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.
Novozym 188
Manufactured by Novozymes
Sourced in Denmark
Novozym 188 is a lab equipment product manufactured by Novozymes. It is a cellulase enzyme preparation derived from a selected strain of Trichoderma reesei. The core function of Novozym 188 is to hydrolyze cellulose into glucose.
Automatically generated - may contain errors
Lab products found in correlation
Celluclast 1.5 l, by Novozymes (8 mentions)
Ttlpmo9e, by Novozymes (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Talpmo9a, by Novozymes (1 mentions)
Hydroquinone, by Merck Group (1 mentions)
3 hydroxyanthranilic acid 3haa, by Merck Group (1 mentions)
Abts 2 2 azino bis 3 ethylbenzthiazoline 6 sulfonate, by Merck Group (1 mentions)
Resveratrol, by Merck Group (1 mentions)
Catechin, by Merck Group (1 mentions)
Sinapic acid, by Merck Group (1 mentions)
Caffeic acid, by Merck Group (1 mentions)
Ferulic acid, by Merck Group (1 mentions)
Tannic acid, by Merck Group (1 mentions)
D gluconic acid, by Merck Group (1 mentions)
Almond β glucosidase, by Merck Group (1 mentions)
3 methylcatechol, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
β glucosidase from aspergillus niger, by Megazyme (1 mentions)
Cellic ctec2, by Novozymes (1 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (1 mentions)
12 protocols using novozym 188
1
Characterization of Fungal LPMOs
2
Enzymatic Hydrolysis of Pretreated Bagasse
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The WIS fraction of pretreated bagasse samples was subjected to enzymatic hydrolysis to evaluate the effect of the pretreatment and the differences between sugarcane varieties on the hydrolysis of bagasse. These experiments were conducted in 250 ml Erlenmeyer flasks. The flasks were loaded with 1 g (dry weight) of WIS and 50 ml of 0.05 M citrate buffer (pH 4.8) with the enzyme solution, to give a solids loading of 2% (w/v). Sodium azide was added at a concentration of 0.02% (w/v) to prevent microbial growth. Two commercial enzymes preparations were used: Spezyme CP (Genencor-Danisco, Brabrand, Denmark) with cellulase activity of 65 FPU/ml and Novozym 188 (Novozymes A/S, Bagsvaerd, Denmark) with β-glucosidase activity of 995 IU/ml. Enzyme activities were determined according to Ghose [34 ]. Cellulase loading of 0.2308 mL/ g WIS (15 FPU/g WIS) of Spezyme CP supplemented with β-glucosidase of 0.01508 mL/g WIS (15 IU/g WIS) was applied in all the experiments. Flasks loaded with the mixtures were placed in water bath maintained at 50°C with shaking at 90 rpm. Samples were withdrawn after 72 h and prepared for analysis as described below.
3
Enzymatic Hydrolysis with Cellulolytic Cocktails
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Three enzyme cocktails (Celluclast, Novozym 188, and Cellic CTec2) were used for enzymatic hydrolysis; all were kindly provided by Novozymes A/S (Bagsværd, Denmark). The enzymes were loaded based on activity measurements. The cellulolytic activity in Celluclast and Cellic CTec2 was determined using the filter paper unit (FPU) assay [50 (link)] with some adjustments [51 (link)]. The β-glucosidase activity of Novozym 188 was determined as described previously [52 (link)] with some adjustments [13 (link)]. Protein concentration was measured with the Bradford method using bovine serum albumin to generate a calibration curve [53 (link)]. Prior protein precipitation was performed as described hereafter. For every 200 µL of sample, 20 µL of 500 mM K3PO4, 20 µL of 250 mM CaCl2, and 500 µL of pure ethanol were added. After mixing, the samples were centrifuged at 14,000 rpm for 1 min, the supernatant was removed, and the Bradford assay was performed on the dissolved pellet.
4
Enzymatic Lignocellulose Hydrolysis Reagents
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Ascorbic acid, hydroquinone, 3-hydroxyanthranilic acid (3HAA), resveratrol, catechin, caffeic acid, tannic acid, ferulic acid, sinapic acid, and ABTS (2, 2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) were obtained from Sigma Aldrich (Saint Louis, USA). Stock solutions of 100 mM were made in either water (Ascorbic acid, hydroquinone, ABTS and tannic acid) or pure ethanol (3HAA, resveratrol, catechin, caffeic acid, ferulic acid and sinapic acid) and kept at -20 °C in the dark. Commercial cellulase mixtures Celluclast 1.5L and Novozym188 were obtained from Novozymes A/S, Denmark. The Celluclast 1.5L mixture had a protein content of 127 mg/g, containing 62 FPU/g cellulase activity and 15 U/g β-glucosidase activity. Novozym188 had a protein content of 220 mg/g, containing 231 U/g β-glucosidase activity. Purified Thielavia terrestris LPMO (TtLPMO9E, previously TtGH61E17 ), was obtained from Novozymes A/S. PcLPMO9D (previously known as PcGH61D) from Phanerochaete chrysosporium was cloned and expressed in Pichia pastoris and purified according to34 (link).
5
Regenerated Amorphous Cellulose Preparation
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Regenerated amorphous cellulose (RAC) was prepared from Avicel PH-101 as previously described (Zhang and Lynd 2004 (link); Frommhagen et al. 2015 (link)). d -Glucose, d -gluconic acid, ascorbic acid, and 3-methylcatechol were purchased from Sigma-Aldrich (Steinheim, Germany). d -Cellobionic acid ammonium salt was obtained from Toronto Research Chemicals (Toronto, Ontario, Canada). Almond β-glucosidase was purchased from Sigma-Aldrich and had, according to the supplier’s information, a specific activity of 6 U mg−1 lyophilized powder. Commercial cellulase mixtures Celluclast 1.5 L and Novozym 188 were obtained from Novozymes A/S (Bagsværd, Denmark).
6
Enzymatic Hydrolysis of Cellulose
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TtLPMO9E was donated by Novozymes A/S (Bagsværd, Denmark). The TtLPMO9E concentration was determined by absorbance at 280 nm using a molar extinction coefficient of 58,120 M−1 cm−1. Novozym 188 was obtained from Novozymes A/S (Bagsværd, Denmark). β-glucosidase from Aspergillus niger and cellobiohydrolase I (CBHI) from Trichoderma longibrachiatum were purchased from Megazyme with a specified activity of 40 U ml−1 and a concentration of 10 mg ml−1, respectively.
7
Enzymatic Pretreatment of Forest Residues
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Untreated forest residues were provided by SLU (Umeå, Sweden). The total solids (TS) and volatile solids (VS) of the materials were as follows (w/w): spruce, 90.81% TS and 90.49% VS; pine, 91.45% TS and 91.26% VS; birch, 92.07% TS and 91.86% VS. The thermophilic anaerobic sludge used during this work was collected from a biogas plant in Boden, Sweden, where sewage sludge and food waste are co-digested.
The cellulolytic enzymes used during this work were the commercial enzyme solutions Celluclast® 1.5L and Novozym® 188 (Novozymes A/S, Bagsværd, Denmark) at a ratio of 5:1 v/v. The activity of the mixture was measured to be equal to 83 filter paper units (FPU)/mL. The enzymatic detoxification was performed using a laccase from the fungus Pycnoporus cinnabarinus, which was kindly provided by Beldem (Belgium) with a declared activity of 13 IU/mL.
The cellulolytic enzymes used during this work were the commercial enzyme solutions Celluclast® 1.5L and Novozym® 188 (Novozymes A/S, Bagsværd, Denmark) at a ratio of 5:1 v/v. The activity of the mixture was measured to be equal to 83 filter paper units (FPU)/mL. The enzymatic detoxification was performed using a laccase from the fungus Pycnoporus cinnabarinus, which was kindly provided by Beldem (Belgium) with a declared activity of 13 IU/mL.
8
Enhancing Methane Yields via Thermal and Enzymatic Pretreatment
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During this work two different treatments were applied to improve methane yields, one thermal and one enzymatic, alone or in combination. Thermal treatment was performed using an autoclave apparatus at 105°C for 1 hour with sweet sorghum's concentration of 20% w/w.
During enzymatic treatment, a mixture of the commercial enzyme solutions Celluclast 1.5 L and Novozym 188 (Novozymes A/S, Denmark) at 5 : 1 v/v ratio was used at the same enzyme loading that was previously found optimal for sweet sorghum saccharification during ethanol production [16 ]. Two different process configurations were evaluated during the enzymatic treatment, namely, one-step and two-step processes. In one-step process, the enzymes were directly added in the sludge, whereas in the two-step process sweet sorghum was enzymatically presaccharified prior to the addition to sludge. During the two-step process the saccharification was performed at 50°C for 8.6 hours at 20% w/w DM content. In order to avoid the hydrolysis of sucrose by Novozym 188 endogenous invertase activity and subsequent inhibition of cellulases, the enzymatic solution was added in the startup of anaerobic digestion stage.
During enzymatic treatment, a mixture of the commercial enzyme solutions Celluclast 1.5 L and Novozym 188 (Novozymes A/S, Denmark) at 5 : 1 v/v ratio was used at the same enzyme loading that was previously found optimal for sweet sorghum saccharification during ethanol production [16 ]. Two different process configurations were evaluated during the enzymatic treatment, namely, one-step and two-step processes. In one-step process, the enzymes were directly added in the sludge, whereas in the two-step process sweet sorghum was enzymatically presaccharified prior to the addition to sludge. During the two-step process the saccharification was performed at 50°C for 8.6 hours at 20% w/w DM content. In order to avoid the hydrolysis of sucrose by Novozym 188 endogenous invertase activity and subsequent inhibition of cellulases, the enzymatic solution was added in the startup of anaerobic digestion stage.
9
Efficient Bioethanol Production from Yeast Strains
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The common S. cerevisiae (CSC) was purchased from Angel Yeast Company (YiChang, China). The thermophilic S. cerevisiae (TSC) was kindly provided by Microbiology Department of Beijing Forestry University. Among them, the CSC only utilized glucose, and TSC could mainly exploit glucose as well as next xylose. 3% (w/v) dry yeast with different CSC-to-TSC yeast cell mass ratios of 1:3, 1:2, 1:1, 2:1, and 3:0 (w/w) was activated in a 2% (w/v) glucose solution at 35 °C for 1 h before SSF/SSCF, respectively. Cellulolytic enzymes were Cellic Ctec2 with a cellulase activity of 130 FPU/mL and Novozym 188 with a β-glucosidase activity of 48 IU/mL, respectively, which were both kindly donated by Novozymes A/S (Bagsvaerd, Denmark).
10
Enzymatic Hydrolysis of Cellulosic Biomass
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The washed substrates were enzymatically hydrolyzed using Celluclast 1.5 L (Novozymes, DK) supplemented with β-glucosidase (Novozym 188, Novozymes, DK). Enzymatic hydrolysis was conducted at 2% (w/v) solids in sodium acetate buffer (50 mM, pH 4.8), 50 °C, 120 rpm in a rotatory incubator Combi-H12 (FINEPCR, KR). The total volume was 1.5 mL, in 2.0-mL tubes. The protein loadings, based on the Celluclast protein content (130 mg/mL; 60 FPU/mL), were 2.5, 5, 10, 20, and 30 mg/g of cellulose in the PSCB. Novozym 188 (458 UI/mL) was added in order to have a β-glucosidase activity 2 times higher than the filter paper activity in the reaction mixture. After 72 h, the enzymes were inactivated by heating at 100 °C for 5 min. The glucose concentration in the hydrolysate was measured using the glucose oxidase assay [56 (link)]. Hydrolyses were analyzed in triplicates and the standard deviation was calculated.
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