Tannin acyl-hydrolase production by Bacillus subtilis KMS2-2: Purification, characterization, and cytotoxicity studies

Abstract

Objective: Bacterial mediated tannin acyl-hydrolase production was investigated Bacillus subtilis KMS2-2 in this study. Tannin acyl-hydrolase used as catalysts in the production of glucose and gallic acid. Also, it has potential application in beverage and food processing. Methods: This bacterial TAH enzyme was purified by a single methods approach consisting of size exclusion chromatography (sephadex G-100) and characterization of purified enzyme using different methods followed. Result: The molecular mass of purified TAH was determined as (~43 kDa) on 12% (PAGE) and it was confirmed by (MALDI-TOF/MS). Characterization of purified tannin acyl-hydrolase by (HPLC) confirmed that gallic acid was formed as a by-product during hydrolysis of tannic acid as a substrate and (FT-IR) spectroscopy showed the functional groups such as O–H, C-O and C–C. The purified tannin acyl-hydrolase retained the enzyme activity upto 80% under the conditions at 50 °C and pH 6.0 after 60 min incubation. The TAH comprises a typical secondary structure at pH 6.0 and contains α-(16.8%), β-(45.1%), and Turns (38.4%). Also, thermodynamics parameters and different additives like inhibitors, chelators, and metal ions were studied on the tannin acyl hydrolase. Further, the purified TAH enzyme has no cytotoxicity on the vero cell line as well as rat model study. Conclusion: From this experiment, the B. subtilis KMS2-2 would provide a possible resource for more efficient tannin-acyl hydrolase production and can be used for various industrial purposes, in particular food, feed, pharmaceutical industry.

Keywords

• Bacillus subtilis
• Bacterial Tannin acyl-hydrolase (TAH)
• Sephadex G-100
• SDS-PAGE
• In vivo model