The synthetic potential of immobilised cells of Capsicum frutescens Mill cv. annuum

Cells of Capsicum frutescens Mill. cv. annuum, immobilised in reticulate polyurethane foam, produced higher yields of capsaicin, the pungent principle of Chilli pepper fruits, than did freely-suspended cells, when batch-cultured in a medium conducive to culture growth. In the absence of specific precursors to capsaicin, immobilised cells produced between two and three orders of magnitude higher yields than did suspended cells over 5-d or 10-d culture periods (typically up to 4 or 5 mg capsaicin g^-1 dry weight l^-1 medium compared with up to 30 g g^-1l^-1, respectively). These results were reflected by an increased rate and extent of incorporation of L-[U-¹⁴C]phenylalanine into capsaicin in immobilised as compared with freely-suspended cells, and evidence is presented for an inverse relationship between incorporation of [¹⁴C]phenylalanine into protein and capsaicin. The accumulation of capsaicin can be experimentally manipulated and increased by supplementing the medium with precursors of capsaicin such as phenylalanine and isocapric acid and by reducing the growth rate of immobilised cells by omitting growth regulators from the medium. The importance of these observations is discussed.Abbreviations HPLC high-performance liquid chromatography - Phe phenylalanine - TLC thin-layer chromatography     

The binding requirements of monkey brain lysosomal enzymes to their immobilised receptor protein

The lysosomal enzyme binding protein (receptor protein) isolated from monkey brain was immobilised on Sepharose 4B and used to study the binding of brain lysosomal enzymes. The immobilised protein could bind \-D-glucosaminidase, α-D-mannosidase, α-L-fucosidase and2-D-glucuronidase. The bound enzymes could be eluted either at an acid pH of 4.5 or by mannose 6-phosphate but not by a number of other sugars tested. Binding could be abolished by prior treatment of the lysosomal enzymes with sodium periodate. Alkaline phosphatase treatment of the enzymes did not prevent the binding of the lysosomal enzymes to the column but decreased their affinity, as seen by a shift in their elution profile, when a gradient elution with mannose 6-phosphate was employed. These results suggested that an ‘uncovered’ phosphate on the carbohydrate moiety of the enzymes was not essential for binding but can enhance the binding affinity.     

Anti-rabbit immunoglobulin G detection in complex medium by PM-RAIRS and QCM Influence of the antibody immobilisation method

Two antibody immobilisation procedures were compared to set up an immunosensor for goat anti-rabbit immunoglobulin (anti-rIgG), i.e. rIgG covalently bound or immobilised via affinity to protein A (PrA). In both cases, the first layer of protein was covalently bound to a mixed self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) and mercaptohexanol (C6OH) on a gold surface. The elaboration of the sensitive surfaces, as well as their selectivity and sensitivity were studied step by step by polarization modulation-reflection absorption infra-red spectroscopy (PM-RAIRS) and quartz crystal microbalance (QCM) with impedance measurement. QCM measurements showed that the viscoelastic properties of the antibody layer were markedly modified during the antigen recognition when the antibody was bound by affinity to PrA. The specific detection of antigen within a complex medium was assessed by PM-RAIRS thanks to the grafting of cobalt-carbonyl probes. Affinity constants between the immobilised rIgG and the anti-rIgG were determined from PM-RAIRS analysis.     

Determination of inhibitors’ potency (IC50) by a direct high-performance liquid chromatographic method on an immobilised acetylcholinesterase column

An immobilised acetylcholinesterase (AChE) stationary phase was prepared by using an in situ AChE immobilisation procedure. A stainless steel column packed with epoxide silica was connected to the HPLC system and the enzyme solution at pH 5.8 was recycled through the column at a flow-rate of 0.5 ml/min for 24 h. The activity of the immobilised AChE was determined by injecting the substrate acetylthiocholine, using as mobile phase 0.1 M phosphate buffer (pH 7.4) containing Ellman’s reagent [5,5′-dithio-bis(2-nitrobenzoic acid)] and measuring the area of the obtained peak with UV detection at 412 nm. The effect of AChE inhibitors tacrine, edrophonium and donepezil were evaluated by the simultaneous injection of each inhibitor with the substrate. The resulting decrease in the AChE activity, as expressed by the decrease of the peak area detected at 412 nm, was related to the concentration and potency of the solutes. The obtained IC₅₀ values were compared with those derived by the conventional spectrophotometric method. This immobilized enzyme reactor, included in a chromatographic system, can be used for the rapid screening for new inhibitors allowing for the on-line determination of a compound’s inhibitory potency. The advantages over the conventional methods are the increased enzyme stability and system automation which allows a large number of compounds to be analysed continuously.     

Esterification of 2-methylalkanoic acids Catalysed by Lipase from Candida rugosa: Enantioselectivity as a Function of water Activity and Alcohol Chain Length

Esterifications catalysed by immobilised lipase from Candida rugosa (CRL) in cyclohexane at constant water activity (a_w = 0.76) were studied using 2-methyl substituted octa-, nona- or decanoic acids and n-alcohols of varying chain length as substrates. The importance of controlling the water activity and choosing the right alcohol for obtaining maximum enantioselectivity is demonstrated. The immobilised lipase was easily recovered without loss of activity and enantioselectivity.     

Evaluation of magnetic polymer micro-beads as carriers of immobilised biocatalysts for selective and stereoselective transformations

The kinetic, selective and stereoselective properties of enzyme immobilised on magnetic polymer beads with diameters in the range 1 microm was studied with penicillin amidase from E. coli. The enzyme was immobilised on epoxy and glutaraldehyde-activated poly(vinyl alcohol), poly(methylmetacrylate) and poly(vinyl acetate-divinylbenzene) magnetic beads. The amount of covalently bound active protein was dependent on the chemical modification of the matrix and increased at higher ionic strength of the immobilisation buffer. The small size of the magnetic beads, that reduces mass transfer limitations, and the decreased charge density in the electric double layer resulted in lower apparent Km values and higher efficiency for benzylpenicillin hydrolysis, higher stereoselectivity in condensation of R-phenylglycine amide with S- and R-Phe and in hydrolysis of racemic phenylacetyl-Phe and higher selectivity in kinetically controlled synthesis of cephalexin compared to the enzyme immobilised on larger and porous carriers.     

Light-induced immobilisation of biomolecules as an attractive alternative to microdroplet dispensing-based arraying technologies

The present work shows how UV 'light-induced molecular immobilisation' (LIMI) of biomolecules onto thiol reactive surfaces can be used to make biosensors, without the need for traditional microdispensing technologies. Using 'LIMI,' arrays of biomolecules can be created with a high degree of reproducibility. This technology can be used to circumvent the need for often expensive nano/microdispensing technologies. The ultimate size of the immobilised spots is defined by the focal area of the UV beam, which for a diffraction-limited beam can be less than 1 microm in diameter. LIMI has the added benefit that the immobilised molecules will be spatially oriented and covalently bound to the surface. The activity of the sensor molecules is retained. Antibody sensor arrays made using LIMI demonstrated successful antigen binding. In addition, the pattern of immobilised molecules on the surface is not restricted to conventional array formats. The ultimate consequence of the LIMI is that it is possible to write complex protein patterns using bitmaps at high resolution onto substrates. Thus, LIMI of biomolecules provides a new technological platform for biomolecular immobilisation and the potential for replacing present microdispensing arraying technologies.     

Calcium alginate entrapment of the yeast Rhodosporidium toruloides for the kinetic resolution of 1,2-epoxyoctane

Resting cells of the yeast Rhodosporidium toruloides (UOFS Y-0471) were immobilised in calcium alginate beads for the enantioselective kinetic resolution of racemic-1,2-epoxyoctane. The initial activity exhibited by immobilised cells was almost 50% lower than that of the free counterpart but was extremely stable when compared to the free cells. The concentration of the immobilised biomass had no effect on apparent enzyme activity but did lead to a decrease in single cell activity. An increase in both the alginate and CaCl₂ concentrations used for bead preparation led to a decrease in enzyme stability. An increase in the alginate concentration led to an increase in bead diameter. The stoichiometric equation for cross-linking of alginate was only obeyed when CaCl₂ concentrations higher than 0.4 M were utilised for bead preparation.     

Lipase immobilisation on to polymeric membranes

Lipase (EC 3.1.1.3) from Candida rugosa was covalently immobilised on to cellulose, cellulose derivatives (cellulose acetate and cellulose phthalate) and cellulose composite membranes using activating agents such as sodium periodate or carbodiimide. Other non-cellulosic polymeric membranes (nylon, polyurethane, chitosan and hydroxyethyl methacrylate-co-methyl methacrylate) were also prepared and used for lipase immobilisation. The results obtained showed that the expressed activities are of the same order of magnitude for similar enzyme loadings when compared with those obtained from literature.     

Effect of Different Supports on the Reaction Rate of Rhizomucor Miehei Lipase in Organic Media

Five different aluminas, a silica and a zirconia support were used to adsorb lipase (E.C. 3.1.1.3) from Rhizomucor miehei. The activity of the immobilised lipase was measured by esterification of dodecanol and decanoic acid in hexane. The immobilised lipase and the organic phase were pre-equilibrated separately to known water activities before mixing them to commence the reactions. The aluminas, which varied in pore sizes and surface areas, adsorbed similar amounts of enzyme. However, the esterification activities varied about 10-fold, increasing with increasing surface area. The silica and zirconia supports adsorbed about half as much lipase as the aluminas. The esterification reaction rates per unit quantity of enzyme adsorbed were compared with those for aluminas with similar surface areas; this specific rate was about 2 times higher for the zirconia, but the difference with silica was only small. There was no clear correlation between the esterification rates at fixed water activity and the amount of water adsorbed by the support used.     

Esterification of 2-methylalkanoic acids Catalysed by Lipase from Candida rugosa: Enantioselectivity as a Function of water Activity and Alcohol Chain Length

Esterifications catalysed by immobilised lipase from Candida rugosa (CRL) in cyclohexane at constant water activity (a_w = 0.76) were studied using 2-methyl substituted octa-, nona- or decanoic acids and n-alcohols of varying chain length as substrates. The importance of controlling the water activity and choosing the right alcohol for obtaining maximum enantioselectivity is demonstrated. The immobilised lipase was easily recovered without loss of activity and enantioselectivity.     

Rhamnolipid production by Pseudomonas aeruginosa immobilised in polyvinyl alcohol beads

A marine bacterium, Pseudomonas aeruginosa BYK-2 (KCTC 18012P), was immobilised by entrapment in 10% (w/v) polyvinyl alcohol beads and optimized for the continuous production of rhamnolipid. The relative activity of rhamnolipid production was maintained at 80 approximately 90% of the initial production during 15 cycles in a repeated batch culture. Continuous culture was performed in a 1.8 1 airlift bioreactor, yielding 0.1 g rhamnolipid h(-1) at a dilution rate of 0.0 18 h(-1), 25 degrees C, initial pH 7, and 0.5 vvm aeration rate with a 1.21 working volume.     

In Situ Hydration of Enzymes in Non-Polar Organic Media Can Increase the Catalytic Rate

The nature of the buffer species used in the drying process is important when lyophilized enzyme preparations are suspended in organic media. The activity of subtilisin Carlsberg in a transesterification reaction was found to vary depending on the nature of the buffer used. It was postulated that the large excess of salt present in the dried powder could be affecting enzymatic activity by alterations to the microscopic structure of the powder. To establish if this were true, microscopic changes were eliminated by covalently immobilising the enzyme onto a macroporous polymer support so that the counter-ions could be exchanged by washing with dilute salt solutions. It was found that in the immobilised samples no significant effects of salt ions were noted. This was the case even when salt ions were in considerable excess of that needed to balance protein charges. Hence the activity variations noted in freeze-dried powders are probably due to changes to the microscopic structure, rather than to molecular scale interactions. Similarly the previously observed activating effect of crown ether solutions on freeze-dried powders is not repeated on an immobilised preparation suggesting that this too may be due to a microscopic effect on the powder.     

Biotransformation of oleic acid into (E)-10-hydroxy-8-octadecenoic acid and (E)-7,10-dihydroxy-8-octadecenoic acid by Pseudomonas sp. 42A2 in an immobilized system

Oleic acid was transformed into 10-hydroxy-8E-octadecenoic acid and 7,10-dihydroxy-8E-octadecenoic acid using Pseudomonas sp. 42A2 NCIMB 40045 in different immobilised supports. Celite R633 gave highest conversions in 48 h: the cellular yield (Y _p/x) g products g^–1 of cellular protein in the immobilised culture was 5 compared with the free-cell culture Y _p/x of 3.8. Conversion of oleic acid in the immobilised cell culture was 50% of the carbon source supplied.     

Gaseous fluxes of peroxyacetyl nitrate (PAN) into plant leaves

Peroxyactyl nitrate (PAN) is the most abundant of the gaseous organic nitrates produced from the photochemistry of hydrocarbons and NO_x (i.e. ozone and smog production). PAN is known to be toxic to plants and also as a reservoir for the transport nitrogen dioxide in the troposphere. Here, the effect of vegetation on PAN deposition was investigated in four plant species by measuring leaf fluxes of PAN in a dynamic leaf chamber using atmospheric PAN fumigations between 0.7 and 18 nmol mol^?1. A linear relationship was observed between PAN flux and ambient PAN mixing ratio for all species. Depending on the species, measured PAN flux varied between 11 and 24 pmol m^?2 s^?1. Measured fluxes of PAN accounted for 12–48% of the PAN flux predicted solely from modelled stomatal conductance to PAN, suggesting the presence of a mesophyllic resistance to PAN uptake. The brief (approximately 5–10 min) exposure to PAN during uptake measurements did not affect photosynthesis, transpiration or conductance to water vapour. Increasing stomatal resistance by varying the vapour pressure gradient between the leaf chamber and leaf internal air space led to a corresponding drop in PAN uptake. Varying leaf nitrogen and total leaf–ascorbate concentrations did not appear to influence PAN uptake as had been reported for other reactive odd‐nitrogen gases. Measured and model‐predicted PAN fluxes were offset, but correlated suggesting that PAN flux could be estimated using established stomatal conductance algorithms.     

Comparative studies of coordination properties of puromycin and puromycin aminonucleoside towards copper(II) ions

Protonation equilibria of puromycin (PM) and puromycin aminonucleoside (PAN) and their coordination by copper(II) ion were studied in solution by potentiometry, electronic absorption spectroscopy (UV-Vis), circular dichroism (CD), electron paramagnetic resonance (EPR) and mass spectrometry. For puromycin four mononuclear complexes were found, with stoichiometries Cu(PM)2+, CuH(-1)(PM)+, CuH(-2)(PM) and CuH(-3)(PM)(-). In each of them the Cu(II) ion was bound in the peptidic-like manner, the differences of stoichiometries are a consequence of subsequent deprotonations of the sugar C2'-OH group and the coordinated water molecule. The coordination mode for puromycin aminonucleoside was aminosugar-like. Two dimeric complexes, Cu2H(-1)(PAN)2(2+) and Cu2H(-2)(PAN)2+, and one monomeric CuH(-2)(PAN)2 were found. The N6,N6-dimethyladenine moiety of PAN was not involved in the coordination process due to steric hindrance.     

Mucilage acts to adhere cyanobacteria and cultured plant cells to biological and inert surfaces

Abstract The surfaces of cells of several species of cyanobacteria have been studied using low-temperature scanning electron microscopy (SEM), and have been shown to be covered in a layer of hydrated mucilage. This mucilage is observed in specimens of Anabaena azollae adhering to plant cells in their natural symbiotic niche (the cavity of the fronds of Azolla species) and in samples of the various species of cyanobacteria immobilised on polyurethane and polyvinyl support matrices. The mucilage appears to maintain the close contact observed between the cyanobacteria and these surfaces. Comparable films observed surrounding plant cells immobilised on similar polymeric surfaces are considered to be performing a similar function.     

A method for the determination of enzyme mass loading on an electrode surface through radioisotope labelling

A direct method has been developed for the quantitation of the amount of immobilised enzymes on biosensor surfaces. This quantity is of key importance in establishing the activity, kinetics and optimal immobilisation conditions in the construction of both amperometric and optical biosensors. Recombinant L-lactate dehydrogenase incorporating both a biosynthetically introduced radiolabel, 3H-leucine, and a hexahistidine peptide tag was immobilised on a poly(aniline) composite film and then quantitated by liquid scintillation counting. It was found that enzyme mass loading was proportional to the concentration of LDH in solution, and also depended on the morphology of the composite film. The LDH mass loading on the composite film doubled when a surface cysteine containing variant was used, possibly due to the covalent attachment of the cysteine to the diiminoquinoid rings of the poly(aniline).     

Transformations of residual 15N in a coniferous forest soil humus layer in northern Vancouver Island,British Columbia

We studied the effect of ¹⁵N labelling duration on the mineralisation and immobilisation of native and applied (residual) N in the humus layer of a Humo-Ferric Podzol. Ammonium sulphate, labelled with ¹⁵N, was applied to 1 m² plots at a rate of 200 kg N ha^–1. Fertiliser application was timed so that when samples were collected they had been labelled with ¹⁵N for 24 hours, 7 months and 31 months. In a 42-day aerobic incubation of the samples, net mineralisation of total and applied N was greatest in the 24-hr treatment followed by those from the 31-month treatment (p<0.05), indicating that immobilised ¹⁵N was more remineralisable in the samples with ¹⁵N labelled for 24 hours. The percentage of applied N found in the total N mineralised (net) ranged from 76.6 to 87.4%, 13.1 to 42.0% and 10.6 to 14.0% in samples from the 24-hr and 7- and 31-month treatments, respectively, showing reduced relative availability of residual N with increased labelling duration. The carbon mineralisation rate had the following order: 7-month > 24-hr > 31-month treatment. Net mineralisation of C and N was poorly correlated with each other (r=-0.02, p=0.89). Anaerobic incubation showed net mineralisation for the 7- and 31-month treatments but net immobilisation for the 24-hr treatment for both total and applied N, suggesting that immobilisation of inorganic N was encouraged when there was a large pool of mineral N in the soil. Both total and applied N in the extractable organic N fraction and in the N flushed after fumigation with chloroform had the following order: 24-hr > 7-month > 31-month treatment. The results confirmed that N fertiliser was being immobilised within hours after application by the humus material through the microbial population and that the immobilised N had a low mineralisation potential after one growing season.     

Cross-linked enzyme aggregates with enhanced activity: application to lipases

Seven commercially available microbial lipases were immobilised as their cross-linked enzyme aggregates (CLEAs). Preparations with enhanced activity were obtained by a judicious choice of the precipitant [(NH₄)₂SO₄, 1,2-dimethoxyethane or acetone] and by adding either a crown ether or surfactant, depending on the source of the enzyme. Thus, precipitation of the lipases from Thermomyces lanuginosus and Rhizomucor miehei with (NH₄)₂SO₄ in the presence of SDS, followed by cross-linking with glutaraldehyde, afforded CLEAs with three and two times, respectively, the hydrolytic activity of the native enzymes. Preparations with up to ten times enhanced activity in organic medium were similarly prepared.