At the same time that the number of microorganisms was determined, i.e. on days 30 and 60 of the experiment in soil samples, from each repetition in three subsequent replications, the activity of dehydrogenases, catalase, urease, acid phosphatase, alkaline phosphatase, β-glucosidase and arylsulphatase was determined. The substrates used for the determination of the enzyme activity, as well as the units in which the activity of particular enzymes was expressed, are presented in Table 4 . The activity of all enzymes, with the exception of catalase, was determined using a Perkin-Elmer Lambda 25 spectrophotometer (MA, USA). The activity of dehydrogenases was determined at a wavelength (λ) of 485 nm; the activity of urease, acid phosphatase and alkaline phosphatase at 410 nm; the activity of β-glucosidase at 400 nm; and the activity of arylsulphatase at 420 nm. The activity of catalase was determined based on the reaction of hydrogen peroxide decomposition using potassium permanganate.
Methods of determination of soil enzyme activity
| Enzyme | Substrate | Product/unit | References |
|---|---|---|---|
| Deh—dehydrogenases (EC 1.1) | 2,3,5-Triphenyl tetrazolium chloride (TTC) | Triphenyl fomazan (TFF), μmol kg−1 DM of soil h−1 | Öhlinger (1996 ) |
| Cat—catalase (EC 1.11.1.6) | H2O2—aqueous solution | O2, mol kg−1 DM of soil h−1 | Alef and Nannipieri (1998 ) |
| Ure—urease (EC 3.5.1.5) | Urea—aqueous solution | N-NH4, mmol kg−1 DM of soil h−1 | Alef and Nannipieri (1998 ) |
| Glu—β-glucosidase (EC 3.2.1.21) | 4-Nitrophenyl-β-D-glucopyranoside (PNG) | 4-Nitrophenol (PN), mmol kg−1 DM of soil h−1 | Alef and Nannipieri (1998 ) |
| Pac—acid phosphatase (EC 3.1.3.2) | Disodium 4-nitrophenyl phosphate hexahydrate (PNP) | 4-Nitrophenol (PN), mmol kg−1 DM of soil h−1 | Alef and Nannipieri (1998 ) |
| Pal—alkaline phosphatase (EC 3.1.3.1) | Disodium 4-nitrophenyl phosphate hexahydrate (PNP) | 4-Nitrophenol (PN), mmol kg−1 DM of soil h−1 | Alef and Nannipieri (1998 ) |
| Aryl—aryosulphatase (EC 3.1.6.1) | Potassium-4-nitrophenylsulfate (PNS) | 4-Nitrophenol (PN), mmol kg−1 DM of soil h−1 | Alef and Nannipieri (1998 ) |
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