Dataset Information

Screening Collagenase Activity in Bacterial Lysate for Directed Enzyme Applications.

ABSTRACT: Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity.

SUBMITTER: Tohar R

PROVIDER: S-EPMC8395246 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Polymeric nanoparticles provide a non-invasive strategy for enhancing the delivery of labile hydrophilic enzymatic cargo for neurological disease applications. One of the most common polymeric materials, poly(lactic-co-glycolic acid) (PLGA) copolymerized with poly(ethylene glycol) (PEG) is widely studied due to its biocompatible and biodegradable nature. Although PLGA-PEG nanoparticles are generally known to be non-toxic and protect enzymatic cargo from degradative proteases, different formulation parameters including surfactant, organic solvent, sonication times, and formulation method can all impact the final nanoparticle characteristics. We show that 30s sonication double emulsion (DE)-formulated nanoparticles achieved the highest enzymatic activity and provided the greatest enzymatic activity protection in degradative conditions, while nanoprecipitation (NPPT)-formulated nanoparticles exhibited no protection compared to free catalase. However, the same DE nanoparticles also caused significant toxicity on excitotoxicity-induced brain tissue slices, but not on healthy or neuroinflammation-induced tissue. We narrowed the culprit of toxicity to specifically sonication of PLGA-PEG polymer with dichloromethane (DCM) as the organic solvent, independent of surfactant type. We also discovered that toxicity was oxidative stress-dependent, but that increased toxicity was not enacted through increasing oxidative stress. Furthermore, no PEG degradation or aldehyde, alcohol, or carboxylic acid functional groups were detected after sonication. We identified that inclusion of free PEG along with PLGA-PEG polymer during the emulsification phases or replacing DCM with trichloromethane (chloroform) produced biocompatible polymeric nanoparticle formulations that still provided enzymatic protection. This work encourages thorough screening of nanoparticle toxicity and cargo-protective capabilities for the development of enzyme-loaded polymeric nanoparticles for the treatment of disease.

Project description:For the advancement of porcine xenotransplantation for clinical use in type 1 diabetes mellitus, the concerns of a sustainable and safe digestion enzyme blend must be overcome. Incorporating good manufacturing practices (GMP) can facilitate this through utilizing GMP-grade enzymes. In conjunction, still taking into account the cost-effectiveness, a wide concern. We evaluated how GMP-grade enzyme blends impact our piglet islets and their long-term effects. Preweaned porcine islets (PPIs) were isolated from 8- to 10-day-old pigs. Digestion enzyme blends, collagenase type V (Type V), collagenase AF-1 GMP-grade with collagenase NB 6 GMP-grade (AF-1 and NB 6), and collagenase AF-1 GMP-grade with collagenase neutral protease AF GMP-grade (AF-1 and NP AF) were compared. Islet quality control assessments, islet yield, viability, and function, were performed on days 3 and 7, and cell content was performed on day 7. GMP-grade AF-1 and NB 6 (17,209 ± 2,730 islet equivalent per gram of pancreatic tissue [IE/g] on day 3, 9,001 ± 1,034 IE/g on day 7) and AF-1 and NP AF (17,214 ± 3,901 IE/g on day 3, 8,833 ± 2,398 IE/g on day 7) showed a significant increase in islet yield compared to Type V (4,618 ± 1,240 IE/g on day 3, 1,923 ± 704 IE/g on day 7). Islet size, viability, and function showed comparable results in all enzyme blends. There was no significant difference in islet cellular content between enzyme blends. This study demonstrated a comparison of GMP-grade collagenase enzyme blends and a standard crude collagenase enzyme in preweaned-aged porcine, a novel topic in this age. GMP-grade enzyme blends of AF-1 and NB 6 and AF-1 and NP AF resulted in substantially higher yields and as effective PPIs compared to Type V. In the long run, considering costs, integrity, and sustainability, GMP-grade enzyme blends are more favorable for clinical application due to high reproducibility in comparison to undefined manufacturing processes of standard enzymes.

Project description:BackgroundPolyvalent Mechanical Bacterial Lysate (PMBL®) contains antigens of bacteria responsible for respiratory infections. PMBL® has been proven to reduce the number of respiratory infections, and in its use, immunological benefits have been seen in allergic patients. PMBL® activates both innate and specific immune responses. The lysate induces dendritic cells, T and B lymphocytes and IgA secretion, as well as the production of antibodies directed against administered bacterial antigens. Moreover, it increases the response against other bacteria and viruses. The immunologic mechanism of lysate's action is not yet clearly determined. The objective of this study was to assess the effect of PMBL® on T cells in children with allergic asthma.MethodsThis study was a part of the EOLIA study. Herein, 49 children with allergic asthma and house dust mites allergy were included: 21 in PMBL® and 28 in the Placebo group, both, drug and placebo were administered sublingually. The tests were done at baseline and 12 weeks after the last tablet intake. The lymphocytes CD45+, lymphocytes T CD3+, CD3+CD25+, CD3+CD69+, Th CD3+CD4+, CD4+CD25+, CD4+CD25+ high, CD4+CD69+, Treg CD4+CD25+FOXP3, Tc CD3+CD8+, CD8+CD25+, CD8+CD69+, NK-like T CD3+CD16+CD56+ and NK cells CD3-CD16+CD56+ were described.ResultsAt baseline, no significant differences between groups relative to blood count cells were observed, except for eosinophils. After 12 weeks, we observed an increase of T lymphocytes count. In addition, CD4+CD25+FOXP3+, CD8+ and CD3-CD16+CD56+ and (insignificantly) Th count increased. However, CD69+ and CD25+ subset of CD3+ significantly decreased.ConclusionsThe EOLIA study demonstrated that PMBL® administration 10 days per month for 3 months changed the panel of T lymphocytes. Trial registration Clinical Trial Registration: This study was a part of the EOLIA (Efficacy Of mechanical bacterial Lysate In Allergic children), a clinical study NCT02541331. Frederic Durmont, MD Lallemand Pharma International AG. Date of registration 09/08/2013. URL of trial registry record: https://clinicaltrials.gov/ct2/show/study/NCT02541331.

Dataset Information

Screening Collagenase Activity in Bacterial Lysate for Directed Enzyme Applications.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets