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The transesterification activity, autolysis, thermal stability and conformation of subtilisin Carlsberg, made soluble in dioxane by covalent linking to methoxypoly(ethylene glycol) (PEG), were investigated as a function of the concentration of water in the medium. Electrospray mass spectrometry showed that the modified enzyme preparation was a mixture of proteins containing from 2 to 5 covalently linked PEG chains per subtilisin molecule. PEG-subtilisin catalyzed transesterification between vinyl butyrate and 1-hexanol was optimum at 0.55 MH(2)O, while hydrolysis prevailed above 2 MH(2)O. There was a decrease in the overall enzyme activity with increasing water concentration because of autolysis and denaturation of the enzyme. Subtilisin powder and celite-immobilized subtilisin were more stable and less susceptible to autolysis than the PEG-modified enzyme. Circular dichroism and intrinsic protein-fluorescence studies showed that the conformation of PEG-subtilisin did not change as a function of water concentrations between 0 and 9 M. The K(m,app) value of PEG-subtilisin for 1-hexanol was highly influenced by water, which behaved as a competitive inhibitor in the transesterification reaction with an affinity for the enzyme similar to that of the alcohol. The K(m,app) for the acylating agent was not significantly modified by water. Lyoprotectants such as sorbitol and free PEG did not influence the activity of PEG-subtilisin but notably increased the activity of subtilisin powder and celite-immobilized subtilisin. The addition of 1.7-5.5 M water, however, rendered enzyme preparations containing no additives as active as those containing the lyoprotectants.

A chemiluminescent flow-sensing device for the determination of phospholipase D (PLD) activity and/or choline (Ch) in biological samples using choline oxidase (ChO) and horseradish peroxidase (HRP) immobilized on Eupergit C (polymer beads of methacrylamide, N-methylene-bis-methacrylamide, and allyl-glycidyl-ether) was developed. The best results were obtained with immobilized ChO and HRP at a polymer beads wet weight ratio of 16:1. The optimized parameters of the developed sensing device were 56 microM luminol in working solution; sample volume, 60 microliters; flow rate, 0.3 ml/min; and sample throughput, 15/h. The detection limit (3 SD) using a luminescent enhancer was 1.2 microM for Ch, corresponding to 0.167 mIU of PLD activity per milliliter. Without enhancer the values were 3.0 microM and 0.417 mIU, respectively. The Ch recovery varied between 80.4 and 109%. The biological samples quenched the luminescent light to different extents, and this matrix effect was readily overcome by measuring the luminescent signal of added Ch standard. The flow biosensor was used for the determination of PLD in samples of different origin, including rape seeds during maturation.

A highly sensitive and rapid bioluminescent flow sensor was developed for the determination of the content of L-phenylalanine (Phe) in serum by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH), produced by immobilized phenylalanine dehydrogenase (PheDH), with bacterial bioluminescent enzymes immobilized on a separate nylon coil. The L-PheDHs extracted from Bacillus badius, Bacillus sphaericus and Rhodococcus sp. M 4 were investigated and the performances of the three immobilized L-PheDH's were analysed. The B. badius reactor was found to give higher transformation rate and better sensitivity; the response was linear from 1 to 100 microM at 25 degrees , with a detection limit of 10 pmoles (0.5 microM). The intra- and inter-assay coefficients of variation were less than 5% and recoveries ranged from 90 to 101%. The results agreed well with those obtained with a chromatographic method for the Phe determination in serum and with the normal reference values.

The amount of L-lactate in biological fluids (serum, plasma and cerebrospinal fluid) was determined by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH) produced by immobilised lactate dehydrogenase (LDH), with bacterial bioluminescent enzymes immobilised on a separate nylon coil. The LDH catalysed the reaction of L-lactate with NAD; this reaction took place in a nylon coil that preceded the coil for the bioluminescent detection. The co-immobilisation of alanine aminotransferase (ALT) with LDH improved the lactate transformation by 117-183%. The response was linear from 0.1 to 50 micron mol l(-1) at 25 degrees C for the LDH - ALT reactor. The intra- and inter-assay coefficients of variation were less than 5% and the recoveries ranged from 93 to 106%. The results agreed well with those obtained with a spectrophotometric method and with the normal reference values.

Firefly luciferase was immobilized on epoxy methacrylate beads and used for a continuous-flow assay of ATP extracted from platelets. The immobilized luciferase had a half-life of 3 days at 25 degrees C; there was a 25% recovery of luciferase activity upon immobilization, and ca 50 reactors were made from 1 mg of commercial enzyme. The sensitivity of the assay was 0.3 pmol of ATP, and the response was linear between 1 and 500 pmol of ATP. The ATP content of platelets obtained with the present method correlated well with those obtained using soluble luciferase.

The catalytic activity of serum L-lactate dehydrogenase (LDH), was determined by monitoring the NADH produced by LDH with bacterial bioluminescent enzymes immobilized on a nylon coil. The LDH reaction of L-lactate with NAD took place in a flow-through mixing coil that preceded the bioluminescent detector coil. The response was linear from 1 to 5000 U/l at 37 degrees C and from 3 to 2000 U/l at 25 degrees C. The intra- and inter-assay reproducibility (CV%) were less than 10% and recovery range was 92% to 110%. The results agreed well with those obtained with a spectrophotometric method.