Crude Medium Research Articles

Isolation and characterization of a-l-rhamnosidase producing bacterium, Agrococcus sp. bkd37, from a warehouse soil and partial optimization of its culture conditions

The present study was attempted to identify and partially optimize the culture conditions of a potential α-L-rhamnosidase producing bacterium isolated from warehouse soil of Dibrugarh, Assam (India). Twenty eight bacterial strains were isolated and the isolate BKD37 showed maximum rhamnosidase enzyme activity (10.01 ± 0.001 U mL-1) in crude culture medium. The 16S rRNA gene sequencing and phylogenetic analysis of BKD37 strain showed maximum similarity with that of Agrococcus sp. The culture conditions of strain BKD37 for the production of extracellular rhamnosidase enzyme was partially optimized in a basal medium in shake flask. The maximum rhamnosidase production was achieved after 60 h incubation at 30°C and pH 6.0. The rhamnose and NH4NO3 were found to be the best carbon and nitrogen sources, respectively, in basal medium for α-L rhamnosidase enzyme production.

Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad

A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198·H438·E325) was replaced by S198·H438·N322E in silico. Molecular dynamics for 1.5 μs and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293 cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293 cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.

Designing and Development of Rice water based crude media and it's application in fungal isolation and enzyme production

Designing and Development of Rice water based crude media and it's application in fungal isolation and enzyme production

Bacterial consortium expressing surface displayed, intra- and extracellular lipases and pseudopyronine B for the degradation of oil

The lipA gene, encoding a solvent-tolerant extracellular lipase from Proteus sp. SW1, was displayed on the cell surface of Escherichia coli by fusing it to an antigen 43 anchoring motif. The display of LipA on the Escherichia coli cell surface was directly confirmed by immunofluorescence microscopy and flow cytometry. After 6 days of incubation in media containing 1 % used cooking oil, an Escherichia coli strain expressing surface displayed lipase was able to degrade 27 % of the oil. The biosurfactant, pseudopyronine B, was purified from culture supernatants of Pseudomonas sp. SL31. Its critical micelle concentration was determined to be 1400 mg/l, and the surfactant was stable within a temperature range from 0 to 120 °C and a pH range of 3–11. Pseudopyronine B-containing crude media extracts efficiently removed up to 51 % of the cadmium from contaminated water. We demonstrated the oil degradation ability of the mixed culture of four bacterial strains, namely the recombinant Escherichia coli expressing cell surface displayed lipase (pKKJlipA), His-tagged lipase (pETlipA), extracellular lipase-producing Proteus sp. SW1, and pseudopyronine B-producing Pseudomonas sp. SL31 by culturing in LB media containing 1 % oil. The consortium degraded 29 % of oil in one day and reached 84 % after 7 days.

Direct determination of lignin peroxidase released from Phanerochaete chrysosporium by in-capillary enzyme assay using micellar electrokinetic chromatography

Here we describe the application of an in-capillary enzyme assay using micellar electrokinetic chromatography (MEKC) in the determination of enzyme activity in a crude culture medium containing lignin peroxidase released from Phanerochaete chrysosporium (P. chrysosporium). The method consists of a plug–plug reaction between lignin peroxidase and its substrate, veratryl alcohol, the separation of the product, veratraldehyde, from the other components including the enzyme and the culture medium, and the determination of the enzyme activity from the peak area of veratraldehyde produced by the plug–plug reaction. This method is more sensitive than conventional spectrophotometry since the background originates from the enzyme and the culture medium can be removed via MEKC separation. Veratraldehyde was separated at −10kV in a background electrolyte containing 50mM tartrate buffer (pH 2.5) and 50mM sodium dodecyl sulfate (SDS) after a plug–plug reaction in the capillary for 5min. The calibration curve of veratraldehyde was linear up to 4pmol (500μM) with a limit to quantification of 0.026pmol (3.2μM) (SN=10). The activity of lignin peroxidase was directly measured from the peak area of veratraldehyde. The activity of lignin peroxidase released from P. chrysosporium into the medium for 7 days was successfully determined to be 3.40UL−1.

Changes in Venezuelan Orinoco belt crude after different biotechnological approaches

Heavy crude oils from Venezuelan Orinoco belt needs special and costly handling processes due to its high viscosity, sulfur and metals content. Different biotechnological applications have been aimed to improve these properties. However, emulsified systems have not been assessed to increase the bioavailability of crude oil for microorganisms. In the present study were evaluated changes induced in two extra heavy crude oils by bacterial consortium using oil in water emulsified systems. For 5 day incubation period, larger detectable changes were observed for Boscán than Carabobo crude oil. The changes were an increase of the initial boiling point, decrease of the light fractions content, as well as a decrease of total sulfur. These changes were mainly observed after treatment with consortia grown in naphthalene. A 45 day incubation period of Carabobo crude oil treated with consortia grown in naphthalene exhibited an increase of the initial boiling point, CH2 and CH3 groups and partial loss of the lighter crude oil components. A relevant change was found after subsequent thermal treatment, it was a slight decrease of the 550 °C+ residues; also an important biodesulfurization was found under the same conditions. Finally, the study shows that bacterial degradation of extra heavy crude oils using emulsions is a sensitive procedure. The changes could depend on the heavy crude oil, bacterial consortium, culture medium, incubation time and temperature; as well as the implementation of thermal treatments.

Quantitation of encapsidated recombinant adeno-associated virus DNA in crude cell lysates and tissue culture medium by quantitative, real-time PCR

Recombinant AAV vectors are produced by transient transfection of mammalian cells. The virus is usually purified from a combination of lysed cells and spent culture medium by HPLC. We have developed a quantitative, real-time PCR assay for quantifying encapsidated single-stranded viral DNA (i.e. DNA-containing virions) in cell lysates and the spent culture medium. This requires extensive DNaseI digestion to reduce the amount of AAV replicative DNA, as well as plasmid and cellular DNA, to negligible amounts. To demonstrate the utility of this assay, we produced recombinant AAV in HeLa cells and five different types of 293 cells. We used primers to the EGFP transgene to detect the production of a recombinant AAV. We assayed the cell lysates and media by both our quantitative PCR assay and a functional transduction assay. The quantitative PCR assay data correlated well with the transduction assay data. Because this assay only requires standard PCR primers and SYBR Green I dye to detect the amplification of the PCR template, it will readily adapt to any target DNA sequence within the recombinant AAV genome. The recombinant AAV vector does not need to express a reporter gene, such as EGFP or β-galactosidase in order to assay the amount of virus produced.

Assaying phospholipase A2 activity.

Mammalian cells contain many structurally and functionally diverse phospholipases A2 (PLA2) that catalyze the hydrolysis of sn-2 fatty acid from membrane phospholipid. Assays are described for measuring the activity of Group IVA cytosolic PLA2alpha(cPLAalpha) and for secreted PLA2s (sPLA2) that are suitable for purified enzymes and for measuring activity in crude cell lysates and culture medium. The assay for cPLA2alpha involves measuring the calcium-dependent release of radiolabeled sn-2 arachidonic acid from small unilamellar vesicles of phosphatidylcholine. Methods are described for distinguishing cPLA2alpha activity in cell lysates from other PLA2s. sPLA2 activity is monitored using a fluorimetric assay that measures the continuous calcium-dependent formation of albumin-bound pyrene fatty acid from the sn-2 position of phosphatidylglycerol.

An integrated downstream processing strategy for the recovery and partial purification of penicillin acylase from crude media

An integrated process strategy for the recovery of penicillin acylase was developed, based on precipitation of non-enzymatic proteins directly from Escherichia coli homogenates or crude extracts using Rolquat (quaternary ammonium salt) and adsorption of the enzyme on Amp-Seph (3.8 µmole ampicillin cm−3) under pseudo-affinity conditions. The effect of pH, concentrations of ammonium sulfate and Rolquat, and also concentrations of protein and cell debris on the precipitation of non-enzymatic proteins from homogenates and crude extracts of penicillin acylase were analysed. The method of addition of Rolquat to homogenates and crude extracts significantly influenced the size of the precipitated particles. Improved results on the specific activity of penicillin acylase were obtained for 22% and 1% (w/v) of ammonium sulfate and Rolquat, respectively, added sequentially to enzyme solutions and at room temperature. Under these experimental conditions, the specific activity of penicillin acylase in homogenates and crude extracts was enhanced 2.5–3.0-fold. Finally, the integrated process strategy was implemented first by precipitation of non-enzymatic proteins and recovery of penicillin acylase directly from the enzyme solution treated with Rolquat using an adsorption/filtration system with an overall yield of 86%. This system allows simultaneously the filtration of cell debris and fine precipitated particles, in situ recovery of penicillin acylase by its adsorption on Amp-Seph, and selective desorption of the enzyme with a specific activity of 11 IU (mg prot)−1 and a desorption yield of 95%. © 2002 Society of Chemical Industry

Production and characterization of monoclonal antibodies to serine proteinase allergens in Penicillium and Aspergillus species.

Alkaline and/or vacuolar serine proteinases are major allergens in prevalent airborne Penicillium and Aspergillus species. The object of this study is to generate and characterize monoclonal antibodies against these serine proteinase allergens. BALB/c mice were immunized individually with the Penicillium citrinum culture medium or the crude extract and culture medium preparations of Aspergillus fumigatus. Hybridoma cells that secrete monoclonal antibodies against serine proteinase allergens were selected by immunoblotting. Antigens in three different Penicillium (P. citrinum, P. notatum and P. oxalicum) and two different Aspergillus species (A. fumigatus, and A. flavus) recognized by these monoclonal antibodies were analysed by sodium dodecyl sulphate and two-dimensional polyacrylamide gel electrophoresis immunoblotting and N-terminal amino acid sequence analysis. Four (PCM8, PCM10, PCM16 and PCM39) and one (FUM20) monoclonal antibodies against serine proteinase allergens were generated after fusion of NS-1 cells with spleen cells obtained from BALB/c mice immunized with antigens from P. citrinum and A. fumigatus, respectively. Immunoblotting results showed that PCM8 reacted with an alkaline serine proteinase allergen in P. citrinum and P. notatum. PCM10 and PCM39 reacted with the alkaline serine proteinase in two Penicillium (P. citrinum, P. notatum) and two Aspergillus species (A. fumigatus, and A. flavus) tested. PCM16 reacted with the alkaline serine proteinase allergen in P. citrinum, A. fumigatus and A. flavus but not with that in P. notatum. MoAb FUM20 reacted with the alkaline serine proteinase allergen in two Aspergillus species (A. fumigatus and A. flavus) but not with that in two different Penicillium species (P. citrinum, P. notatum) tested. Among these five monoclonal antibodies generated, only PCM39 and FUM20 can react with the vacuolar serine proteinase allergen in P. notatum, P. oxalicum and in A. fumigatus. The 35 kDa P. citrinum component that reacted with FUM20 has an N-terminal amino acid sequence of DSPSVEKNAP. Five monoclonal antibodies against different epitopes of the serine proteinase major allergens in prevalent Penicillium and Aspergillus species were generated in the present study. Antibodies obtained may be useful in the characterization and standardization of serine proteinase allergens in crude fungal extracts.

Biosensor for L-lactate determination as an index of E. coli number in crude culture medium

In a culture medium E. coli produces L-lactate as a metabolite. L-lactate enzyme sensors were assembled and applied to a crude E. coli growth medium for L-lactate determination. The amount of L-lactate was used as an index of the cell number. In the standard solution, L-lactate oxidase (LOD) sensor gave a linear response from 5 to 300 μM L-lactate. Determination of L-lactate in bacteria cultures of different absorbance ( A) was possible from an absorbance at 600 nm, A 600=0.01 and higher. L-lactate measurements were not modified when the LOD electrode was associated with L-lactate dehydrogenase (LDH) in solution. In contrast, the bi-enzyme sensor consisting of LOD coimmobilized with LDH on the electrode, lowered the detection limit to 30 nM L-lactate by amplification of measurements via coupled reactions and substrate recycling. Responses were obtained in growth medium from A=0.0005 (5×10 5 bacteria ml −1). In comparison, the standard spectrophotometer cell estimation was not reliable below A=0.02. The L-lactate detection limit can be decreased further by incubating bacteria with glucose for 1 to 2 h. Measurements of L-lactate by oxygen consumption could be performed in the presence of bacteria and growth medium components. The recycling enzymatic electrode kept 80% of its initial activity with storage overnight in a buffer, at 4°C, during 2 weeks. Such sensors permit fast and sensitive procedures for L-lactate and E. coli assays. The procedure does not disturb the developing cultures as relatively small sample volumes from 1 to 500 μl and no cells are needed for L-lactate determination.

Development of a fluorescence polarization immunoassay (FPIA) for the quantitative determination of paclitaxel

We have developed a fluorescence polarization immunoassay (FPIA) for the quantitative determination of paclitaxel in serum, crude Taxus extracts and Erwinia taxi culture medium. To achieve this, we used an antipaclitaxel monoclonal antibody and an FITC-labeled paclitaxel. Paclitaxel was chemically modified by introducing an amino function to enable coupling with fluorescein isothiocyanate. Paclitaxel competitively inhibited the binding of the monoclonal antibody with FITC-paclitaxel causing a decrease in polarization. We were able to detect paclitaxel in a concentration as low as 2 nM. Fluorescence polarization immunoassay requires only the addition of the fluorescent probe to the antibody, followed by an incubation and measurement of polarization. Both the simplicity and the sensitivity of this method make it useful for estimating the paclitaxel content in yew tree crude extracts and culture medium of paclitaxel producing micro-organisms and a possible assay for monitoring paclitaxel level in patients under treatment.

Hydrophosphination of α,β-unsaturated esters by primary phosphine-boranes; a useful entry to symmetrical and unsymmetrical phosphine-boranes

The reactivity of primary phosphine-boranes RPH 2·BH 3 (R = Ph and Me) towards CH 2CHCO 2Me and CH 2CHP(O)(OMe) 2 is discussed. Hydrophosphination is the major process. The presence of the free phosphine (0–20%) in the crude media indicates that a competitive hydroboration reaction also occurs. The P H addition was found to be controllable to give, in reasonable yields, either the mono or the bis-adducts. All the adducts are stable and are fully characterized. The preparation of an unsymmetrical bis-adduct is also presented.

Constitutive expression in gal7 mutants of Kluyveromyces lactis is due to internal production of galactose as an inducer of the Gal/Lac regulon.

The induction process of the galactose regulon has been intensively studied, but until now the nature of the inducer has remained unknown. We have analyzed a delta gal7 mutant of the yeast Kluyveromyces lactis, which lacks the galactotransferase activity and is able to express the genes of the Gal/Lac regulon also in the absence of galactose. We found that this expression is semiconstitutive and undergoes a strong induction during the stationary phase. The gal1-209 mutant, which has a reduced kinase activity but retains its positive regulatory function, also shows a constitutive expression of beta-galactosidase, suggesting that galactose is the inducer. A gal10 deletion in delta gal7 or gal1-209 mutants reduces the expression to under wild-type levels. The presence of the inducer could be demonstrated in both delta gal7 crude extracts and culture medium by means of a bioassay using the induction in gal1-209 cells. A mutation in the transporter gene LAC12 decreases the level of induction in gal7 cells, indicating that galactose is partly released into the medium and then retransported into the cells. Nuclear magnetic resonance analysis of crude extracts from delta gal7 cells revealed the presence of 50 microM galactose. We conclude that galactose is the inducer of the Gal/Lac regulon and is produced via UDP-galactose through a yet-unknown pathway.

Convenient fluorometric assay for matrix metalloproteinase activity and its application in biological media

Matrix metalloproteinases (MMPs) are involved in physiological tissue remodeling and pathological conditions like tumour metastasis and joint destruction. Until now, no convenient and sensitive MMP-activity assay in crude media like synovial fluid has been available. Therefore, the highly soluble fluorogenic substrate TNO211 (Dabcyl-Gaba-Pro-Gln-Gly-Leu-Glu(EDANS)-Ala-Lys-NH 2), containing the MMP cleavable Gly-Leu bond and EDANS/Dabcyl as fluorophore/quencer combination, was synthesized and characterized as an MMP specific substrate. We show that the fluorogenic assay using TNO211 is sensitive and can detect MMP activity in culture medium from endothelial cells and untreated synovial fluid (SF) from RA and OA patients, and control subjects. MMP activity in SF significantly increased in the order C < OA < RA, thus the frequent use of OA samples as control in studies on RA is debatable.

Detection of neu differentiation factor with a biospecific affinity sensor during chromatography.

A technique using a biospecific affinity sensor, BIAcore, was applied to monitor and determine mammalian cell-derived neu differentiation factor (NDF) in column fractions during chromatography. Specific purified polyclonal antibody against Escherichia coli-derived NDF was chemically bound to the surface of BIAcore sensor chips and the derivatized sensor chips were used to detect the specific binding of NDF. The measurement of NDF at very low levels can be assessed by injecting small volumes of the crude media or column fractions into the BIAcore sensor containing antibody-bound sensor chips. This automated procedure performed under computer programming control allows direct measurement of multiple NDF samples in a short period of time and provides excellent quantitative data, which is not possible using other related methods such as Western blotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stimulatory activity assay on receptor autophosphorylation.

Enzyme immunoassay (ELISA) for the evaluation of antibodies directed to the CD4 receptor-binding site of the HIV gp120 molecule

The interaction between the HIV envelope glycoprotein gp120 and the CD4 molecule is probably the most important primary event determining HIV infection. Reactivity with the native viral envelope has been difficult to measure due to the lack of gp120 ligand purified directly from primary virus cultures. We have developed an ELISA, utilizing Galanthus nivalis agglutinin (GNA) which selectively binds native HIV envelope gp120 in culture medium. The GNA-based ELISA eliminates the need for isotope-labelled reagents, live cells and recombinant non-natively glycosylated envelope proteins and offers an easy way of using gp120 directly from crude HIV culture medium. The reactivities of sera from several categories of HIV infected individuals were assayed for inhibition of the HIV-1 gp120-CD4 binding. 19 32 (59.3%) sera from asymptomatic individuals and 7 10 (70%) sera from ARC/AIDS patients blocked the CD4-gp120 binding. 20 serum samples from uninfected individuals showed a gp120-CD4 interaction blocking capacity of 0–15%. Two monoclonal antibodies, T4.2 directed to CD4 and 1171 directed to the CD4 binding site of gp120 were used as positive controls. Both Mabs inhibited CD4-gp120 binding by 66–90%.

A scatter factor-like factor is produced by a metastatic variant of a rat bladder carcinoma cell line.

The rat bladder carcinoma epithelial NBT-II cell line undergoes, in vitro, a morphological transition to a fibroblast-like state in the presence of different growth factors. We have selected, in vivo, a metastatic clone, designated M-NBT-II, which has a mesenchymal phenotype and secretes into the culture medium a factor able to dissociate epithelial clusters of NBT-II or MDCK cells. This factor was designated scatter factor-like (SFL) by analogy to the HGF/SF, which has the same dissociating effect in these two cell lines. Here, we show that SFL factor and HGF/SF are different factors: (i) no HGF/SF transcripts could be detected using either specific rat HGF/SF cDNA probes or PCR; (ii) blocking antibodies against rat HGF/SF do not inhibit the SFL activity; and (iii) crude culture medium or partially purified SFL factor-containing fractions do not induce MDCK tubulogenesis, a biological assay that is specific for HGF/SF activity in vitro. We report the partial purification of the SFL factor, based on ion exchange and reverse-phase chromatography. The results indicate that the M-NBT-II metastatic variant secretes a dissociating factor sharing some common biological properties with the HGF/SF, which suggests that the SFL factor is a member of the HGF/SF family and may be involved in tumor progression.

Dissociation of collagenase-tissue inhibitor of metalloproteinases-1 (TIMP-1) complex - its application for the independent measurements of TIMP-1 and collagenase activity in crude culture media and body fluids

Collagenase-tissue inhibitor of metalloproteinases-1 (TIMP-1) complex was prepared from activated collagenase and TIMP-1 purified from culture media of human skin fibroblasts. After having been confirmed to be a complex by zinc chelate chromatography, the complex was demonstrated to dissociate by passage through an anti-TIMP-1 monoclonal antibody-affinity column. On the basis of above evidence, a simple strategy was set up for the independent measurements of TIMP-1 concentration, and both active and total collagenase activities in crude culture media and body fluids.

Porphyrin Rings and Phospholipids: Stimulators of Cloning Efficiency in Certain Species of Tetrahymena

Species of Tetrahymena, including T. vorax, T. thermophila, T. pyriformis, and T. pigmentosa, were tested for cloning efficiency in proteose peptone and in synthetic nutrient media to which were added hemin, protoporphyrin IX, chlorophyllin, or asolectin, an impure mixture of phospholipids. All species could be cloned with high efficiency in the crude media. In unsupplemented synthetic medium the cloning efficiencies were 0-10%, around 50%, around 50%, and 90-100% for T. thermophila, T. vorax, T. pyriformis, and T. pigmentosa, respectively. The first three were all stimulated to 90-100% by addition of the porphyrin or phospholipid compounds mentioned above. Uroporphyrin III and coproporphyrin I and III had no effect. We suggest that cells unable to form clones suffer from a lack of cellular energy. This situation may be alleviated by our additions, certain porphyrin rings may be built into cytochromes and phospholipids may be used as fuel. Thus, the synthetic media used so far for these ciliates have not been optimal.