A diluted alkali pretreatment was applied using a slight modification of the method of Santoro et al. [35 ]. Weighed AIR powder (1.5‒2.0 mg) was mixed with 870 μL of 100 mM NaOH solution and incubated for 2 h at 90 °C. After cooling to room temperature, the solution was neutralized with 20 μL of 30% (v/v) HCl, and saccharification was then carried out with 110 μL of neutralized solution containing cellulase [100 mM citrate (pH 4.5), 0.02% (w/v) sodium azide, 0.2 CPU cellulase (Celluclast® 1.5 L, Merck Inc.), and 0.4 CBU cellobiase (Novozyme 188, Merck Inc.)]. After incubation for 24 h at 50 °C with shaking at 200 rpm, the amount of liberated glucose and xylose was determined with a Glucose CII test kit (Wako Inc.) and Xylose Assay kit (Megazyme Inc.), respectively. For non-pretreated samples, AIR powder was digested with 1,000 μL of cellulase solution [10 mM citrate (pH 4.5), 0.02% (w/v) sodium azide, 0.2 CPU cellulase (Celluclast® 1.5 L, Merck Inc.), and 0.4 CBU cellobiase (Novozyme 188, Merck Inc.)] without any pretreatment.
Novozyme 188
Manufactured by Merck Group
Sourced in United States
Novozyme 188 is a commercial enzyme preparation derived from the fungus Aspergillus niger. It is a multi-functional enzyme with a primary activity as a β-glucosidase, capable of hydrolyzing cellulose to glucose.
Automatically generated - may contain errors
Lab products found in correlation
Celluclast 1.5 l, by Merck Group (13 mentions)
Celluclast, by Merck Group (5 mentions)
Cellic ctec2, by Novozymes (2 mentions)
Avicel, by Merck Group (2 mentions)
Initiator exp, by Biotage (1 mentions)
Gopod assay kit k gluc, by Megazyme (1 mentions)
K xylose, by Megazyme (1 mentions)
Atcc 26921, by American Type Culture Collection (1 mentions)
Thermomixer comfort, by Eppendorf (1 mentions)
Glucoamylase, by Merck Group (1 mentions)
Sulfuric acid, by Merck Group (1 mentions)
α amylase, by Merck Group (1 mentions)
Ecotron incubator shaker, by Infors (1 mentions)
Gopod assay kit, by Megazyme (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Cellic ctec2, by Merck Group (1 mentions)
Avicel ph 101, by Merck Group (1 mentions)
Restriction endonuclease, by Takara Bio (1 mentions)
Salicin, by Merck Group (1 mentions)
Restriction endonuclease, by Promega (1 mentions)
33 protocols using novozyme 188
1
Alkali Pretreatment and Saccharification Protocol
2
Microwave-Assisted Acid Pretreatment and Enzymatic Hydrolysis
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Rough wood powder was fractionated and processed as described in Additional file 12 . Acid pretreatment was performed with 1% sulphuric acid (w/w) using a single-mode microwave system (Initiator Exp, Biotage, Uppsala, Sweden) at 165 °C for 10 min. Enzymatic hydrolysis was performed at 45 °C for 72 h with 50 mg of a 1:1 (w/w) mixture of Cellulase 1.5 L (a cellulase-rich liquid enzyme preparation from Trichoderma reesei ATCC 26921 with a stated activity of 700 endoglucanase units per g) and Novozyme 188 (a cellobiase-rich liquid enzyme preparation from A. niger with a stated activity of 250 cellobiase units per g (both from Sigma Aldrich). The liquid fractions were analyzed using high-performance anion-exchange chromatography (HPAEC), as previously described [37 (link)].
3
Enzymatic Hydrolysis of SECS Biomass
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Accellerase 1500 was a generous gift from Genencor (Jiangsu, China). Novozyme 188 was purchased from Sigma-Aldrich (St Louis, Missouri, United States). The filter paper unit of Accellerase 1500 is 77 FPU/mL, while xylanase and β-xylosidase is 72 IU/mL and 23 IU/mL, respectively. The β-glucosidase activity of Novozyme 188 is 250 p-nitrophenol-β-D- glucopyranoside units (pNPGU)/mL. SECS was hydrolyzed at different glucan loadings in a 0.05 M citrate buffer solution (pH 4.8) with an Accellerase 1500 loading of 15 FPU, 30 FPU, or 60 FPU/g glucan and a β-glucosidase loading of 64 pNPGU/g glucan. The experiments were conducted at 50°C and 200 rpm for 168 hours. Hydrolyzate was collected by centrifuging at 10,000 rpm for 10 minutes. The residues were washed with a volume of water equal to 15 times the dry weight of initial SECS. Composition in hydrolyzate and washed liquid were analyzed using HPLC. All these experiments were conducted with two replicates. Glucan conversion was calculated based on that glucan dissolved into the liquor divided by glucan content in SECS.
4
Enzymatic hydrolysis of pretreated biomass
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Enzymatic hydrolysis of raw YP and pretreated solids was conducted in triplicate on a shaking incubator at 150 rpm and 50 °C following NREL standard protocols27 . A substrate amount equivalent to 0.1 g glucan (based on dry weight) was loaded into a 15 mL glass vial. Sodium azide solution (100 µL of 20 mg/mL) was added as an antimicrobial. Citrate buffer (50 mM, pH 4.8) was added to make the final liquid volume of 10 mL. The slurry was put into the incubator at 50 °C for 1 h before addition of enzyme. Cellulase (Celluclast 1.5 L, Sigma, USA, 5 or 30 FPU/ g glucan) and β–glucosidase (Novozyme 188, Sigma, 10 or 30 pNPGU/ g glucan) were loaded into each glass vial. The activities of cellulase and β–glucosidase were 319 FPU/mL and 118 pNPGU/mL, respectively. Aliquots (200 µL) were removed at specific intervals for sugar yield determination by HPLC. The glucose yield (%) was calculated from the amount of glucose generated by enzymatic hydrolysis divided by amount of glucan in each pretreated solid, adjusted for the weight increase upon hydrolysis, and multiplied by 100%.
5
Enzymatic Hydrolysis of Alkaline-Pretreated Corn Stover
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Corn stover was pretreated by 1 % NaOH at 121 °C for 30 min in an autoclaver, washed with ddH2O, oven-dried, and mixed with 20 ml of 100 mM Na2HPO4-citric acid buffer (pH 5.0) in 50 ml shake flasks at the concentration of 2 % (dry material). The enzyme(s), i.e., Celluclast 1.5L (Cellulase from Trichoderma reesei ATCC 26921, C2730, Sigma-Aldrich) alone (5 FPU/g dry matter) or supplemented with identical units of purified wild type Bgl3A, mutant M1 or commercial liquid Novozyme 188 (Cellobiase from Aspergillus niger, C6105, Sigma-Aldrich) (10 BGU/g dry material), respectively, were added into the cellulosic preparations and incubated at 50 °C with an agitation rate of 200 rpm for 96 h. Hydrolyzates were collected at several intervals and centrifuged at 12,000 rpm, 4 °C for 10 min. The amounts of reducing sugars and glucose released in the supernatants were determined using the DNS [62 (link)] and GOD-POD methods, respectively. All these experiments were conducted with three replicates.
6
Enzymatic Saccharification of Lignocellulosic Biomass
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The saccharifying enzyme mixture of Celluclast (Sigma) and Novozyme 188 (Sigma) was prepared as a 1:1 mixture. 30 mg of sample was saccharified with an enzyme loading of 6 EGU (based on the manufacturer’s measurement) (~0.6 FPU) per 30 mg of sample in 50 mM citrate buffer pH 4.8 with 0.02% w/v NaN3 in a total volume of 1.5 ml for 72 h at 50 °C with shaking. The reactions for each of the untreated and pretreated substrates were performed in triplicate. After 72 h, the glucose released was quantified using the GOPOD assay kit (K-GLUC) (Megazyme) and the xylose with a xylose assay kit (K-XYLOSE) (Megazyme) and expressed per mg of initial sample. As a control, Whatman filter paper was saccharified and the saccharification efficiency was >90%. Corrections were made for any sugars present in the enzymes mixture or substrates controls where no enzymes were added.
7
Ionic Liquid Pretreatment and Compositional Analysis of Miscanthus Biomass
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Miscanthus (cv. Giganteus) was ground to pass an 0.5 mm sieve as described previously [31 (link)]. The ionic liquid (IL) pretreatment was performed using 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) for 2 h at 160 °C and hydrothermal treatment was performed at 200 °C for 2 h, as described in detail previously for wheat straw [31 (link)]. The compositional and structural analysis of substrates was performed as described in detail previously for wheat straw [31 (link)], employing total acid hydrolysis assay followed by analysis of soluble sugar monomers in the hydrolysate by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and wide-angle X-ray diffraction. Digestibility of the substrates was assessed in saccharification assays with a commercial enzyme mixture (Celluclast (Sigma) and Novozyme 188 (Sigma), 1:1 v/v), as well as by measurement of residual xylose in solids of shake-flask cultures during fungal exposure [31 (link)].
8
Enzymatic Hydrolysis of Cellulose
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Enzymatic hydrolysis was carried out using cellulase (Celluclast 1.5 L, Sigma) at 20 filter paper units (FPU)/g cellulose and β-glucosidase (Novozyme 188, Sigma) at 40 cellobiase units (CBU)/g cellulose. The solid phase was hydrolyzed at 10% (w/v) water-insoluble content (WIS) in a total volume of 250 mL at 50 °C and 175 rotations per minute (rpm) in a shaker. 50 mM citrate buffer was added to maintain the pH at 4.8. After 48 h of enzymatic hydrolysis, the flask temperature was reduced to 30 °C and the pH increased to 6.0 using 1.0 M sodium hydroxide (NaOH) in preparation for fermentation as described in the following sections.
9
Enzymatic Hydrolysis of Cellulosic Biomass
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The dilute acid was aspirated and 1 mL of water was added to each tube. After vigorous vortexing, the tubes were centrifuged at 14,000 g for 5 min. The water was aspirated and replaced with 0.9 mL of 50 mM sodium citrate (pH = 4.8). Twenty microliters of 0.1× Celluclast (Sigma) and 80 μL of 0.1× Novozyme 188 (Sigma) were added to each tube to bring the final volume to 1 mL. The optimal activities of the enzymes were determined to be 111 FPU/mL and 500 U/mL, respectively (Ghose, 1987 (link)). After vortexing, the tubes were placed in a water bath kept at 50oC for 48 h. Periodical vortexing was done. The tubes were then centrifuged at 14,000 g for 5 min and 5 μL of the supernatant were placed into 45 μL of water in a 96-well spectrophotometric plate. The reducing sugars were quantified using 0.2% anthrone as described above.
10
Cellulase Enzyme Characterization
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Celluclast 1.5 L and Novozyme 188, were obtained from Sigma Aldrich. Filter-paper and Carboxymethyl cellulose activities were used as a measure of cellulase activity. FPA 4.49 FPU/mL and CMCase to 20.6 U/mL was measured after 7 days of fermentation. β-glucosidase activity on pNPG was measured as 4.77 U/mL and xylanase activity as 6.61 nkat/mL. Commercial cellulase (celluclast 1.5 L) showed 81.8 FPU/mL filter paper activity, β-glucosidase activity of 58.66 U/mL and xylanase activity 107.3 nkat/mL. PNPG activities (β-glucosidase) in A. saccharolyticus and in commercial enzyme Novozym 188 were 339.9 U/mL and 698.3 U/mL, respectively.
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