Use Of Whole Cells Research Articles

Optimisation of the biocatalytic resolution of styrene oxide by whole cells of Rhodotorula glutinis

Various studies have described the optimisation of the hydrolysis of epoxides through epoxide hydrolase, however, far fewer have investigated the specific application of whole cells containing the enzyme. For this reason the enantioselective biocatalytic hydrolysis of styrene oxide by Rhodotorula glutinis UOFS Y-0653 through the use of whole cells was explored. It was found that a pH of 7.2, temperature of 45 °C and an initial substrate concentration of 50 mM led to maximum enzymatic activity. The whole cells were resistant to a changing environment. High temperatures were found to increase enzymatic activity but decrease enantioselectivity. At low temperatures (15 °C) enantioselectivity was significantly increased leading to an increase in both enantiopure substrate yield and the enantiomeric excess of both the substrate and product. No substrate inhibition was observed at initial substrate concentrations as high as 100 mM. The low deactivation energy (85.2 kJ/mol) obtained for this hydrolysis reaction suggests thermal instability of the enzyme. No significant effect on the reaction was observed when using unbuffered water instead of phosphate buffer as reaction medium.

Surface display of transglucosidase onEscherichia coli by using the ice nucleation protein ofXanthomonas campestris and its application in glucosylation of hydroquinone

A surface anchoring motif using the ice nucleation protein (INP) of Xanthomonas campestris pv. campestris BCRC 12,846 for display of transglucosidase has been developed. The transglucosidase gene from Xanthomonas campestris pv. campestris BCRC 12,608 was fused to the truncated ina gene. This truncated INP consisting of N- and C-terminal domains (INPNC) was able to direct the expressed transglucosidase fusion protein to the cell surface of E. coli with apparent high enzymatic activity. The localization of the truncated INPNC-transglucosidase fusion protein was examined by Western blot analysis and immunofluorescence labeling, and by whole-cell enzyme activity in the glucosylation of hydroquinone. The glucosylation reaction was carried out at 40 degrees C for 1 h, which gave 23 g/L of alpha-arbutin, and the molar conversion based on the amount of hydroquinone reached 83%. The use of whole-cells of the wild type strain resulted in an alpha-arbutin concentration of 4 g/L and a molar conversion of 16% only under the same conditions. The results suggested that E. coli displaying transglucosidase using truncated INPNC as an anchoring motif can be employed as a whole-cell biocatalyst in glucosylation.

Directed evolution strategies for improved enzymatic performance

The engineering of enzymes with altered activity, specificity and stability, using directed evolution techniques that mimic evolution on a laboratory timescale, is now well established. However, the general acceptance of these methods as a route to new biocatalysts for organic synthesis requires further improvement of the methods for both ease-of-use and also for obtaining more significant changes in enzyme properties than is currently possible. Recent advances in library design, and methods of random mutagenesis, combined with new screening and selection tools, continue to push forward the potential of directed evolution. For example, protein engineers are now beginning to apply the vast body of knowledge and understanding of protein structure and function, to the design of focussed directed evolution libraries, with striking results compared to the previously favoured random mutagenesis and recombination of entire genes. Significant progress in computational design techniques which mimic the experimental process of library screening is also now enabling searches of much greater regions of sequence-space for those catalytic reactions that are broadly understood and, therefore, possible to model.Biocatalysis for organic synthesis frequently makes use of whole-cells, in addition to isolated enzymes, either for a single reaction or for transformations via entire metabolic pathways. As many new whole-cell biocatalysts are being developed by metabolic engineering, the potential of directed evolution to improve these initial designs is also beginning to be realised.

Therapeutic potential of microbes and microbial products in the management of human allergic asthma

The intellectual property literature concerning novel therapeutic applications of microbes and their products in the management of human allergic asthma during the period of 2001 – 2004 is reviewed. In contrast to the primary literature, surprisingly few patent citations (< 30) have been identified in this period, and the majority of the citations describe the application of Mycobacterium spp. and their novel components, bacterial unmethylated CpG dideoxynucleotides and endotoxin. In addition to the use of whole cells, several potentially therapeutic bacterial components have been characterised from Mycobacterium spp. and shown to be promising in the suppression of allergic and asthma-like responses when tested in mouse models. However, in most of these studies, the bacteria or their products were given before, concurrently, or shortly after allergen sensitisation, and hence their efficacy in the therapeutic application is not clear. Although the availability of microbe-based immunotherapy for clinical use in the management of allergic asthma remains a challenge to the research community, it can be viewed with cautious optimism that, in the foreseeable future, microorganisms and their products have the potential to become an important treatment option for asthmatic patients.

D-Amino acid production by E. coli co-expressed three genes encoding hydantoin racemase, d-hydantoinase and N-carbamoyl- d-amino acid amidohydrolase

Three genes respectively encoding d-specific hydantoinase (DHHase), N-carbamoyl- d-amino acid amidohydrolase (DCHase) and hydantoin racemase (HRase) were co-expressed in E. coli in a system designed for the efficient enzymatic production of d-amino acids via a combination of hydantoin hydrolysis and hydantoin racemization. With the use of whole cells, the d-forms of eight amino acids – d-phenylalanine, d-tyrosine, d-tryptophan, O-benzyl- d-serine, d-valine, d-norvaline, d-leucine and d-norleucine – were efficiently converted from the corresponding dl-5-monosubtituted hydantoin compounds.

Strain improvement for fermentation and biocatalysis processes by genetic engineering technology.

Twenty years ago, the first complete gene cluster encoding the actinorhodin biosynthetic pathway was cloned and characterized. Subsequently, the gene clusters encoding the biosynthetic pathways for many antibiotics were isolated. In the past decade, breakthroughs in technology brought that generation of rationally designed or new hybrid metabolites to fruition. Now, the development of high-throughput DNA sequencing and DNA microarray techniques enables researchers to identify the regulatory mechanisms for the overproduction of secondary metabolites and to monitor gene expression during the fermentation cycle, accelerating the rational application of metabolic pathway engineering. How are the new tools of biotechnology currently being applied to improve the production of secondary metabolites? Where will this progress lead us tomorrow? The use of whole cells or partially purified enzymes as catalysts has been increased significantly for chemical synthesis in pharmaceutical and fine-chemical industries. The development of PCR technologies for protein engineering and DNA shuffling is leading to the generation of new enzymes with increased stability to a wide range of pHs, temperatures and solvents and with increased substrate specificity, reaction rate and enantioselectivity. Where will this emerging technology lead us in the twenty-first century?

Expression of Sulfolobus solfataricus α-glucosidase in Lactococcus lactis

The industrial potential to use extreme thermophilic microorganisms and their enzymes lies in applications in which the temperature cannot be adjusted (cooled) at will. The production of enzymes from wild-type thermophiles is very low, therefore, for industrial applications, it is necessary to use recombinant microorganisms. In this paper, the cloning of a heat-stable alpha-glucosidase from Sulfolobus solfataricus using lactic acid bacteria as expression system is reported. The extremophilic alpha-glucosidase was cloned in Lactococcus lactis and correctly folded despite being expressed at a lower temperature. The recombinant cells were assayed for enzyme residual activity at 75 degrees C in order to analyze the direct use of whole cells as biocatalysts. Maximum activity corresponded to 40 U/l in static cultures. The protein yield was further improved by optimizing fermentation and reached 600 U/l in batch mode. Microfiltration led to an even higher enzyme production of 850 U/l as a result of increased biomass. The overall production of alpha-glucosidase using the engineered L. lactis strain in microfiltration fermentation is 1,000-fold higher than obtained using the wild-type.

Postsynaptic K+ current induced by nociceptin in medullary dorsal horn neurons.

The actions of the endogenous ORL1 receptor (opioid receptor-like1) ligand nociceptin on the membrane properties of rat trigeminal nucleus caudalis neurons were examined by use of whole cell and perforated patch clamp recording in brain slices. Nociceptin produced an outward current in all neurons tested (EC50 112 nM). The outward current produced by nociceptin was completely reversed with the addition of the non-peptide ORL1 antagonist J-113397. Outward currents reversed polarity at -99+/-2 mV, close to the potential for K+ of -102 mV, suggesting that they were mediated by an increased K+ conductance. These results suggest that the analgesic action of nociceptin might be mediated by direct postsynaptic inhibition within the dorsal horn.

Purification and characterization of an l-amino acid deaminase used to prepare unnatural amino acids

l-Amino acid deaminase (l-AAD) from Proteus myxofaciens was cloned and over-expressed in Escherichia coli K12. This enzyme has a broad substrate specificity, working on both natural and unnatural l-amino acids. Of the 20 naturally occurring l-amino acids, l-AAD prefers amino acid substrates that have aliphatic, aromatic or sulfur-containing side chains; those with charged side chains (–CO2− or –NH3+) are poor or non-substrates. Enzyme activity was monitored using a microtiter-plate-based assay, which measures the formation of phenylpyruvic acid from l-phenylalanine. The reaction has an absolute requirement for O2, releases NH3 and does not produce H2O2. Substrate comparisons were carried out by using an O2 electrode to measure the O2 utilization rates. Studies on partially purified enzyme show a pH optimum of 7.5 with a subunit molecular weight of approximately 51 kDa. Additional purification and characterization strategies will be presented. The use of whole cells containing l-AAD will be discussed to prepare chiral pharmaceutical intermediates.

Nitrile biotransformations using free and immobilized cells of a thermophilic Bacillus spp.

A thermophilic Bacillus spp. capable of transforming aliphatic nitriles, cyclic nitriles and dinitriles was used as a free cell suspension and immobilized in alginate beads to study the utilization of acetonitrile and acrylonitrile in a buffered biotransformation medium. The cells grew optimally at 65°C and contained a nitrile hydratase–amidase enzyme system that transformed nitrile compounds stoichiometrically to the corresponding carboxylic acids. In the presence of urea or chloroacetone, amidase activity was inhibited and the amide intermediate was accumulated. Mass transfer limitation of nitrile utilization rates was observed with immobilized cells, but the alginate afforded the cells some degree of additional thermal stability and potential advantage in re-use. In vitro inhibition of the partially purified amidase was confirmed and the use of whole cells of this organism in a continuous bioreactor to generate amide products from nitrile substrates was demonstrated.

Characterization of the Enantioselective Properties of the Quinohemoprotein Alcohol Dehydrogenase of Acetobacter pasteurianus LMG 1635. 1. Different Enantiomeric Ratios of Whole Cells and Purified Enzyme in the Kinetic Resolution of Racemic Glycidol.

Resting cells of Acetobacter pasteurianus LMG 1635 (ATCC 12874) show appreciable enantioselectivity (E=16-18) in the oxidative kinetic resolution of racemic 2,3-epoxy-1-propanol, glycidol. Distinctly lower values (E=7-9) are observed for the ferricyanide-coupled oxidation of glycidol by the isolated quinohemoprotein alcohol dehydrogenase, QH-ADH, which is responsible for the enantiospecific oxidation step in whole cells. The accuracy of E-values from conversion experiments could be verified using complementary methods for the measurement of enantiomeric ratios. Effects of pH, detergent, the use of artificial electron acceptors, and the presence of intermediate aldehydes, could be accounted for. Measurements of E-values at successive stages of the purification showed that the drop in enantioselectivity correlates with the separation of QH-ADH from the cytoplasmic membrane. It is argued that the native arrangement of QH-ADH in the membrane-associated complex favors the higher E-values. The consequences of these findings for the use of whole cells versus purified enzymes in biocatalytic kinetic resolutions of chiral alcohols are discussed.

Whole-cell biosensor for determination of volatile organic compounds in the form of aerosols

A biosensor using Chlorella microalgae immobilized on the membrane of an oxygen electrode has been designed to operate in the gas phase for determination of volatile organic compounds in the form of aerosols. Perchloroethylene (tetrachloroethylene) is used as an example to produce aerosols and vapours since it is toxic to the people working in dry-cleaning atmospheres and municipal treatment plants. A home-made controlled atmosphere chamber is constructed for perchloroethylene detection. Monitoring of perchloroethylene is achieved by the measurement of oxygen production during the algae photosynthetic process. The use of whole cells as bioreceptors allows the detection of a toxic aerosol in the gas phase without preliminary trapping of the analyte in a solution.

Whole-cell biosensor for direct determination of solvent vapours

A whole-cell biosensor has been designed to operate in the gas phase for determination of vapours of solvents. Methanol was chosen as an example of a volatile organic solvent to evaluate the algae sensor response. The use of whole cells as bioreceptors allows detection of those compounds in the gas phase without preliminary trapping of the analytes in a solution. In addition, near real-time monitoring is possible by the measurement of the algae photosynthetic activity with an oxygen electrode.

Prolonged physiological entrapment of glutamate in the synaptic cleft of cerebellar unipolar brush cells.

The cellular mechanism underlying the genesis of the long-lasting alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor-mediated excitatory postsynaptic currents (EPSCs) at the mossy fiber (MF)-unipolar brush cell (UBC) synapse in rat vestibular cerebellum was examined with the use of whole cell and excised patch-clamp recording methods in thin cerebellar slices. Activation of MFs evokes an all-or-none biphasic AMPA-receptor-mediated synaptic current with a late component that peaks at 100-800 ms, which has been proposed to originate from an entrapment of glutamate in the MF-UBC synaptic cleft and is generated by the steady-state activation of AMPA receptors. Bath application of cyclothiazide, which blocks desensitization of AMPA receptors, produced a dose-dependent enhancement of the amplitude of the synaptic current (median effective dose 30 microM) and slowing of the rise time of the fast EPSC. N-methyl-D-aspartate-receptor-mediated EPSCs in UBCs were not potentiated in amplitude or time course by cyclothiazide (100 microM). The dose-response relations for the steady-state current evoked by glutamate acting at AMPA receptors in excised outside-out patches from UBC and granule somatic membranes was biphasic, peaking at 50 microM and declining to 50-70% of this value at 1 mM glutamate. When glutamate was slowly washed from patches to simulate the gradual decline of glutamate in the synapse, a late hump in the transmembrane current was observed in patches from both cell types. The delivery of a second MF stimulus at the peak of the slow EPSC evoked a fast EPSC of reduced amplitude followed by an undershoot of the subsequent slow current, consistent with the hypothesis that the peak of the slow EPSC reflects the peak of the biphasic steady-state dose-response curve. Estimates of receptor occupancy and glutamate concentration derived from the ratio of fast EPSC amplitudes, and the amplitude and polarity of the initial steady-state current in paired-pulse experiments, predict a slow decline of glutamate with a time constant of 800 ms, declining to ineffective concentrations at 5.4 s. Manipulation of cleft glutamate concentration by lowered extracellular calcium or delivery of brief stimulus trains abolished the slow EPSC and restored the undershoot to paired stimuli, respectively, in a manner consistent with a prolonged lifetime of glutamate in the cleft. The slow component of the EPSC was prolonged in duration by the glutamate reuptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate, suggesting that glutamate transport contributes to the time course of the synaptic current in UBCs. The data support the notion that the MF-UBC synapse represents an ultrastructural specialization to effectively entrap glutamate for unusually prolonged periods of time following release from MF terminals. The properties of the postsynaptic receptors and constraints on diffusional escape of glutamate imposed by synaptic ultrastructure and glutamate transporters act in concert to sculpt the time course of the resulting slow EPSC. This in turn drives a long-lasting train of action potentials in response to single presynaptic stimuli.

Bakers' yeast oxidation of methyl para-tolylsuifide: Synthesis of a chiral intermediate in the preparation of the mevinic acid-type hypocholestemic agents

The use of ( R)-methyl para-tolylsulfoxide as a chiral auxiliary in a novel synthesis of a key intermediate en route to mevinic acid-type hypocholestemic agents is described. The synthesis is short and simple consisting of eight steps to yield enantiomerically pure β-silyloxy-δ-lactone. The chiral sulfoxide used in the synthesis was obtained via a straightforward biooxidation of methyl para-tolylsulfide using bakers' yeast ( Saccharomyces cerevisiae NCYC 73). The biotransformation involves the use of whole cells and affords the sulfoxide in good yield and with high stereoselectivity.

Voltage dependence of conductance changes evoked by glycine release in the zebrafish brain.

1. The kinetics and mechanisms underlying the voltage dependence of inhibitory postsynaptic currents (IPSCs) recorded in the Mauthner cell (M cell) were investigated in the isolated medulla of 52-h-old zebrafish larvae, with the use of whole cell and outside-out patch-clamp recordings. 2. Spontaneous miniature IPSCs (mIPSCs) were recorded in the presence of 10(-6) M tetrodotoxin (TTX), 10 mM MgCl2, and 0.1 mM [CaCl2]o. Depolarizing the cell from -50 to +50 mV did not evoke any significant change in the distribution of mIPSC amplitudes, whereas synaptic currents were prolonged at positive voltages. The average decay time constant was increased twofold at +50 mV. 3. The voltage dependence of the kinetics of glycine-activated channels was first investigated during whole cell recording experiments. Currents evoked by voltage steps in the presence of glycine (50 microM) were compared with those obtained without glycine. The increase in chloride conductance (gCl-) evoked by glycine was time and voltage dependent. Inactivation and reactivation of the chloride current were observed during voltage pulses from 0 to -50 mV and from -50 to 0 mV, respectively, and they occurred with similar time constants (2-3 s). During glycine application, voltage-ramp analysis revealed a shift in the reversal potential (ECl-) occurring at all [Cl-]i tested. 4. The basis of the voltage sensitivity of glycine-evoked gCl- was first analyzed by measuring the relative changes in the total open probability (NPo) of glycine-activated channels with voltage.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosorption of cadmium, copper and lead by isolated mother cell walls and whole cells of Chlorella fusca

Using a new method for the isolation of released mother cell walls of Chlorella fusca, the biosorption of cadmium, copper and lead by purified cell wall isolates and whole cell suspensions was comparatively characterized. In all cases whole cells accumulated more metal ions than isolated cell walls. Both the Langmuir and Freundlich isotherm models were suitable for describing the short-term adsorption of cadmium, copper and lead by cell walls and the cadmium and copper adsorption by whole cells. However, neither model could sufficiently explain the lead accumulation by whole cells. The feasibility of a practical use of whole cells or isolated cell walls as biosorbents is discussed.

New method for detecting methylmercury by its enzymic conversion to methane

A new method is suggested for determining methylmercury in biological samples. The determination is based on the use of whole cells of a broad-spectrum mercury-resistant bacterium, Pseudomonas putida, strain FB1, which is induced to produce the enzymes organomercurial lyase and mercuric reductase. These biocatalysts convert organomercurials to elemental mercury and their derivative hydrocarbons. The extraction procedure for methylmercury in biological samples follows the conventional toluene extraction method, which is retained until methylmercury is concentrated in an aqueous phase containing 0.01 M Na{sub 2}S{sub 2}O{sub 3}. This solution is mixed with bacterial cells in exponential growth phase and then incubated in microreaction vessels. A complete biological degradation of methylmercury to methane occurs. The efficiency of derivatization depends on the organomercurial concentration, the incubation time, and the cell density. The detection limit is 15 ng of methylmercury in 1 g of biological tissue and the coefficient of variation is 1.9% in 10 replicate samples with 100 ng/mL.

Evidence for Multiple Protein Constituents of the Somatostatin Receptor in Pituitary Tumor Cells: Affinity Cross-Linking and Molecular Characterization*

Previous studies have shown that somatostatin receptors on AtT-20 and GH3 pituitary tumor cells show relative preference for binding somatostatin-28 (S-28) and somatostatin-14 (S-14), respectively. Here we have attempted to determine whether this selectivity can be explained by molecular heterogeneity of the receptor. Cells were incubated with [125I-Tyr11]S-14, [125I-Leu8-D-Trp22,Tyr25]S-28, and [125I-Tyr3]SMS, and the bound ligand was chemically cross-linked with bis-[2-succinimido-oxycarbonyloxy)ethyl]sulfone, disuccinimidyl suberate, or dithiobis (succinimidyl propionate). The solubilized cross-linked material was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography. [125I-Tyr11]S-14 labeled three specific receptor proteins of 57K, 42K, and 27K mol wt in AtT-20 cells. The relative proportions of the protein bands were unaltered by the use of whole cells or cell membranes or by the inclusion of dithiothreitol or antiproteolytic agents. With both [125I-Tyr11]S-14 and [125I-LTT]S-28, the 57K protein constituted the major labeled component, representing 70-75% of the total cross-linked proteins. Labeling of the three protein species by [125I-Tyr11]S-14 and [125I-LTT]S-28 was inhibited by both S-14 and S-28 in a dose-dependent manner. S-28 was 10-20 times more potent than S-14 for inhibiting the labeling by both ligands of the principal receptor species of 57K. By contrast, when a radioiodinated derivative of the octapeptide analog octreotide ([125I-Tyr3]SMS) was used as ligand, the 27K protein was preferentially labeled, whereas the 57K and 42K bands were detected only as minor components. Labeling of GH3 cells with [125I-Tyr11]S-14 and [125I-LTT]S-28 revealed three cross-linked proteins of 57K, 42K, and 27K mol wt similar to those observed in AtT-20 cells. However, in this cell line the 27K protein, not the 57K species, was the dominant component identified with these two ligands, comprising 40-50% of the total cross-linked proteins. These results suggest that there are three somatostatin receptor proteins of 57K, 42K, and 27K in pituitary cells. In AtT-20 cells, the 57K protein constitutes the major receptor protein labeled by [125I-Tyr11]S-14 and [125I-LTT]S-28, whereas the 27K protein is the major species labeled by [125I-Tyr3]SMS. The 27K, not the 57K, moiety is the principal receptor form in GH3 cells. Such ligand- and tissue-selective binding by the somatostatin receptor provides strong evidence for receptor molecular heterogeneity.

Histamine-induced inward currents in cultured endothelial cells from human umbilical vein.

1. The membrane response to applied histamine of cultured endothelial cells from human umbilical vein was studied by use of whole cell and single channel patch clamp techniques. A value of -27 +/- 1.4 mV was found for the resting potential under whole cell current clamp. No voltage-gated currents were seen at either the macroscopic or single channel levels. 2. At holding potentials of -20 to -40 mV, histamine evoked slow rising, long lasting whole cell inward currents. The inward current was associated with depolarization and decreased input resistance. The calcium ionophore A23187 provoked similar whole cell inward currents. 3. Single channel currents were observed in cell-attached and inside-out patches for both histamine and A23187. The single channel conductance was about 20 pS with a mean open time of 5 ms and a reversal potential of 0 mV in symmetrical potassium solutions. Internal sodium blocked outward going currents. 4. For cell-attached patches, histamine-dependent channel activity required external calcium and was also seen when histamine was present in the bath but not the pipette. Recording from inside-out patches revealed that decreases in 'internal' calcium resulted in the disappearance of channel activity. 5. The histamine-dependent inward current appears to involve calcium-dependent activation of cationic channels.