Peptidases from basidiomycetes
A technology of basidiomycetes and peptidases, applied in a method of producing them and involving them in the field of protein hydrolysis, can solve the problems of incomplete reaction and carcinogenicity of enzymatic processes
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Embodiment 1
[0120] Example 1 - Peptidases from six Basidiomycetes
[0121] Culture of Basidiomycetes
[0122] During processing, all media and equipment are autoclaved and standard aseptic technique is used before use. Pre-cultivation of Basidiomycetes (Table 2) at 150 rpm and 24°C in a standard nutrient medium (30 g / L glucose monohydrate, 3 g / L yeast extract, 4.5 g / L asparagine monohydrate, 0.5g / L MgSO 4 , 1.5g / L KH 2 PO 4 , 1mL trace element solution, pH6.0[0.08g / LFeCl 3 , 0.09g / L ZnSO 4 wxya 2 O, 0.005g / L CuSO 4 x5 2 O, 0.027g / L MnSO 4 , 0.4g / L Titriplex III]) for seven days.
[0123] 20 mL of mycelial culture was separated by centrifugation (10 min, 3000xg, 4°C) and replaced with 20 mL of mineral salt medium (MM) lacking additional carbon and nitrogen sources (0.5 g / L MgSO 4 , 1.5g / LKH 2 PO 4 , 1mL trace element solution, pH 6.0) and washed twice. For maincultures, 250 mL of fresh MM was inoculated with 20 mL of prewashed mycelium.
[0124] Basidiomycetes cultivated f...
Embodiment 2
[0128] Example 2 - Enzyme Preparation Using Grifola Phyllostachys
[0129] After 6 days of cultivation with 4% gluten, the Gfr culture was filtered and the extracellular enzyme-containing supernatant (200 mL) was precipitated with 800 mL of ethanol (96%), stored at -20°C for 3 hours, and then centrifuged at 12,000xg for 20 minutes . Resuspend an aliquot of the pellet in 0.1M K 2 HPO 4 / KH 2 PO 4 buffer (pH 6.0) for 30 minutes at 4°C, and the activity of the supernatant was assayed.
[0130] Most of the initially present hydrolytic activity was restored, indicating that this method yielded useful enzyme concentrates.
Embodiment 3
[0131] Embodiment 3-hydrolysis experiment
[0132] Suspend 0.5 g of gluten in 20 mL of 0.1 M K 2 HPO 4 / KH 2 PO 4 18.9 kU of peptidase precipitate obtained from Pfl (Pleurotus florida) was added to the buffer (pH 6.0), and the sample was incubated at 40° C., 110 rpm, in a water bath for 18 hours. The samples were centrifuged at 3000 xg for 15 minutes at 4°C. Blanks were prepared with heat-inactivated enzyme preparations. 5 mL of supernatant was used for Kjeldahl analysis and 7 μL of supernatant for AA-TLC. To visualize proteolysis, SDS-PAGE of samples was performed.
[0133] The analytical evidence indicated a gradual enzymatic hydrolysis of the difficult substrate wheat glutenin.
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