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Sarcosine dehydrogenase was purified to homogeneity from Pseudomonas fluorescens induced in TSB medium by sarcosine. The enzyme in a cell-free extract was initially bound to the hydrophobic matrix phenyl-Sepharose in low ionic strength buffer and eluted with Triton X-100. FPLC anion-exchange and gel-filtration was performed using Mono-Q and Superose-12 matrices respectively. Final chromatofocusing with Mono-P gave homogeneous enzyme, as shown by a single protein band silver-stained on a PAGE gel.

A method for assaying glucose in serum or plasma samples using a thermostable glucokinase was developed. Glucokinase from Bacillus Stearothermophilus was coupled with glucose-6-phosphate dehydrogenase to produce NADPH, which reduced the tetrazolium dye MTT to its formazan. Detection of the product at 660 nm allowed samples containing up to 30 mmol/L glucose to be assayed with an endpoint method. Use of the optimal wavelength for formazan detection, 570 nm, increased sensitivity for NADPH detection by over threefold compared to UV detection. The stability of glucokinase assay mixtures was extensively studied, with variation in buffers, salt and enzyme stabilizers. Maximal half life for reagent stability at room temperature was approximately 30 days, with storage of assay mixtures in two solutions. Various drugs and metabolites were tested for interference in the method and no significant interferences were found.

We evaluated a new method for measurement of acetaminophen. From acetaminophen, acetate and p-aminophenol are enzymically produced, and the latter product is determined colorimetrically by reaction with o-cresol and ammoniacal copper sulfate. The method is rapid and precise, and results compare well with those by spectrophotometric and liquid-chromatographic techniques. Major metabolites of acetaminophen, some commonly used drugs, or phenolic substances that may be present in patients with liver or renal disease do not interfere.

For an enzyme to be employed as a reagent in any field, be it clinical chemistry or organic synthesis, it must first be purified to a degree that removes any other enzyme capable of catalysing undesirable side-reactions. This may or may not mean purification to homogeneity. For the enzyme to be commercially viable, purification must yield tens or hundreds of grams of protein. On this scale, the availability of sufficient starting material may pose a problem, particularly for animal tissues. For this and other reasons, bacteria may provide the most valuable source of enzymes. Bacteria are readily produced in enormous quantities and, by virtue of their diverse metabolism, contain many enzymes not present in other organisms. Furthermore, the enzymes from thermophilic species often show a desirable increase in stability under the arduous conditions which may be encountered in a reactor. The preparation of enzymes on this scale from bacteria, or any other source, can usually be accomplished by the application of the normal range of techniques available to the protein chemist. Thus precipitation methods, gel filtration, ion exchange and affinity chromatography are all valuable. A major difference between this and normal laboratory-scale scale purification is cost; large-scale enzyme purification requires a considerable investment in equipment, materials and manpower. This cost must be viewed in terms of the value of the product.

We evaluated a new method for measurement of acetaminophen. From acetaminophen, acetate and p-aminophenol are enzymically produced, and the latter product is determined colorimetrically by reaction with o-cresol and ammoniacal copper sulfate. The method is rapid and precise, and results compare well with those by spectrophotometric and liquid-chromatographic techniques. Major metabolites of acetaminophen, some commonly used drugs, or phenolic substances that may be present in patients with liver or renal disease do not interfere.

Homogeneous beta-lactamase (beta-lactam hydrolase, E.C. 3.5.2.6) from Enterobacter cloacae P99, an enzyme that has an important function in antibiotic resistance, was prepared using a single cation-exchange chromatographic step with CM-Sepharose fast-flow. A 6-g amount of the enzyme was isolated from 5 kg of cell paste, with 84% of the enzyme activity in the cell homogenate being recovered by the single cation-exchange step. The specific activity of the beta-lactamase was 587 U/mg protein. The relative molecular mass of the enzyme was determined to be 45 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and the isoelectric point was 8.95.