Recombinant production of a highly efficient photolyase from Thermus thermophilus.

Ultraviolet (UV) radiation from sunlight can damage DNA, inducing mutagenesis and eventually leading to skin cancer. Topical sunscreens are used to avoid the effect of UV irradiation, but the topical application of DNA repair enzymes, such as photolyase, can provide active photoprotection by DNA recovery. Here we produced a recombinant Thermus thermophilus photolyase expressed in Escherichia coli, evaluated the kinetic parameters of bacterial growth and the kinetics and stability of the enzyme. The maximum biomass (𝑋𝑚𝑎𝑥 ) of 2.0 g L-1 was reached after 5 h of cultivation, corresponding to 𝑃X  = 0.4 g L-1 h. The µð‘šð‘Žð‘¥ corresponded to 1.0 h-1 . Photolyase was purified by affinity chromatography and high amounts of pure enzyme were obtained (3.25 mg L-1 of cultivation). Two different methods demonstrated the enzyme activity on DNA samples and very low enzyme concentrations, such as 15 µg mL-1 , already resulted in 90% of CPD photodamage removal. We also determined photolyase kM of 9.5 nM, confirming the potential of the enzyme at very low concentrations, and demonstrated conservation of enzyme activity after freezing (-20°C) and lyophilization. Therefore, we demonstrate T. thermophilus photolyase capacity of CPD damage repair and its potential as an active ingredient to be incorporated in dermatological products.

Glutaminase-free L-asparaginase production by Leucosporidium muscorum isolated from Antarctic marine-sediment.

L-asparaginase (ASNase) is an essential drug in the treatment of acute lymphoblastic leukemia (ALL). Commercial bacterial ASNases increase patient survival, but the consequent immunological reactions remain a challenge. Yeasts ASNase is closer to human congeners and could lead to lower side effects. Among 134 yeast strains isolated from marine-sediments in King George Island, Antarctica, nine were L-asparaginase producing yeasts and glutaminase-free. Leucosporidium muscorum CRM 1648 yielded the highest ASNase activity (490.41 U.L-1) and volumetric productivity (5.12 U.L-1 h-1). Sucrose, yeast extract and proline were the best carbon and nitrogen sources to support growth and ASNase production. A full factorial design analysis pointed the optimum media condition for yeast growth and ASNase yield: 20 g L-1 sucrose, 15 g L-1 yeast extract and 20 g L-1 proline, which resulted in 4582.5 U L-1 and 63.64 U L-1 h-1 of ASNase and volumetric productivity, respectively. Analysis of temperature, pH, inoculum and addition of seawater indicated the best condition for ASNase production by this yeast: 12-15 °C, pH 5.5-6.5 and seawater >25% (v/v). Inoculum concentration seems not to interfere. This work is pioneer on the production of ASNase by cold-adapted yeasts, highlighting the potential of these microbial resources as a source of glutaminase-free L-asparaginase for commercial purposes.

Quality-by-Design Approach for Biological API Encapsulation into Polymersomes Using "Off-the-Shelf" Materials: a Study on L-Asparaginase.

Polymersomes are versatile nanostructures for protein delivery with hydrophilic core suitable for large biomolecule encapsulation and protective stable corona. Nonetheless, pharmaceutical products based on polymersomes are not available in the market, yet. Here, using commercially available copolymers, we investigated the encapsulation of the active pharmaceutical ingredient (API) L-asparaginase, an enzyme used to treat acute lymphoblastic leukemia, in polymersomes through a quality-by-design (QbD) approach. This allows for streamlining of processes required for improved bioavailability and pharmaceutical activity. Polymersomes were prepared by bottom-up (temperature switch) and top-down (film hydration) methods employing the diblock copolymers poly(ethylene oxide)-poly(lactic acid) (PEG45-PLA69, PEG114-PLA153, and PEG114-PLA180) and the triblock Pluronic® L-121 (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), PEG5-PPO68-PEG5). Quality Target Product Profile (QTPP), Critical Quality Attributes (CQAs), Critical Process Parameters (CPPs), and the risk assessment were discussed for the early phase of polymersome development. An Ishikawa diagram was elaborated focusing on analytical methods, raw materials, and processes for polymersome preparation and L-asparaginase encapsulation. PEG-PLA resulted in diluted polymersomes systems. Nonetheless, a much higher yield of Pluronic® L-121 polymersomes of 200 nm were produced by temperature switch, reaching 5% encapsulation efficiency. Based on these results, a risk estimation matrix was created for an initial risk assessment, which can help in the future development of other polymersome systems with biological APIs nanoencapsulated.