Isolation of Restriction Enzyme from Sewage Samples Derived Bacillus sp.

Abstract

Objectives:The aim is to identify and characterize the restriction enzyme activity of bacteria from a sewage sample. Methods: The sample was plated on Nutrient Agar, Luria Bertani, and Tryptone Soy Broth Agar. The selected bacteria were screened for restriction enzyme activity and mass cultured. Extraction and purification of the enzyme were performed using column chromatography (PC 11 column, HTP column, and HPS column) and dialysis. Bacterial identification was performed using Gram's staining and 16S rRNA gene sequencing. Findings: A total of 2138 bacterial colonies were retrieved using Nutrient Agar, Luria Bertani, and Tryptone Soy Broth Agar. From the enzymatic screening, bacterial isolate strain no. 8 was selected, and the extraction and purification of restriction enzymes from the selected bacterial strain were performed using column chromatography. After dialysis, the enzyme showed complete activity in 1:6 dilutions; hence, the unit of the enzyme is 6 units. The isolated bacterial strain was found to be a gram-positive rodshaped Bacillus after Gram's staining was performed. The strain was identified using 16S rRNA gene sequencing technology, and it was found out that it has 97.91% homology with Bacillus sp. Novelty: The study reported sewage water derived Bacillus sp. having restriction enzyme.