Cellic ctec2

Manufactured by Merck Group

Sourced in United States

Cellic CTec2 is a cellulase enzyme complex developed by Novozymes, a subsidiary of Merck Group. It is designed to facilitate the hydrolysis of cellulose, a key component of lignocellulosic biomass, during the process of biofuel production or other industrial applications.

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Lab products found in correlation

19 protocols using cellic ctec2

Poplar Biomass Pretreatment and Enzymatic Hydrolysis

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Poplar processing
residues as woody biomass were collected from a pilot-scale plant
located in the campus of Washington State University in Pullman, WA,
USA. The poplar samples were dried at 105 °C in an oven for 48
h and cut, ground, and sieved through 60 mesh size sieving. The resultant
poplar samples were stored at 4 °C prior to use. Enzymes of Cellic
CTec2 and Cellic HTec2 were obtained from Sigma (cellulase from Aspergillus niger; catalogue no. C1184). The total
cellulase activity of commercial cellulase was 100 FPU g^–1 based on the filter paper assay.

Shi F., Wang Y., Davaritouchaee M., Yao Y, & Kang K. (2020). Directional Structure Modification of Poplar Biomass-Inspired High Efficacy of Enzymatic Hydrolysis by Sequential Dilute Acid–Alkali Treatment. ACS Omega, 5(38), 24780-24789.

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Enzymatic Hydrolysis of Switchgrass Biomass

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Around 50 mg (DM) of raw and extract-free SMS was suspended in 900 µL of 50 mM sodium citrate buffer (pH 5.2) at a 5% (w/w) solids content in Eppendorf tubes. The tubes with the suspensions were mixed at 100 rpm for one hour in a Thermo Scientific compact digital waving rotator (Thermo Fischer Scientific, Waltham, MA, USA) placed in a Termaks B4115 incubator set at 45 °C. After that, 50 µL of a previously prepared stock solution of the cellulase blend Cellic CTec2 (Sigma-Aldrich, Steinheim, Germany) was added at a load of 80 CMCase units/g biomass. The reaction mixtures were then incubated under the same conditions for 72 h. Enzyme-free substrate controls and substrate-free enzyme blanks were run in parallel. After elapsing the reaction time, the resulting slurry was separated using a Heraeus Pico 21 centrifuge (Thermo Fisher Scientific, Osterode, Germany) at 20,200× g for 5 min at 4 °C. The supernatant, hereafter referred to as hydrolysate, was transferred to new Eppendorf tubes and stored frozen until further analysis, and the solid material was discarded. Glucose in the hydrolysates was determined by high-pressure liquid chromatography (HPLC). Glucose concentration was used for calculating the enzymatic digestibility. The enzymatic digestibility was calculated as the mass percentage of the cellulose contained in the AES assay saccharified to glucose.

Klausen S.J., Falck-Ytter A.B., Strætkvern K.O, & Martin C. (2023). Evaluation of the Extraction of Bioactive Compounds and the Saccharification of Cellulose as a Route for the Valorization of Spent Mushroom Substrate. Molecules, 28(13), 5140.

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Steam Explosion Pretreatment of Cardoon Biomass

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Defatted cardoon pretreated by steam explosion
(EC) was provided by University of Perugia. It was collected
from the same batch of EC used in previous work.^{25 (link)} The particle size of the EC fibers was in the range of
2.0–6.0 mm. Analytical grade solvents such as ammonium hydroxide
solution 30% (NH₃ 30% solution), anhydrous absolute ethanol
(EtOH), 2-methyltetrahydrofuran (MeTHF), ethyl acetate (EtOAc), cyclohexane,
chloroform (CHCl₃), anhydrous pyridine, dimethylsulfoxide
(DMSO), and citric acid were purchased from Sigma-Aldrich. Ammonium
hydroxide solution 10% (NH_3,aq 10% solution) was obtained
by dilution of commercial ammonium hydroxide solution 30% with deionized
water. Sulfuric acid 97% (H₂SO₄) and Whatman
glass microfiber filters (grade GF/A) were purchased from Sigma-Aldrich.
Reagents such as cholesterol, 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane
(TMDP), and chromium(III) acetylacetonate were also purchased from
Sigma-Aldrich. DMSO-d₆ and CDCl₃ for NMR spectra with 99.9 atom % D-enrichment were purchased from
Acros Organics.
The enzymatic mixture Cellic CTec3 HS was kindly
provided by Novozymes (Denmark) and used as received. The enzyme Cellic
CTec2 was purchased from Sigma-Aldrich.

D’Orsi R., Di Fidio N., Antonetti C., Raspolli Galletti A.M, & Operamolla A. (2023). Isolation of Pure Lignin and Highly Digestible Cellulose from Defatted and Steam-Exploded Cynara cardunculus. ACS Sustainable Chemistry & Engineering, 11(5), 1875-1887.

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Lignocellulosic Biomass Conversion Protocol

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Sodium hydroxide (NaOH), citric acid monohydrate, ammonium sulfate, dibasic potassium phosphate, magnesium sulfate heptahydrate, hydrochloric acid (HCl), and sulfuric acid (H₂SO₄) were purchased from Duksan Chemical (Korea). Acetic acid, formic acid, 5-hydroxymethylfurfural (5-HMF), furfural, Celluclast 1.5 L, Cellic CTec2, glucose, and corn steep liquor (CSL) were purchased from Sigma-Aldrich (USA). Yeast extract and peptone were purchased from Difco (USA). All chemicals and reagents used in this study were of analytical grade.

Lee J., Lee K.H., Kim S., Son H., Chun Y., Park C, & Yoo H.Y. (2022). Microbial Production of Bacterial Cellulose Using Chestnut Shell Hydrolysates by Gluconacetobacter xylinus ATCC 53524. Journal of Microbiology and Biotechnology, 32(11), 1479-1484.

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Enzymatic Hydrolysis Optimization for Ruminal Fermentation

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The exogeneous cellulase (Cellic CTec2) used in the ruminal fermentation is purchased from Sigma-Aldrich (St. Louis, MO, United States). The 10 FPU/(g dry substance) enzyme loading was optimized in previous enzymatic hydrolysis experiments (Zhang et al., 2018 (link)). All hydrolysis conditions were the same as those of the experiments in the above ruminal fermentation (Sub-Section 2.5). The VFA and glucose contents in the supernate were measured after hydrolysis. The glucose yields were analyzed based on the standard method of NREL (NREL/TP-510-42623) using an HPLC system equipped with a Bio-Rad HPX-87H column (Sluiter et al., 2008 ). The mobile phase was 0.005M H₂SO₄ with a flow rate of 0.6 mL/min at the column temperature of 55 C and the detection time for 30 min.

Zhang H., Zhang W., Wang S., Zhu Z, & Dong H. (2024). Microbial composition play the leading role in volatile fatty acid production in the fermentation of different scale of corn stover with rumen fluid. Frontiers in Bioengineering and Biotechnology, 11, 1275454.

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Enzymatic Hydrolysis of Biomass

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Enzymatic hydrolysis of the solid fraction was done for both biomasses. The biomass (2% w/v, 100 mL total volume) was mixed with 0.05 M sodium citrate buffer (pH 4.8) in Erlenmeyer flasks. Then, 50 FPU per g biomass of enzyme (Cellic CTec2, Sigma Aldrich code SAE0020) were added to the flasks in order to initiate the reactions. The experiments were maintained in an incubator at 50 °C and 200 rpm, for 72–96 h. At the end of the saccharification, the hydrolysis was stopped by heating at 100 °C and the samples were centrifuged (15 000 rpm, 10 min) and washed two times. The solid residue was analyzed according to the NREL Laboratory Analytical Procedure.⁴¹

Vázquez V., Giorgi V., Bonfiglio F., Menéndez P., Gioia L, & Ovsejevi K. (2023). Lignocellulosic residues from bioethanol production: a novel source of biopolymers for laccase immobilization. RSC Advances, 13(20), 13463-13471.

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Corn Stover Depolymerization and Hydrolysis

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The corn stover was collected from Lianyungang City, China. It was firstly cut and sieved to a particle size of ~ 0.4 mm. The fine corn stover was then dried in an oven (GZX-9140MBE, Shanghai Boxun Medical Biological Instrument Corp., China) at 60 °C for 12 h to remove the moisture and stored in plastic bags at 4 °C. Three kinds of inorganic acids, including dibasic acid (hydrochloric acid, 37 wt%), binary acid (sulfuric acid, 98 wt%), and ternary acid (phosphoric acid, 85 wt%) were used as pretreatment agents for corn stover depolymerization. The inorganic acids were purchased from Sinopharm Chemical Reagent Co., Ltd. All of the chemicals were used as received without other specified purification. A commercial cellulase Cellic CTec2 (enzyme blend, SAE0020-50 mL solution) was obtained from Sigma-Aldrich (St. Louis, MO, USA), and used for hydrolyzing the pretreated corn stover to obtain glucose.

Luo H., Gao L., Liu Z., Shi Y., Xie F., Bilal M., Yang R, & Taherzadeh M.J. (2021). Prediction of phenolic compounds and glucose content from dilute inorganic acid pretreatment of lignocellulosic biomass using artificial neural network modeling. Bioresources and Bioprocessing, 8(1), 134.

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Enzymatic Degradation of Cellulose

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Cellulose Arbocel^® was kindly supplied by JRS (Rosenberg, Germany). Two samples of cellulose were employed, with average fibers length of 700 and 200 µm. These samples are indicated in the text as C700 and C200, respectively. Carboxymethyl cellulose (CMC) was purchased from Sigma (St. Louis, MO, USA). The enzymes used in this work were either single enzymes (all from Megazyme, Wicklow, Ireland) i.e., endo-1-4-beta-D-glucanase (Thermotoga maritima, Megazyme), endo-1-4-beta-D-glucanase (Bacillus amyloliquefaciens, Megazyme,), endo-1,4-β-D-glucanase (Thermobifida halotolerans, Megazyme) or enzymatic cocktails (all from Sigma, St. Louise, Missouri, USA), i.e., cellulases from Trichoderma reesei ATCC 26921, Viscozyme^® L, and Cellic Ctec2. Polyethylene flexible packaging films (PE) were supplied by Manuli Films (Sessa Aurunca, Italy).

Aulitto M., Castaldo R., Avolio R., Errico M.E., Xu Y.Q., Gentile G, & Contursi P. (2023). Sustainable and Green Production of Nanostructured Cellulose by a 2-Step Mechano-Enzymatic Process. Polymers, 15(5), 1115.

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Heterologous Expression and Purification of CDH

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CDH (EC 1.1.99.18) from Phanerochaete
chrysosporium was heterologously expressed in Trichoderma reesei and purified as described in a
previous study.^{20 (link)} The RZ value (A₄₂₀/A₂₈₀) was 0.62
and indicated homogeneous enzyme preparation, exhibiting a specific
activity of 17.5 U mg^–1 determined with substrate
cellobiose and 2,6-dichloroindophenol as the electron acceptor at
pH 5.0. Cellulase from T. reesei (Cellulase,
9012-54-8, 0.7 U mg^–1), β-glucosidase from Aspergillus niger (9033-06-1, 0.75 U mg^–1), Cellic CTec2 (Cellulase, enzyme blend, ∼1.15 g/mL), and
GOx (9001-37-0) from A. niger with
a specific activity of 100–250 U mg^–1 were
all purchased from Sigma-Aldrich.

Chang H., Wohlschlager L., Csarman F., Ruff A., Schuhmann W., Scheiblbrandner S, & Ludwig R. (2021). Real-Time Measurement of Cellobiose and Glucose Formation during Enzymatic Biomass Hydrolysis. Analytical Chemistry, 93(21), 7732-7738.

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Characterization of Industrial Hemp

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Industrial hemp was cultivated at the Chicken of the Woods Permaculture Farm (Gaithersburg, MD, 39°07′02.4′′N latitude, 77°16′46.9′′W longitude, https://chickenwoods.com) in 2019, and they kindly donated hemp stems and branches after harvest and extraction of flowers for their market business. Hemp samples were dried in an oven overnight at 50 °C and ground using a Wiley mill with a 30-mesh screen (0.595 mm). The milled stem and hemp were then collected and oven-dried for further use. The moisture content of each sample was measured (<3%) using a Halogen moisture analyzer (Mettler Toledo HB 43, Columbia, OH, USA). Cellic CTec2 (enzyme blend, SAE0020) and Cellulase (Celluclast 1.5L, C2730) were purchased from Sigma-Aldrich (St. Louis, MO). Multipect pectinase enzyme was provided from Genencor, Danisco Division (Palo Alto, CA). All chemical reagents such as hydrochloric acid and 72% sulphuric acid (H₂SO₄) were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated.

Kim D., Yoo C.G., Schwarz J., Dhekney S., Kozak R., Laufer C., Ferrier D., Mackay S., Ashcraft M., Williams R, & Kim S. (2021). Effect of lignin-blocking agent on enzyme hydrolysis of acid pretreated hemp waste. RSC Advances, 11(36), 22025-22033.

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