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2.6 Protease Assay
Activity was determined according to the method described by Polley et al. using
casein as substrate (Polley et al. 2016). The proteolytic unit was defined as the amount
of enzyme that released 1 μg of tyrosine per minute under the assay condition.
2.7 Immobilization Yield
Initial activity of enzyme used for immobilization and is expressed as follows.
Yield(%) =
Total Immobilized activity × 100
Starting activity
(1)
Total activity of immobilized enzyme was calculated by subtracting the residual
enzyme activity remaining in the enzyme solution after immobilization.
Efficiency(%) =
Observed activity × 100
Immobilized activity
(2)
2.8 Optimization of Amount of Solid Matrix
on Immobilization of Alkaline Protease
Effect of amount of solid matrix on immobilization of protease was studied by carring
out immobilization with (100, 200, 300, 400, and 500 mg) of used tea. Each case the
enzyme yield was determined.
2.9 Optimization of Incubation Time on Immobilization
of Alkaline Protease
This effect was studied by incubating the mixture for the time period (2, 4, 6, 8, and
10 h) and for each sample enzyme assay was done.