ASTU

Adama Science and Technology University

Department of Chemical Engineering
ChE 4201 Fundamentals of
Biochemical Engineering
By Eba A
December 2019
 Contents
1. Biotechnology and Biochemical Engineering
2. Enzyme Kinetics
3. Immobilized Enzyme
4. Industrial Applications of Enzymes
5. Cell Kinetics and Fermenter Design
6. Sterilization
7. Agitation and Aeration
8.Downstream Processing
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 Course Assessments Breakdown
1. Continuous Assessment for the remaining
chapters (Chapter 4-8): 50%
Quiz and Tests: 30% (~ 6% /chapter)
Group Assignment: 15% (Max 5 students/Group)
Class attendance: 5%
2. Final Exam: 50% ( covers all chapters)

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Objectives & Competences to be Acquired
Upon completion of this chapter, the students
will be able to
 explain uses of major industrial enzymes
 explain different types of carbohydrate
substrates in selected industrial processes
 familiarize themselves with typical process
parameters for the enzyme action in industrial
processes
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 4. Industrial Application of Enzymes
 The applications of enzymes can be classified into three major
categories:
Industrial enzymes: transform its substrate into the desired
product
Analytical enzymes; determine the concentration of their
substrates (as analytes)
Medical enzymes: potential therapeutic(disease treatment)
applications
Today, most application for industrial enzymes are in
Food industry
 Beverage industry
 Detergent industry, tanning and textile industry 5
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Industrial Enzymes & Application in d/t Sectors

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 Common Industrial Enzyme Substrates
In most industrial applications, the common substrates are
considered to be Carbohydrates
Carbohydrates constitute a major class of naturally occurring
organic compounds, including sugars, starches, and celluloses.
Carbohydrates are classified into three major groups:
1. monosaccharides, 2. oligosaccharides, and
3. polysaccharides.
• Monosaccharides are the simplest carbohydrate units.
• Oligosaccharides contain two or more of these simple mono
saccharides units, and
• Polysaccharides contain hundreds or thousands of
monosaccharides
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 Common Industrial Enzyme Substrates
The basic carbohydrate molecules are simple sugars, or
monosaccharides,
 All simple monosaccharides have the general empirical formula,
(CH2O)n, where n is the whole number ranging 3 to 8.
 All monosaccharides can be grouped into two general classes as:
1. Aldoses: contain a functional aldehyde grouping (-CHO), or
2. ketoses: contain a functional ketone grouping (>CO)
 Monosaccharides have asymmetric carbon(a carbon atom that is
attached to four different types of atoms or groups of atoms)
The number of possible optical isomers for a compound can be
determined by the formula 2n , where n stands for the number of
asymmetric carbons. Friday, December 6, 2019 8
Common Industrial Enzyme Substrates
 As an example, aldohexose (see below) has four asymmetric carbon
atoms, second carbon through fifth carbon from the top.
• Therefore, it has 16 possible isometric forms, with eight L forms and
eight D forms.
• The D form has OH on the right side of the highest-numbered
asymmetric carbon (fifth carbon for aldohexos) and rotates polarized
light in the +direction, while the L form has OH on the left side and
rotates polarized light in the - direction.

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 Common Industrial Enzyme Substrates Cont’d
 Glucose (or dextrose) is one of aldohexoses which has two isometric forms (Figure below): D and L.

 The D form predominates in the nature.

 Glucose is the most common and most important hexose and is found in most sweet fruits and in
blood sugar.
 In solution, very few sugar molecules exist with free aldehyde or ketone functional groups
 Aldehydes and hydroxyls in a sugar molecule can react in a solution so that the H from the OH at
the fifth carbon joins the aldehyde and the O from the same OH bonds to the first carbon, as shown
in Figure below

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Common Industrial Enzyme Substrates Cont’d
Two sugars can link to each other by losing water from OHs to form disaccharides.
Disaccharides may hydrolyze to form two monosaccharide molecules.

Sucrose, known as table sugar, is comprised of α -D-glucose and β -D-fructose.

 The aldehyde group (1' carbon) of glucose is linked with the ketone group (2'
carbon) of fructose (1' - 2'), so that no carbonyl group (-CO) from either
monosaccharides portion is available as reducing agent.
• For this reason sucrose is termed as a nonreducing sugar.
Lactose, sugar present in milk, is a dimer of β -D-galactose bonded (1' - 4') with D-
glucose.
Polysaccharides consist of many simple sugar units linked together.
One of the most important polysaccharides is starch, which is produced by plants
for food storage.
 Animals produce a related material calledFriday,glycogen.
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Starch Structure
• Amylose is a linear polysaccharide  Amylopectin is a branched-chain
composed entirely of D-glucose polysaccharide composed of glucose
units joined by the α-1,4-glycosidic units linked primarily by α-1,4-
linkages. glycosidic bonds but with
 Degraded by amylase enzymes occasional α-1,6-glycosidic bonds,
which are responsible for the
branching

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Case study 1 Production of HFCS
Starch comprises a large percentage of cereals, potatoes, corn, and
rice.
 Complete hydrolysis of starch yields glucose, but partial hydrolysis
gives maltose as well.
This shows that starch is a polymer of glucose units, joined by α -
glycosidic linkage.
Starch can be separated into two fractions by treatment with hot
water.
 The insoluble component (10 percent to 25 percent) is amylose, the
soluble component (75 percent to 90 percent) is amylopectin.
Amylose and amylopectin are degraded by α - and β -amylase, which
are found in the pancreatic juice and saliva of animals,
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Case Study 1 Production of HFCS
α -amylase is an endoglycosidase which attacks the amylose and
amylopectin randomly along the a (1' - 4') bonds.
In recent years, the conversion of starch to fructose has become a
very important commercial process.
 High-fructose corn syrup (HFCS) is approximately twice as sweet as
sucrose.
 HFCS is used in soft drinks, canned fruits, lactic acid beverages, juice,
bread, ice cream, frozen candies, and so on.

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Corn wet milling and Refining
1. Corn cereal is first cleaned, steeped and degerminated to get a
mixture of starch , proteins and other organic compounds. Starch is
separated from protein in a centrifuge and purified to protein level
of 0.3% in hydrocyclone.
2. Starch slurry is now fed into Corn refining Process. Corn refining
process comprises the following major units.
- Starch gelatinization
- Saccharification
- Purification
- Concentration
- Isomerization
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Corn Refining Process
- Starch slurry is cooked at 104 deg C- 107 deg C for about 8 minutes in
the presence of alpha amylase enzyme.
- They liquefy starch slurry in to dextrin and maltose
The contents is then hydrolyzed to glucose in the presence of fungal
glucoamylase. 95% of glucose is produced at this level.
Impurities are successively removed by filtration and activated carbon
adsorption.
The content is concentrated to 60% solids in an evaporator.
Glucose Isomerization: Glucose syrup is isomerized to fructose by
passing through an immobilized isomerase column for a residence time
of thirty minutes.
This produces High Fructose Corn Syrup (HFCS) that is 42% fructose
and 50% glucose.
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Case Study 1 Production of HFCS

Corn refining process

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Scenario 2: Brew Mashing

Mashing is the process by which starch is converted in to

simple sugars by the use of enzymes.
The enzymes required for mashing are;
• alpha & beta amylase,
• proteases and
• beta-glucanase
These are produced during malting and from the time of
kilning they lay dormant waiting to be reactivated by the
moisture and heat of the mash vessel.
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Scenario 2: Brew Mashing

 It is imperative that the correct mashing temperature is

achieved during mashing in,
 High temperature can kill the enzymes resulting in poor
conversion or no conversion taking place;
• Enzymes known as proteases break down large protein
molecules into amino acids and these amino acids are
themselves an essential yeast nutrient.
• The yeast can only digest small sugar particles.
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Scenario 2: Brew Mashing

 Glucans are carbohydrate polymers of glucose and are largely

present in the cell walls of the endosperm. They are broken
down by beta-glucanases ( Ceramix ) at about 50 - 55°.
 This too is a prerequisite for successful mashing and takes
place mostly during malting. Betaglucanase / Ceramix (an
enzyme) is added to assist in the break down of beta glucans
and improve the filterability of the beer.
 They are sticky and can block the fillter or will pass through
the filter and show as haze.Friday, December 6, 2019 20
Case Study 2: Brew Mashing
The most significant process taking place in the mash conversion
however, is the breakdown of starch into fermentable sugars;
The range of sugars produced during conversion determines the
fermentability of the wort.
If the enzyme attack is incomplete, the starch will not be
completely converted into fermentable sugar and so the desired
final gravity will not be reached at fermentation.

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 Case Study 2: Brew Mashing
Enzymes are sensitive to the conditions that they work in, they
are affected by how much water is present, temperature and
pH or mash acidity.
The optimum pH in MCV is 5.2 - 5.4.
They take time to work, so the length of time that is allowed
for mash conversion will affect the degree of conversion.

This would be detected in the Iodine test as 'black brew‘ or

Positive brew.

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Enzyme Activity

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Factors Affecting Enzymes Actions
 Enzymes are affected by changes in temperature and pH.
 The most favourable pH value - the point where the enzyme
is most active - is known as the optimum pH.
 Extremely high or low pH values generally result in complete
loss of activity for most enzymes.
pH is also a factor in the stability of enzymes
• As with activity, for each enzyme there is also a region of pH
optimal stability. The optimum pH value will vary greatly
from one enzyme to another.
• Most of the brewing enzymes have an optimum pH in the
range 4.5 to 6 which is the operating range of most brewing
processes. Friday, December 6, 201924
 Enzyme optimum pH
Enzyme pH of optimum activity Action

Alpha–amylase 5.2 Breaks down starch to glucose,

maltose and a wide range of dextrins

Beta–amylase 5.5 Breaks down starch mainly to maltose

but some glucose and maltotriose

Proteases 5.5 Breaks down proteins to smaller

proteins and amino acids

Beta–glucanase 6.0 Breaks down cell walls (i.e. beta

glucan)

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 Enzyme activity with temperature
Like most chemical reactions, the rate of an enzyme-catalysed reaction
increases as the temperature is raised
Enzyme Temperature of activity Action

Proteases Aprox 50 degrees C Breaks down proteins into smaller

proteins and amino acids

Beta-glucanase Aprox 55 degrees C Breaks down cell walls and frees the
starch that is inside the cells

Beta-amylase Aprox 63 degrees C Breaks down starch mainly into

maltose but some glucose and
maltotriose
Alpha-amylase Aprox 65 degrees C Breaks down starch to glucose,
maltose and a range of dextrins

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Case Study 2: Brew Mashing
To assist the natural enzymes in the malted barley to convert
the starch into fermentable sugars, the following enzyme
supplements are added;

Ceramix

Amagase( Brewdiase )

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Case Study 3: Cellulose Conversion
Lignocellulosic materials are a potential feedstock for producing fuels and chemicals.
• are renewable material
• obtained from fibrils
• Have two components: cellulose(30-60%) and hemicellulose (10-30%)
Cellulase is an enzyme used to hydrolyze cellulose
 Present mostly in the fungus
 3 types of them are used from Trichoderma fungus
1) endo- β – 1,4 glucanases
2) Cellobiohydrolysases: cleave cellobiose and glucose from a non reducing end.
3) β-1,4 glucosidase : Convert cello biose to glucose
(Reading Assignment: Enzymatic Lignocellulose Conversions)

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THANK YOU!

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