Kinetic and modeling analyses of lactose‑hydrolyzing β‑galactosidase from Lactiplantibacillus plantarum GV54

Abstract

The enzyme β‑galactosidase plays a very essential role in food and in the pharmaceutical industry. The aim of the present study was to identify the most effective lactic acid bacteria isolated from homemade fermented milk samples and screened for β‑galactosidase production using the substrate, 5‑bromo‑4‑chloro‑3‑indolyl β‑D‑galactopyranoside (X‑gal). For the optimization of the enzyme production with carbon and nitrogen sources, four lactic acid bacterial isolates were used. Of these, only the <em>Lactiplantibacillus plantarum</em> (<em>L. plantarum</em>) GV54 and <em>L. plantarum</em> GV64 isolates significantly increased the production of β‑galactosidase with lactose as the carbon source. Similar results were obtained when nitrogen sources, such as ammonium sulfate and beef extract were used. The extraction of β‑galactosidase using the toluene/acetone method and SDS‑PAGE were performed to determine the molecular weight. The <em>L. plantarum</em> GV54 isolate exhibited the maximum β‑galactosidase activity (162.960±0.36 Miller units) compared with the other isolates with O‑nitrophenyl‑β‑D‑galactopyranoside (ONPG) as the substrate. The β‑galactosidase activity was high at pH 7.0 and a temperature of 37˚C. In addition, Mg²⁺, Mn²⁺ and Fe²⁺ had an indirect effect on the activity of β‑galactosidase. The kinetic constant was found to be 27.37574 mM, and its maximum velocity was 0.259289 U/min for pure β‑galactosidase in relation to ONPG. Comparative modeling and structural annotation analysis were performed, and the structural comparison of β‑galactosidase enzyme from <em>L. plantarum</em> and the crystal structure of <em>Bifidobactarum bifidium</em> was also performed. It was observed that the isolated enzyme shared the highest degree of similarity. Considering these characteristics of β‑galactosidase isolated from <em>L. plantarum</em> GV54, this isolate may be a promising commercial enzyme for various industrial and other biotechnological applications.