4-nitrophenyl

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.Table 4

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 )

Chitinolytic activity was determined using the synthetic chromogenic substrate, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside (Sigma-Aldrich), at a concentration of 200 µM. Each reaction was done in triplicate. All enzymatic reactions for the determination of optimum pH and temperature were conducted in a volume of 50 µL containing E. coli- or CHO-expressed protein in McIlvaine’s buffer [14] (link) or 0.1 M Gly-HCl buffer. Reactions for optimum pH and kinetic assays were conducted for 30 min at 37°C. Reactions were halted with the addition of 20 µL of 1 M sodium carbonate to the reaction mixture. The absorbance of the liberated 4-nitrophenolate ion was measured at 405 nm. A molar extinction coefficient for 4-nitrophenol of 17,700 M⁻¹ cm⁻¹ was used in the calculations. One enzyme unit (U) was defined as 1 µmol of 4-nitrophenol released from 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside per min at 37°C in Gly-HCl buffer (pH 2.0).

Cellulase was produced in a 500 mL flask that contained 100 mL of fluid medium through a two-step cultivation procedure. Strains were first grown at 30°C in 100 mL of medium that contained 2 g of glucose as a carbon source and were then regulated at pH 5.5 and 200 rpm for 20 hours. The cultures were collected through vacuum drum filtration during this second step, and 0.5 g vegetative mycelia was added to 100 mL of Vogel’s medium that contained 2% cellulose as carbon source or wheat bran medium at an initial pH of 5.5 at 30°C and 200 rpm. Culture supernatants (crude enzyme) were diluted with sodium acetate buffer solution (SABF, 0.2 M, pH 4.8). Enzymatic hydrolyses of the polysaccharides were also performed in SABF (0.2 M, pH 4.8). The filter paper enzyme (FPA), endoglucanase (CMCase), xylanase, and amylase activities of the culture supernatants (diluted samples) were assayed using a DNS reagent (10 g 3, 5-dinitrosalicylic acid, 20 g sodium hydroxide, 200 g sodium potassium tartrate, 2.0 g redistilled phenol, and 0.50 g sodium sulfite anhydrous per 1000 mL DNS reagent) against Whatman No. 1 filter paper, carboxymethylcellulose sodium salt (CMC-Na), xylan (from beechwood), and soluble starch. CMC-Na, xylan, or starch was dissolved in SABF to a final concentration of 1% (mass/volume percent, m/v %), and then the mixture was left overnight and was shaken well before using. The following components were added in a 2.0 mL reaction mixture: 0.5 mL diluted culture supernatants and 1.5 mL CMC-Na, xylan, or starch solution for CMCase, xylanase, or amylase activity assays, respectively; and 2.0 mL diluted culture supernatants and 50 mg Whatman No. 1 filter paper for FPA assay into 25 mL colorimetric tube. The mixture was mixed gently and the reaction mixture was incubated for FPA measurement in a 50°C water bath for 1 hour, for CMCase and xylanase activity measurements at 50°C for 30 min, and for amylase activity measurement at 40°C for 10 min. Three milliliters of DNS reagent were then added to stop the reaction. A blank tube (with boiled crude enzyme) was used as control to correct any reducing sugar present in the crude enzyme samples. The tubes were placed in boiling water for 10 min, 20 mL distilled water was added, 200 μL of reaction mixture was pipetted, and the absorbance was determined at 540 nm. The cellobiohydrolase (pNPCase) and β-glucosidase (pNPGase) activities were measured by using 4-Nitrophenyl β-D-cellobioside (pNPC) and 4-Nitrophenyl β-D-glucopyranoside (pNPG) as substrates, respectively. The pNPC or pNPG was dissolved in SABF to a final concentration of 1 mg/mL. Moreover, 50 μL of pNPC solution (containing 1 mg/mL D-Glucono-δ-lactone) or 50 μL of pNPG solution and 100 μL of diluted culture supernatants were mixed, and then the mixtures were incubated in a 50°C water bath for 30 min. The reaction was stopped by adding 0.15 mL of sodium carbonate solution (10%, m/v), then 200 μL of these reaction mixtures was pipetted, and the absorbance was measured at 420 nm. One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol of glycoside bonds of the substrate per minute under defined assay conditions. Independent triplicate cultures were sampled and analyzed.
The total protein was determined using a Bradford assay kit according to the instructions of the manufacturer.