Purified T. terrestris LPMO (TtLPMO9E, previously TtGH61E) and T. auranticus (TaLPMO9A) were donated from Novozymes A/S (Denmark). The enzymes are produced by expression in a host organism and subsequently purified. T. fusca AA10 (TfLPMO10A) cloned and expressed in Escherichia coli was purchased from Nzytech Ltd (Portugal). All LPMOs were free of any residual cellulase or hemicellulose activities. Commercial cellulase mixtures Celluclast 1.5l and Novozym 188 were obtained from Novozymes A/S. The Celluclast 1.5l mixture had a protein content of 127 mg g−1, containing 62 filter paper units (FPU) per g, and a β-glucosidase activity of 15 U per g. Novozym 188 had a protein content of 220 mg g−1, containing a β-glucosidase activity of 231 U per g.
Celluclast 1.5 l
Manufactured by Novozymes
Sourced in Denmark, United States
Celluclast 1.5 L is a laboratory-grade enzyme product developed by Novozymes. It is a liquid formulation containing cellulase enzymes derived from the fungus Trichoderma reesei. The core function of Celluclast 1.5 L is to hydrolyze cellulose, the primary structural component of plant cell walls.
Automatically generated - may contain errors
Lab products found in correlation
Novozyme 188, by Novozymes (11 mentions)
Novozym 188, by Novozymes (8 mentions)
Cellic ctec2, by Novozymes (7 mentions)
Viscozyme l, by Novozymes (6 mentions)
Ascorbic acid, by Merck Group (3 mentions)
Glucose cii test kit, by Fujifilm (3 mentions)
Ultraflo l, by Novozymes (3 mentions)
Pectinex ultra sp l, by Novozymes (2 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (2 mentions)
Ethanol, by Thermo Fisher Scientific (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Galacturonic acid, by Merck Group (2 mentions)
Ultraflo max, by Novozymes (2 mentions)
Ultraflo xl, by Novozymes (2 mentions)
Ultimase bwl 40, by Novozymes (2 mentions)
Pectinex ultra tropical, by Novozymes (2 mentions)
Shearzyme plus 2x, by Novozymes (2 mentions)
Alcalase 2.4 l fg, by Novozymes (2 mentions)
Avicel ph 101, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
57 protocols using celluclast 1.5 l
1
Characterization of Fungal LPMOs
2
Carrot Callus Protoplast Isolation
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Protoplast isolation was performed using 300 mg of carrot callus with addition of 3 mL digestion solution (Table S1 ), then immersed into a 55 rpm shaker for 12 h. To find the Celluclast® 1.5 L (Novozymes, Bagsværd, Denmark) concentration for cell wall digestion, various concentrations of Celluclast® 1.5 L were investigated ranging 0–3% (v/v) in the cell wall digestion solution. For effective protoplast isolation, digestion times of 10–14 h were tested using a digestion solution containing 3% Celluclast® 1.5 L. After digestion, the protoplast solution with removed cell wall was filtered through a 100 μm nylon sieve (SPL, Pocheon, Korea) to remove debris and centrifuged at 100× g for 5 min, followed by discarding of the supernatant and retention of the protoplast pellet. The protoplast pellet was gently resuspended in washing solution (W5 solution, Table S1 ) via pipetting and centrifuged at 100× g for 5 min. That washing step was repeated twice. After washing, the protoplast pellet was resuspended in 1 mL of protoplast culture medium (PCM, Table S1 ). Protoplast isolation efficiency was determined using a hemocytometer (Sigma-Aldrich, St. Louis, MO, USA) loaded with 10 µL of resuspended protoplasts, and the protoplasts were counted under an optical microscope (DMi8, Leica, Wetzlar, Germany).
3
Enzymatic Production of Crocetin
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The commercial enzyme source-Celluclast® 1.5 L (batch No. 04070012) obtained from Novozymes (Bagsværd, Denmark) was applied for the hydrolysis of crocins in the production of crocetin. The macroporous resin HPD-100 was purchased from Cangzhou Bonchem Co., Ltd. (Cangzhou, Hebei, China). Crocin (crocetin digentiobiose ester) and geniposide were from Sigma Aldrich (St. Louis, MO, USA).
4
Padina gymnospora Bioactive Extraction
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Brown seaweed Padina gymnospora was obtained from Phu Quoc City (Kien Giang, Vietnam). The seaweed was washed with water and dried in an oven at a temperature of 40 °C for 48 h. The seaweed sample was then ground using a blender and stored at 4 °C for further experiments. Commercial enzymes, Celluclast® 1.5 L (a cellulase that hydrolyses (1,4)-β-D-glycosidic linkages in cellulose and other β-D-glucans; activity of 700 endo-glucanase units (EGU)/g), Pectinex® Ultra SP-L (a polygalacturonase that hydrolyzes (1,4)-α-D-galactosiduronic linkages in pectate and other galacturonans; activity of 3800 polygalacturonase units (PGNU)/mL), and Alcalase® 2.5 L PF (a serine endoprotease that hydrolyses internal peptide bonds; activity of 2.5 Anson unit (AU-A)/g) were provided by Novozymes A/S (Bagsvaerd, Denmark). Ascorbic acid (99%), DPPH, ethanol, methanol, and chloroform were obtained from Sigma-Aldrich (Singapore).
5
Wheat Germ Enzymatic Extraction
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Wheat germ was provided by Sajo DongA One (Dangjin, South Korea). The germ was separated during the milling of Triticum aestivum (Australia Standard White Wheat). Unground wheat germ (50 g) was incubated in 250 mL of water with or without 0.5% Celluclast 1.5L (Novozymes, Bagsvaerd, Denmark) for 24 h at 30 ℃ in a water bath. After centrifugation, the supernatant was freeze-dried using a vacuum freeze-dryer (Eyela, Tokyo, Japan).
6
Nannochloropsis gaditana Enzymatic Extraction
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The lyophilized microalgal biomass of Nannochloropsis gaditana (batch L3250520) was purchased from Cianoalgae SI (Gipuzkoa, Spain). Enzymatic solutions (Viscozyme® L, Celluclast® 1.5 L, Saczyme® Yield) were kindly donated by Novozymes A/S (Bagsvaerd, Denmark). The main components of the enzymatic preparations and their activities are reported in Table 2 .
Chloroform, methanol, and ethanol were purchased from Fisher Scientific GmbH (Wien, Austria). Sodium hydrogen carbonate, potassium hydroxide, Triton X, and dimethylsulfoxide (DMSO) were purchased from Carl Roth (Karlsruhe, Germany). Cell culture media and respective supplements were obtained from Gibco Thermo Fisher Scientific (Waltham, MA, USA) and Szabo Scandic (Vienna, Austria). CellTiter-Blue (CTB) 10× concentrate was purchased from Promega (Waldorf, Germany). Sulforhodamine B sodium salt (SRB) and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich (München, Germany). Invitrogen CyQuant LDH Cytotoxicity Assay Kit was bought from Thermo Fisher Scientific (Waltham, MA, USA). Fatty acid methyl esters standard (Supelco 37 FAME Mix) was from Supelco (Bellefonte, PA, USA).
Chloroform, methanol, and ethanol were purchased from Fisher Scientific GmbH (Wien, Austria). Sodium hydrogen carbonate, potassium hydroxide, Triton X, and dimethylsulfoxide (DMSO) were purchased from Carl Roth (Karlsruhe, Germany). Cell culture media and respective supplements were obtained from Gibco Thermo Fisher Scientific (Waltham, MA, USA) and Szabo Scandic (Vienna, Austria). CellTiter-Blue (CTB) 10× concentrate was purchased from Promega (Waldorf, Germany). Sulforhodamine B sodium salt (SRB) and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich (München, Germany). Invitrogen CyQuant LDH Cytotoxicity Assay Kit was bought from Thermo Fisher Scientific (Waltham, MA, USA). Fatty acid methyl esters standard (Supelco 37 FAME Mix) was from Supelco (Bellefonte, PA, USA).
7
Enzymatic Liquefaction and Saccharification
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All chemicals were of analytical grade. During this work a mixture of the commercial enzyme solutions from Novozymes A/S (Bagsværd, Denmark) Celluclast® 1.5 L (cellulases) and Novozym 188 (β-glucosidase) at a ratio of 5:1 v/v has been applied for the liquefaction/saccharification process. The activity of the mixture was measured according to the standard filter paper assay [49 (link)] and found to be 83 FPU/mL.
8
Enzymatic Hydrolysis of Cellulose and Pectin
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Celluclast 1.5L (cellulase), Novozyme 188 (β-glucosidase) and Pectinex Ultra SP-L (pectinase) preparations were kindly donated by Novozymes A/S (Bagsvaerd, Denmark). The cellulase and β-glucosidase activities of the above enzyme preparations were determined using standard assays (Ghose 1987 ); cellulase activity was expressed as filter paper units, FPU ml−1, whereas the β-glucosidase activity was expressed as international units (IU), where 1 IU is equal to 1 μmol min−1 of substrate converted. Pectinase activity was determined as described elsewhere (Phutela et al. 2005 (link)), using citrus pectin (Sigma-Aldrich, St. Louis, MO, USA) as substrate and galacturonic acid (Sigma-Aldrich) as standard and expressed as units (U) where 1 U is equal to the amount of enzyme required to release 1 μmol min−1 of galacturonic acid equivalents.
9
Saccharification of Pretreated OPEFB Using Cellulase
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Commercial cellulase (Celluclast 1.5 L, Novozymes, Denmark) was used for the saccharification of pretreated OPEFB into fermentable sugar. The saccharification was conducted at 1.5 L working volume in a 2-L bioreactor (EYELA, Japan) with an agitation speed of 150 rpm, the temperature at 35 °C for 96 h. The process was conducted by adding 5% of pretreated OPEFB into the 2-L bioreactor and autoclaved at 121 °C for 15 min. Approximately 1.5 L of sterilized 0.05 M acetate buffer, pH 5.5 was added into the bioreactor. Then, 15 FPU/mL of prepared cellulase solution sterilized via filtration using 0.22 mm sterilized nylon driven filters (Millipore, Denmark) was added into the bioreactor before sparged with nitrogen gas for about 1 h until no oxygen was detected. Samples were collected every 24 h and kept in the freezer of −20 °C for sugar analysis.
10
Cellulose Hydrolysis Protocol
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Cellic® CTec2 and Celluclast 1.5L were provided by Novozymes A/S (Bagsværd, Denmark). An LPMO from Neurospora crassa (NcLPMO9C) was expressed and purified as described by Müller et al. [21 (link)]. Protein concentrations in all enzyme preparations was determined using the Bio-Rad Protein Assay (Bio-Rad, USA), which is based on the Bradford method [39 (link)], using Bovine Serum Albumin (BSA) as standard. Cellic Ctec2 contained 109 mg/mL protein while Celluclast contained 26.7 mg/mL. A mixture with 85% Celluclast 1.5L and 15% purified NcLPMO9C (Cell + 9C) was prepared based on protein ratio and used where needed. Boiled enzymes were prepared by incubating the enzymes at 100 °C for at least 15 min.
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