Sacred Grove Soil as a Novel Source of Protease-Producing Alishewanella aestuarii B11: A First Report from North Gujarat, India

Abstract Sacred groves are forest patches that local communities have protected for generations on religious grounds. Their soils have seen little tillage, grazing or chemical input, yet few studies have screened them for bacteria of industrial use. We collected soil from 25 sacred groves in five districts of North Gujarat, India (Sabarkantha, Banaskantha, Patan, Mehsana and Aravalli) and screened it for protease-producing bacteria by serial dilution, spread-plating and clearance-zone assays on skim-milk agar. Of the 98 morphologically and biochemically distinct isolates recovered, the isolate with the largest clearance zone in each district was taken forward for quantitative work. The best producer overall, BKNR-1 from a Banaskantha grove, gave a 29 mm clearance zone on casein agar and was identified by 16S rRNA gene sequencing and phylogenetic analysis as Alishewanella aestuarii B11, a Gram-negative, oxidase- and catalase-positive bacterium. In submerged fermentation, one-factor-at-a-time (OFAT) optimization gave maximum protease production after 48 h with 2% (w/v) fructose as carbon source, 2% (w/v) urea as nitrogen source, an initial pH of 7.0 and incubation at 37°C. Activity under these conditions reached about 655–700 U mL⁻¹ min⁻¹, roughly 4.3 times that of the unoptimized medium. Ammonium sulfate fractionation of the crude extract raised specific activity, in line with reports for other bacterial proteases purified by stepwise salting-out. To our knowledge, this is the first report of a strongly proteolytic A. aestuarii strain from Indian sacred-grove soil. The genus has rarely been studied as an enzyme producer, and the result supports further bioprospecting of traditionally conserved forest soils for hydrolytic enzymes with uses in the detergent, leather, food and waste-management sectors.