Chitinase, glucanase, and protease activity of the Lysobacter strains were tested as described in De Bruijn et al. (in press ). In brief, 2–5 μl of Lysobacter suspensions (of stationary phase of growth) was spot-inoculated in the center of different media containing 1.5–2% agar. For chitinase activity, R2A (Oxoid) and 1/10th strength TSB agar Petri dishes were used containing 0.2% colloidal chitin prepared from crab shell chitin (Sigma) and Petri dishes were incubated for 3–7 days at 25°C. For glucanase activity, R2A medium containing 0.5% laminarin was used and Petri dishes were incubated for 3 days at 25°C. The colonies were removed by washing with water and the medium was stained with 1% congo red. After destaining, coloration of the medium was determined. For protease activity, bacteria were inoculated on 15 g/l skimmed milk powder, 4 g/l blood agar base and 0.5 g/l yeast extract and Petri dishes were incubated for 3–7 days at 25°C.
Crab shell chitin
Manufactured by Merck Group
Sourced in United States
Crab shell chitin is a naturally occurring polysaccharide derived from the exoskeletons of crustaceans. It is a versatile material with a wide range of applications in various industries.
Automatically generated - may contain errors
Lab products found in correlation
Cellulose, by Merck Group (4 mentions)
Chitin beads, by New England Biolabs (4 mentions)
Xylan, by Merck Group (3 mentions)
Laminarin, by Merck Group (2 mentions)
Chitosan, by Merck Group (2 mentions)
Luminol, by Merck Group (1 mentions)
Colloidal chitin, by Merck Group (1 mentions)
N acetylglucosamine nag, by Merck Group (1 mentions)
Anti gst antibody, by Thermo Fisher Scientific (1 mentions)
Avicel, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Nα benzoyl dl arginine p nitroanilide, by Merck Group (1 mentions)
Pglu phe leu pnitroanilide, by Merck Group (1 mentions)
N acetyl d glucosamine, by Merck Group (1 mentions)
N succinyl ala ala pro phe p nitroanilide, by Merck Group (1 mentions)
Bovine pancreatic trypsin, by Sisco Research Laboratories (1 mentions)
Bromelain, by Sisco Research Laboratories (1 mentions)
Zorbax carbohydrate analysis column, by Agilent Technologies (1 mentions)
20a prominence, by Shimadzu (1 mentions)
Trichloroacetic acid, by HiMedia (1 mentions)
16 protocols using crab shell chitin
1
Enzymatic Activities of Lysobacter Strains
2
Screening Extremophilic Bacteria for Cell Wall Degrading Enzymes
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Extremophilic bacteria were screened for the production of three CWDEs: β−1,4-glucanase, cellulase, and chitinase. β−1,4-glucanase and cellulase activities were estimated in minimal medium [18 ,19 ] with barley flour and carboxymethylcellulose as the sole carbon source, respectively. The plates were inoculated with overnight bacterial cultures and incubated at 28±2 °C for 48 h. The formation of translucent halos around the colonies confirmed the ability of the bacteria to use the substrates as the sole carbon source. To visualize the halos for the cellulose activity, the plates were flooded with an aqueous solution of congo red 1% (w/v) for 30 min and washed with 1 M NaCl [20] .
The chitinase production was evaluated in minimal medium [21] (link) containing chitin as the sole carbon source. Colloidal chitin was prepared by dissolving 10 g of crab shell chitin (Sigma-Aldrich) in 150 mL of concentrated HCl [22] (link). The plates were inoculated (109 CFU/mL) and incubated at 28 ± 2 °C for 8 days. The chitinolytic activity was evidenced through the formation of clear halos around the colonies.
The enzymatic activity indexes (EI) were calculated for the three CWDEs by measuring the clearance zone and using the following expression: Each experiment was performed in triplicate.
The chitinase production was evaluated in minimal medium [21] (link) containing chitin as the sole carbon source. Colloidal chitin was prepared by dissolving 10 g of crab shell chitin (Sigma-Aldrich) in 150 mL of concentrated HCl [22] (link). The plates were inoculated (109 CFU/mL) and incubated at 28 ± 2 °C for 8 days. The chitinolytic activity was evidenced through the formation of clear halos around the colonies.
The enzymatic activity indexes (EI) were calculated for the three CWDEs by measuring the clearance zone and using the following expression: Each experiment was performed in triplicate.
3
Chitin Purification and Cell Activation
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Crab shell chitin was purchased from Sigma-Aldrich, Stockholm, Sweden. Chitin molecules are insoluble in PBS. To assure that chitin preparations were not contaminated with soluble compounds that may interfere with activation of cells, chitin supernatants were prepared as described for setae (see above) and used in control cultures.
4
Chitin, Cellulose, and Xylan Binding Assay
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The affinity of the complete AtlA LysM domains (L1–L6 in Fig. 1a ) for various polysaccharides was determined by incubating 32 μg of protein with 1 mg of crab shell chitin (Sigma, Ref. C-7170), cellulose (Sigma, Ref. 8002) or xylan (Sigma, Ref. X-0502) in a total volume of 150 μl of phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). After 30 min of gentle rocking at ambient temperature, the insoluble fraction was harvested by centrifugation (5 min, 20,000 g) and 50 μl of the supernatant was collected (unbound fraction). The remaining supernatant was discarded and the insoluble fraction was washed three times with 1.5 ml of PBS. The washed pellet was resuspended in 150 μl of Laemmli buffer (bound fraction) and boiled for 5 min. Five microliters of the unbound and bound fractions were loaded on a 13% (w/v) SDS–PAGE and stained with Coomassie brilliant blue.
5
Oxidative Burst Assay in Barley and Arabidopsis
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Leaf disks from 3-week-old barley plants (HvPIIN_08944 or EV control) or 4-week-old Arabidopsis plants (AtPIIN_08944 or WT control) were cut and pre-incubated overnight in sterile distil water in 96 well micro-titer plate. Water is carefully discarded and the leaf disks are treated with luminol (Sigma, A8511-5 g) 40 μl luminol buffer [15 mg/ml], 400 μl horseradish peroxidase [1 mg/ml] and elicitor [100 nM flg22, 200 mg/ml crab shell chitin (sigma–aldrich) or water control]. Immediately after treatment, luminescence was measured in a TECAN infinite® F200 micro plate reader (TECAN, Switzerland). The relative light units over time as a result of the production of oxygen radicals were measured for 50 min.
6
Fungal Glucanase and Chitinase Production
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Glucanase production of both isolates was evaluated on growth medium consisting of an agar synthetic medium (NaNO3, 0.2; KH2PO4, 0.1; MgSO47H2O, 0.05; KCl, 0.05; agar 15 g/L and deionized water) supplemented with laminarin 1% (laminarin from Laminaria digitata, Sigma Aldrich, México) as a sole carbon source. Chitinase production was evaluated on the same synthetic basal medium but supplemented with colloidal chitin 1% rather than laminarin. Colloidal chitin was prepared using 10 g of purified crab shell chitin (Sigma Aldrich, México) suspended in 100 mL concentrated HCl for 2.5 h at 4°C and then was washed with cold deionized water and NaOH overnight at 4°C, followed by re-washing with cold deionized water at approximately pH 7.0. Mycelia discs (4 mm) were removed from PDA purified cultures with a sterilized needle and transferred to specific carbon-source media. After incubation at 24°C for 15 days, growth was evaluated either by measurement of the diameter of the developing colonies in comparison with a negative control (basal medium without carbon source), or by comparison with a positive control (basal medium supplemented with D-glucose 1%) in threefold replication.
7
Screening for Hydrolytic Enzyme Activities
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The cells from pre-cultures were washed 3x with 0.9 % NaCl and the cell density was adjusted to OD600 of 1. Subsequently, 2–5 μl droplets were spotted on different media containing 1.5-2 % agar. For chitinase activity, R2A and 1/10th strength TSB agar plates were used containing 0.2 % colloidal chitin prepared form crab shell chitin (Sigma) according to [47 (link)]. After incubation for 3–7 days at 25 °C, clearing zones surrounding the colonies could be observed. For glucanase activity, R2A medium containing 0.5 % laminarin was used. After growth for 3 days at 25 °C, the colonies were removed by washing with water and the medium was stained with 1 % congo red. After destaining, coloration of the medium was determined. R2A with and without laminarin was included as controls. For lipase activity, the strains were inoculated on R2A medium supplemented with 0.01 % (w/w) CaCl2 and 1 % (w/v) Tween 80 and after 3–5 days of incubation at 25 °C, plates were investigated for opaque halos surrounding the colonies, which indicates lipase activity [48 (link)]. For protease activity, a medium was prepared containing 15 g/l skimmed milk powder, 4 g/l blood agar base, 0.5 g/l yeast extract. After incubation for 3–7 days at 25 °C, clearing zones surrounding the colonies could be observed.
8
Trichoderma, Botrytis, and Rhizoctonia Cultivation
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T. atroviride strain IMI206040 (WT) and mutants derived from it, Botrytis cinerea strain B05.10 and R. solani strain SA1 were maintained on potato dextrose agar (PDA; Oxoid, Cambridge, UK) medium at 25 °C. Synthetic minimal salt (SMS) liquid medium (Dubey et al. 2012 (link)) supplemented with 1% glucose was used for submerged liquid culture unless otherwise specified. Culture medium for different nutrient conditions was prepared by substituting 1% glucose in SMS medium with colloidal chitin (1%), R. solani cell wall material (RsCW) (1%), or N-acetylglucosamine (NAG; Sigma-Aldrich, St. Louis, MO, USA) (10 mM). Limitation media for carbon (C lim), nitrogen (N lim), and carbon + nitrogen (C + N lim) were prepared as described before (Dubey et al. 2012 (link)). Starvation for iron (Fe lim) was induced by a tenfold reduction of FeSO4 × 7H2O concentration in SMS medium. colloidal chitin was prepared from crab-shell chitin (Sigma-Aldrich, St. Louis, MO, USA) as described previously (Roberts and Selitrennikoff 1988 ). R. solani cell wall material was prepared as described previously (Inglis and Kawchuk 2002 (link)).
9
Chitin-Binding Assay for Recombinant Proteins
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The recombinant proteins (GST-UvCBP1 and GST) were purified from Escherichia coli and used for chitin-binding assay as described earlier with modifications (Han et al., 2019 (link)). Briefly, the recombinant proteins were incubated with chitin beads (NEB), crab shell chitin (Sigma), cellulose (Sigma), and chitosan (Sigma) in 800 μL ddH2O at 4°C. After 4 h, the insoluble pellet fraction was centrifuged (4°C, 12000 rpm, 10 min), and the supernatant was collected. The insoluble pellets were rinsed three times with ddH2O. Both the supernatants and the pellets were boiled in 1% sodium dodecyl sulfate (SDS) for extraction of proteins, which were then separated in 10% SDS-polyacrylamide gel electrophoresis (PAGE) gels and immunoblotted with an anti-GST antibody (Invitrogen).
10
Screening Endophytic Isolates for CWDE and Chitinase Activities
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All endophytic isolates obtained were evaluated for their potential to produce clear zones on F. solani mycelial fragment agar (MFA) as an indicator of preliminary production of CWDEs [37 (link)]. Plates were incubated in the dark at 28 °C for 7 days. Large diameters of clear zones (>30 mm) represented high CWDE activities. We also examined all obtained isolates for their abilities to secrete chitinase enzyme. Each isolate was inoculated onto colloidal chitin agar (CCA) plates and incubated in the dark at 28 °C for 7 days [38 (link)]. Colloidal chitin was prepared from crab shell chitin (Sigma-Aldrich) [39 (link)].
To detect the antagonistic isolates of highly active CWDEs and chitinase activities, large clear zone diameters (>30 mm) on both MFA and CCA plates, respectively, were selected. Six plates were used for each actinobacterial isolate.
To detect the antagonistic isolates of highly active CWDEs and chitinase activities, large clear zone diameters (>30 mm) on both MFA and CCA plates, respectively, were selected. Six plates were used for each actinobacterial isolate.
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