Alpha Amylase

Alpha Amylase is an enzyme which aids in the breakdown of starch to maltose.  Alpha-amylase hydrolyzes bonds between glucose repeats.   Carbohydrates and sugars are the major energy storage molecules used by living organisms.  Plants store the energy of the sun in sugars using photosynthesis.

Alpha Amylase's official name is 1,4-a-D-Glucan glucanohydrolase; EC 3.2.1.1.  The official names of enzymes are maintained by a commission on enzyme nomenclature

Alpha amylase is breaks down starch by hydrolysis to release maltose. Starch is a complex molecule mostly made in plants and bacteria.  It consists of two types of polysaccharide amylose and amylopectin. 

ChemCards are a site which reviews prinicipals of chemistry. They have a useful sequence on Carbohydrates that helps connect the chemistry with the biotechnology.

This is a chain of amylose.  The simple glucose molecules have a bond between the 1 carbon in the ring and the 4 carbon in the ring.  They are joined by an oxygen. 

amylose

AMYLOSE

Below is the building block of starch, glucose.  Notice that when the single oxygen is left in the bond in the amylose, the two glucose molecules must have lost two hydrogen and an oxygen in the formation of this bond- or one H2O molecule.  This chemical reaction is called dehydration, because it generates water. 

glucose

GLUCOSE

Similarly, when this bond breaks to generate a free glucose or pairs of free maltose,there is the addition of a water molecule to make the OH at carbon 1 and carbon 4.  This is called hydrolysis or Hydro (water) + Lysis (breaking).  One of the disaccharides or monosaccharides gets an OH- from the water molecule and the other part gets an H+ from the water.  Hydration is adding a water without splitting the molecule apart.

 maltose

MALTOSE

Amylopectin is another component of starch and it also consists entirely of repeating chains of glucose.  It is different from amylose because it has two different types of linkages between the glucose subunits: the 1C-4C and the 1C-6C.  The 1:6 linkage makes a branched structure instead of a linear structure.  The branches occur every 24 to 30 glucose units.  The molecules of Amylopectin are composed of between 2000 and 200,000 units of glucose.

amylopectin 

AMYLOPECTIN

Starch is very important molecule for energy storage in nature.  Starch is very stable and is usually insoluble in cold water.  Heating starch can make it go into solution and form a jelly-like solid consistency when it cools.  The ratios of the two molecules amylose and amylopectin, are different depending on the plant which made them  High-amylose corn starch is 85% amylose, while waxy corn is more than 99% amylopectin.  In plants, starch is made in specialized cellular organs called amyloplasts.  The starch forms granules with consistent size and shape, depending on the plant that made them.

bean starch

STARCH GRAINS FROM BEAN EMBRYO

Seed Sprouting

Plant seeds tend to be rich in starch, oils, and proteins.

link

  "The seed develops from the ovule and contains the embryo and endosperm, surrounded by the maternally derived seed coat. The function of the seed is to protect the embryo, to sense environmental conditions favorable to germination and to nourish the germinating seedling." link

The sprouting seed can nourish the embryo using the energy from these stored carbohydrates until it can make it's own glucose with photosynthesis.  It also needs a source of amino acids to make the new, unique proteins which are part of cell growth.  Sprouting plants need  make new cells through cell divisions and elongation of existing cells to push the plant through the soil so it reaches sunlight.  The first leaves are the endosperm leaves of cotyledons. 

At the same time, seeds must maintain stable carbohydrates, lipids and proteins that won't break down if the seeds are waiting around until the next season to sprout.  Enzymes are turned on during seed sprouting to mobilize the stored resources of the seeds.  When we eat seeds (like corn, wheat and rice) we are taking the unborn plant resources while eating its embryos.  If we eat wheat germ, we are just eating the embryos.

Cellulose vs. Starch

Another molecule made in plants completely of repeating glucose units is Cellulose.  The big difference is the bond.  In Starch this is the 1:4 Alpha bond.   Notice both Hs are on the same side of the projection in the pink box.  In Cellulose this is the 1:4 Beta bond.  The H are on opposite sides of the projection in the blue box. 

cellulose_starch

CELLULOSE VS STARCH

While cellulose and starch are both glucose chains, starches can be used as energy sources by the humans while cellulose cannot be digested. Cellulose molecules are usually straight chains which form strong, supporting fibers in plants.  The a-1-4 linkages in starch give the polysaccharide a helical twist.

The b-1-4 linkages in cellulose give the polysaccharide an extended ribbon conformation.  More than one cellulose chain can align with Hydrogen bonds.  Cellulose is more resistant to hydrolysis than is starch. 

Enzymes are important in the breakdown of these two molecules into simpler sugars, which can serve as energy sources.  The enzymes that break down these molecules very specific.  The enzymes that break down starch are called amylases and those that break down cellulose are cellulases.   Humans have amylases, but we lack cellulases.  Bacteria make cellulases, and animals that have a lot of bacteria in their guts like cow and termites can digest cellulose.  That is why celery is so low calorie and potatoes aren't!

Additional link on carbohydrate biochemistry

Alpha Amylase is present in almost all plants, animals and microorganisms. Different organisms have differences in physical, chemical and catalytic properties of their enzymes, but they are all doing the same biochemical reaction. We will focus on bacterial amylases.  Porcine pancreas and human saliva have been extensively studied.  Serum alpha amylase activity is measured in individuals to diagnose acute pancreatitis, mumps, renal disease and abdominal disorders such as cholecystitis (gall bladder disease)

Uses for Alpha Amylase

Alpha amylase is widely used in the food industry and in laundry detergents. 

Industrial Enzymes

Low Temperature Alpha Amylase

High Temperature Alpha Amylase

Worthington Biochemistry Alpha Amylase Assay is listed below.

Assay

Method: That of Bernfield (1951) wherein the reducing groups released from starch are measured by the reduction of 3,5-dinitrosalicylic acid. One unit releases from soluble starch one micromole of reducing groups (calculated as maltose) per minute at 25C and pH 6.9 under the specified conditions.

Reagents

  • 0.02 M Sodium phosphate buffer, pH 6.9 with 0.006 M sodium chloride
  • 2 N Sodium hydroxide
  • Dinitrosalicylic acid color reagent. Prepare by dissolving 1.0 gm of 3,5-dinitrosalicylic acid in 50 ml of reagent grade water. Add slowly 30.0 gms sodium potassium tartrate tetrahydrate. Add 20 ml of 2 N NaOH. Dilute to a final volume of 100 ml with reagent grade water. Protect from carbon dioxide and store no longer than 2 weeks.
  • 1% Starch. Prepare by dissolving 1.0 gm soluble starch, (Merck) in 100 ml 0.02 M sodium phosphate buffer, pH 6.9 with 0.006 M sodium chloride. Bring to a gentle boil to dissolve. Cool and bring volume to 100 ml, with water, if necessary. Incubate at 25C for 4-5 minutes prior to assay.
  • Maltose Stock Solution. Prepare by dissolving 180 mg maltose (MW 360.3) in 100 ml reagent grade water in a volumetric flask.

Enzyme

Dilute to a concentration of 1-10 micrograms/ml. A minimum of three different concentrations in this range should be run.

calculation mg_ml

Procedure

Adjust spectrophotometer to 540 nm and 25C.

Using the maltose stock solution prepare a maltose standard curve as follows: In numbered tubes, prepare 10 maltose dilutions ranging from 0.3 to 5 micromoles per ml. Include two blank tubes with reagent grade water only. Into a series of corresponding numbered tubes pipette 1 ml of each dilution of maltose. Add 1 ml of dinitrosalicylic acid color reagent. Incubate in boiling water bath for 5 minutes and cool to room temperature. Add 10 ml distilled water to each tube and mix well. Read A540 versus micromoles maltose.

Enzyme assay: Pipette 0.5 ml of respective enzyme dilutions into a series of numbered test tubes. Include a blank with 0.5 ml reagent grade water. Incubate tubes at 25C for 3-4 minutes to achieve temperature equilibration. At timed intervals, add 0.5 ml starch solution (at 25C). Incubate exactly 3 minutes and at timed intervals add 1 ml dinitrosalicylic acid color reagent to each tube. Incubate all tubes in a boiling water bath for 5 minutes. Cool to room temperature and add 10 ml reagent grade water. Mix well and read A540 versus blank. Determine micromoles maltose released from standard curve.

Calculation

calculation