I have been trying to adapt the enzymatic assay method (Sharma et al., 2000) to final reaction volume of 1 ml as described here (Kanpiengjai et al., 2019). I add water to the control reaction mix (buffer + water + substrate) in the place of inactivated enzyme. I prepare methyl gallate substrate in water and my final substrate concentration will be 25 mM ( and final buffer concentration is 100 mM) in the 100 ul reaction mix. However, I find higher values for my control (without enzyme) or no difference than the test samples absorbance values. We brought commercial tannase enzyme and tested it. However, the absorbance values are similar to test samples. Please help me with your inputs. Thank you.

P.S.: I have attached my protocol below and have listed the papers mentioned in the description.

1). Shweta Sharma, Talapady N Bhat, Rajinder Dawra

A Spectrophotometric Method for Assay of Tannase Using Rhodanine

DOI: 10.1006/abio.1999.4405

2). Apinun Kanpiengjai, Kridsada Unban, Thu-Ha Nguyen, Dietmar Haltrich, Chartchai Khanongnuch,

Expression and biochemical characterization of a new alkaline tannase from Lactobacillus pentosus,

https://doi.org/10.1016/j.pep.2019.01.005.

(http://www.sciencedirect.com/science/article/pii/S1046592818305928)