Alginate lyase

Manufactured by Merck Group

Sourced in United States, Japan, United Kingdom

Alginate lyase is an enzyme that cleaves the glycosidic bonds in alginate, a polysaccharide found in the cell walls of brown algae. It catalyzes the depolymerization of alginate, breaking down the polymer into smaller units. The core function of alginate lyase is to facilitate the degradation and modification of alginate molecules.

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Lab products found in correlation

Trypsin edta, by Thermo Fisher Scientific (6 mentions) Sodium alginate, by Merck Group (5 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Collagenase, by Merck Group (2 mentions) 3d discovery printer, by RegenHU (2 mentions) Biocad drawing software, by RegenHU (2 mentions) Biocam software, by RegenHU (2 mentions) Eclipse ti, by Nikon (2 mentions) Eclipse ni e, by Nikon (2 mentions) Triton x 100, by Merck Group (2 mentions) Fetal bovine serum (fbs), by Merck Group (2 mentions) Blebbistatin, by Cayman Chemical (2 mentions) Y 27632, by Cayman Chemical (2 mentions) Fitc dextran, by Merck Group (2 mentions) Collagenase p, by Merck Group (2 mentions) Trifluoroacetic acid, by Thermo Fisher Scientific (2 mentions) Triisopropylsilane, by Merck Group (2 mentions) Ethanedithiol, by Merck Group (2 mentions) Nb maleimidopropionic acid hydrazide bmph, by Thermo Fisher Scientific (2 mentions)

51 protocols using alginate lyase

Alginate Oligosaccharide Production Protocol

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To prepare Type 1 AOs, Type 2 AOs, and Type 3 AOs, alginate lyase (Sigma-Aldrich, A1603) was used to catalyze the cleavage of alginate. To make Type 1 AOs, 2% (w/v) of sodium alginate (Sigma-Aldrich, A0682) was prepared by dissolving sodium alginate in 100 ml of distilled water (DW) followed by incubation at 37 °C for 6 h in the presence of 20 U/mg alginate lyase in a rotary shaker at 150 rpm. After the incubation, alginate lyase in the sample was inactivated at 100 °C for 10 min and removed after centrifugation at 12,000×g for 10 min at 4 °C^{33 (link)}. Type 2 and Type 3 AOs were prepared with the same method except that alginate lyase treatment time was changed from 6 to 12 h for Type 2 AOs and 24 h for Type 3 AOs. Fractions of AOs were collected, freeze-dried, and store at − 4 °C.

Park R.M., Nguyen N.H., Lee S.M., Kim Y.H, & Min J. (2021). Alginate oligosaccharides can maintain activities of lysosomes under low pH condition. Scientific Reports, 11, 11504.

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Antimicrobial Susceptibility Testing Protocol

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MICs were determined by broth microdilution in cation-adjusted Mueller-Hinton broth (CA-MHB; BD Bioscience, Franklin Lakes, NJ, USA) [5] and RPMI-1640 (Invitrogen, Paisley, UK) adjusted to pH 7.4 and complemented with 10% fetal calf serum [8] . MICs were also measured in the presence of 1) 20 mg•L -1 Phe-Arg-β-naphthylamide (PAβN; a broad-spectrum efflux pump inhibitor) and 1 mM MgSO 4 (to strengthen the outer membrane and thereby limit PAβN toxicity [20] ), or 2) 20 U•mL -1 alginate lyase, 0.02% DNase and 20 mM MgCl 2 (all from Sigma-Aldrich, St Louis, MO, USA), or 100 mg•L -1 Proteinase K (Thermo Fisher, Waltham, MA, USA).

Mustafa M.H., Khandekar S., Tunney M.M., Elborn J.S., Kahl B.C., Denis O., Plésiat P., Traore H., Tulkens P.M., Vanderbist F, & Van Bambeke F. (2017). Acquired resistance to macrolides in Pseudomonas aeruginosa from cystic fibrosis patients. The European respiratory journal, 49(5).

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Isolation and Viability Evaluation of Primary Rat Hepatocytes

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Counting primary rat hepatocytes and evaluating their viability per cell fiber were performed three times and averaged. Prior to those procedures, the alginate shells were removed, and the primary rat hepatocytes were retrieved. To remove the shells of the cell fibers, 4 mg mL⁻¹ of alginate lyase (Sigma Aldrich) in Dulbecco's Phosphate-Buffered Saline (DPBS) (+) was added at a 1:100 ratio to the culture media, and the media were incubated for 15 min. To retrieve primary rat hepatocytes from ECM, 4 mg mL⁻¹ of collagenase (Sigma-Aldrich) in DPBS (+) was added at a 1:50 ratio to the culture media and the media were incubated for 5 min. The cell number of primary rat hepatocytes per cell fiber was counted by using cell-counting plate (WakenBtech, Japan). The cellular viabilities were examined by the trypan blue dye-exclusion test.

Mazari-Arrighi E., Okitsu T., Teramae H., Aoyagi H., Kiyosawa M., Yano M., Chatelain F., Fuchs A, & Takeuchi S. (2022). In vitro proliferation and long-term preservation of functional primary rat hepatocytes in cell fibers. Scientific Reports, 12, 8813.

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3D Bioprinting of Anatomical Heart Structures

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Support media were vortexed vigorously, briefly centrifuged to remove air bubbles and transferred into a transparent, open sterile plastic box. Printing was performed within one hour from vortexing, unless indicated otherwise. Constructs were printed using 3DDiscovery® printer (regenHU, Villaz-Saint-Pierre, Switzerland) by extrusion (through 30G needles, unless stated otherwise) according to designs generated by BioCAD™ drawing software (regenHU) or according to data from STL files (sliced and processed by BioCAM™ software (regenHU)) which were downloaded from Thingiverse (www.thingiverse.com “Anatomical Human Heart” by 517860 (modified), under the Creative Commons - Attribution - Share Alike license - CC BY-SA 3.0 https://creativecommons.org/licenses/by-sa/3.0/). Printouts were visualized by an inverted fluorescence microscope (Nikon Eclipse TI) or an upright confocal microscope (Nikon ECLIPSE NI-E). The printing process was captured from a side-view using a long working distance Dino-Light digital microscopes (AnMo Electronics, Taiwan). For extraction of the printed structures from the support medium, the box was incubated at 37°C for 30 min to cure the bioink. Then, the support medium was supplemented with alginate lyase (Sigma-Aldrich, 1U/ml) and incubated at 37°C. The digested support medium was gradually aspirated and replaced with growth medium.

Shapira A., Noor N., Oved H, & Dvir T. (2020). Transparent support media for high resolution 3D printing of volumetric cell-containing ECM structures. Biomedical materials (Bristol, England), 15(4), 045018.

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Hydrogel Cell Isolation with Enzymes

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Hydrogels were digested with 100–150 μl of 300 U ml⁻¹ collagenase IV (Worthington) and 34 U ml⁻¹ of alginate lyase (Sigma-Aldrich) in DBPS + Ca/Mg, 0.5% BSA at 37 °C for 15 min, followed by addition of another 100–150 μl of 300 U ml⁻¹ collagenase IV and incubation at 37 °C for 15 min. Next, 0.5 ml wash buffer (DPBS without Ca/Mg, 2 mM EDTA, 0.5% BSA) was added, and the samples were transferred to a deep-bottom 1.2 ml 96-well plate. The plate was centrifuged at 400g for 5 min at 4 °C and the supernatant discarded. A final set of washes was performed with cold 0.2 ml wash buffer on ice, followed by centrifugation at 400g for 5 min at 4 °C to obtain a clean cell pellet. Cell counts and viabilities were measured by viability staining and flow cytometry (MUSE) to confirm successful isolation and to quantify cell yields.

Green R.M., Seth A, & Connell N.D. (2000). A Peptide Permease Mutant of Mycobacterium bovis BCG Resistant to the Toxic Peptides Glutathione and S-Nitrosoglutathione. Infection and Immunity, 68(2), 429-436.

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Biofilm Water Loss Quantification

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We grow biofilms as we do for rheology. We spread 250μL from liquid overnight onto LB agar plates of standard size, 100mm x 15mm, and let these grow overnight at 37°C. The biofilm is then gently scraped from the plate onto a weigh boat. For ΔmucA (Alg+) biofilms, we add 100μL of treatment such that the concentration is 200 U/mL alginate lyase (Sigma) in deionized water and for ΔwspF Δpsl (Pel+) biofilms, we add 100μL of treatment such that the concentration is 500 U/mL deoxyribonuclease I from bovine pancreas (Sigma) in 0.15M NaCl. The weigh boat with biofilm and treatment is then weighed. The biofilm is moved from the weigh boat onto a clean LB agar plate, which acts as a sink for the excess water in the biofilm. This parallels treatment conditions for our rheology measurements, and we found that this gave rise to faster drying than allowing the biofilm to dry on a weigh boat without an agar sink. This plate is then moved to an incubator at 37°C for one hour. After the hour, the biofilm is returned to its weigh boat and weighed. The alteration in weight is then calculated as water loss by

% W a t e r L o s s = \frac{H y d r a t e d w e i g h t - D r i e d W e i g h t}{H y d r a t e d W e i g h t} \times 100.

Kovach K.N., Fleming D., Wells M.J., Rumbaugh K.P, & Gordon V.D. (2020). Specific Disruption of Established P. aeruginosa Biofilms Using Polymer-Attacking Enzymes. Langmuir : the ACS journal of surfaces and colloids, 36(6), 1585-1595.

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Phenolic Biopolymer Synthesis and Characterization

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Sodium alginate (MW: 70,000) was obtained from Kimica (Tokyo, Japan). Alginate lyase (from Sphingobacterium multivorum), gelatin (type A from porcine skin), and amylopectin (from maize) were purchased from Sigma-Aldrich (St Louis, MO, USA). Lecithin (from soybean), horseradish peroxidase (HRP), and H₂O₂ aqueous solution [30% (w/w)] were obtained from Wako Pure Chemical Industries (Osaka, Japan). Liquid paraffin and tyramine hydrochloride were purchased from Tokyo Chemical Industry (Tokyo, Japan). The derivatives of alginate, gelatin, and amylopectin possessing phenolic hydroxyl moieties (denoted as Alg-Ph, gelatin-Ph, and AP-Ph, respectively) were synthesized based on reported methods by the conjugation with tyramine hydrochloride using carbodiimide for synthesis of Alg-Ph [23] (link) and gelatin-Ph [24] (link), and carbonylimidazole for synthesis of AP-Ph [25] . The amounts of Ph moieties in Alg-Ph, gelatin-Ph, and AP-Ph were 1.5 × 10⁻⁴ mol Ph/g alginate, 9.4 × 10⁻⁵ mol Ph/g gelatin, and 3.6 × 10⁻⁶ mol Ph/g amylopectin, respectively.

Liu Y., Sakai S, & Taya M. (2016). Engineering tissues with a perfusable vessel-like network using endothelialized alginate hydrogel fiber and spheroid-enclosing microcapsules. Heliyon, 2(2), e00067.

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Alginate-Based Microparticle Fabrication

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Sodium alginate was purchased from Acros Organics (Geel, Belgium). Calcium chloride dihydrate (CaCl₂•2H₂O) was purchased from Fisher Scientific (Nepean, ON, Canada). Alginate lyase (≥10,000 units/g), bis-(2-ethylhexyl) sulfosuccinate sodium salt (AOT), toluene, ethanol (95%), acetone, and hydrochloric acid were purchased from Sigma-Aldrich (Markham, ON, Canada).

Van Bavel N., Lewrenz A.M., Issler T., Pang L., Anikovskiy M, & Prenner E.J. (2023). Synthesis of Alginate Nanoparticles Using Hydrolyzed and Enzyme-Digested Alginate Using the Ionic Gelation and Water-in-Oil Emulsion Method. Polymers, 15(5), 1319.

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Visualizing Angiogenesis in HUVEC Scaffold

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To observe migration, morphology, and angiogenesis of the embedded HUVEC, F-actin, CD31, and nuclei were stained using rhodamine-phalloidin, anti-CD31, and DAPI, respectively. First, the formulated scaffold was fixed with 4% paraformaldehyde (P6148, Sigma-Aldrich, U.S.A.) for 40 min at room temperature (RT). The fixed scaffold was immersed in alginate lyase (Sigma-Aldrich, U.S.A.) solution to remove alginate at 37°C. The alginate removed HUVEC core was immersed in a collagen matrix and incubated at 37°C for gelation. Subsequently, the HUVEC core in collagen was permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, U.S.A.) for 5 min at RT. Primary antibody of anti-CD31 (MA5-13188, Invitrogen, U.S.A.) was incubated at 4°C overnight. Then, secondary antibodies (Alexa Fluor 488, Invitrogen, U.S.A.) and Phalloidin (Alexa Fluor 488, Invitrogen, U.S.A.) were applied for 2 h at RT. Besides, nuclei of the HUVEC core were stained with DAPI (D1396, Invitrogen, U.S.A.) for 5 min. After every chemical treating step, the treated sample was washed 3 times with PBS for 5 min. The stained samples were observed using an IX53 inverted fluorescent microscope (Olympus, Japan) and a FV1000 laser scanning confocal microscope (Olympus, Japan).

Nguyen C.T., Duong V.T., Hwang C.H, & Koo K.I. (2022). Angiogenesis in Free-Standing Two-Vasculature-Embedded Scaffold Extruded by Two-Core Laminar Flow Device. International Journal of Bioprinting, 8(3), 557.

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Quantification of Alginate Production

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Production of alginate by CPA-7 was evaluated using supernatants from overnight cultures in TSB at 25 °C. Alginate detection was carried out using the Periodic acid Schiffs (PAS) procedure adapted to 96-well microplate format (Houghton et al., 2014) .
Cell-free supernatants, cell pellets and fresh medium were subjected to hydrolysis with alginate-lyase (0.05 mg/mL) (Sigma-Aldrich, St Louis, USA) for 2 h at 30 °C.
Quantification of the exopolysaccharides that were present in the samples before and after hydrolysis was carried out by interpolating OD 550nm values in a sodium alginate (PubChem CID: 5102882) (Sigma-Aldrich, St Louis, USA) standard curve. Three independent assays including three replicates of each sample were performed.

Collazo C., Abadias M., Aguiló-Aguayo I., Alegre I., Chenoll E, & Viñas I. (2017). Studies on the biocontrol mechanisms of Pseudomonas graminis strain CPA-7 against food-borne pathogens in vitro and on fresh-cut melon. Lebensmittel-Wissenschaft + [i.e. und] Technologie. Food science + technology. Science + technologie alimentaire, 85.

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