Enzymatic Conversion of Starch Into Glucose Syrup

 

This is my first laboratory activity: converting starch into glucose syrup using enzymes.

Starch is a polysaccharide carbohydrate; a polymer of glucose joined together by glycosidic bonds. Starch consists mainly of amylose and amylopectin; amylose being a relatively linear polymer of glucose joined by α-1,4-glycosidic bond, and amylopectin being a branched polymer of glucose joined by both α-1,4-glycosidic bond (linear) and α-1,6-glycosidic bond (branching) (Caballero, 2003).

To convert this polymer into its monomer, the amylase enzyme is used. The amylase enzyme can be classified into three categories: α-amylase, β-amylase, and glucoamylase. α-amylase will break the α-1,4-glycosidic bond randomly, giving molecules of dextrins. α-amylase can also break the α-1,6-glycosidic bond, but at a much slower rate (usually the enzym pullulanase is added to accelerate the breakage of α-1,6-glycosidic bond). β-amylase breaks the α-1,4-glycosidic bond from the non-reducing end, giving molecules of maltoses. And glucoamylase breaks the α-1,4-glycosidic bond also from the non-reducing end, giving molecules of glucose (Wiseman, 1985).

The α-amylase used is obtained from the bacteria B. subtilis or B. licheniformis, whereas the β-amylase is obtained from Aspergillus sp. and Rhizopus sp.

This conversion took place in a couple of steps:

• First, we make a solution from the starch. In Wiseman (1985), a 30-40% solution w/w is preferred, which will -after the conversion reaction- give a 94-97% glucose in equilibrium mixture.
• Then, we gelatinized this solution. Gelatinization is the process of breaking down the intermolecular bonds if starch molecules in the presence of water and heat.
• After the starch solution is gelatinized (by heating), the solution became very viscous, just like the starch (kanji) we used to stiffen our clothing items. This is where the α-amylase is added, at 90 degrees Celcius and stirred for approximately 2 hours. This is the process of liquefying the starch. See, this α-amylase will break down the α-1,4-glycosidic bond, but not the α-1,6-glycosidic bond. Therefore, the reaction yields molecules of branched but short glucose. Branched molecules are soluble in water, whereas linear ones are insoluble. In other words, the branched molecule will make a less viscous solution that the linear ones. Hence the viscosity of the starch solution will decrease as the α-amylase works (Wiseman, 1985).
• Liquefying can also be done with acid (HCl), in room temperature and acidic condition (pH 4.5-5). The downside of using acid is that acid can hydrolyze protein into amino acid, which will cause the browning reaction (or the Maillard reaction: reaction between amino acid and reducing sugar which will result in the presence of flavour. www.wikipedia.org/wiki/Maillard_reaction). This is of course not desirable, because in order to get a pure glucose (or high concentrated, at least), the starch has to be really rid of impurities, where as an enzymatic reaction is specific and only the starch will be converted.
• After the liquefying process, saccharifying is done with glucoamylase. The temperature for this step is 55-60 degrees Celcius. Saccharifying literally meant to convert into sugar (saccharose). Or, in more scientific words, saccharifying is the process of converting a sugar derivative or complex carbohydrate into a simple soluble fermentable sugar by hydrolysis.
• The conversion is assumed to be done. A sample of the sugar is to be taken repeatedly at a time interval, and analyzed. Here we use the Fehling’s reagent. The Fehling’s reagen consisted of Fehling A (blue, copper sulfate solution) and Fehling B (colorless, potassium hydroxide and potassium sodium tartrate solution). This reagent specifically oxidize reducing sugars (glucose is a reducing sugar), and will result in a change of color from blue to red (cuprous sulfate). A standard solution of pure glucose is used to standarize the Fehling’s reagent.

After obtaining the glucose concentration at every interval of the reaction, these data is plotted according to the Michaelis-Menten kinetics.