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Amylase Production Immobilization Characterization
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You said:
full protocol of research 1.pdf
PDF
By following this add specific and required values in
production ,immobilization and Characterization of amylase enzyme
ChatGPT said:
To make the provided research protocol more specific and production-ready, we can
incorporate detailed and required values in the following key sections related
to production, immobilization, and characterization of the amylase enzyme:
✅ Amylase Enzyme Production
Medium Composition (example for Bacillus sp.):
Starch: 1.0%
Peptone: 0.5%
Yeast extract: 0.2%
NaCl: 0.5%
KH₂PO₄: 0.1%
MgSO₄: 0.05%
pH: 7.0
Nanoparticle Incorporation:
Use synthesized NPs at 0.1–1.0 mM concentrations.
Optimal temperature: 37°C
Incubation time: 48 hours
Shaking at 120 rpm
✅ Amylase Enzyme Immobilization
Entrapment in Sodium Alginate Beads:
Sodium alginate: 2.5% (w/v)
Enzyme solution: Mix 1:1 (v/v) with alginate
Calcium chloride solution: 0.1 M (for bead formation)
Dropping method: Use syringe to drop mixture into CaCl₂ with stirring
Adsorption on Solid Supports:
Support: Silica gel or chitosan beads (washed and dried)
Enzyme loading: 10 mg/mL
Contact time: 1–2 hours at 25°C
Wash with phosphate buffer (pH 7.0)
Storage:
Store immobilized enzyme at 4°C in phosphate buffer (pH 7.0)
✅ Characterization of Amylase Enzyme
1. Activity Assay (DNS Method):
Substrate: 1% soluble starch in 0.1M phosphate buffer (pH 6.8)
Incubation: 10 min at 37°C
DNS reagent: Add 1 mL, then boil for 5 min
Absorbance: Measure at 540 nm
Express in Units: 1 U = 1 μmol of maltose released/min
2. Optimum Conditions:
pH range: 4–9 (test in citrate, phosphate, and Tris buffers)
Temperature range: 20–70°C
3. Kinetic Parameters:
Substrate concentrations: 0.1–1.0% starch
Plot: Lineweaver–Burk to determine Km and Vmax
4. SDS-PAGE:
Use 12% polyacrylamide gel
Standard marker: 10–100 kDa
Visualize with Coomassie Blue
5. Metal NP Influence:
Compare enzyme kinetics (with/without NPs)
Metal NP concentrations: 0.1–1.0 mM
Would you like me to prepare this in a formatted protocol or printable document format? I
can also help with charts and graphs, sample calculations, or enzyme assay templates.
Tools
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Skip to content
Chat history
New chat
Search chats
Library
GPTs
Today
Amylase Production Immobilization Characterization
CT Recruitment Test Answers
Chemical Protocol Summary
Previous 30 Days
Green Synthesis ZnONPs Review
Metal NPs Plant Enzymes
Nanoparticles Effects on Enzymes
Upgrade plan
More access to the best models
ChatGPT
Share
SA
You said:
full protocol of research 1.pdf
PDF
By following this add specific and required values in
production ,immobilization and Characterization of amylase enzyme
ChatGPT said:
To make the provided research protocol more specific and production-ready, we can
incorporate detailed and required values in the following key sections related
to production, immobilization, and characterization of the amylase enzyme:
✅ Amylase Enzyme Production
Medium Composition (example for Bacillus sp.):
Starch: 1.0%
Peptone: 0.5%
Yeast extract: 0.2%
NaCl: 0.5%
KH₂PO₄: 0.1%
MgSO₄: 0.05%
pH: 7.0
Nanoparticle Incorporation:
Use synthesized NPs at 0.1–1.0 mM concentrations.
Optimal temperature: 37°C
Incubation time: 48 hours
Shaking at 120 rpm
✅ Amylase Enzyme Immobilization
Entrapment in Sodium Alginate Beads:
Sodium alginate: 2.5% (w/v)
Enzyme solution: Mix 1:1 (v/v) with alginate
Calcium chloride solution: 0.1 M (for bead formation)
Dropping method: Use syringe to drop mixture into CaCl₂ with stirring
Adsorption on Solid Supports:
Support: Silica gel or chitosan beads (washed and dried)
Enzyme loading: 10 mg/mL
Contact time: 1–2 hours at 25°C
Wash with phosphate buffer (pH 7.0)
Storage:
Store immobilized enzyme at 4°C in phosphate buffer (pH 7.0)
✅ Characterization of Amylase Enzyme
1. Activity Assay (DNS Method):
Substrate: 1% soluble starch in 0.1M phosphate buffer (pH 6.8)
Incubation: 10 min at 37°C
DNS reagent: Add 1 mL, then boil for 5 min
Absorbance: Measure at 540 nm
Express in Units: 1 U = 1 μmol of maltose released/min
2. Optimum Conditions:
pH range: 4–9 (test in citrate, phosphate, and Tris buffers)
Temperature range: 20–70°C
3. Kinetic Parameters:
Substrate concentrations: 0.1–1.0% starch
Plot: Lineweaver–Burk to determine Km and Vmax
4. SDS-PAGE:
Use 12% polyacrylamide gel
Standard marker: 10–100 kDa
Visualize with Coomassie Blue
5. Metal NP Influence:
Compare enzyme kinetics (with/without NPs)
Metal NP concentrations: 0.1–1.0 mM
Would you like me to prepare this in a formatted protocol or printable document format? I
can also help with charts and graphs, sample calculations, or enzyme assay templates.
Tools
ChatGPT can make mistakes. Check important info.
