Showing papers in "Biotechnology Letters in 2006"

Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications.

Abstract: Hyaluronic acid (hyaluronan, HA) is a linear polysaccharide formed from disaccharide units containing N-acetyl-D-glucosamine and glucuronic acid. It has a high molecular mass, usually in the order of millions of Daltons, and interesting viscoelastic properties influenced by its polymeric and polyelectrolyte characteristics. HA is present in almost all biological fluids and tissues. In clinical medicine, it is used as a diagnostic marker for many diseases including cancer, rheumatoid arthritis and liver pathologies, as well as for supplementation of impaired synovial fluid in arthritic patients by means of intra-articular injections. It is also used in certain ophthalmological and otological surgeries and cosmetic regeneration and reconstruction of soft tissue. Herein we present an overview of the occurrence and physiological properties of HA, as well as of the recent advances in production biotechnology and preparation of the HA-based materials for medical application.

Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR.

Abstract: Relative quantification in quantitative real-time RT-PCR is increasingly used to quantify gene expression changes. In general, two different relative mRNA quantification models exist: the delta-delta Ct and the efficiency-corrected Ct model. Both models have their advantages and disadvantages in terms of simplification on the one hand and efficiency correction on the other. The particular problem of RNA integrity and its effect on relative quantification in qRT-PCR performance was tested in different bovine tissues and cell lines (n = 11). Therefore different artificial and standardized RNA degradation levels were used. Currently fully automated capillary electrophoresis systems have become the new standard in RNA quality assessment. RNA quality was rated according the RNA integrity number (RIN). Furthermore, the effect of different length of amplified products and RNA integrity on expression analyses was investigated. We found significant impact of RNA integrity on relative expression results, mainly on cycle threshold (Ct) values and a minor effect on PCR efficiency. To minimize the interference of RNA integrity on relative quantification models, we can recommend to normalize gene expression by an internal reference gene and to perform an efficiency correction. Results demonstrate that innovative new quantification methods and normalization models can improve future mRNA quantification.

Involvement of polyamines in plant response to abiotic stress

Abstract: Environmental stresses are the major cause of crop loss worldwide Polyamines are involved in plant stress responses However, the precise role(s) of polyamine metabolism in these processes remain ill-defined Transgenic approaches demonstrate that polyamines play essential roles in stress tolerance and open up the possibility to exploit this strategy to improve plant tolerance to multiple environmental stresses The use of Arabidopsis as a model plant enables us to carry out global expression studies of the polyamine metabolic genes under different stress conditions, as well as genome-wide expression analyses of insertional-mutants and plants over-expressing these genes These studies are essential to dissect the polyamine mechanism of action in order to design new strategies to increase plant survival in adverse environments

Bacterial alginates: from biosynthesis to applications.

Abstract: Alginate is a polysaccharide belonging to the family of linear (unbranched), non-repeating copolymers, consisting of variable amounts of beta-D-mannuronic acid and its C5-epimer alpha- L-guluronic acid linked via beta-1,4-glycosidic bonds. Like DNA, alginate is a negatively charged polymer, imparting material properties ranging from viscous solutions to gel-like structures in the presence of divalent cations. Bacterial alginates are synthesized by only two bacterial genera, Pseudomonas and Azotobacter, and have been extensively studied over the last 40 years. While primarily synthesized in form of polymannuronic acid, alginate undergoes chemical modifications comprising acetylation and epimerization, which occurs during periplasmic transfer and before final export through the outer membrane. Alginate with its unique material properties and characteristics has been increasingly considered as biomaterial for medical applications. The genetic modification of alginate producing microorganisms could enable biotechnological production of new alginates with unique, tailor-made properties, suitable for medical and industrial applications.

Extremely rapid extraction of DNA from bacteria and yeasts.

Abstract: A very simple and rapid method for extracting genomic DNA from Gram-negative bacteria, Gram-positive bacteria and yeasts is presented. In this method, bacteria or yeasts are lysed directly by phenol and the supernatant is extracted with chloroform to remove traces of phenol. The supernatant contains DNA that is suitable for molecular analyses, such as PCR, restriction enzyme digestion and genomic library construction. This method is reproducible and simple for the routine DNA extraction from bacteria and yeasts.

Microbial production of 1,3-propanediol by Klebsiella pneumoniae using crude glycerol from biodiesel preparations.

Abstract: 1,3-Propanediol (1,3-PD) was produced by Klebsiella pneumoniae using crude glycerol obtained from biodiesel production. The 1,3-PD concentration of 51.3 g/l−1 on crude glycerol from alkali-catalyzed methanolysis of soybean oil was comparable to that of 53 g/l−1 on crude glycerol derived from a lipase-catalyzed process. The productivities of 1.7 g l−1 h−1 on crude glycerol were comparable to that of 2 g l−1 h−1 on pure glycerol. It could be concluded that the crude glycerol could be directly converted to 1,3-PD without any prior purification.

Immobilized yeast cell systems for continuous fermentation applications

Abstract: In several yeast-related industries, continuous fermentation systems offer important economical advantages in comparison with traditional systems. Fermentation rates are significantly improved, especially when continuous fermentation is combined with cell immobilization techniques to increase the yeast concentration in the fermentor. Hence the technique holds a great promise for the efficient production of fermented beverages, such as beer, wine and cider as well as bio-ethanol. However, there are some important pitfalls, and few industrial-scale continuous systems have been implemented. Here, we first review the various cell immobilization techniques and reactor setups. Then, the impact of immobilization on cell physiology and fermentation performance is discussed. In a last part, we focus on the practical use of continuous fermentation and cell immobilization systems for beer production.

Bioreactors for tissue engineering.

Abstract: Bioreactors are essential in tissue engineering, not only because they provide an in vitro environment mimicking in vivo conditions for the growth of tissue substitutes, but also because they enable systematic studies of the responses of living tissues to various mechanical and biochemical cues. The basic principles of bioreactor design are reviewed, the bioreactors commonly used for the tissue engineering of cartilage, bone and cardiovascular systems are assessed in terms of their performance and usefulness. Several novel bioreactor types are also reviewed.

Response of Growth and Fatty Acid Compositions of Nannochloropsis sp. to Environmental Factors Under Elevated CO2 Concentration

Abstract: Nannochloropsis sp. was grown with different levels of nitrate, phosphate, salinity and temperature with CO(2) at 2,800 microl l(-1). Increased levels of NaNO(3) and KH(2)PO(4) raised protein and polyunsaturated fatty acids (PUFAs) contents but decreased carbohydrate, total lipid and total fatty acids (TFA) contents. Nannochloropsis sp. grew well at salinities from 22 to 49 g l(-1), and lowering salinity enhanced TFA and PUFAs contents. TFA contents increased with the increasing temperature but PUFAs contents decreased. The highest eicosapentaenoic acid (EPA, 20:5 omega3) content based on the dry mass was above 3% under low N (150 microM NaNO(3)) or high N (3000 microM NaNO(3)) condition. Excessive nitrate, low salinity and temperature are thus favorable factors for improving EPA yields in Nannochloropsis sp.

Growing phototrophic cells without light.

Abstract: Many phototrophic microorganisms contain large quantities of high-value products such as n-3 polyunsaturated fatty acids and carotenoids but phototrophic growth is often slow due to light limitation. Some phototrophic microorganisms can also grow on cheap organic substrate heterotrophically. Heterotrophic cultivation can be well controlled and provides the possibility to achieve fast growth and high yield of valuable products on a large scale. Several strategies have been investigated for cultivation of phototrophic microorganisms without light. These include trophic conversion of obligate photoautotrophic microorganisms by genetic engineering, development of efficient cultivation systems and optimization of culture conditions. This paper reviews recent advances in heterotrophic cultivation of phototrophic cells with an emphasis on microalgae.

Reduction of Cr(VI) by a Bacillus sp.

Abstract: A Bacillus sp. RE was resistant to chromium and reduced Cr(VI) without accumulating chromium inside the cell. When Cr(VI) was 10 and 40 μg ml−1, >95% of the total Cr(VI) was reduced in 24 and 72 h of growth, respectively, whereas at 80 μg Cr(VI) ml−1 only 50% of Cr(VI) was reduced. However growth was not affected; the cell mass was 0.7–0.8 mg ml−1 in all cases. The cell-free extract showed Cr(VI) reducing enzyme activity which was enhanced (>5 fold) by NADH and NADPH. Like whole cells the enzyme also reduced Cr(VI) with decreasing efficiency on increasing Cr(VI) concentration. The enzyme activity was optimal at pH 6.0 and 30 °C. The enzyme was stable up to 30 °C and from pH 5.5 to 8, but from pH 4 to 5 the enzyme was severely destabilized. Its Km and Vmax were 14 μm and 3.8 nmol min−1 mg−1 respectively. The enzyme activity was enhanced by Cu2+ and Ni2+ and inhibited by Hg2+.

Biological Synthesis of Semiconductor Zinc Sulfide Nanoparticles by Immobilized Rhodobacter sphaeroides

Abstract: A novel, clean biological transformation reaction by immobilized Rhodobacter sphaeroides has been developed for the synthesis of zinc sulfide (ZnS) nanoparticles with an average diameter of 8 nm. The nanoparticles were examined by X-ray diffraction, transmission electron microscopy, energy dispersive analyses of X-rays, UV-vis optical absorption and photoluminescence spectra. The average diameter of ZnS nanoparticles varied according to the culture time.

Immobilization of beta-glucosidase on Eupergit C for lignocellulose hydrolysis

Abstract: beta-Glucosidase is frequently used to supplement cellulase preparations for hydrolysis of cellulosic and lignocellulosic substrates in order to accelerate the conversion of cellobiose to glucose. Typically, commercial cellulase preparations are deficient in this enzyme and accumulation of cellobiose leads to product inhibition. This study evaluates the potential for recycling beta-glucosidase by immobilization on a methacrylamide polymer carrier, Eupergit C. The immobilized beta-glucosidase had improved stability at 65 degrees C, relative to the free enzyme, while the profile of activity versus pH was unchanged. Immobilization resulted in an increase in the apparent Km from 1.1 to 11 mM: and an increase in Vmax from 296 to 2430 micromol mg(-1) min(-1). The effect of immobilized beta-glucosidase on the hydrolysis of cellulosic and lignocellulosic substrates was comparable to that of the free enzyme when used at the same level of protein. Operational stability of the immobilized beta-glucosidase was demonstrated during six rounds of lignocellulose hydrolysis.

Methylglyoxal Bypass Identified as Source of Chiral Contamination in l (+) and d (−)-lactate Fermentations by Recombinant Escherichia coli

Abstract: Two new strains of Escherichia coli B were engineered for the production of lactate with no detectable chiral impurity. All chiral impurities were eliminated by deleting the synthase gene (msgA) that converts dihydroxyacetone-phosphate to methylglyoxal, a precursor for both l(+)- and d(−)-lactate. Strain TG113 contains only native genes and produced optically pure d(−)-lactate. Strain TG108 contains the ldhL gene from Pediococcus acidilactici and produced only l(+)-lactate. In mineral salts medium containing 1 mM betaine, both strains produced over 115 g (1.3 mol) lactate from 12% (w/v) glucose, >95% theoretical yield.

Palladium and gold removal and recovery from precious metal solutions and electronic scrap leachates by Desulfovibrio desulfuricans

Abstract: Biomass of Desulfovibrio desulfuricans was used to recover Au(III) as Au(0) from test solutions and from waste electronic scrap leachate. Au(0) was precipitated extracellularly by a different mechanism from the biodeposition of Pd(0). The presence of Cu(2+) ( approximately 2000 mg/l) in the leachate inhibited the hydrogenase-mediated removal of Pd(II) but pre-palladisation of the cells in the absence of added Cu(2+) facilitated removal of Pd(II) from the leachate and more than 95% of the Pd(II) was removed autocatalytically from a test solution supplemented with Cu(II) and Pd(II). Metal recovery was demonstrated in a gas-lift electrobioreactor with electrochemically generated hydrogen, followed by precipitation of recovered metal under gravity. A 3-stage bioseparation process for the recovery of Au(III), Pd(II) and Cu(II) is proposed.

Improvement of Biohydrogen Production Under Decreased Partial Pressure of H2 by Enterobacter cloacae

Abstract: When the partial pressure of H(2) was decreased by lowering the total pressure in the headspace of the reactor in a batch fermentation process from 760 mm Hg to 380 mm Hg containing Enterobacter cloacae, the molar yield of H(2) increased from 1.9 mol to 3.9 mol H(2)/mol glucose. The maximum production rate was 0.017 mmol H(2)/h l at 380 mm Hg. The lag period as well as total batch time of H(2) production decreased using a decreased partial pressure.

Genetics and biochemistry of polyhydroxyalkanoate granule self-assembly: The key role of polyester synthases.

Abstract: PHAs (polyhydroxyalkanoates = biopolyester) composed of hydroxy fatty acids represent a rather complex class of storage polymers synthesized by various bacteria and archaea and are deposited as water-insoluble cytoplasmic nano-sized inclusions. These spherical particles are composed of a polyester core surrounded by phospholipids and proteins. The key enzymes of polyester biosynthesis and polyester particle formation are the polyester synthases, which catalyze the formation of polyesters. Various metabolic routes have been identified and established in bacteria to provide substrate for polyester synthases. Although not essential for particle formation, non-covalently attached proteins, the so-called phasins, can be found at the particle surface and are considered as structural proteins. Protein engineering of polyester synthases and phasins was used to shed light into the topology of these granule attached proteins. Biopolyesters and the respective micro-/nano-structures are currently considered as biocompatible and biodegradable biomaterials with numerous potential applications particularly in the medical field.

Purification and characterization of a family 5 endoglucanase from a moderately thermophilic strain of Bacillus licheniformis.

Abstract: Strains of thermophilic bacilli were screened for cellulolytic activity by gel diffusion assay on selective medium at 55°C. Strain B-41361, identified as a strain of Bacilluslicheniformis, displayed activity against carboxymethylcellulose. Zymogram analysis demonstrated several catalytically active polypeptides with the most prominent species having a mass of 37 kDa. The enzyme was purified 60-fold with a 17% yield and specific activity of 183 U/mg. The amino terminal sequence was homologous to members of glycoside hydrolase family 5. Optimal temperature was 65°C (measured over 30 min), but the enzyme was most stable at 60°C, retaining greater than 90% activity after one hour. The enzyme had a broad pH range, with maximal activity at pH 6.0, 75% maximal activity at pH 4.5, and 40% at pH 10. The enzyme hydrolyzed p-nitrophenylcellobioside, barley β-glucan, and lichenan, but no activity was detected against avicel or acid-swollen cellulose.

Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics

Abstract: Genetically-coded, fluorescence resonance energy transfer (FRET) biosensors are widely used to study molecular events from single cells to whole organisms. They are unique among biosensors because of their spontaneous fluorescence and targeting specificity to both organelles and tissues. In this review, we discuss the theoretical basis of FRET with a focus on key parameters responsible for designing FRET biosensors that have the highest sensitivity. Next, we discuss recent applications that are grouped into four common biosensor design patterns--intermolecular FRET, intramolecular FRET, FRET from substrate cleavage and FRET using multiple colour fluorescent proteins. Lastly, we discuss recent progress in creating fluorescent proteins suitable for FRET purposes. Together these advances in the development of FRET biosensors are beginning to unravel the interconnected and intricate signalling processes as they are occurring in living cells and organisms.

Biosynthesis of medium-chain-length poly(hydroxyalkanoates) from soy molasses.

Abstract: Pseudomonas corrugata was selected from a screening process for the bioconversion of inexpensive soy molasses into medium-chain-length poly(hydroxyalkanoates) (mcl-PHA). We obtained yields of 1.5 g cell dry weight (CDW)/l culture with growth medium supplemented with 2% (w/v) soy molasses, and of an average of 3.4 g CDW/l with 5% (w/v) soy molasses. Crude PHAs were obtained at 5–17% of CDW. The most prominent repeat-unit monomers in the PHAs were 3-hydroxydodecanoate, 3-hydroxyoctanoate, 3-hydroxydodecanoate, and 3-hydroxytetradecenoate. This work represents the first description of fermentative mcl-PHA production from the soy molasses.

Anti-apoptotic activity of laminarin polysaccharides and their enzymatically hydrolyzed oligosaccharides from Laminaria japonica.

Abstract: Laminarin polysaccharides (LP1) were prepared from Laminaria japonica, a marine brown alga with potential biological activities, by hot water extraction, ultrafiltration and gel chromatography; the molecular weights of the LP1s were between 5 and 10 kDa. Laminarin oligosaccharides (LO) derived by hydrolyzing LP1 with an endo-beta-(1-->3)-glucanase from Bacillus circulans were mainly di- and penta-oligosaccharides. Treatment of mouse thymocytes with LO or LP1 (1-4 mg ml(-1)) suppressed apoptotic death around 3- or 2-fold and extended cell survival in culture at a rate of about 30 or 20%. A mouse cDNA microarray showing the genes coding for immune response proteins were induced and apoptotic cell death proteins were reduced significantly by LO provided preliminary information regarding the immunomodulatory mechanism of LO. These results suggest that laminarin oligosaccharides and polysaccharides can be utilized to develop new immunopotentiating substances and functional alternative medicines.

Increase of the hydrophilicity of polyethylene terephthalate fibres by hydrolases from Thermomonospora fusca and Fusarium solani f. sp. pisi.

Abstract: Treatment of polyethylene terephthalate fibres with hydrolase preparations from Thermomonospora (Thermobifida) fusca and Fusarium solani f. sp. pisi resulted in an increase of the hydrophilicity of the fibres determined by measurement of their dyeing behaviour with reactive dyes and their water absorption ability. Reflectance spectrometry of treated fibres dyed with a reactive dye showed that the colour became more intense corresponding to an increase of hydroxyl groups on the fibre surfaces and indicated a stepwise peeling of the fibres by the enzymes comparable to the effects obtained by alkaline treatments. The synthetic fibres treated with the hydrolase from T. fusca also showed enhanced water absorption ability further confirming the increased surface hydrophilicity caused by the enzyme.

Catabolic pathways and biotechnological applications of microbial caffeine degradation

Abstract: Catabolism of caffeine (1,3,7-trimethylxanthine) in microorganisms commences via two possible mechanisms: demethylation and oxidation. Through the demethylation route, the major metabolite formed in fungi is theophylline (1,3-dimethylxanthine), whereas theobromine (3,7-dimethylxanthine) is the major metabolite in bacteria. In certain bacterial species, caffeine has also been oxidized directly to trimethyl uric acid in a single step. The conversion of caffeine to its metabolites is primarily brought about by N-demethylases (such as caffeine demethylase, theobromine demethylase and heteroxanthinedemethylase), caffeine oxidase and xanthine oxidase that are produced by several caffeine-degrading bacterial species such as Pseudomonas putida and species within the genera Alcaligenes, Rhodococcus and Klebsiella. Development of biodecaffeination techniques using these enzymes or using whole cells offers an attractive alternative to the present existing chemical and physical methods removal of caffeine, which are costly, toxic and non-specific to caffeine. This review mainly focuses on the biochemistry of microbial caffeine degradation, presenting recent advances and the potential biotechnological application of caffeine-degrading enzymes.

Growth of Escherichia coli, Pichia pastoris and Bacillus cereus in the presence of the ionic liquids [BMIM][BF4] and [BMIM][PF6] and Organic Solvents.

Abstract: The influence of the two most commonly used ionic liquids (1-butyl-3-methyl imidazolium tetrafluoroborate, [BMIM][BF4], 1-butyl-3-methyl imidazolium hexafluorophosphate, [BMIM][PF6]) and three selected organic solvents (dimethylsulfoxide, ethanol, methanol) on the growth of Escherichia coli, Pichia pastoris and Bacillus cereus was investigated. [BMIM][BF4] was toxic at 1% (v/v) on all three microorganisms. The minimal inhibitory concentration (MIC) of [BMIM][BF4] on E. coli growth was between 0.7 and 1% (v/v). In contrast, [BMIM][PF6] was less toxic for P. pastoris and B. cereus, whereas E. coli was not able to tolerate [BMIM][PF6] (MIC value: 0.3–0.7% v/v). Growth of P. pastoris was unaffected by [BMIM][PF6] at 10% (v/v). Similar results were found for dimethylsulfoxide. Thus, ionic liquids (ILs) can have substantial inhibitory effects on the growth of microorganisms, which should be taken into account for environmental reasons as well as for the use of ILs as co-solvents in biotransformations.

Expression of poplar chitinase in tomato leads to inhibition of development in Colorado potato beetle

Abstract: The previously described poplar chitinase, WIN6, is induced during infestation by gypsy moth (Lymantria dispar L.) larvae, thus suggesting a role in defense against insect pests. To test this hypothesis, we produced tomato seedlings infected with a recombinant potato virus X (PVX), which produces WIN6, and tested its insecticidal properties on Colorado potato beetle [CPB; Leptinotarsa decemlineata (Say)], which is a serious pest of tomatoes and other crops. The advantage of PVX is that plant material is ready for insect bioassay within 3-4 weeks of constructing the recombinant virus. Considering that production of transgenic tomato seedlings using Agrobacterium takes at least 6 months, this hastens the rate at which genes can be examined. Upon insect bioassay, only 47% CPB neonates feeding on leaves containing >0.3% w/w WIN6 developed to 2nd instar while 93% of controls reached 2nd instar. To our knowledge this is the first plant chitinase that retards development of an insect pest.

Fermentation of 12% (w/v) glucose to 1.2 M lactate by Escherichia coli strain SZ194 using mineral salts medium.

Abstract: A non-recombinant mutant of Escherichia coli B, strain SZ194, was developed that produces over 1 M D-lactate from glucose (or sucrose) in 72 h using mineral salts medium supplemented with 1 mM: betaine in simple anaerobic fermentations. Rates and yields were highest at pH 7.5. Yields approached the theoretical maximum with only trace amounts of co-products. Chiral purity of D-lactate was estimated to be 95%. Specific and volumetric productivities for SZ194 in mineral salts medium (pH 7.5) with betaine were equivalent to those in Luria broth.

A polysaccharide isolated from the medicinal herb Bletilla striata induces endothelial cells proliferation and vascular endothelial growth factor expression in vitro

Abstract: A polysaccharide from the traditional Chinese medicinal herb, Bletilla striata (Thunb.) Reichb. f., was isolated, purified and characterized. It induced the proliferation of human umbilical vascular endothelial cells and the expression of vascular endothelial growth factor up to 156% and 147% of control after 72 h, respectively.

Supersaturation of dissolved H2 and CO2 during fermentative hydrogen production with N2 sparging

Abstract: Dissolved H2 and CO2 were measured by an improved manual headspace-gas chromatographic method during fermentative H2 production with N2 sparging. Sparging increased the yield from 1.3 to 1.8 mol H2/mol glucose converted, although H2 and CO2 were still supersaturated regardless of sparging. The common assumption that sparging increases the H2 yield because of lower dissolved H2 concentrations may be incorrect, because H2 was not lowered into the range necessary to affect the relevant enzymes. More likely, N2 sparging decreased the rate of H2 consumption via lower substrate concentrations.

Production of high-content galacto-oligosaccharide by enzyme catalysis and fermentation with Kluyveromyces marxianus

Abstract: Of three beta-galactosidases from Aspergillus oryzae, Kluyveromyces lactis and Bacillus sp., used for the production of low-content galacto- oligosaccharides (GOS) from lactose, the latter produced the highest yield of trisaccharides and tetrasaccharides. GOS production was enhanced by mixing beta-galactosidase glucose oxidase. The low-content GOS syrups, produced either by beta-galactosidase alone or by the mixed enzyme system, were subjected to the fermentation by Kluyveromyces marxianus, whereby glucose, galactose, lactose and other disaccharides were depleted, resulting in up to 97% and 98% on a dry weight basis of high-content GOS with the yields of 31% and 32%, respectively.

Natural and engineered ribonucleases as potential cancer therapeutics.

Abstract: By reason of their cytotoxicity, ribonucleases (RNases) are potential anti-tumor drugs. Particularly members from the RNase A and RNase T1 superfamilies have shown promising results. Among these enzymes, Onconase, an RNase from the Northern Leopard frog, is furthest along in clinical trials. A general model for the mechanism of the cytotoxic action of RNases includes the interaction of the enzyme with the cellular membrane, internalization, translocation to the cytosol, and degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the capability of the RNase to evade the intracellular RNase inhibitor has not yet been fully elucidated. This paper discusses the various approaches to exploit RNases as cytotoxic agents.