This study used conservative one variable-at-a-time study and statistical surface response methods to increase the yields of an extracellular thermostable protease secreted by a newly identified thermophilic Bacillus subtilis BSP strain. Using conventional optimization techniques, physical parameters in submerged fermentation were adjusted at the shake flask level to reach 184 U/mL. These physicochemical parameters were further optimized by statistical surface response methodology using Box Behnken design, and the protease yield increased to 295 U/mL. The protease was purified and characterized biochemically. Both Ca²⁺ and Fe²⁺ increased the activity of the 36 kDa protease enzyme. Based on its strong inhibition by ethylenediaminetetracetate (EDTA), the enzyme was confirmed to be a metalloprotease. The protease was also resistant to various organic solvents (benzene, ethanol, methanol), surfactants (Triton X-100), sodium dodecyl sulfate (SDS), Tween 20, Tween-80 and oxidants hydrogen per oxide (H₂O₂). Characteristics, such as tolerance to high SDS and H₂O₂ concentrations, indicate that this protease has potential applications in the pharmaceutical and detergent industries.

Despite plastics constituting a vital part of our live, such as food items, or home utensils, they have also earned notoriety among researchers due to their high greenhouse gas emissions, increased accumulation in landfills, and their sedimentation on the marine environment causing hazardous health concerns for mankind by entering into our food web as micro plastics. Hence while looking for a substitute for plastics, researchers came across bio-plastics, plastics made from biomass (renewable resources) that are even biodegradable and are now getting incorporated into the global world in the form of rigid packaging, consumers goods, textiles, in the agricultural and horticulture, and in automotive and transport, with an expected growth rate of 200% in the near future. The ability of bio-plastic to biodegrade makes it eco-friendly, and new innovations in its compositions increase its versatility as packaging or consumer products. This review article discusses the misconceptions about the biodegradability of bio-plastics and gaps regarding the basic concept of ‘the rate of biodegradability in bioplastics’; all bioplastics are not biodegradable and some require different conditions such as aeration time, composting environment, etc., along with an overview of the biodegradability of various bio-plastics under different standards and conditions such as aeration time, days, a comparison between conventional plastics and bio-plastics, types of bio-plastics based on raw materials, and future perspectives that despite being a sustainable solution for greener environment, bio-plastics still lack in certain areas as production cost, recycling infrastructure, mechanical properties, and quick bio-degradation standards that are more suitable for industry and natural environment. This review article also provides suggestions to improve bio-plastics towards a more sustainable solution using various techniques as polymer mixing, use of additives such as plasticizers or surfactants etc. Graphical Abstract

Thermophilic bacteria represent a valuable source of robust enzymes for industrial applications, yet their high growth temperatures often limit large-scale cultivation. In this study, the MprT gene encoding a thermostable metalloprotease was cloned from the thermophilic Bacillus subtilis BSP and heterologously expressed in both Escherichia coli BL21(DE3) and the protease-deficient B. subtilis 1A751. The full-length MprT open reading frame (1638 bp), including its native signal and pro-peptide sequences, was amplified by PCR and inserted into the pET29b vector under the control of the T7 promoter for expression in E. coli. The same gene was subsequently cloned into the integration vectors pDR111 (IPTG-inducible hyper-spank promoter) and pSG1154 (xylose-inducible promoter) for chromosomal integration and extracellular secretion in B. subtilis 1A751. Recombinant constructs were verified by restriction digestion, Sanger sequencing, and SDS-PAGE, which confirmed production of a 36 kDa polypeptide. Functional expression was demonstrated on casein-supplemented agar plates containing appropriate inducers, with clear hydrolytic halos appearing after incubation at 60 °C. The purified recombinant MprT protease displayed optimal activity at 50 °C and pH 7–8, retained 82% of its initial activity after 30 min at 60 °C, and remained stable across a broad pH range (4–11). Activity was strongly stimulated by Ca²⁺, Fe²⁺, and Mn²⁺ ions and was completely abolished by EDTA, consistent with its classification as a metalloprotease. Compared with previous reports on thermolysin-like proteases, this work provides the first successful functional overexpression of the BSP-derived MprT gene in a protease-deficient B. subtilis host, enabling efficient extracellular production at moderate cultivation temperatures while preserving the enzyme’s native thermostability and alkaline tolerance. These properties position the recombinant MprT as a promising candidate for detergent formulations and waste-to-biofuel processes conducted under harsh industrial conditions.