Application of Used Tea as Solid Matrix for Immobilization …
221
2.2 Solid-State Fermentation
SSF was carried out in 250 ml conical flask containing 5 gm of substrate with 5 ml of
distilled water (w/v) and it was autoclaved at 121 °C for 15 min. After sterilization,
the flasks were inoculated with 5 ml of inoculum and incubated at 30 °C for 7 days.
At the end of fermentation, fermented substrate was extracted with 50 ml distilled
water for 2 h. The filtrate obtained was centrifuged at 4000 rpm for 20 min at room
temperature. The supernatant was used as crude enzyme extract.
2.3 Partial Purification of the Enzyme
The crude enzyme obtained after centrifugation was concentrated to half of its initial
volume using rotator vacuum evaporator (EYELA CCA-1110). Ammonium sulfate
precipitation was done according to Abidi et al. 2011. To remove the high amount of
salt from the precipitated protein, dialysis of the precipitate was performed. The 80%
ammonium sulfate precipitate was used for dialysis against 1 mM Tris Hcl buffer
(pH 8.0) till removal of sulfate from the protein fraction. The dialyzed protein was
then subjected to further purification. The partially purified enzyme was used for
further studies.
2.4 Preparation of Solid Matrix
The tea residues obtained after preparation of tea beverage were treated for
polyphenol compound extraction (Pavlovic et al. 2013). The solid separated out
of extraction process was collected and further treated with chlorine dioxide solution
(1:6 w/v) as an oxidizing agent. The oxidation reaction was continued for 30 min.
After this reaction the treated used tea was separated by filtration washed thoroughly
with distilled water and dried at 80 °C for overnight. This polyphenol extracted used
tea was employed as solid matrix in the immobilization process.
2.5 Immobilization of Alkaline Protease by Used Tea
1gm of used tea was mixed with a predetermined initial concentration of enzyme
(1:20 w/v). Then the immobilized samples were collected after filtration, washed
with distilled water, and enzyme activity was measured.
221
2.2 Solid-State Fermentation
SSF was carried out in 250 ml conical flask containing 5 gm of substrate with 5 ml of
distilled water (w/v) and it was autoclaved at 121 °C for 15 min. After sterilization,
the flasks were inoculated with 5 ml of inoculum and incubated at 30 °C for 7 days.
At the end of fermentation, fermented substrate was extracted with 50 ml distilled
water for 2 h. The filtrate obtained was centrifuged at 4000 rpm for 20 min at room
temperature. The supernatant was used as crude enzyme extract.
2.3 Partial Purification of the Enzyme
The crude enzyme obtained after centrifugation was concentrated to half of its initial
volume using rotator vacuum evaporator (EYELA CCA-1110). Ammonium sulfate
precipitation was done according to Abidi et al. 2011. To remove the high amount of
salt from the precipitated protein, dialysis of the precipitate was performed. The 80%
ammonium sulfate precipitate was used for dialysis against 1 mM Tris Hcl buffer
(pH 8.0) till removal of sulfate from the protein fraction. The dialyzed protein was
then subjected to further purification. The partially purified enzyme was used for
further studies.
2.4 Preparation of Solid Matrix
The tea residues obtained after preparation of tea beverage were treated for
polyphenol compound extraction (Pavlovic et al. 2013). The solid separated out
of extraction process was collected and further treated with chlorine dioxide solution
(1:6 w/v) as an oxidizing agent. The oxidation reaction was continued for 30 min.
After this reaction the treated used tea was separated by filtration washed thoroughly
with distilled water and dried at 80 °C for overnight. This polyphenol extracted used
tea was employed as solid matrix in the immobilization process.
2.5 Immobilization of Alkaline Protease by Used Tea
1gm of used tea was mixed with a predetermined initial concentration of enzyme
(1:20 w/v). Then the immobilized samples were collected after filtration, washed
with distilled water, and enzyme activity was measured.