Spectrophotometric Methods

Many substrates and products of enzymatic reactions absorb light either in the visible or in the ultraviolet region of the spectrum or can be measured by a simple color reaction. Due to their higher sensitivity, methods based on the analysis of the released product are more frequently used than methods based on the analysis of substrate depletion. Analyzing enzyme activities involves incubating soils with

FIGURE 3.4 Locations of enzymes (from Burns, 1982, adapted by Klose, 2003). (i) Intracellular enzymes. (ii) Periplasmatic enzymes. (iii) Enzymes attached to outer surface of cell membranes. (iv) Enzymes released during cell growth and division. (v) Enzymes within nonproliferating cells (spores, cysts, seeds, endospores). (vi) Enzymes attached to dead cells and cell debris. (vii) Enzymes leaking from intact cells or released from lysed cells. (viii) Enzymes temporarily associated in enzyme- substrate complexes. (ix) Enzymes absorbed to surfaces of clay minerals. (x) Enzymes complexed with humic colloids. (Reproduced with permission from Taylor & Francis Group, LLC.)

Humus-enzyme complex Clay-enzyme complex

FIGURE 3.4 Locations of enzymes (from Burns, 1982, adapted by Klose, 2003). (i) Intracellular enzymes. (ii) Periplasmatic enzymes. (iii) Enzymes attached to outer surface of cell membranes. (iv) Enzymes released during cell growth and division. (v) Enzymes within nonproliferating cells (spores, cysts, seeds, endospores). (vi) Enzymes attached to dead cells and cell debris. (vii) Enzymes leaking from intact cells or released from lysed cells. (viii) Enzymes temporarily associated in enzyme- substrate complexes. (ix) Enzymes absorbed to surfaces of clay minerals. (x) Enzymes complexed with humic colloids. (Reproduced with permission from Taylor & Francis Group, LLC.)

the respective substrate at specific temperature, pH, and time; subsequently extracting the product; and then colorimetrically determining its concentration.

Dehydrogenases, which are intracellular enzymes catalyzing oxidation-reduction reactions, can be detected using a water-soluble, almost colorless tetrazolium salt, which forms a reddish formazan product after an incubation period of several hours. A commonly used tetrazolium salt is 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT), which is transformed into an intensely colored, water-insoluble formazan (INT-formazan). The production of INT-formazan can be detected spectrophotometrically by quantifying total INT-formazan production. Another enzyme assay system used to reflect general or total microbial activity in soil samples is based on the hydrolysis of fluorescein diacetate. Fluorescein diacetate is hydrolyzed by a wide variety of enzymes including esterases, proteases, and lipases.

Enzymes involved in C cycling (xylanase, cellulase, invertase, and trehalase) are measured based on the release of sugars after incubating soils with a buffered solution (pH 5.5) containing their corresponding substrates (xylan, carboxymethyl-cellulose, sucrose, or trehalose). The incubation period depends on the substrate used: high-molecular-weight substrates are incubated for 24 h, whereas low-molecular-weight substrates are incubated for only 1 to 3 h. Reducing sugars released during the incubation period cause the reduction of potassium hexa-cyanoferrate(III) in an alkaline solution. Reduced potassium hexacyanoferrate(II) reacts with ferric ammonium sulfate in an acid solution to form a complex of ferric hexacyanoferrate(II) (Prussian blue), which is determined colorimetrically.

Enzymes involved in P cycling (phosphomonoesterase, phosphodiesterase, phosphotriesterase) are preferably determined after the addition of a substrate analog, p-nitrophenyl phosphate. Phosphomonoesterase hydrolyzes the phosphateester bond of the p-nitrophenyl phosphate, and the p-nitrophenol released is yellow under alkaline conditions. The concentration can be determined spectrophotomet-rically against a standard curve. For determining arylsulfatase, which catalyzes the hydrolysis of organic sulfate esters, p-nitrophenyl sulfate is used as a substrate analog. The widespread use of p-nitrophenyl substrates for different enzyme assays is due to the possibility of quantitatively extracting the released product p-nitrophenol. Urease is an important extracellular enzyme that hydrolyzes urea into CO2 and NH3. The urease assay involves measuring the released ammonia either by a colorimetric procedure or by steam distillation followed by a titration assay of ammonia.

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