Browsing by Author "Blazquez-Sanchez, Paula"

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    An Open One-Step RT-qPCR for SARS-CoV-2 detection
    (2024) Cerda, Ariel; Rivera, Maira; Armijo, Grace; Ibarra-Henriquez, Catalina; Reyes, Javiera; Blazquez-Sanchez, Paula; Aviles, Javiera; Arce, Anibal; Seguel, Aldo; Brown, Alexander J.; Vasquez, Yesseny; Cortez-San Martin, Marcelo; Cubillos, Francisco A.; Garcia, Patricia; Ferres, Marcela; Ramirez-Sarmiento, Cesar A.; Federici, Fernan; Gutierrez, Rodrigo A.
    The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.
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    Antarctic Polyester Hydrolases Degrade Aliphatic and Aromatic Polyesters at Moderate Temperatures
    (2022) Blazquez-Sanchez, Paula; Engelberger, Felipe; Cifuentes-Anticevic, Jeronimo; Sonnendecker, Christian; Grinen, Aransa; Reyes, Javiera; Diez, Beatriz; Guixe, Victoria; Richter, P. Konstantin; Zimmermann, Wolfgang; Ramirez-Sarmiento, Cesar A.
    Polyethylene terephthalate (PET) is one of the most widely used synthetic plastics in the packaging industry, and consequently has become one of the main components of plastic waste found in the environment. However, several microorganisms have been described to encode enzymes that catalyze the depolymerization of PET. While most known PET hydrolases are thermophilic and require reaction temperatures between 60 degrees C and 70 degrees C for an efficient hydrolysis of PET, a partial hydrolysis of amorphous PET at lower temperatures by the polyester hydrolase IsPETase from the mesophilic bacterium Ideonella sakaiensis has also been reported. We show that polyester hydrolases from the Antarctic bacteria Moraxella sp. strain TA144 (Mors1) and Oleispira antarctica RB-8 (OaCut) were able to hydrolyze the aliphatic polyester polycaprolactone as well as the aromatic polyester PET at a reaction temperature of 25 degrees C. Mors1 caused a weight loss of amorphous PET films and thus constitutes a PET-degrading psychrophilic enzyme. Comparative modeling of Mors1 showed that the amino acid composition of its active site resembled both thermophilic and mesophilic PET hydrolases. Lastly, bioinformatic analysis of Antarctic metagenomic samples demonstrated that members of the Moraxellaceae family carry candidate genes coding for further potential psychrophilic PET hydrolases.
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    Engineering the catalytic activity of an Antarctic PET-degrading enzyme by loop exchange
    (2023) Blazquez-Sanchez, Paula; Vargas, Jhon A.; Furtado, Adriano A.; Grinen, Aransa; Leonardo, Diego A.; Sculaccio, Susana A.; Pereira, Humberto D'Muniz; Sonnendecker, Christian; Zimmermann, Wolfgang; Diez, Beatriz; Garratt, Richard C.; Ramirez-Sarmiento, Cesar A.
    Several hydrolases have been described to degrade polyethylene terephthalate (PET) at moderate temperatures ranging from 25 degrees C to 40 degrees C. These mesophilic PET hydrolases (PETases) are less efficient in degrading this plastic polymer than their thermophilic homologs and have, therefore, been the subject of many protein engineering campaigns. However, enhancing their enzymatic activity through rational design or directed evolution poses a formidable challenge due to the need for exploring a large number of mutations. Additionally, evaluating the improvements in both activity and stability requires screening numerous variants, either individually or using high-throughput screening methods. Here, we utilize instead the design of chimeras as a protein engineering strategy to increase the activity and stability of Mors1, an Antarctic PETase active at 25 degrees C. First, we obtained the crystal structure of Mors1 at 1.6 A resolution, which we used as a scaffold for structure- and sequence-based chimeric design. Then, we designed a Mors1 chimera via loop exchange of a highly divergent active site loop from the thermophilic leaf-branch compost cutinase (LCC) into the equivalent region in Mors1. After restitution of an active site disulfide bond into this chimera, the enzyme exhibited a shift in optimal temperature for activity to 45 degrees C and an increase in fivefold in PET hydrolysis when compared with wild-type Mors1 at 25 degrees C. Our results serve as a proof of concept of the utility of chimeric design to further improve the activity and stability of PETases active at moderate temperatures.
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    Low Carbon Footprint Recycling of Post-Consumer PET Plastic with a Metagenomic Polyester Hydrolase
    (2022) Sonnendecker, Christian; Oeser, Juliane; Richter, P. Konstantin; Hille, Patrick; Zhao, Ziyue; Fischer, Cornelius; Lippold, Holger; Blazquez-Sanchez, Paula; Engelberger, Felipe; Ramirez-Sarmiento, Cesar A.; Oeser, Thorsten; Lihanova, Yuliia; Frank, Ronny; Jahnke, Heinz-Georg; Billig, Susan; Abel, Bernd; Straeter, Norbert; Matysik, Joerg; Zimmermann, Wolfgang
    Earth is flooded with plastics and the need for sustainable recycling strategies for polymers has become increasingly urgent. Enzyme-based hydrolysis of post-consumer plastic is an emerging strategy for closed-loop recycling of polyethylene terephthalate (PET). The polyester hydrolase PHL7, isolated from a compost metagenome, completely hydrolyzes amorphous PET films, releasing 91 mg of terephthalic acid per hour and mg of enzyme. Vertical scanning interferometry shows degradation rates of the PET film of 6.8 mu m h(-1). Structural analysis indicates the importance of leucine at position 210 for the extraordinarily high PET-hydrolyzing activity of PHL7. Within 24 h, 0.6 mg(enzyme) g(PET)(-1) completely degrades post-consumer thermoform PET packaging in an aqueous buffer at 70 degrees C without any energy-intensive pretreatments. Terephthalic acid recovered from the enzymatic hydrolysate is then used to synthesize virgin PET, demonstrating the potential of polyester hydrolases as catalysts in sustainable PET recycling processes with a low carbon footprint.