Activity and Composition of Hydrolases Secreted by Thermophilic Strain Bacillus sp. Grown on Plant Flour Media

Plant raw materials are an accessible and efficient substrate for multipurpose enzymes and multi-enzyme complexes obtaining. Thermophilic bacteria are reliable producer strains for industrial enzymes with valuable physicochemical properties, e.g., heat resistance, stability at extreme pH, and chemical stability. Food scientists are on the lookout for new thermophilic strains capable of producing high yields of industrially valuable enzymes from cheap plant raw materials. In this research, various plant cultivation media affected the complex hydrolytic enzymes produced by a thermophilic strain of Bacillus subtilis Kb.12.Gl.35. The research objective was to select the optimal raw materials for the synthesis of thermostable hydrolases, i.e., proteases, amylases, and hemicellulases. The thermophilic strain was isolated from a compost sample, identified by 16S rRNA sequencing, and tested using standard microbiological and biochemical methods. The strain grew on different media with plant flour combined with yeast or corn extract. The activity of extracellular enzymes made it possible to study the composition of the hydrolase complex. The proteolytic activity was determined using casein as the substrate and by zymography. The 3,5-dinitrosalicylic acid (DNS) method revealed the carbohydrase activity. When culturing the hydrolase producer on nutrient media with plant flour (1%) and corn extract (0.5%), the highest level of protease synthesis (≥ 40 units/mL) was detected in the samples with amaranth, oat, and rice flour. The highest level of amylase (≥ 1,300 units/mL) belonged to the chickpea, oat, and rice flour samples. The biggest yields of galactomannanase (≥ 200 units/mL) and xylanases (≥ 60 units/mL) was found in the samples with amaranth, pea, and chickpea samples. The biggest amount of arabinogalactanase (≥ 35 units/mL) belonged to the amaranth, chickpea, and rice flour. Based on the zymographic analysis, the enzymes of 24.7–28.2 kDa and the proteases of 62.7–75.0 kDa appeared to be the most efficient proteolytic agents. Their activity was obvious on plant raw materials, in contrast to the standard LB medium. The secretion level of 15.1 kDa proteases was the same on the plant media as on the standard medium. Bacillus subtilis Kb.12.Gl.35 proved to be an effective producer of proteases, amylases, and hemicellulases with plant flour and corn extract in the medium. Further studies are needed to optimize the composition of nutrient media and cultivation conditions to increase the yield of the target hydrolytic enzymes.