describe the breakage of a glycosidic bond in polysaccharides and disaccharides by hydrolysis, with reference to the non-reducing sugar test

Carbohydrates & Lipids – Glycosidic‑Bond Hydrolysis (Cambridge AS & A‑Level Biology 9700)

Learning Objective (AO1 + AO2)

Describe how a glycosidic bond in polysaccharides and disaccharides is broken by hydrolysis (acid‑catalysed and enzymatic) and explain how the non‑reducing‑sugar test (hydrolysis + Benedict’s test) demonstrates this process.

1. Core Definitions (AO1)

• Monosaccharide: the simplest carbohydrate unit (e.g., glucose, fructose). Possesses a free hemiacetal (aldose) or hemiketal (ketose) carbonyl at the anomeric carbon (C1 of an aldose, C2 of a ketose).
• Disaccharide: two monosaccharides linked by a glycosidic bond (e.g., sucrose, maltose, lactose).
• Polysaccharide: long chains of monosaccharide residues (e.g., starch, glycogen, cellulose).
• Triglyceride: a lipid formed when glycerol is esterified with three fatty‑acid chains.

2. Major Carbohydrate & Lipid Structures (Why They Matter)

Biomolecule	Structure (labelled sketch)	Key Functional Groups	Relevance to Hydrolysis
Starch (amylose + amylopectin)		α‑1,4 (linear) & α‑1,6 (branch) glycosidic bonds; many free –OH groups.	Rapidly hydrolysed by amylase to supply glucose for metabolism.
Glycogen		α‑1,4 backbone, α‑1,6 branches every ~8‑10 residues.	Hydrolysed by glycogen phosphorylase and debranching enzymes to release glucose during fasting or exercise.
Cellulose		β‑1,4 glycosidic bonds; three free –OH groups per glucose for inter‑chain H‑bonding.	Not hydrolysed by human enzymes (no cellulase) → dietary fibre.
Triglyceride (e.g., triolein)		Ester (–COO–) bonds, long hydrocarbon tails, glycerol –OH groups.	Hydrolysed by lipase (enzyme) or by strong acid/alkali to give glycerol + fatty acids – an example of hydrolysis of a non‑carbohydrate ester bond.

3. Glycosidic Bonds (AO1)

A glycosidic bond joins the anomeric carbon of one sugar to a hydroxyl group of another. The bond can be:

• α or β (orientation of the –OH on the anomeric carbon)
• 1‑4, 1‑2, 1‑6, etc. (position of the carbon atoms involved)

4. Hydrolysis of Glycosidic Bonds (AO2)

4.1 General Reaction

\[

\text{R–O–C}1\text{–C}2\text{–OH} \;+\; \text{H}_2\text{O}

\;\xrightarrow{\text{catalyst}}\;

\text{R–OH} \;+\; \text{HO–C}1\text{–C}2\text{–OH}

\]

Water supplies the nucleophilic –OH that attaches to one sugar and the H⁺ that attaches to the other, regenerating the free carbonyl (aldehyde or ketone) on each monosaccharide.

4.2 Acid‑Catalysed Hydrolysis

1. Protonation of the glycosidic oxygen makes it a better leaving group.
2. Water attacks the anomeric carbon, forming a protonated hemiacetal.
3. De‑protonation restores the neutral carbonyl, giving two free monosaccharides.

Typical laboratory conditions: 0.1 M HCl, 60–80 °C, 5–10 min. The reaction is reversible; a large excess of water drives it toward the monosaccharides.

4.3 Enzymatic Hydrolysis (Physiological Conditions)

• Specific enzymes (e.g., amylase, sucrase, lactase, cellulase, lipase) bind the substrate at an active site.
• The active site positions the glycosidic (or ester) bond for nucleophilic attack by a water molecule that is activated by amino‑acid side chains (often a catalytic Asp/Glu and a His).
• The enzyme stabilises the transition state, thereby lowering the activation energy and allowing the reaction to proceed at ~37 °C and neutral pH.
• Enzyme specificity illustrates the lock‑and‑key model, refined by the induced‑fit concept.

4.4 Role of Water (AO1 – properties of water)

• Water is a polar solvent; its ability to form hydrogen bonds solvates both reactants and products, increasing reaction rates.
• Its high dielectric constant weakens ionic interactions, facilitating the proton transfers required in both acid‑catalysed and enzymatic mechanisms.
• In the Benedict’s test, water is the medium that allows Cu²⁺ ions to be reduced by the aldehyde group of a reducing sugar.

5. Reducing vs. Non‑Reducing Sugars (AO1)

• Reducing sugar: possesses a free hemiacetal/hemiketal carbon that can be oxidised (e.g., glucose, maltose, lactose). Gives a positive Benedict’s test without prior treatment.
• Non‑reducing sugar: both anomeric carbons are involved in glycosidic linkages, so no free carbonyl is available (e.g., sucrose, trehalose). Gives a negative Benedict’s test until the bond is hydrolysed.
• Hydrolysis of a non‑reducing disaccharide releases two reducing monosaccharides.

6. Non‑Reducing‑Sugar Test (Hydrolysis + Benedict’s Test) (AO2)

6.1 Materials

• Disaccharide sample (0.5 g sucrose, or any non‑reducing sugar)
• Dilute HCl (0.1 M) – for acid hydrolysis OR a specific enzyme solution (e.g., sucrase, 5 U mL⁻¹)
• 0.1 M NaOH (for neutralising the acid route)
• Benedict’s reagent (blue CuSO₄ solution)
• Boiling water bath, test tubes, pipettes, pH paper

6.2 Procedure

1. Prepare the sample: dissolve the disaccharide in 10 mL distilled water.
2. Hydrolysis:

   • Acid route – add 2 mL 0.1 M HCl, heat in a water bath for 5 min.
   • Enzyme route – add 2 mL sucrase solution, incubate at 37 °C for 5 min.

3. Neutralise (acid route only) – add 2 mL 0.1 M NaOH; check that pH ≈ 7.
4. Benedict’s test:

   • Add 5 mL Benedict’s reagent to the hydrolysed mixture.
   • Heat in a boiling water bath for 2–3 min.
   • Observe the colour change (blue → green → yellow → orange → brick‑red precipitate).

6.3 Safety Checklist

• Wear lab coat, safety goggles, and nitrile gloves.
• Handle HCl and NaOH in a fume cupboard; always add acid to water.
• Use heat‑resistant gloves when handling the boiling water bath.
• Collect copper‑containing waste in designated heavy‑metal containers.

6.4 Data‑Analysis (AO2)

• Record the observed colour against a standard colour chart (e.g., blue = 0 % reducing sugar, brick‑red = 100 %).
• Calculate the % reducing sugar produced and compare with the theoretical yield (100 % for complete hydrolysis of sucrose).
• Plot % reducing sugar versus hydrolysis time to illustrate reaction kinetics for both acid and enzymatic routes.

6.5 Interpretation

• Before hydrolysis: sucrose (non‑reducing) gives no colour change – solution remains blue.
• After hydrolysis: a coloured precipitate appears, confirming cleavage of the glycosidic bond and formation of glucose and fructose, both of which are reducing sugars.
• Thus the test demonstrates that hydrolysis converts a non‑reducing disaccharide into reducing monosaccharides.

7. Example Reaction – Hydrolysis of Sucrose

\[

\text{C}{12}\text{H}{22}\text{O}{11} \;+\; \text{H}2\text{O}

\;\xrightarrow{\text{acid or sucrase}}\;

\text{C}6\text{H}{12}\text{O}_6\;(\text{glucose}) \;+\;

\text{C}6\text{H}{12}\text{O}_6\;(\text{fructose})

\]

• Both products possess a free carbonyl group → give a positive Benedict’s test.

8. Cross‑Topic Box – Enzymes are Proteins (Topic 3 linkage)

Enzymes that catalyse hydrolysis are globular proteins. Their three‑dimensional active site arranges specific amino‑acid side chains (e.g., Asp, Glu, His) to:

• Activate a water molecule for nucleophilic attack.
• Stabilise the high‑energy transition state, thereby lowering the activation energy.
• Provide substrate specificity through the lock‑and‑key model, refined by induced‑fit adjustments.

This connects carbohydrate hydrolysis to the syllabus outcomes for protein structure and enzyme function.

9. Summary Comparison of Reducing & Non‑Reducing Sugars

10. Key Points to Remember (AO1 + AO2)

• Hydrolysis adds water across a glycosidic (or ester) bond, regenerating free carbonyl groups that act as reducing agents.
• Acid catalysis and enzymatic catalysis achieve the same net reaction but under very different conditions (high temperature & low pH vs. physiological pH and temperature).
• Water’s polarity and hydrogen‑bonding ability make it an excellent reactant and solvent for both hydrolysis and the redox step of the Benedict’s test.
• The non‑reducing‑sugar test shows that a previously non‑reducing disaccharide becomes reducing after its bond is cleaved.
• Enzymes lower activation energy by stabilising the transition state; this exemplifies the lock‑and‑key/induced‑fit concepts taught in Topic 3.
• Understanding glycosidic‑bond hydrolysis underpins digestion, cellular metabolism, and industrial processing of carbohydrates and lipids.