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Applied Microbiology and Biotechnology (v.70, #6)
Flavin-dependent halogenases involved in secondary metabolism in bacteria by Karl-Heinz van Pée; Eugenio P. Patallo (pp. 631-641).
The understanding of biological halogenation has increased during the last few years. While haloperoxidases were the only halogenating enzymes known until 1997, it is now clear that haloperoxidases are hardly, if at all, involved in biosynthesis of more complex halogenated compounds in microorganisms. A novel type of halogenating enzymes, flavin-dependent halogenases, has been identified as a major player in the introduction of chloride and bromide into activated organic molecules. Flavin-dependent halogenases require the activity of a flavin reductase for the production of reduced flavin, required by the actual halogenase. A number of flavin-dependent tryptophan halogenases have been investigated in some detail, and the first three-dimensional structure of a member of this enzyme subfamily, tryptophan 7-halogenase, has been elucidated. This structure suggests a mechanism involving the formation of hypohalous acid, which is used inside the enzyme for regioselective halogenation of the respective substrate. The introduction of halogen atoms into non-activated alkyl groups is catalysed by non-heme FeII α-ketoglutarate- and O2-dependent halogenases. Examples for the use of flavin-dependent halogenases for the formation of novel halogenated compounds in in vitro and in vivo reactions promise a bright future for the application of biological halogenation reactions.
Molecular plant breeding: achievements in green biotechnology and future perspectives by Gerhard Wenzel (pp. 642-650).
Since one decade ago, transgenic crop plants are globally grown; in 2004, it was estimated to cover a total of 81 Mio ha in 17 countries. At present, four plant species (soybean, maize, cotton and rapeseed) dominate with two traits (herbicide tolerance and insect resistance). The traits on which research concentrates and the constructs which might come next onto the market are outlined. The procedure on how to clone such genes of interest, e.g. via map-based cloning, and some other helpful approaches of green biotechnology, like high throughput techniques and functional markers, are summarised, and a rough calculation about the market value of transgenic crops in US dollars is quoted.
Environmental monitoring in stem cell banks by Fernando Cobo; Glyn N. Stacey; José Luis Cortés; Ángel Concha (pp. 651-662).
The processing of stem cell lines for application in human therapy requires a physical environment in which air quality (i.e., the number of airborne particles) is controlled to minimize risk of contamination. The processing facility should be constructed and operated to minimise the introduction, generation and retention of particles and microorganisms. A formal program of environmental monitoring should be maintained in each stem cell bank to specify and assess key factors and their influence on the microbiological quality of the process and product. This program should assure the manipulation of cells involved in the derivation of stem cell lines and their culture under established limits for airborne particles and for microbial contamination of the air and surfaces. Environmental monitoring should also address the regulatory requirements in the countries in which the cells will be used. The monitoring programme will depend on local conditions in each processing centre or cell bank. Each centre will need to evaluate its specific needs and establish appropriate monitoring procedures which should not become intrusive to the extent that they might compromise the quality of the cell banks or products.
Microbial production of optically active β-phenylalanine ethyl ester through stereoselective hydrolysis of racemic β-phenylalanine ethyl ester by Jun Ogawa; Junichi Mano; Sakayu Shimizu (pp. 663-669).
The ability to produce (R)- or (S)-β-phenylalanine ethyl ester (3-amino-3-phenylpropionic acid ethyl ester, BPAE) from racemic BPAE through stereoselective hydrolysis was screened for in BPAE-assimilating microorganisms. Sphingobacterium sp. 238C5 and Arthrobacter sp. 219D2 were found to be potential catalysts for (R)- and (S)-BPAE production, respectively. On a 24-h reaction, with 2.5% (w/v) racemic BPAE (130 mM) as the substrate and wet cells of Sphingobacterium sp. 238C5 as the catalyst, 1.15% (w/v) (R)-BPAE (60 mM) with enantiomeric purity of 99% e.e. was obtained, the molar yield as to racemic BPAE being 46%. On a 48-h reaction, with 2.5% (w/v) racemic BPAE (130 mM) as the substrate and wet cells of Arthrobacter sp. 219D2 as the catalyst, 0.87% (w/v) (S)-BPAE (45 mM) with enantiomeric purity of 99% e.e. was obtained, the molar yield as to racemic BPAE being 35%. The enzyme stereoselectively hydrolyzing (S)-BPAE was purified to homogeneity from the cell-free extract of Sphingobacterium sp. 238C5. The enzyme was a monomeric protein with a molecular mass of about 42,000. The enzyme catalyzed hydrolysis of β-phenylalanine esters, while the common aliphatic and aromatic carboxylate esters were not catalyzed.
Statistical analysis of inactivation of Listeria monocytogenes subjected to high hydrostatic pressure and heat in milk buffer by Yu-Long Gao; Xing-Rong Ju; Han-Hu Jiang (pp. 670-678).
Previous unpublished experimental results of fractional factorial experiments showed that significant external factors affecting high-pressure processing (HPP) inactivation were pressure, temperature, and pressure holding time. Based on these results, response surface methodology (RSM) was employed in the present work, and a quadratic equation for HPP inactivation was built with RSM. By analyzing response surface plots and their corresponding contour plots and by solving the quadratic equation, experimental values were shown to be significantly in good agreement with predicted values, since the adjusted determination coefficient (R Adj 2) was 0.9812 and the level of significance was P<0.0001. Optimum process parameters for a 6-log cycle reduction of Listeria monocytogenes were obtained: pressure, 448.0 MPa; temperature, 41°C; and pressure holding time, 11 min. The adequacy of the model equation in predicting optimum response values was verified effectively by validation data.
A simple activity staining protocol for lipases and esterases by Rajni Singh; Namita Gupta; Vineet Kumar Goswami; Rani Gupta (pp. 679-682).
A simple activity staining protocol for rapid detection and differentiation of lipases and esterases was developed based on pH drop due to fatty acids released following lipolysis. Though the detection of lipolysis as a function of drop in pH is not new, the present method has been made more sensitive by the judicious selection of the initial pH of the chromogenic substrate, which has been set near the end point of the dye so that even a slight drop in pH results in immediate color change. In the present case, the dye phenol red was taken, which has the end point at pH 7.3–7.4 where the color is pink. A slight drop due to fatty acid release results in yellow coloration. The assay has high reproducibility and can detect as low as 0.5 p-NPP enzyme units within 15 min. In addition, this method can be used for various lipidic substrates such as oils and tributyrin, making it suitable for both lipases and esterases.
Keywords: Activity staining; Chromogenic substrate; Esterases; Lipases; Zymogram
Expression, purification, and immobilization of His-tagged d-amino acid oxidase of Trigonopsis variabilis in Pichia pastoris by Huabao Zheng; Xiaolan Wang; Jun Chen; Ke Zhu; Yuhua Zhao; Yunliu Yang; Sheng Yang; Weihong Jiang (pp. 683-689).
High-level expression of d-amino acid oxidase (DAO) has been reported in Pichia pastoris by integrating the DAO gene under the control of the alcohol oxidase promoter (PAOX1). However, the time taken to reach peak product concentration is usually long (∼43 h), and cultivation requires tight regulation of methanol feeding. In this paper, we describe the expression of His-tagged DAO (HDAO) in P. pastoris using the glyceraldehydes-3-phosphate dehydrogenase promoter (PGAP). The maximal level of HDAO expression using the PGAP integrant is attained in 13 h and is equal to that obtained using the PAOX1 integrant in 43 h. We also explored the possibility of secreting HDAO in P. pastoris. In-frame fusion of Saccharomyces cerevisiae α-factor secretion signal under a PGAP or PAOX1 resulted in low-level secretion of active HDAO, which was not of practical use. The intracellularly expressed HDAO under PGAP was purified by agar-based affinity support and then immobilized on Amberzyme oxirane resin. The immobilized HDAO, with specific activity of 75 U g−1 (wet weight), could be recycled more than 14 times without significant loss of activity. The data suggest that intracellular production of HDAO under PGAP, followed by affinity purification and immobilization on oxirane resin, may serve as an effective process for the manufacture of immobilized DAO for industrial application.
Construction of a new reporter system to study the NaCl-dependent dnaK promoter activity of Lactobacillus sanfranciscensis by Sebastian Hörmann; Rudi F. Vogel; Matthias Ehrmann (pp. 690-697).
A reporter system was developed to study gene expression in Lactobacillus sanfranciscensis. It was based on the Escherichia coli/Lactobacillus shuttle vector pLP3537 and the melA gene encoding α-galactosidase originating from Lactobacillus plantarum. melA was functionally expressed in E. coli and L. sanfranciscensis, and activity was easily monitored in vivo as well as in vitro by applying an optimized enzyme assay. The reporter system was validated by demonstrating the induction of the dnaK operon of L. sanfranciscensis by NaCl stress. The complete operon, which was composed of hrcA, grpE, dnaK, and dnaJ, was sequenced. A 299-bp sequence upstream of this operon, including a putative sigmaA-type promoter and a single conserved Controlling Inverted Repeat of Chaperone Expression element, was amplified. This amplicon was cloned directly upstream of melA. Both reporter enzyme activity and Northern hybridization analyses of dnaK and melA revealed a transcriptional induction, reaching its maximum when the culture was exposed to 0.75 M NaCl.
Recombinant expression of indolicidin concatamers in Escherichia coli by K. M. Morin; S. Arcidiacono; R. Beckwitt; C. M. Mello (pp. 698-704).
Antimicrobial peptides are part of the innate immune system of vertebrates and invertebrates. They are active against gram-negative and gram-positive bacteria, fungi, and protozoa. Currently, most antimicrobial peptides are extracted from host organisms or produced by solid-phase peptide synthesis. Recombinant protein expression in Escherichia coli is a tool for greater production yields at a decreased cost and reduces the use of hazardous materials. We have constructed a concatamer of indolicidin and successfully expressed a fusion product with thioredoxin in E. coli BL21DE3. Codons for methionine residues flanking individual indolicidin genes were incorporated for cyanogen bromide cleavage of the fusion protein and liberation of active monomeric indolicidin. Peptide yields of 150 μg/l monomeric indolicidin were achieved in this first report of recombinant production of indolicidin with demonstrated antimicrobial activity.
Development of a multifunctional and efficient conjugal plasmid for use in Streptomyces spp by X. Y. Chen; H. B. Mo; Y. Liu; F. Xia (pp. 705-710).
A plasmid, pGB112, has recently been developed to transfer DNA from Escherichia coli to Streptomyces spp via conjugation. This technique made use of (A) E. coli replicon, (B) ampicillin (amp) resistance gene for selection in E. coli and thiostrepton (tsr) resistance gene for selection in Streptomyces, (C) a fragment of SCP2* replicon, (D) a 2.6 kb fragment of tra-cassette which consists of pIJ101 transfer gene (tra) and two ermE promoters, (E) a 0.8 kb fragment of oriT of (IncP) RK2. The results showed that this plasmid was able to transfer plasmid DNA from E. coli to Streptomyces coelicolor via conjugation, and that it could also transfer DNA between Streptomyces strains. Since this plasmid has both pBR322 and SCP2* replicons, it may provide a novel and useful method for genetic operation in E. coli and Streptomyces.
Functional characterization of Δ9 and ω9 desaturase genes in Mortierella alpina 1S-4 and its derivative mutants by Takahiro Abe; Eiji Sakuradani; Takahiro Asano; Hiroyuki Kanamaru; Sakayu Shimizu (pp. 711-719).
Cloning and characterization of the Δ9 desaturase (Δ9I) gene of a fungus, Mortierella alpina 1S-4, was previously reported. In this study, two genes encoding Δ9 desaturase homologs were isolated from this fungus. One is a Δ9 desaturase (Δ9II) that exhibits 86% amino acid sequence similarity to Δ9I. Functional analysis involving expression of the encoding gene in Aspergillus oryzae revealed that Δ9II exhibits Δ9 desaturase activity, 18:0 being converted to 18:1Δ9. However, unlike Δ9I, the Δ9II transformant accumulated a low amount of 16:1Δ9. The other homolog is a ω9 desaturase (ω9) that exhibits 56 and 58% amino acid sequence similarity to Δ9I and Δ9II, respectively. On functional analysis with the Aspergillus transformant, it was found that ω9 does not convert 18:0 to 18:1Δ9, but converts 24:0 and 26:0 to 24:1ω9 and 26:1ω9, respectively. On the other hand, Δ9 desaturation-defective mutants characterized by accumulation of 18:0 were derived from M. alpina 1S-4 with a chemical mutagen, and the mutated sites of the Δ9 desaturase genes were identified. The mutation on the Δ9I gene was assumed to cause an amino acid replacement (W136Stop, G265D, and W360Stop) in the mutants (HR222, T4, and ST56), respectively. In these mutants, there was no mutated site on the Δ9II and ω9 genes. Real-time quantitative PCR (RTQ-PCR) analysis revealed that (1) the transcriptional level of the Δ9I gene in HR222 and T4 was much higher than that in the wild strain until the fifth day of the cultivation periods, (2) the Δ9II gene of the mutants was transcribed until the fourth day at the same level as the Δ9I gene of the wild strain, whereas the Δ9II gene of the wild strain was transcribed at a lower level, and (3) the transcriptional level of the ω9 gene in both the mutants and the wild strain was low, i.e., as low as that of the Δ9II gene of the wild strain. In these Δ9 desaturation-defective mutants, Δ9II is likely to play an important role in Δ9 desaturation.
Emergence of two types of nondechlorinating variants in the tetrachloroethene-halorespiring Desulfitobacterium sp. strain Y51 by Taiki Futagami; Yoshinori Tsuboi; Akiko Suyama; Masatoshi Goto; Kensuke Furukawa (pp. 720-728).
Desulfitobacterium sp. strain Y51 exhibits a strong dechlorinating activity for tetrachloroethene (PCE), converting it to cis-1,2-dichloroethene via trichloroethene by the action of the PceA reductive dehalogenase (encoded by pceA). The gene organization around the pceA gene cluster was determined to be in the following order: orf4, orf3, ISDesp1, pceA-B-C-T-mcpA, and ISDesp2, where the pceA gene cluster is surrounded by two nearly identical copies of the ISDesp insertion sequence. Serial subculture of strain Y51 gave rise to variants that abolished the PCE-dechlorination activity. Southern hybridization analysis revealed two types of variants termed small deletion (SD) and large deletion (LD). The characterization of both variants revealed a genetic rearrangement around the pceAB gene cluster. In variant SD, ISDesp1 comprised of 1,572 bp was deleted, which includes the tnpAa encoding IS256 family transposase and unknown orf1. The ISDesp1 contained the inverted terminal repeat sequence and a −35 promoter stretch just upstream of the pceA gene, indicating that this IS element is involved in the formation of the variant SD. Loss of the pceA transcription changed the variant SD to the PCE-nondechlorinating phenotype. The variant LD lost the 6.5-kb region, including one copy of ISDesp and the pceABCT–mcpA gene cluster, confirming that the homologous recombination is associated with the emergence of this variant.
Induction of defense responses against Alternaria rot by different elicitors in harvested pear fruit by Shiping Tian; Yakun Wan; Guozheng Qin; Yong Xu (pp. 729-734).
Pear fruit (Pyrus pyrifolia L. cv. Yali) treated by different elicitors, such as salicylic acid (SA), oxalic acid, calcium chloride, and antagonistic yeast Cryptococcus laurentii, were investigated to determine the induction of defense responses. The possible mechanism by which elicitors induced the resistance of pear fruit against postharvest disease was also evaluated. The results indicated that all the elicitors could significantly enhance defense-related enzyme activities, such as β-1,3-glucanase, phenylalanine ammonia lyase, peroxidase, and polyphenol oxidase activity, and reduce the disease incidence caused by Alternaria alternata in pear fruit (P=0.05). Among these different elicitors, SA treatment showed the best result in inducing the defense responses and reducing the decay in pear fruit.
Controlling food-contaminating fungi by targeting their antioxidative stress-response system with natural phenolic compounds by Jong H. Kim; Noreen Mahoney; Kathleen L. Chan; Russell J. Molyneux; Bruce C. Campbell (pp. 735-739).
The antioxidative stress-response system is essential to fungi for tolerating exposure to phenolic compounds. We show how this system can be targeted to improve fungal control by using compounds that inhibit the fungal mitochondrial respiratory chain. Targeting mitochondrial superoxide dismutase with selected phenolic acid derivatives (e.g., vanillyl acetone) resulted in a 100- to 1,000-fold greater sensitivity to strobilurin or carboxin fungicides. This synergism is significantly greater with strobilurin than with carboxin, suggesting that complex III of the mitochondrial respiratory chain is a better target than complex II for fungal control, using phenolics. These results show certain natural compounds are effective synergists to commercial fungicides and can be used for improving control of food-contaminating pathogens. These results suggest that the use of such compounds for fungal control can reduce environmental and health risks associated with commercial fungicides, lower cost for control, and the probability for development of resistance.
Use of 2-deoxyglucose in liquid media for the selection of mutant strains of Penicillium echinulatum producing increased cellulase and β-glucosidase activities by Aldo J. P. Dillon; Cinthia Zorgi; Marli Camassola; João Antônio P. Henriques (pp. 740-746).
Mutagenesis and selection were applied to a strain of Penicillium echinulatum by treating conidia with hydrogen peroxide or 1,2,7,8-diepoxyoctane and then by incubating the conidia for 48 h in broth containing microcrystalline cellulose washed in 0.5% (w/v) aqueous 2-deoxyglucose before plating them onto cellulose agar containing 1.5% (w/v) glucose from which colonies showing the fastest production of halos of cellulose hydrolysis were selected. This process resulted in the isolation of two new cellulase-secreting P. echinulatum mutants: strain 9A02S1 showing increased cellulase secretion (2 IU ml−1, measured as filter paper activity) in submerged culture in agitated flasks containing a mineral salts medium and 1% of cellulose, and strain 9A02D1, which proved more suitable for the production of cellulases in semisolid bran culture where it produced 23 IU of β-glucosidase per gram of wheat bran.
Metabolism of fluoranthene by Mycobacterium sp. strain AP1 by Zaira López; Joaquim Vila; Cristina Minguillón; Magdalena Grifoll (pp. 747-756).
The pyrene-degrading Mycobacterium strain AP1 was found to utilize fluoranthene as a sole source of carbon and energy. Identification of metabolites formed from fluoranthene (by growing cells and washed-cell suspensions), the kinetics of metabolite accumulation, and metabolite-feeding studies all indicated that strain AP1 oxidizes fluoranthene using three alternative routes. The first route is initiated by dioxygenation at C-7 and C-8 and, following meta cleavage and pyruvate release, produces a hydroxyacenaphthoic acid that is decarboxylated to acenaphthenone (V). Monooxygenation of this ketone to the quinone and subsequent hydrolysis generates naphthalene-1,8-dicarboxylic acid (IV), which is further degraded via benzene-1,2,3-tricarboxylic acid (III). A second route involves dioxygenation at C-1 and C-2, followed by dehydrogenation and meta cleavage of the resulting diol. A two-carbon fragment excision of the meta cleavage product yields 9-fluorenone-1-carboxylic acid (II), which appears to undergo angular dioxygenation and further degradation to produce benzene-1,2,3-tricarboxylic acid (III), merging this route with the 7,8-dioxygenation route. Decarboxylation of benzene-1,2,3-tricarboxylic acid to phthalate (VIII), as well as further oxidation of the latter, would connect both routes with the central metabolism. The identification of Z-9-carboxymethylenefluorene-1-carboxylic acid (I) suggests a third route for fluoranthene degradation involving dioxygenation at C-2, C-3, and ortho cleavage. There is no evidence of any further degradation of this compound.
Monitoring Arthrobacter protophormiae RKJ100 in a ‘tag and chase’ method during p-nitrophenol bio-remediation in soil microcosms by Gunjan Pandey; Janmejay Pandey; Rakesh K. Jain (pp. 757-760).
Monitoring of micro-organisms released deliberately into the environment is essential to assess their movement during the bio-remediation process. During the last few years, DNA-based genetic methods have emerged as the preferred method for such monitoring; however, their use is restricted in cases where organisms used for bio-remediation are not well characterized or where the public domain databases do not provide sufficient information regarding their sequence. For monitoring of such micro-organisms, alternate approaches have to be undertaken. In this study, we have specifically monitored a p-nitrophenol (PNP)-degrading organism, Arthrobacter protophormiae RKJ100, using molecular methods during PNP degradation in soil microcosm. Cells were tagged with a transposon-based foreign DNA sequence prior to their introduction into PNP-contaminated microcosms. Later, this artificially introduced DNA sequence was PCR-amplified to distinguish the bio-augmented organism from the indigenous microflora during PNP bio-remediation.
Response surface methodology as an approach to determine the optimal activities of xylose reductase and xylitol dehydrogenase enzymes from Candida Mogii by Zea D. V. L. Mayerhoff; Inês C. Roberto; Telma T. Franco (pp. 761-767).
A central composite experimental design leading to a set of 16 experiments with different combinations of pH and temperature was performed to attain the optimal activities of xylose reductase (XR) and xylitol dehydrogenase (XDH) enzymes from Candida mogii cell extract. Under optimized conditions (pH 6.5 and 38°C), the XR and XDH activities were found to be 0.48 U/ml and 0.22 U/ml, respectively, resulting in an XR to XDH ratio of 2.2. Stability, cofactor specificity and kinetic parameters of the enzyme XR were also evaluated. XR activity remained stable for 3 h under 4 and 38°C and for 4 months of storage at −18°C. Studies on cofactor specificity showed that only NADPH-dependent XR was obtained under the cultivation conditions employed. The XR present in C. mogii extracts showed a superior K m value for xylose when compared with other yeast strains. Besides, this parameter was not modified after enzyme extraction by aqueous two-phase system.