The production of recombinant enzymes, primarily used for obtaining pure and functional target molecules, holds significant importance in modern biotechnology. This study aimed to obtain and characterize recombinant, extracellularly expressed α-amylases (Amy3500 and Amy1974), derived from B. amyloliquefaciens MDC1974 and B. subtilis MDC3500, respectively, using the pBE-S shuttle vector. Both α-amylase genes were molecularly cloned into the E. coli/B. subtilis pBE-S shuttle vector, both with (Amy1974sig and Amy3500sig) and without their signal peptides (Amy1974 and Amy3500), along with a signal peptide originating from the plasmid, and tested in flask fermentations. For recombinant Amy3500, the amylase variants resulted in similar levels of volumetric activity (700–750 U/mL). In contrast, the expression of Amy1974 nearly doubled compared to Amy1974sig with double signal peptides, reaching 2000 U/mL. SDS-PAGE estimated the molecular weight of Amy1974 α-amylase to be 54.6 kDa, which is consistent with the theoretical molecular mass calculations. However, the estimated molecular weight of Amy3500 α-amylase was significantly lower upon exiting the producer cells. Ca²⁺ ions exhibit a modest activating effect on the activities of Amy1974 and Amy3500 amylases, likely due to their tight binding to the protein scaffold. Both enzymes exhibited broad activity peaks between 45 and 70 °C, with a maximum at 65 °C. The Amy1974 and Amy3500 α-amylases demonstrated broad pH optima and pH-dependent thermostability, with optimum pH values at 6.5 and 5.8, and thermal stability peaks at pH 7.6 and 5.9, respectively. Both α-amylases displayed high relative activity against various starches, including corn amylopectin and potato amylose, while showing comparatively lower activity towards α-, β-, and γ-cyclodextrins. The Amy1974 amylase is effective in converting starch into dextrins of varying lengths, while Amy3500 primarily converts starch into glucose. These characteristics make them promising candidate enzymes for industrial applications.

Laminarinase, an enzyme with a specific affinity for laminarin—a complex polysaccharide found in the cell walls of brown algae and select marine orga

The α-amylase gene from Bacillus amyloliquefaciens MDC1974 was cloned into the pBE-S shuttle vector and expressed extracellularly using Bacillus subtilis RIK1285. Following the optimization of fermentation conditions, a pilot-scale production of 100 liters was conducted, achieving a maximum volumetric activity of 1969 U/ml, which is 1.4 times higher than the maximum activity obtained in flask fermentation. Additionally, enzyme concentration, drying, and preservation conditions were studied and optimized.

N-carbamoyl-beta-alanine amidohydrolase (C beta AA) constitutes one of the most important groups of industrially relevant enzymes used in the production of optically pure amino acids and derivatives. In this study, a C beta AA-encoding gene from Rhizobium radiobacter strain MDC 8606 was cloned and overexpressed in Escherichia coli. The purified recombinant enzyme (RrC beta AA) showed a specific activity of 14 U.mg(-1) using N-carbamoyl-beta-alanine as a substrate with an optimum activity at 55 degrees C and pH 8.0. In this work, we report also the first prokaryotic C beta AA structure at a resolution of 2.0 angstrom. A discontinuous catalytic domain and a dimerisation domain attached through a flexible hinge region at the domain interface have been revealed. We identify key ligand binding residues, including a conserved glutamic acid (Glu131), histidine (H385) and arginine (Arg291). Our results allowed us to explain the preference of the enzyme for linear carbamoyl substrates, as large and branched carbamoyl substrates cannot fit in the active site of the enzyme. This work envisages the use of RrC beta AA from R. radiobacter MDC 8606 for the industrial production of L-alpha-, L-beta- and L-gamma-amino acids. The structural analysis provides new insights on enzyme-substrate interaction, which shed light on engineering of C beta AAs for high catalytic activity and broad substrate specificity.

The rapid development of biodegradable polymers has intensified the need for reliable, standardized methodologies that can accurately assess their environmental fate. However, biodegradability is not an intrinsic property of materials, but rather the result of complex interactions between polymer chemistry, physicochemical characteristics, environmental conditions, and microbial activity. Consequently, biodegradation performance determined under one set of conditions cannot be directly extrapolated to other environments, highlighting the importance of selecting appropriate testing methodologies. This review provides a comprehensive and critical evaluation of the main international standards used to assess polymer biodegradability, including ISO, ASTM, EN, and OECD methods applicable to soil, industrial composting, freshwater, and marine environments. It discusses the fundamental mechanisms of polymer biodegradation, together with the major factors governing degradation kinetics, and comparatively analyzes the biodegradation behavior of polyesters, polysaccharides, polyamides, and polyesteramides across different environmental compartments. Unlike previous reviews, this work critically compares the applicability, strengths, limitations, and biodegradation endpoints of standardized methodologies, identifies current methodological gaps, and proposes a practical decision-making framework for selecting appropriate tests according to the intended end-of-life scenario of polymeric materials. Finally, it highlights the need for more harmonized and environmentally relevant testing approaches to better distinguish compostability from true environmental biodegradability.