Oxidizing enzymes in the manufacture of paper materials
A paper material, fatty acid oxidase technology, applied in the field of oxidase in paper material manufacturing
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Embodiment 1
[0081] Measuring the activity of fatty acid oxidase on linoleic acid
[0082] An "Oxi 3000 Oximeter" (WTW, Weilheim, Germany) containing a TriOxmatic 300 oxygen electrode and a standard reaction volume of 4 ml was used.
[0083] 10 mg of linoleic acid (10 ml of 60% linoleic acid) was dissolved in 1 ml of ethanol, then 2 microliters of Tween20 was added. From this raw matrix solution, 50 μl was added to a reaction beaker containing 3.85 ml of a buffer solution (Britton-Robinson: 100 mM phosphoric acid, acetic acid, and boric acid; pH was adjusted with NaOH) using a small stirring bar, stirred to mix the solution thoroughly, and The oxygen electrode is inserted into the reaction beaker. Add 100 microliters of purified enzyme solution, i.e. (a) lipoxygenase derived from Magnaporthe salvinii at a concentration of about 0.4 mg / ml; (b) lipoxygenase derived from Gaeumannomyces at a concentration of about 0.76 mg / ml (in It was approximately 0.02 mg / ml in the final reaction). The prep...
Embodiment 2
[0087] fatty acid oxidase
[0088] Four enzymes, two laccases and two lipoxygenases, were tested as shown below. Laccase derived from Polyporus pinsitus has a MW of 65 kDa by SDS-Page, 3.5 pl of IEF, and a temperature optimum of 60°C at pH 5.5. Laccase derived from Coprinus cinereus has a MW of 67-68 kDa by SDS-Page, 3.5-3.8 pl by IEF, and a temperature optimum of 65°C at pH 7.5. Enzymes were prepared and purified as described in WO96 / 00290 and US Patent 6,008,029. Two lipoxygenases were derived from Magnaporthe salvinii and Gaeumannomyces graminis and were prepared as described above.
[0089] The dosage of enzyme was adjusted to ensure that the maximum increase in absorbance per minute was obtained at 234nm / 530nm, ie in the range of 0.1-0.25 absorbance units / minute.
[0090] Matrix solution: 11.65 mg linoleic acid (60% Sigma), and 12.5 ml 0.56 mM syringaldazine (Sigma) in ethanol were mixed with deionized water and the total volume brought to 25 ml.
[0091] Transfer 50 ...
Embodiment 3
[0096] Pitch reduction with fatty acid oxidase
[0097] Asphalt samples were prepared as follows:
[0098] 50% Linoleic Acid 60% (Sigma L-1626),
[0099] 20% Abietic Acid (Sigma A9424),
[0100] 20% Oleic Acid (Merck 471),
[0101] 5% Cholesteryl Linoleate (Sigma C-0289),
[0102] 5% olive oil (Sigma O-1500),
[0103] Stir at 65°C for 30 minutes and store in the refrigerator for no more than 30 days.
[0104] To prepare a 0.1% asphalt suspension:
[0105] 50mg bitumen sample,
[0106] 1ml ethanol,
[0107] 1ml 0.1M NaOH,
[0108] 48ml buffer (50mM borate pH9.0),
[0109] Stir at room temperature for 10 minutes.
[0110] The circular paper (diameter = 5.5-6mm; Multicopy 80g / m 2 ) into wells of two 96-well microtiter plates (ID 269620 from NUNC) designated A and B. Two other similar microtiter plates C and D were also used, but without the discs. 100 microliters of the 0.1% bitumen suspension was added to each well of these four microtiter plates. The above-mention...
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