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Applied Microbiology and Biotechnology (v.72, #1)
Microbial degradation of styrene: biochemistry, molecular genetics, and perspectives for biotechnological applications by Aisling Mooney; Patrick G. Ward; Kevin E. O’Connor (pp. 1-10).
Large quantities of the potentially toxic compound styrene are produced and used annually by the petrochemical and polymer-processing industries. It is as a direct consequence of this that significant volumes of styrene are released into the environment in both the liquid and the gaseous forms. Styrene and its metabolites are known to have serious negative effects on human health and therefore, strategies to prevent its release, remove it from the environment, and understand its route of degradation were the subject of much research. There are a large number of microbial genera capable of metabolizing styrene as a sole source of carbon and energy and therefore, the possibility of applying these organisms to bioremediation strategies was extensively investigated. From the multitude of biodegradation studies, the application of styrene-degrading organisms or single enzymes for the synthesis of value-added products such as epoxides has emerged.
Artemisinin: current state and perspectives for biotechnological production of an antimalarial drug by Chunzhao Liu; Yan Zhao; Yuchun Wang (pp. 11-20).
Artemisinin isolated from the aerial parts of Artemisia annua L. is a promising and potent antimalarial drug which has a remarkable activity against chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum, and is useful in treatment of cerebral malaria. Because the low content (0.01–1 %) of artemisinin in A. annua is a limitation to the commercial production of the drug, many research groups have been focusing their researches on enhancing the production of artemisinin in tissue culture or in the whole plant of A. annua. This review mainly focuses on the progresses made in the production of artemisinin from A. annua by biotechnological strategies including in vitro tissue culture, metabolic regulation of artemisinin biosynthesis, genetic engineering, and bioreactor technology.
A novel regulation on developmental gene expression of fruiting body formation in Myxobacteria by Toshiyuki Ueki; Sumiko Inouye (pp. 21-29).
Myxobacteria are Gram-negative soil microorganisms that prey on other microorganisms. Myxobacteria have significant potential for applications in biotechnology because of their extraordinary ability to produce natural products such as secondary metabolites. Myxobacteria also stand out as model organisms for the study of cell–cell interactions and multicellular development during their complex life cycle. Cellular morphogenesis during multicellular development in myxobacteria is very similar to that in the eukaryotic soil amoebae. Recent studies have started uncovering molecular mechanisms directing the myxobacterial life cycle. We describe recent studies on signal transduction and gene expression during multicellular development in the myxobacterium Myxococcus xanthus. We provide our current model for signal transduction pathways mediated by a two-component His–Asp phosphorelay system and a Ser/Thr kinase cascade.
Keywords: M. xanthus; FruA; MrpC; Developmental gene expression; Protein Ser/Thr kunase
Recent progress in understanding thiamin biosynthesis and its genetic regulation in Saccharomyces cerevisiae by Kazuto Nosaka (pp. 30-40).
The yeast Saccharomyces cerevisiae is able to synthesize thiamin pyrophosphate (TPP) de novo, which involves the independent formation of two ring structures, 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-β-hydroxyethylthiazole, in the early steps. In addition, this organism can efficiently utilize thiamin from the extracellular environment to produce TPP. Nineteen genes involved in the synthesis of TPP and the utilization of thiamin (THI genes) have been identified, and the function of several THI genes has been elucidated. All THI genes participating in the synthesis of the pyrimidine unit belong to multigene families. It is also intriguing that some thiamin biosynthetic proteins are composed of two distinct domains or form an enzyme complex. The expression of THI genes is coordinately induced in response to thiamin starvation. It is likely that the induction of THI genes is activated by a positive regulatory factor complex and that the protein–protein interaction among the factors is disturbed by TPP. Thiamin-hyperproducing yeast and fermented food containing a high content of thiamin are expected to be available in the future based on the progress in understanding thiamin biosynthesis and its genetic regulation in S. cerevisiae.
High-level heterologous production and functional expression of the sec-dependent enterocin P from Enterococcus faecium P13 in Lactococcus lactis by Jorge Gutiérrez; Rasmus Larsen; Luis M. Cintas; Jan Kok; Pablo E. Hernández (pp. 41-51).
Enterocin P (EntP), a sec-dependent bacteriocin from Enterococcus faecium P13, was produced by Lactococcus lactis. The EntP structural gene (entP) with or without the EntP immunity gene (entiP) was cloned in (1), plasmid pMG36c under control of the lactococcal constitutive promoter P32, (2) in plasmid pNG8048e under control of the inducible PnisA promoter, and (3) in the integration vector pINT29. Introduction of the recombinant vectors in L. lactis resulted in production of biologically active EntP in the supernatants of L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris NZ9000, and the coproduction of nisin A and EntP in L. lactis subsp. lactis DPC5598. The level of production of EntP, detected and quantified by specific anti-EntP antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay, by the recombinant L. lactis strains depended on the host strain, the expression vector, and the presence of the entiP gene in the constructs of the recombinant L. lactis strains. The highest amount of EntP was produced with derivatives containing entP and entiP, for both L. lactis IL1403 and L. lactis NZ9000. These derivatives produced up to five- to six-fold more EntP than E. faecium P13. Mass spectrometry analysis revealed that EntP purified from L. lactis IL1403 (pJP214) has a molecular mass identical to that purified from E. faecium P13, suggesting that the synthesis, processing, and secretion of EntP progresses efficiently in recombinant L. lactis hosts.
Sophorolipid produced from the new yeast strain Wickerhamiella domercqiae induces apoptosis in H7402 human liver cancer cells by Jing Chen; Xin Song; Hui Zhang; Yin-bo Qu; Jun-ying Miao (pp. 52-59).
The effects of sophorolipid on the growth and apoptosis of H7402 human liver cancer cells were investigated. By treatment with sophorolipid, a dose- and time-dependent inhibition of cell proliferation was observed. The cells developed many features of apoptosis, including condensation of chromatin, nuclear fragmentation, and appearance of apoptotic bodies, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positive cells were stained dark brown. Sophorolipid treatment induced apoptosis in H7402 cells by blocking cell cycle at G1 phase and partly at S phase, activating caspase-3, and increasing Ca2+ concentration in cytoplasm. These findings may suggest a potential use of sophorolipid for liver cancer treatment.
Heterologous expression and characterization of a novel branching enzyme from the thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii by Volker Thiemann; Bodo Saake; Angela Vollstedt; Thomas Schäfer; Jürgen Puls; Costanzo Bertoldo; Roland Freudl; Garabed Antranikian (pp. 60-71).
The gene encoding the branching enzyme (BE) from the thermoalkaliphilic, anaerobic bacterium Anaerobranca gottschalkii was fused with a twin arginine translocation protein secretory-pathway-dependent signal sequence from Escherichia coli and expressed in Staphylococcus carnosus. The secreted BE was purified using hydrophobic interaction and gel filtration chromatography. The monomeric enzyme (72 kDa) shows maximal activity at 50°C and pH 7.0. With amylose the BE displays high transglycosylation and extremely low hydrolytic activity. The conversion of amylose and linear dextrins was analysed by applying high-performance anion exchange chromatography and quantitative size-exclusion chromatography. Amylose (104–4×107 g/mol) was converted to a major extent to products displaying molecular masses of 104–4×105 g/mol, indicating that the enzyme could be applicable for the production of starch or dextrins with narrow molecular mass distributions. The majority of the transferred oligosaccharides, determined after enzymatic hydrolysis of the newly synthesized α-1,6 linkages, ranged between 103 and 104 g/mol, which corresponds to a degree of polymerisation (DP) of 6–60. The minimal donor chain length is DP 16. Furthermore, the obtained results support the hypotheses of a random endocleavage mechanism of BE and the occurrence of interchain branching.
Recombinant production of fibrinogenase IV from Agkistrodon acutus venom and its preliminary evaluation by Li-Peng Xu; Tao Ma; Wei-Jian Jiang; Peng-Xin Qiu; Xia Wang; Xing-Wen Su; Guang-Mei Yan (pp. 72-76).
A novel metalloproteinase, recombinant fibrinogenase IV (rFIVa), was expressed and purified from Agkistrodon acutus venom. It is a single-chain protein with an apparent molecular weight of 27 kDa. Western blot showed that it had a good immunological reaction against anti-FIVa rabbit serum. The kinetic parameters Km and Kcat of rFIVa on the substrate T6140 were 7.471×10−4 mol/l and 5.103×10−5 s−1. RFIVa cleaved preferentially the α-chain, and the β- and γ-chains of fibrinogen were also cleaved when the incubation time was prolonged. The administration of rFIVa (1.8 and 5.4 mg/kg) to animals with acute blood-stasis model produced a decrease in fibrinogen to control values. To our knowledge, this is the first report of the expression, purification, and evaluation of recombinant fibrinogenase IV, which belongs to class P-I metalloproteinase from A. acutus venom.
Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis by Anirban Banerjee; Praveen Kaul; U. C. Banerjee (pp. 77-87).
(R)-mandelic acid was produced from racemic mandelonitrile using free and immobilized cells of Pseudomonas putida MTCC 5110 harbouring a stereoselective nitrilase. In addition to the optimization of culture conditions and medium components, an inducer feeding approach is suggested to achieve enhanced enzyme production and therefore higher degree of conversion of mandelonitrile. The relationship between cell growth periodicity and enzyme accumulation was also studied, and the addition of the inducer was delayed by 6 h to achieve maximum nitrilase activity. The nitrilase expression was also authenticated by the sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis. P. putida MTCC 5110 cells were further immobilized in calcium alginate, and the immobilized biocatalyst preparation was used for the enantioselective hydrolysis of mandelonitrile. The immobilized system was characterized based on the Thiele modulus (ϕ). Efficient biocatalyst recycling was achieved as a result of immobilization with immobilized cells exhibiting 88% conversion even after 20 batch recycles. Finally, a fed batch reaction was set up on a preparative scale to produce 1.95 g of (R)-(-)-mandelic acid with an enantiomeric excess of 98.8%.
New tool for spreading proteins to the environment: Cry1Ab toxin immobilized to bioplastics by Cristina Moldes; Gema P. Farinós; Laura I. de Eugenio; Pedro García; José L. García; Félix Ortego; Pedro Hernández-Crespo; Pedro Castañera; María A. Prieto (pp. 88-93).
A new tool to provide an environmentally friendly way to deliver active proteins to the environment has been developed, based on the use of polyhydroxyalkanoate (PHA, bioplastic) granules. To illustrate this novel approach, a derived Cry1Ab insect-specific toxin protein was in vivo immobilized into PHA granules through the polypeptide tag BioF. The new toxin, named Fk-Bt1, was shown to be active against Sesamia nonagrioides (Lepidoptera: Noctuidae). The dose–mortality responses of the new toxin granule formulation (PFk-Bt1) and purified Cry1Ab have been compared, demonstrating the effectiveness of PFk-Bt1 and suggesting a common mode of action.
Expression of Vitreoscilla hemoglobin enhances growth and levels of α-amylase in Schwanniomyces occidentalis by Devesh H. Suthar; Bharat B. Chattoo (pp. 94-102).
A metabolic engineering approach was exploited to improve growth and protein secretion in the non-conventional yeast, Schwanniomyces occidentalis. Vitreoscilla hemoglobin (VHb) gene was expressed in S. occidentalis under the control of the native α-amylase (AMY1) promoter. Expression of VHb was confirmed by reverse transcriptase polymerase chain reaction and Western blot hybridization analysis. Effect of VHb on growth and protein secretion was studied in synthetic medium under both limiting and non-limiting dissolved oxygen conditions. Under both conditions, VHb-expressing cells exhibited higher oxygen uptake and higher specific growth rates. Levels of extracellular α-amylase were also elevated in the VHb-transformed strain relative to the control strain. In amylase production medium, VHb-expressing cells showed 3-fold elevated levels of α-amylase and a 31% increase in the total secreted protein under oxygen-limiting environment. VHb was found to localize in the mitochondria in addition to its cytoplasmic location. Inhibition of respiration by antimycin A resulted in the loss of the growth-enhancing effects of VHb. A 2.5-fold increase in the cytochrome c oxidase (COX) activity was observed in VHb-expressing cells relative to the control. In addition to this, exogenously added VHb in the assay mixture augmented COX activity.
High-level expression of recombinant human paraoxonase 1 Q in silkworm larvae (Bombyx mori) by Jie Zhu; Yuguan Ze; Chi Zhang; Yuhui Zang; Haiqin Lu; Ping Chu; Manji Sun; Junchuan Qin (pp. 103-108).
Human serum paraoxonase 1 (hPON1) belongs to a family of enzymes that catalyze the hydrolysis of a broad range of esters and lactones. Although the very first identification of hPON1 might have been as a calcium-dependent paraoxonase/arylesterase, PON1 is in fact a lactonase associated with high-density lipoprotein and strongly stimulated by apoA-I. PON1 hydrolyzes various organophosphates, including insecticides and nerve gases. PON1 also plays a key role in prevention of atherosclerosis. Mediation of cholesterol efflux from macrophage is a key in vivo function of PON1. In present study, the hPON1 Q gene was cloned into baculovirus transfer vector pVL1392 and expressed in silkworm expression system. The rhPON1 Q presented two bands with every near molecular weight of about 40 and 43 kDa according to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis. The expression level was up to 1,256 mg/L in haemolymph, about 50 times as high as that from BmN cells (24.8 mg/L). After purified by two chromatography steps (DEAE-Sepharose and HiTrap Chelating HP), the purity of rhPON1 Q was up to 90%, and the enzymatic properties are similar to serum hPON1.
Assessment of prokaryotic collagen-like sequences derived from streptococcal Scl1 and Scl2 proteins as a source of recombinant GXY polymers by Runlin Han; Antoni Zwiefka; Clayton C. Caswell; Yi Xu; Douglas R. Keene; Ewa Lukomska; Zhihong Zhao; Magnus Höök; Slawomir Lukomski (pp. 109-115).
Collagen triple helix, composed of the repeating Gly–Xaa–Yaa (GXY) sequence, is a structural element found in all multicellular animals and also in some prokaryotes. Long GXY polymers are highly regarded components used in food, cosmetic, biomedical, and pharmaceutical industries. In this study, we explore a new concept for the production of recombinant GXY polymers which are based on the sequence of “prokaryotic collagens”, the streptococcal collagen-like proteins Scl1 and Scl2. Analysis of 50 Scl variants identified the amino acid distribution and GXY-repeat usage that are involved in the stabilization of the triple helix in Scls. Using circular dichroism spectroscopy and electron microscopy, we show that significantly different recombinant rScl polypeptides form stable, unhydroxylated homotrimeric triple helices that can be produced both intra- and extracellularly in the Escherichia coli. These rScl constructs containing 20 to 129 GXY repeats had mid-point melting temperatures between 32 and 39°C. Altogether, Scl-derived collagens, which are different from the mammalian collagens, can form stable triple helices under physiological conditions and can be used for the production of recombinant GXY polymers with a wide variety of potential applications.
Proteomic analysis of a distilling strain of Saccharomyces cerevisiae during industrial grain fermentation by Rasmus Hansen; Stephen Y. Pearson; James M. Brosnan; Philip G. Meaden; Derek J. Jamieson (pp. 116-125).
The fermentation performance of industrial yeast strains is influenced, among other things, by their genetic composition and the nature of the fermentable sugar, availability of nitrogen, and temperature. Therefore, to manipulate the fermentation process, it is important to understand, at a molecular level, the changes occurring in the yeast cell throughout industrial fermentation processes. With this aim in mind, using two-dimensional gel electrophoresis and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS), we have examined the proteome of distillers yeast in an industrial context. Using yeast sampled from a local grain whisky distillery, we have prepared a detailed reference map of the proteome of distillers yeast and have examined in some detail the alterations in protein levels that occur throughout fermentation. In particular, as fermentation progresses, there is a significant increase in the levels of a variety of proteins involved in protecting against stress and nitrogen limitation. These results therefore give an insight into the stresses that yeast are exposed to in industrial fermentations and reveal some of the proteins and enzymes that are either necessary or important for efficient fermentation.
Keywords: Fermentation; Yeast; Proteomics; Distillers yeast
Relationship between the extracellular polymeric substances and surface characteristics of Rhodopseudomonas acidophila by Guo-Ping Sheng; Han-Qing Yu (pp. 126-131).
The relationship between the extracellular polymeric substances (EPS) and surface characteristics of Rhodopseudomonas acidophila in its different growth phases was established. The equilibrium constant of partition (K par) and the Gibbs energies of partition (△G par) between hexadecane and aqueous phases were also calculated according to the microbial adhesion to hexadecane (MATH) testing. The EPS content decreased with cultivation time at the logarithmic phase, but kept almost unchanged around 22.9 mg g−1 dry cell at the stationary phase. The EPS production of R. acidophila had a significant effect on its surface characteristics. The relative hydrophobicity and the K par values of R. acidophila before EPS extraction were both lower than those after extraction. Both EPS content and ratio of proteins to carbohydrates had a negative effect on the water contact angle of the bacterium, but had a positive influence on the bacterial surface free energy and its polar component. On the other hand, the EPS were not related with MATH% or the Gibbs energy of partition between hexadecane and aqueous phase.
Rhamnolipid production by a novel thermophilic hydrocarbon-degrading Pseudomonas aeruginosa AP02-1 by Amedea Perfumo; Ibrahim M. Banat; Francesco Canganella; Roger Marchant (pp. 132-138).
Thermophilic bacterial cultures were isolated from a hot spring environment on hydrocarbon containing mineral salts media. One strain identified as Pseudomonas aeruginosa AP02-1 was tested for the ability to utilize a range of hydrocarbons both n-alkanes and polycyclic aromatic hydrocarbons as sole carbon source. Strain AP02-1 had an optimum growth temperature of 45°C and degraded 99% of crude oil 1% (v/v) and diesel oil 2% (v/v) when added to a basal mineral medium within 7 days of incubation. Surface activity measurements indicated that biosurfactants, mainly glycolipid in nature, were produced during the microbial growth on hydrocarbons as well as on both water-soluble and insoluble substrates. Mass spectrometry analysis showed different types of rhamnolipid production depending on the carbon substrate and culture conditions. Grown on glycerol, P. aeruginosa AP02-1 produced a mixture of ten rhamnolipid homologues, of which Rha-Rha-C10-C10 and Rha-C10-C10 were predominant. Rhamnolipid-containing culture broths reduced the surface tension to ≈28 mN and gave stable emulsions with a number of hydrocarbons and remained effective after sterilization. Microscopic observations of the emulsions suggested that hydrophobic cells acted as emulsion-stabilizing agents.
Glycerophosphate as a phosphorus source in a defined medium for Pichia pastoris fermentation by Wenhui Zhang; Jayanta Sinha; Michael M. Meagher (pp. 139-144).
Pichia pastoris has emerged as a commercially important yeast for the production of a vast majority of recombinant therapeutic proteins and vaccines. The organism can be grown to very high cell densities using a defined basal salts media (BSM). However, BSM contains bi-cation or tri-cation phosphate, which precipitates out of the medium at pH above 5.5, although the optimal fermentation pH of most recombinant protein fermentation varies between 5.5 and 7.0. In this article, the application of glycerophosphates was investigated as a substitute phosphate source in an effort to eliminate precipitation. The solubility of BSM containing sodium or potassium glycerophosphates was examined before and after autoclaving at various pHs. Sodium glycerophosphate was found stable at autoclave temperature but formed complexes with coexisting magnesium and calcium ions that were insoluble above pH 7.0. Medium where sodium glycerophosphate was autoclaved separately and then added to the growth medium did not produce any precipitate up to pH 10.5. The performance of P. pastoris fermentations expressing α-galactosidase and ovine interferon-τ using a glycerolphosphate-based medium was found to be comparable to a conventional BSM. The results from this work demonstrate that sodium glycerophosphate can be assimilated by the P. pastoris strains and can be employed as a reliable phosphorus source for both cell growth and recombinant protein production.
Characterization of Streptomyces MITKK-103, a newly isolated actinomycin X2-producer by K. Kurosawa; V. P. Bui; J. L. VanEssendelft; L. B. Willis; P. A. Lessard; I. Ghiviriga; T. G. Sambandan; C. K. Rha; A. J. Sinskey (pp. 145-154).
A new actinomycete strain designated MITKK-103 was isolated from the soil of a flowerpot using a humic acid agar medium. The newly isolated strain was able to produce a large amount of actinomycin X2 even under nonoptimized growing conditions and serves as a promising source of this antibiotic. Actinomycin X2 has higher cytotoxicity toward cultured human leukemia (HL-60) cells than does actinomycin D, and it induces cell death via apoptosis. A nearly complete 16S ribosomal DNA (rDNA) sequence from the isolate was determined and found to have high identity (98.5–100%) with Streptomyces galbus, Streptomyces griseofuscus, and Streptomyces padanus, indicating that MITKK-103 belongs to the genus Streptomyces. The isolate clustered with species belonging to the S. padanus clade in a 16S-rDNA-based phylogenetic tree and showed 75% overall homology to S. padanus ATCC 25646 in DNA–DNA relatedness analysis. Although the growth of the isolate was somewhat different from the three species mentioned, the strain MITKK-103 most closely resembles S. padanus on the basis of the morphological and phenotypic characteristics, phylogenetic analysis, and genotypic data. As such, this is the first report of a strain of S. padanus capable of producing actinomycins.
Generation of aroma compounds from Ditaxis heterantha by Saccharomyces cerevisiae by L. Del Toro-Sánchez; S. Sánchez; M. A. Ortiz; S. Villanueva; E. Lugo-Cervantes (pp. 155-162).
Ditaxis heterantha, a plant of the Euphorbiaceae family, is growing wild in the semiarid regions of Mexico. The seed endosperm contains yellow pigments (carotenoids). By high-pressure liquid chromatography the total pigment (TP) was separated into seven fractions: two of them, heterathin (F4) and ditaxin (F5), characterized as apocarotenoids, represent 80% of TP. Both molecules have double bonds, which seem to be the target for degradation and aroma formation. In this work, TP, F4, and F5 were supplied to nine cultures able to degrade lutein. From these strains, only one (identified as Saccharomyces cerevisiae) was able to produce aromas from either TP or F4. Using TP as substrate, the produced aromas were 4-oxo-isophorone (1), isophorone (2), cinnamic aldehyde (6), 3-hydroxy-β-cyclocitral (7), safranal (8), geranyl (9), 3-oxo-α-ionone (10), 3-oxo-α-ionol (11), 3-oxo-7,8-dihydro-α-ionone (12), and eugenol (13). Of these aromas, only seven were produced from F4: (1), (2), (7), (8), (10), (11), and (12). In both cases, safranal was the main degradation product (30%). The enzymatic activity responsible for this effect was found in the cytosolic fraction and detected only when S. cerevisiae was grown in the presence of TP or F4.
Cellulase production by Penicillium echinulatum on lactose by Nicole Teixeira Sehnem; Luciano Ramos de Bittencourt; Marli Camassola; Aldo J. P. Dillon (pp. 163-167).
The inducer effect of lactose on cellulase activity in Penicillium echinulatum 9A02S1 was studied. Submerged cultivation was performed using different concentrations of lactose and cellulose, in which the pH, mycelial mass, soluble proteins, filter paper activity (FPA), and activity of β-glucosidases were measured. The cultures containing lactose only presented low FPAs (0.1 FPU/ml). The cultures with associated cellulose and lactose and those containing cellulose only presented similar enzymatic activities (1.5 FPU/ml), suggesting the possibility of up to 75% reduction in the cellulose concentration. In relation to the β-glucosidases, increasing the lactose/cellulose ratio results in a proportional increase of enzymatic activity. In the cultures using both inducers, there is a longer duration of the acid phase in relation to treatments using only cellulose or lactose, indicating diauxia and catabolic repression.
A serum-free medium for colony growth and hyaluronic acid production by Streptococcus zooepidemicus NJUST01 by Jianfa Zhang; Xia Ding; Liuyan Yang; Zhiming Kong (pp. 168-172).
A hyaluronic acid (HA)-producing strain, Streptococcus zooepidemicus NJUST01, can grow in a serum-free agar medium, with starch as exclusive carbon source, but not glucose, sucrose, dextrine, xylose, or lactose. In this starch medium, the strain NJUST01 reproduced successively at 37°C for 60 generations, with no obvious variation on morphology and physiology, but colonies of the strain after 60th generation could not produce a clear hemolytic zone on sheep blood agar plates. Hyaluronic acid production by the strain NJUST01 was analyzed relative to the starch medium. Employing a multifactor cross experiment, an optimum medium revealed for hyaluronic acid production was composed of 5% starch, 0.3% glucose, 0.5% peptone, 0.15% MgSO4, and 2.0% K2HPO4. The amount of HA 6.7 g/l was obtained in batch fermentation on a rotary shaker at 37°C, 220 rpm for 36 h.
Biological function of the pld gene product that degrades -poly-l-lysine in Streptomyces albulus by Y. Hamano; T. Yoshida; M. Kito; S. Nakamori; T. Nagasawa; H. Takagi (pp. 173-181).
ε-Poly-l-lysine (ε-PL) is one of the few naturally occurring biopolymers and is characterized by a peptide bond between the α-carboxyl and ε-amino groups. Previously, we purified and characterized the ε-PL-degrading enzyme (Pld) from Streptomyces albulus, which is an ε-PL producer, and this enzyme was expected to confer self-resistance to the ε-PL produced by the organism itself. The gene encoding Pld was cloned based on the N-terminal amino acid sequence determined in this study, and a sequencing analysis revealed eight open reading frames (ORFs), i.e., ORF1 to ORF8 in the flanking region surrounding the pld gene (present in ORF5). To investigate the biological function of Pld, we constructed a knockout mutant in which the pld gene is inactivated. Studies on ε-PL susceptibility, ε-PL-degrading activity, and ε-PL productivity demonstrated that the pld gene does play a partial role in self-resistance and that S. albulus was found to produce other ε-PL-degrading enzyme(s) in addition to Pld. To the best of our knowledge, this is the first report on a self-resistance gene for a biopolymer possessing antibacterial activity.
Molecular analysis of halophilic bacterial community for high-rate denitrification of saline industrial wastewater by Sachiko Yoshie; Hiroshi Makino; Hidenobu Hirosawa; Kosuke Shirotani; Satoshi Tsuneda; Akira Hirata (pp. 182-189).
A denitrification system for saline wastewater utilizing halophilic denitrifying bacteria has not been developed so far. In this study, denitrification performance and microbial community under various saline conditions were investigated using denitrifying sludge acclimated under low-salinity condition for a few years as seed sludge. A continuous denitrification experiment showed that denitrification performance and microbial community at 10% salinity was higher than that at 1% salinity. The microbial community in the denitrification sludge that was acclimated under low salinity was monitored by terminal-restriction fragment length polymorphism (T-RFLP) analysis during acclimation to high-salinity condition. T-RFLP profiles and clone analysis based on 16S rRNA-encoding genes in the sludge of the denitrification system with 10% salinity indicated that the γ-Proteobacteria, particularly Halomonas spp., were predominant species, suggesting that these bacterial members were possibly responsible for a high denitrification activity under high-salinity conditions. Furthermore, the investigation of denitrification performance under various saline conditions revealed that 4–10% salinity results in the highest denitrification rate, indicating that this salinity was optimal for predominant bacterial species to exhibit denitrification activity. These results indicate the possibility that an appropriate denitrification system for saline wastewater can be designed using acclimated sludge with a halophilic community.
Kinetic behaviors between acetone and composite bead in biofilter by Wu-Chung Chan; Liang-Yuan Chang (pp. 190-196).
In this study, the kinetic behaviors between acetone and composite bead were investigated. The microbial growth rate decreased with increasing average inlet concentration and increased with increasing operation temperature at average inlet concentration ranging from 50 to 300 ppm and operation temperature ranging from 30 to 40°C. The microbial growth rate would be inhibited with increasing average inlet concentration, and the inhibitive effect was more pronounced at higher operation temperature. The microbial growth rate would be enhanced with increasing operation temperature, and the enhancing effect was more pronounced at higher average inlet concentration. The values of maximum reaction rate V m and half-saturation constant K s ranged from 0.04 to 0.05 g-C/h-kg packing material and from 37.19 to 42.77 ppm, respectively. The biochemical reaction model could be regarded as the zero-order kinetic with the diffusion rate limitation. The biochemical reaction rate decreased with increasing average inlet concentration and increased with increasing operation temperature. The biochemical reaction rate would be inhibited with increasing average inlet concentration, and the inhibitive effect was more pronounced at lower operation temperature. The biochemical reaction rate would be enhanced with increasing the operation temperature, and the enhancing effect was more pronounced at higher average inlet concentration. The maximum elimination capacity of biofilter increased with increasing operation temperature. The values of critical and maximum elimination capacity ranged from 0.07 to 0.15 and from 0.13 to 0.16 g-C/h-kg packing material, respectively.
Expression of mercuric reductase from Bacillus megaterium MB1 in eukaryotic microalga Chlorella sp. DT: an approach for mercury phytoremediation by Chieh-Chen Huang; Meng-Wei Chen; Ju-Liang Hsieh; Wen-Hao Lin; Pei-Chung Chen; Lee-Feng Chien (pp. 197-205).
A eukaryotic microalga, Chlorella sp. DT, was transformed with the Bacillus megaterium strain MB1 merA gene, encoding mercuric reductase (MerA), which mediates the reduction of Hg2+ to volatile elemental Hg0. The transformed Chlorella cells were selected first by hygromycin B and then by HgCl2. The existence of merA gene in the genomic DNA of transgenic strains was shown by polymerase chain reaction amplification, while the stable integration of merA into genomic DNA of transgenic strains was confirmed by Southern blot analysis. The ability to remove Hg2+ in merA transgenic strains was higher than that in the wild type. The merA transgenic strains showed higher growth rate and photosynthetic activity than the wild type did in the presence of a toxic concentration of Hg2+. Cultured with Hg2+, the expression level of superoxide dismutase in transgenic strains was lower than that in the wild type, suggesting that the transgenic strains faced a lower level of oxidative stress. All the results indicated that merA gene was successfully integrated into the genome of transgenic strains and functionally expressed to promote the removal of Hg2+.
Novel blastospore-based transformation system for integration of phosphinothricin resistance and green fluorescence protein genes into Beauveria bassiana by Sheng-Hua Ying; Ming-Guang Feng (pp. 206-210).
A novel system was developed for efficient transformation of the fungal biocontrol agent Beauveria bassiana. Competent blastospores were prepared and stored in LiAc- and glycerol-inclusive suspension at −76 °C for sequential use in transformation. The system was successfully applied to integrating phosphinothricin resistance gene bar and enhanced green fluorescence protein gene egfp into B. bassiana via blastospore absorption of a plasmid vectoring bar and egfp. A frequency of 24 transformants per microgram of DNA was achieved. The blastospore-based transformation system has proven to be very convenient and would be highly potential for use in genetic manipulation of B. bassiana and other filamentous species.