacillus pumilus ARA and its over-expression in Escherichia coli by Jianjun Pei; Weilan Shao (pp. 115-121).

The α-l-arabinosidase, AraB, was induced when Bacillus pumilus ARA was grown at 50°C in a minimal medium containing xylan. A 56-kDa protein with α-l-arabinosidase activity was purified from culture supernatant to gel electrophoretic homogeneity. The optimal activity was at pH 6.4 and 60°C over a 10-min assay. The purified enzyme was stable over a pH range of 5.2–7.6 and had a 1-h half life at 70°C. The enzyme released arabinose from oat spelt xylan. Kinetic experiments at 60°C with p-nitrophenyl α-l-arabinofuranoside as substrate gave a K _m, and V _max of 1.05 mM and 240 U per mg of protein. The NH₂-terminal amino acid sequence of the enzyme was determined, and its gene araB was subsequently cloned, sequenced, and over-expressed in Escherichia coli. The open reading frame of araB consists of a 1,479-bp fragment encoding a protein of 472 amino acids, which belonged to family 51 of the glycoside hydrolases with an identity of 67% to the protein encoded by abfB of Bacillus subtilis 168.

High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage by Jung Min Kim; Su A Jang; Byung Jo Yu; Bong Hyun Sung; Ju Hyun Cho; Sun Chang Kim (pp. 123-130).

Direct expression of an antimicrobial peptide (AMP) in Escherichia coli causes several problems such as the toxicity of AMP to the host cell, its susceptibility to proteolytic degradation, and decreased antimicrobial activity due to the additional residue(s) introduced after cleavage of AMPs from fusion partners. To overcome these problems and produce a large quantity of a potent AMP histonin (RAGLQFPVGKLLKKLLKRLKR) in E. coli, an efficient expression system was developed, in which the toxicity of histonin was neutralized by a fusion partner F4 (a truncated fragment of PurF protein) and the productivity was increased by a multimeric expression of a histonin gene. The expression level of the fusion proteins reached a maximum with a 12-mer of a histonin gene. In addition, because of the RLKR residues present at the C terminus of histonin, furin cleavage of the multimeric histonin expressed produces an intact, natural histonin. The AMP activity of the histonin produced in E. coli was identical to that of a synthetic histonin. With our expression system, 167 mg of histonin was obtained from 1 l of E. coli culture. These results may lead to a cost-effective solution for the mass production of AMPs that are toxic to a host.

Keywords: Antimicrobial peptide; Histonin; Multimerization; Fusion; Furin

Lycopene over-accumulation by disruption of the negative regulator gene crgA in Mucor circinelloides by Francisco E. Nicolás-Molina; Eusebio Navarro; Rosa M. Ruiz-Vázquez (pp. 131-137).

Lycopene has become one of the most interesting antioxidant compounds, especially in relation to human health. This work describes a genetic strategy to modify the carotenoid biosynthesis pathway to develop a lycopene-overproducing strain. The crgA gene, a negative regulator of carotenogenesis, was disrupted in the Mucor circinelloides strain MU202, which lacks the lycopene cyclase activity and accumulates lycopene instead of β-carotene. The resultant strain, MU224, demonstrated increased transcriptional levels of the carotenogenesis structural genes carRP and carB compared to the parental strain MU202. As a consequence, strain MU224 accumulated 5 mg/g of dry weight of cells in liquid cultures, a sevenfold increase with respect to the parental strain. Moreover, when lycopene production was examined in a complex enriched medium, biomass increased tenfold compared to that obtained in synthetic minimal medium. In this complex medium, the production rate of lycopene by strain MU224 reached 54 g/l. These results illustrate how a combination of genetic manipulation and optimized culture conditions can be utilized to enhance the production of commercially desirable compounds such as lycopene.

Keywords: Lycopene; crgA ; Mucor circinelloides ; Gene regulation

Changing the N-terminal sequence protects recombinant Plasmodium falciparum circumsporozoite protein from degradation in Pichia pastoris by Qingfeng Zhang; Feng Ding; Xiangyang Xue; Xindong Xu; Weiqing Pan (pp. 139-145).

Proteolytic degradation is the primary obstacle in the use of the yeast Pichia pastoris for the expression of recombinant proteins. During the production of a recombinant Plasmodium falciparum circumsporozoite protein in this system, the (NANP) n repeats region at the N-terminus were completely proteolytically degraded. To remove the potential proteolytic site within the recombinant protein, different strategies were tried, including adjusting the cultivation conditions and mutating the sequence at the junction of the repeat domain and C-terminal region, but the degradation continued. However, modification of the N-terminal sequence by adding an epitope-based peptide to the N-terminus not only protected the repeat domain from cleavage by native proteases during longer induction in the yeast host and purification process, but also stabilized this recombinant protein emulsified with adjuvant ISA720 for at least 6 months. The results showed that proteolytic degradation of the recombinant circumsporozoite protein produced in P. pastoris was amino acid sequence (NANP)-specific, and that this effect was likely dependent on the conformation of the recombinant protein.

Keywords: Pichia pastoris ; Recombinant protein; Proteolytic degradation; Plasmodium falciparum ; Circumsporozoite protein

Electricity generation by thermophilic microorganisms from marine sediment by B. J. Mathis; C. W. Marshall; C. E. Milliken; R. S. Makkar; S. E. Creager; H. D. May (pp. 147-155).

The search for microorganisms that are capable of catalyzing the reduction of an electrode within a fuel cell has primarily been focused on bacteria that operate mesobiotically. Bacteria that function optimally under extreme conditions are beginning to be examined because they may serve as more effective catalysts (higher activity, greater stability, longer life, capable of utilizing a broader range of fuels) in microbial fuel cells. An examination of marine sediment from temperate waters (Charleston, SC) proved to be a good source of thermophilic electrode-reducing bacteria. Electric current normalized to the surface area of graphite electrodes was approximately ten times greater when sediment fuel cells were incubated at 60°C (209 to 254 mA/m²) vs 22°C (10 to 22 mA/m²). Electricity-generating communities were selected in sediment fuel cells and then maintained without sediment or synthetic electron-carrying mediators in single-chambered fuel cells. Current was generated when cellulose or acetate was added as a substrate to the cells. The 16S ribosomal ribonucleic acid genes from the heavy biofilms that formed on the graphite anodes of acetate-fed fuel cells were cloned and sequenced. The preponderance of the clones (54 of 80) was most related to a Gram-positive thermophile, Thermincola carboxydophila (99% similarity). The remainder of clones from the community was most related to T. carboxydophila, or uncultured Firmicutes and Deferribacteres. Overall, the data indicate that temperate aquatic sediments are a good source of thermophilic electrode-reducing bacteria.

Keywords: Thermophiles; Microbial fuel cells; Electricity; Thermincola ; Deferribacteres

Substrate specificity of Stenotrophomonas nitritireducens in the hydroxylation of unsaturated fatty acid by In-Sik Yu; Soo-Jin Yeom; Hye-Jung Kim; Jung-Kul Lee; Yong-Hwi Kim; Deok-Kun Oh (pp. 157-163).

An isolated bacterium that converted unsaturated fatty acids to hydroxy fatty acids was identified as Stenotrophomonas nitritireducens by API analysis, cellular fatty acids compositions, sequencing the full 16S ribosomal ribonucleic acid, and evaluating its nitrite reduction ability. S. nitritireducens has unique regio-specificity for C16 and C18 cis-9 unsaturated fatty acids. These fatty acids are converted to their 10-hydroxy fatty acids without detectable byproducts. Among the cis-9-unsaturated fatty acids, S. nitritireducens showed the highest specificity for linoleic acid. The cells converted 20 mM linoleic acid to 13.5 mM 10-hydroxy-12(Z)-octadecenoic acid at 30°C and pH 7.5 with a yield of 67.5% (mol/mol).

Keywords: 10-Hydroxy fatty acids; Substrate specificity; Stenotrophomonas nitritireducens ; cis-9-Unsaturated fatty acids

Simultaneous biodegradation of creosote-polycyclic aromatic hydrocarbons by a pyrene-degrading Mycobacterium by Zaira López; Joaquim Vila; José-Julio Ortega-Calvo; Magdalena Grifoll (pp. 165-172).

When incubated with a creosote-polycyclic aromatic hydrocarbons (PAHs) mixture, the pyrene-degrading strain Mycobacterium sp. AP1 acted on three- and four-ring components, causing the simultaneous depletion of 25% of the total PAHs in 30 days. The kinetics of disappearance of individual PAHs was consistent with differences in aqueous solubility. During the incubation, a number of acid metabolites indicative of distinctive reactions carried out by high-molecular-weight PAH-degrading mycobacteria accumulated in the medium. Most of these metabolites were dicarboxylic aromatic acids formed as a result of the utilization of growth substrates (phenanthrene, pyrene, or fluoranthene) by multibranched pathways including meta- and ortho-ring-cleavage reactions: phthalic acid, naphthalene-1,8-dicarboxylic acid, phenanthrene-4,5-dicarboxylic acid, diphenic acid, Z-9-carboxymethylenefluorene-1-carboxylic acid, and 6,6′-dihydroxy-2,2′-biphenyl dicarboxylic acid. Others were dead-end products resulting from cometabolic oxidations on nongrowth substrates (fluorene meta-cleavage product). These results contribute to the general knowledge of the biochemical processes that determine the fate of the individual components of PAH mixtures in polluted soils. The identification of the partially oxidized compounds will facilitate to develop analytical methods to determine their potential formation and accumulation in contaminated sites.

Keywords: Biodegradation; PAHs; Mycobacterium ; Metabolites

One-base excess adaptor ligation method for walking uncloned genomic DNA by Yuki Tonooka; Yoichi Mizukami; Masahiro Fujishima (pp. 173-180).

This report describes a novel and efficient method for walking the sequence of a genomic deoxyribonucleic acid (DNA) from a known region to an unknown region based on an oligodeoxynucleotide (oligo) cassette-mediated polymerase chain reaction technique. In this method, genomic DNA is digested by a restriction enzyme that generates a sticky 5′-end, followed by ligation of a one-base excess oligo-adaptor using T4 DNA ligase. The adaptor consists of two complementary oligos that form the same sticky end as the digested genomic DNA fragments, except that the 5′-overhang base overlaps the corresponding 3′-end base of the restriction site. This overhanging terminal base prevents ligation between the adaptors, and the appropriate molar ratio of adaptor to genomic DNA enables specific amplification of the target sequence. T4 DNA ligase catalyzes both the ligation of the phosphorylated overhang base of the adaptor to genomic DNA and the excision of the corresponding 3′-terminal base of the genomic DNA. This sequence-specific exonuclease activity of T4 DNA ligase was confirmed by ligation of an alternative adaptor in which the 5′-terminal base was not consistent with the corresponding 3′-terminal base. Using this technique, the 3′- and 5′-flanking sequences of the catalase gene of the ciliate Paramecium bursaria were determined.

Keywords: Oligo-cassette PCR; Sticky end; Ligase; Paramecium bursaria

Generation of readily transformable Bacillus licheniformis mutants by Bianca Waschkau; Jens Waldeck; Susanne Wieland; Renèe Eichstädt; Friedhelm Meinhardt (pp. 181-188).

A set of mutants was generated by targeted deletion of the hsdR loci of two type I restriction modification systems (RMS) identified in Bacillus licheniformis DSM13. Single as well as double knock-outs resulted in strains being readily transformable with plasmids isolated from Bacilli. Introduction of shuttle plasmids isolated from Escherichia coli was routinely possible when the double mutant B. licheniformis MW3 (ΔhsdR1, ΔhsdR2) was used in transformation experiments. Growth and secretion of extracellular enzymes were not affected in any of the mutants. Thus, along with an optimized transformation protocol, this study makes available an urgently needed transformation system for this industrially exploited species.

Keywords: Bacillus licheniformis ; Targeted gene deletion; Restriction modification system; Restriction-negative mutants; Improved transformation; Transformation

Sections

Personal tools

Applied Microbiology and Biotechnology (v.78, #1)

Sections

Personal tools

Local resources

Applied Microbiology and Biotechnology (v.78, #1)