Subtilisin carlsberg

Manufactured by Merck Group

Sourced in United Kingdom, United States, Germany

Subtilisin Carlsberg is a serine protease enzyme produced by the bacterium Bacillus licheniformis. It is a commonly used laboratory reagent due to its broad catalytic activity and stability.

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Subtilisin (44 citations)

7 protocols using subtilisin carlsberg

Quantification of Imidacloprid in Animal Health

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IMD injection (85 mg/100 mL) was provided by Qilu Animal Health Products Corp. Ltd. (Jinan, China). The reference drug (the marketed IMD, 98.5%) was manufactured and provided by J&K Scientific Corp. LTD (Beijing, China). Acetonitrile (ACN) and methanol from Fisher Scientific (Fair Lawn, NJ, USA) were liquid chromatography/mass spectrometer (LC/MS)-grade. Formic acid (HPLC) was purchased from ERA (Austin, TX, USA). Trifluoroacetic acid (AR) and ammonia (AR) were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). The SPE column was purchased from WATERS Oasis (USA). Subtilisin Carlsberg was from Sigma (Poole, Dorset, UK). A cylinder-type microporous filter membrane (WAT200810, 0.22 μm) was obtained from WATERS Oasis (USA). Ultrapure water was prepared by PALL Cascada II I (Show Low, AZ, USA).

Tang Y., Yu N., Liu C., Han M., Wang H., Chen X., Kang J., Li X, & Liu Y. (2022). Residue Depletion of Imidocarb in Bovine Tissues by UPLC-MS/MS. Animals : an Open Access Journal from MDPI, 13(1), 104.

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Subtilisin Carlsberg Digestion and Protein Identification

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Pure protein (5 µg) in 120 µl of 100 mM NaCl, 50 mM Tris, pH 7.5, 1 mM β-mercaptoethanol was digested with subtilisin Carlsberg (Sigma-Aldrich) at 30°C (1∶100 w/w protein:protease). Aliquots of 20 µl were removed at 0, 15, 45, 60, 90, and 120 min, the protease inactivated with 1 µl each of 1 M phenylmethylsulfonyl fluoride (PMSF) and benzamidine, and analyzed in 15% SDS-PAGE gels with Coomassie Blue staining. A 60-min digest of an identical 40 µl sample was divided into two, and subjected to SDS-PAGE; with one sample, subtilisin-resistant bands identified by Coomasie Blue staining were excised from the gel and analyzed by MALDI-TOF mass spectrometry. The other sample was electrotransferred to Immobilon PSQ membrane (Millipore, MA), and the bands excised after staining with Coomasie Blue were subject to N-terminal sequencing.

Gallego-García A., Mirassou Y., García-Moreno D., Elías-Arnanz M., Jiménez M.A, & Padmanabhan S. (2014). Structural Insights into RNA Polymerase Recognition and Essential Function of Myxococcus xanthus CdnL. PLoS ONE, 9(10), e108946.

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Cytoskeletal Protein Preparation Protocol

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EGTA (ethylene glycol-bis(β-amino ethylether)-N,N,N′,N′-tetraacetic acid) Tris (hydroxymethyl)aminomethane), Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), phalloidin, subtilisin Carlsberg, sucrose (Sigma Ultra), dithiothreitol, and ATP were from Sigma Chemical Co. (St Louis, MO, USA). Dansyl ethylenediamine (DED) was purchased from Molecular Probes (Eugene, OR, USA). All other chemicals were of analytical grade. Microbial transglutaminase was a generous gift from Drs. M. Motoki and K. Seguro.

Gruszczynska-Biegala J., Stefan A., Kasprzak A.A., Dobryszycki P., Khaitlina S, & Strzelecka-Gołaszewska H. (2021). Myopathy-Sensitive G-Actin Segment 227-235 Is Involved in Salt-Induced Stabilization of Contacts within the Actin Filament. International Journal of Molecular Sciences, 22(5), 2327.

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Assaying Protease Activity of Hip1 Proteins

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Hip1wt, Hip1SeMet, Hip1S228A, or Hip1T466A proteins (2 μg in 25 μL) were incubated for 30 min at 37 °C before the addition of the substrate. BSA (Thermo Scientific) and the protease Subtilisin Carlsberg (Sigma-Aldrich) were used as negative and positive controls, respectively, and treated the same as the Hip1 proteins. Azocasein (Sigma-Aldrich) was reconstituted in 1× phosphate buffered saline, pH 7.4 (Boston BioProducts), to produce a 5% solution and then passed through a 0.22 μm filter. Addition of the Hip1 proteins, BSA, or the protease Subtilisin Carlsberg resulted in a final concentration of 4% Azocasein in 200 μL of total volume. After incubation for 1 h at 37 °C, the reactions were terminated by the addition of 200 μL of 10% trichloroacetic acid and incubated on ice for 30 min. The reactions were then centrifuged at 13,200 rpm for 10 min at 4 °C. The supernatant (200 μL) was transferred to a 96 well plate to which was added 50 μL of 1.8 N NaOH, and the absorbance was read at 440 nm. The enzyme activities are expressed as units of enzyme/mg protein (one enzyme unit is the quantity of enzyme required to increase the absorbance by 0.01 at 440 nm). The assay was performed in triplicate and graphed using GraphPad Prism 7.0a.

Naffin-Olivos J.L., Daab A., White A., Goldfarb N.E., Milne A.C., Liu D., Baikovitz J., Dunn B.M., Rengarajan J., Petsko G.A, & Ringe D. (2017). Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c). Biochemistry, 56(17), 2304-2314.

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Comprehensive Enzyme and Reagent Sourcing

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Polymerase chain reactions (PCR) were performed with Phusion® Hot Start II High‐Fidelity Green Master Mix from Thermo Fisher Scientific GmbH (Karlsruhe, Germany). Oligonucleotides were synthesized by Eurofins Genomics GmbH (Ebersberg, Germany). Restriction enzymes, T4 DNA Ligase, and GeneRuler™ 1 kb DNA ladder were purchased from Thermo Fisher Scientific GmbH. Peptide protease substrates were purchased from BACHEM (Bubendorf, Switzerland). Azocasein, α‐CHCA (alpha‐cyano‐4‐hydroxycinnamic acid), subtilisin Carlsberg, and Savinase were purchased from Sigma‐Aldrich (Schnelldorf, Germany). BPN' was from DuPont (Wilmington, NC, USA). MALDI‐TOF MS protein standards were purchased from LaserBio Labs (Valbonne, France). Lysozyme from chicken and materials for isoelectric focusing (IEF) were purchased from SERVA (Heidelberg, Germany). Centrifugal spin columns and PMSF (phenylmethylsulfonyl fluoride) were purchased from Avantor VWR (Radnor, PA, USA). Molecular weight marker for use with SDS/PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) was purchased from Bio‐Rad (Hercules, CA, USA). All other chemicals were acquired from Carl Roth (Karlsruhe, Germany).

Falkenberg F., Rahba J., Fischer D., Bott M., Bongaerts J, & Siegert P. (2022). Biochemical characterization of a novel oxidatively stable, halotolerant, and high‐alkaline subtilisin from Alkalihalobacillus okhensis Kh10‐101T. FEBS Open Bio, 12(10), 1729-1746.

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Kinetic Analysis of Subtilisin Enzyme Activity

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The activity of the subtilisin enzyme was tested in a solution-phase with a peptide (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) that, upon proteolysis, releases 4-nitroanaline, which absorbs light at wavelength of 410 nm. Initial substrate concentration was 0.8 mM with an enzyme concentration of 0.12 mg/L. The reaction was conducted at 37°C and a consistent pH of 7.8. pH was maintained by using a 50 mM Phosphate buffer. Activity was characterized with calculated values of K_M and V_max. Wavelength readings were obtained using the UV-6300PC Double Beam Spectrophotometer. Subtilisin Carlsberg was purchased from Sigma Aldrich (P5380) and N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide was purchased from Sigma Aldrich (S7388). 4-nitroanaline (to make standard curve for concentration analysis to test enzyme activity) was purchased from Millipore Sigma (185310).

Mills R., Vogler R.J., Bernard M., Concolino J., Hersh L.B., Wei Y., Hastings J.T., Dziubla T., Baldridge K.C, & Bhattacharyya D. (2022). Aerosol capture and coronavirus spike protein deactivation by enzyme functionalized antiviral membranes. Communications materials, 3(1), 34.

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Enzymatic Characterization of Proteases

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Human neutrophil elastase, cathepsin G, and α₂-macroglobulin were purchased from Athens Research and Technology. Succinyl-A–A–P–F-pNA, batimastat, ecotin, subtilisin Carlsberg, bovine pancreatic trypsin, porcine pancreatic elastase, B. thermoproteolyticus thermolysin, and bovine pancreatic α-chymotrypsin were from Sigma-Aldrich. Serratia sp. serralysin and protealysin, P. aeruginosa LasB and aeruginolysin, F. nucleatum fusolisin, T. denticola dentilysin, and S. gordonii challisin were commercially expressed and purified by Creative Enzymes. Azocoll was from EMD-Millipore, fluorescein-labelled casein (FTC-casein), the PageRuler pre-stained protein ladder (10–180 kDa), restriction endonucleases BamHI and XhoI, and Phusion DNA polymerase were from Thermo Fisher Scientific. The expression vector pGEX-6P-1, glutathione-Sepharose 4 fast flow resin, and 3C (PreScission) protease were from Cytiva. Full-length human MMPs 1, 2, 3, 7, 8, 9, 10, 12, 13, 14, and full-length murine MMP-12 were purchased from R&D Systems Europe. The catalytic domain of human MMP-20 was from Enzo Fischer Scientific. Primers (Table S1†) were synthesized by Genomed. All other chemical reagents, unless stated otherwise, were from BioShop Canada.

Książek M., Goulas T., Mizgalska D., Rodríguez-Banqueri A., Eckhard U., Veillard F., Waligórska I., Benedyk-Machaczka M., Sochaj-Gregorczyk A.M., Madej M., Thøgersen I.B., Enghild J.J., Cuppari A., Arolas J.L., de Diego I., López-Pelegrín M., Garcia-Ferrer I., Guevara T., Dive V., Zani M.L., Moreau T., Potempa J, & Gomis-Rüth F.X. (2022). A unique network of attack, defence and competence on the outer membrane of the periodontitis pathogen Tannerella forsythia. Chemical Science, 14(4), 869-888.

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