Sustainable bioprocessing of kitchen waste for amylase production by an autochthonous Bacillus paralicheniformis AOLT3: process optimization and enzyme characterization

Abstract

Amylase is an industrially important enzyme that continues to be explored from novel microbial sources to enable sustainable, eco-friendly, and cost-effective production processes. In this study, an amylolytic bacterium was isolated from a waste disposal site and identified as Bacillus paralicheniformis AOLT3 (GenBank accession no. OR478155). The strain was evaluated for the biotechnological valorization of food wastes as low-cost substrates for amylase production under submerged fermentation. Medium components were initially screened using a one-variable-at-a-time (OVAT) approach, followed by statistical optimization using response surface methodology based on a central composite design (RSM–CCD). Maximum amylase production [(76.9 ± 2.3) U/mL] was achieved using 12.5 g/L kitchen waste, 7 g/L peptone, and 4 g/L yeast extract. The optimized quadratic model demonstrated high reliability, with R² = 99.28%, adjusted R² = 98.63%, and strong statistical significance (P ≤ 0.005). Partial purification by ammonium sulfate precipitation and Amicon desalting resulted in a 2.08-fold purification with a specific activity of 5.33 U/mg. SDS–PAGE revealed three amylase isoforms with molecular weights of 82, 56, and 48 kDa, which were further confirmed by zymogram analysis. The enzyme showed maximum activity at 45 °C and pH 7.5, with K_m and V_max values of (3.13 ± 0.03) mg/mL and (6.71 ± 0.14) U/mg, respectively, and retained approximately 80% activity after 1 h at 60 °C and pH 7.5–9.0. Overall, this study highlights the potential of B. paralicheniformis AOLT3 for efficient amylase production via kitchen waste valorization, supporting sustainable bioprocess development and alignment with United Nations Sustainable Development Goal (SDG) 12.

Graphical abstract

Keywords

Bacillus paralicheniformis / Amylase / Kitchen waste / Response surface methodology / Zymogram

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