Industrial Enzymes

Learning objectives:

Describe the different market sectors where industrial enzymes are used
Describe the basic properties of enzymes, explain how they differ from
chemical catalysts and how enzymes catalyse biochemical reactions
Understand the basic principles of enzyme kinetics, explain how enzyme
activity is measured. Explain and analyse enzyme kinetic data (Michaelis-
Menten kinetics)
Understand the principles of biological detergents and explain the benefits
of enzymes in industrial detergents
Describe the different classes of enzymes used in detergents and the roles
they play
Using Lipolase as a case study describe the genetic engineering and protein
engineering strategies to produce and optimise this industrial enzyme
Describe a typical industrial enzyme production process including upstream
and downstream unit operations.
 Industrial Enzymes
The global market for industrial
enzymes is forecast to reach US$5.6
billion in 2018.

Food & Beverages is the largest

market for Industrial Enzymes with
an estimated share of 26% that is
equivalent to US$1.4 billion in 2017

Biofuels and Detergents with 18% Industrial enzymes

(US$969.3 million) and 14%
(US$754.4 million) respectively in
the same year.

Industrial Enzymes in Biofuels is

expected the fastest growing segment
through to 2024.
 Enzymes
Enzymes are catalysts that perform a wide variety of biochemical reactions in
the cell

They differ from chemical catalysts;

1) Rates are typically 105 1012 greater than un-catalysed reactions and several
orders of magnitude greater than chemical catalysed reactions
2) Enzymes catalyse reactions under mild conditions (atm pressure,
physiological pH etc) in contrast to chemical catalysts
3) Enzymes have high degree of specificity with respect to their substrates
(reactants) and products. Enzyme reactions rarely yield side products
4) Enzyme activity is regulated within the cell. Catalytic activity is regulated
in response to conc of substances other than their substrates. Mechanism
of control includes allosteric control, chemical modification of the
enzyme, varying the amounts of enzyme present in the cell
Enzymes
 Substrate recognition
•Enzymes bind substrates through series
of non covalent forces similar to those
that determine the conformation of
proteins

•Van der Waals, electrostatic, hydrogen

bonding and hydrophobic interactions

•Amino acids that form the binding site

are arranged to specifically interact with
the substrate in a favourable manner (eg
electronic complementarity)

•Substrate binding sites most enzymes

are pre-formed but most exhibit some
Enzyme substrate complex demonstrating degree of induced fit upon binding the
geometrical and physical complementarity
between enzyme and substrates substrate
Catalysts lower the activation barrier for the reaction being catalysed

If an catalyst lowers the

*
activation barrier by ΔΔG*cat then the rate of reaction is
G / RT
enhanced by e cat
Therefore a 10 fold rate enhancement requires that ΔΔG*cat =5.71 kJ mole-1

(less than half the energy of typical h-bond). A million fold rate increase occurs
when ΔΔG*cat =32.25 kJ mole-1 a small fraction of energy of most co-valent bonds.
The rate enhancement is therefore sensitive to ΔΔG*cat

NB kinetic barrier is lowered for

both forward and reverse
reactions.

Catalyst accelerates both

forward and reverse reactions,
hence eqb constant for reaction
is unaltered.
https://pdb101.rcsb.org/learn/videos/how-enzymes-work
 Industrial Enzymes
Learning objectives:

Describe the different market sectors where industrial enzymes are used
Describe the basic properties of enzymes, explain how they differ from
chemical catalysts and how enzymes catalyse biochemical reactions
Understand the basic principles of enzyme kinetics, explain how enzyme
activity is measured. Explain and analyse enzyme kinetic data (Michaelis-
Menten kinetics)
Understand the principles of biological detergents and explain the benefits
of enzymes in industrial detergents
Describe the different classes of enzymes used in detergents and the roles
they play
Using Lipolase as a case study describe the genetic engineering and protein
engineering strategies to produce and optimise this industrial enzyme
Describe a typical industrial enzyme production process including upstream
and downstream unit operations.
 Enzyme activity
• Catalytic potential of enzyme = enzyme activity
• Reduction in energy barrier leads to increase in reaction rate
• Activity is expressed and measured as a rate of reaction typically referred
as “initial rate of reaction “
• Represents the maximum catalytic potential of the enzyme

For conversion of substrate S into Product P (s is molar conc of species S)

𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦=𝑣 𝑡= 0=− ( )
𝑑𝑠
𝑑𝑡 𝑡=0
Production of product recorded
curve with decreasing slope
produced due to enzyme
desaturation, enzyme inactivation,
equilibrium displacement, product
inhibition.

All will regulate catalytic potential

of an enzyme.
 Enzyme activity
Enzyme kinetics quantifies these effects. Therefore determination of enzyme
activity is important to evaluate any enzymatic process.
Enzyme activity can measure substrate (S) depletion or product (P) production.

Measuring product production is preferred – why?

Activity can also be measure by coupling the enzymatic reaction to others

which transform the product into a more detectable analyte (Q) – provided
the transformation of S into P remains the limiting step of the overall
reaction.
=

As initial rate is a measure of enzyme activity – must ensure that this rate
is accurately measured.
 Enzyme activity

Adrian Brown first proposed enzyme catalysed reaction as:

k1 k2
E  S  ES  E  P
k 1

E, ES, ES and P correspond to enzyme, substrate, enzyme-

substrate complex and products.

Using this model when [S] is high enough to convert E to ES form

second step of reaction is rate limiting and overall reaction is
insensitive to further increases in [S]
This is the Michaelis-Menten (MM) equation-basic equation of enzyme
kinetics

Vmax [ S ]
vO  Have you seen a similar equation before?
K M  [S ]
 Plot of vo of a simple MM reaction vs [S]

MM eq describes rectangular
hyperbola as shown.

•Definition of KM. At substrate conc where [S] = KM eq () yields vo=Vmax/2

So KM is substrate conc at which the reaction velocity is half its maximum

•Enzymes with low KM achieve max catalytic efficiency at low substrate

conc

•KM varies with the enzyme, temp and pH.

 Analysis of kinetic data
•At high [S] vo approaches Vmax.
•In practice difficult to asses Vmax from plots of vo against [S]
•At high [S] = 10KM indicates that vo =91 Vmax
•Leads to underestimation if extrapolated
1  KM  1 1
   
Can use a reciprocal plot v o  Vmax  [ S ] Vmax

Linear eq in 1/vo and 1/[S].

Therefore plot enables slope
= KM/Vmax and y intercept is
1/vo and x intercept is -1/KM

Double reciprocal or Lineweaver-Burke plot

 Eadie-Hofstee plot
•Plot of kinetic enzyme data that gives a straight line for
reactions obeying Michaelis-Menten kinetics.

•Reaction velocity (V) versus velocity/substrate concentration

(V/[S]).

•The slope of the line is equal to -KM and the x-intercept is

Vmax
•Eadie-Hofstee is more robust against error-prone data than
the Lineweaver-Burke plot.

•Gives equal weight to data points in any range of substrate

concentration or reaction velocity. (The Lineweaver–Burk
plot unevenly weights such points.)

•One drawback from the Eadie–Hofstee approach is that

neither ordinate nor abscissa (y, x) represent independent
variables: both are dependent on reaction velocity
Example questions:

1) Calculate the initial velocity for an enzymatic reaction when Vmax = 9 x 10–5 mol•sec–1,
[S] = 2.0 x 10–3 M, and KM = 8.6 x 10–3 M.

2) The equation describing a typical enzyme catalysed reaction is shown below.

k1 k2
E  S  ES  E  P
k 1

Using the assumption of equilibrium (second step of the reaction is rate limiting) write
an equation that describes the equilibrium dissociation constant of the Michaelis
complex (KS) for the above reaction
 Industrial Enzymes
Learning objectives:

Describe the different market sectors where industrial enzymes are used
Describe the basic properties of enzymes, explain how they differ from
chemical catalysts and how enzymes catalyse biochemical reactions
Understand the basic principles of enzyme kinetics, explain how enzyme
activity is measured. Explain and analyse enzyme kinetic data (Michaelis-
Menten kinetics)
Understand the principles of biological detergents and explain the benefits
of enzymes in industrial detergents
Describe the different classes of enzymes used in detergents and the roles
they play
Using Lipolase as a case study describe the genetic engineering and protein
engineering strategies to produce and optimise this industrial enzyme
Describe a typical industrial enzyme production process including upstream
and downstream unit operations.
 Case Study- Industrial Enzymes
Bio-based Solutions for Laundry Detergents
The best known application of enzymes is in the manufacture of enzymatic
washing agents (‘biological’ detergents). There are two major reasons for
this.

• Firstly, since the mid­60s the use of enzymes in detergents has been the
largest of all enzyme applications.

• Secondly, consumers of washing agents are actually users of an

enzymatic product. In the vast majority of other applications, enzymes
are used as auxiliary agents at some point in the manufacturing process
and are not, as a rule, present in the finished product
 Case Study- Industrial Enzymes
History:
First detergent containing a bacterial protease (“Biotex”) was introduced by
Novo Industry A/S (now Novozymes) in 1956. It contained an alcalase
produced by Bacillus licheniformis.

In 1994, Novo Nordisk introduced LipolaseTM, the first commercial

recombinant lipase for use in a detergent, by cloning the Humicola
lanuginose lipase into the A. oryzae genome.

In 1995, Genencor International introduced two bacterial lipases, one from

Pseudomonas mendocina (LumafastTM), and another from Pseudomonas
alcaligenes (LipomaxTM). An enzyme added recently to detergents is
MannawayTM, a Bacillus mannanase which removes food stains containing
guar gum
 Case Study- Industrial Enzymes
Dirt comes in many forms and includes proteins, starches and lipids. In
addition, clothes that have been starched must be freed of the starch.

Using detergents in water at high temperatures and with vigorous

mixing, it is possible to remove most types of dirt but the cost of
heating the water is high and lengthy mixing or beating will shorten
the life of clothing and other materials.

If you could engineer the

perfect laundry detergent
what properties would this
have?

Discuss
http://biosciences.dupont.com/fileadmin/user_upload/Editor/
Industrial_Biosciences/infographics/enzym_explainer/
 Case Study- Industrial Enzymes
Proteases, lipases, amylases, oxidases, peroxidases and cellulases are added to
detergents where they catalyze the breakdown of chemical bonds on the addition
of water. To be suitable, they must be active under thermophilic (60 °C) and
alkalophilic (pH 9–11) conditions, as well as in the presence of the various
components of washing powders.
Proteases
Proteases are the most widely used enzymes in the detergent industry. They
remove protein stains such as grass, blood, egg and human sweat.

Proteases hydrolyze proteins and break them down into more soluble polypeptides
or free amino acids. As a result of the combined effect of surfactants and enzymes,
stubborn stains can be removed from fibres.
Novozymes’s first detergent protease was Alcalase® developed in the 1960s.

Amylases
Amylases are used to remove residues of starchy foods such as mashed potatoes,
spaghetti, oatmeal porridge, custards, gravies and chocolate. This type of enzyme,
e.g. Termamyl®, can be used in laundry detergents as well as in chlorine ­free
automatic dishwashing detergents.
 Case Study- Industrial Enzymes
Cellulases
• The development of detergent enzymes has focused mainly on enzymes
capable of removing stains.
• Cellulases modify the structure of cellulose fibrils such as those found on
cotton and cotton blends enabling colour brightening and softening.
Lipases

• Oily and fatty stains have always been troublesome to remove. The trend
towards lower washing temperatures has made the removal of grease spots
an even bigger problem. This applies particularly to materials made up of a
blend of cotton and polyester.

• 1988 Novo launched Lipolase® for the detergent industry – the first
industrial enzyme developed by the application of genetic engineering.
Lipolase is capable of removing fatty stains such as lipstick, frying fats,
butter, salad oil, sauces and the tough stains on collars and cuffs.
https://www.youtube.com/watch?v=GQ1R-mFu2jw
 Case Study- Industrial Enzymes
Liquid detergents=Increasing interest in liquid household washing
detergents.

Why are these more expensive or less common?? Can you get liquid
dishwasher tablets yet?

It is more difficult to preserve the stability of the enzyme in liquid detergents

than in washing powders.
Novozymes enzymes are now formulated in a liquid form that can be used in
practically all types of liquid detergent.

In recent years, the application of enzymes for washing purposes

has been given further impetus by endeavours to save energy. By
optimizing application parameters, a satisfactory effect can be
achieved even at very low temperatures, either by increasing the
application time (pre­soaking) or by increasing the enzyme
concentration (pre­spotting).
 Case Study- Industrial Enzymes
Detergent enzymes must be cost-effective and safe to use.

Enzymes are used in surprisingly small amounts in most detergent

preparations, only 0.4 - 0.8% crude enzyme by weight (about 1% by cost).

The ability to withstand the conditions of use is a more important criterion

than extreme cheapness.

Once released from its granulated form the enzyme must withstand anionic
and non-ionic detergents, soaps, oxidants such as sodium perborate which
generate hydrogen peroxide, optical brighteners and various less-reactive
materials, all at pH values between 8.0 and 10.5.

Although one effect of incorporating enzymes is that lower washing

temperatures may be employed with consequent savings in energy
consumption, the enzymes must retain activity up to 60°C.
 Biomanufacturing Industrial Enzymes
The detergent enzymes used are all produced using species of Bacillus,
mainly by just two companies. Novozymes (NovoNorisk) and Danisco
(Dupont),

Alcalase, from B. licheniformis, Esperase, from an alkalophilic strain of a B.

licheniformis and Savinase, from an alkalophilic strain of B.
amyloliquefaciens

GistBrocades produce and supply Maxatase, from B. licheniformis. Alcalase

and Maxatase (both mainly subtilisin) are recommended for use at 10-65⁰C
and pH 7-10.5.

Savinase and Esperase may be used at up to pH 11 and 12, respectively.

The a-amylase supplied for detergent use is Termamyl, the enzyme from B.
licheniformis which is also used in the production of glucose syrups. a-
Amylase is particularly useful in dish-washing and de-starching detergents.
 Cell factories for production
of biobased products
https://www.basf.com/global/en/products/segments/nutrition_and_care/
nutrition_and_health/enzymes.html

https://www.novozymes.com/en/biology
 Case Study- Lipolase

https://www.ncbi.nlm.nih.gov/Structure/pdb/1DT3

https://www.youtube.com/watch?v=o8Cj-1WZfOg
 Case Study- Lipolase
Genetic Engineering
Novo Nordisk introduced LipolaseTM as the first detergent lipase with a commercially
relevant cost performance based on the use of rDNA technology in an industrial
scale[1].

The gene encoding LipolaseTM was cloned from Humicola lanuginose into Aspergillus
oryzae enabling large scale production of active recombinant Humicola lipase

It has also made it possible to alter the DNA sequence randomly or at specific sites
of a lipase enzyme, in order to change the amino acid sequence producing a variant
lipase.
Protein Engineering
• Engineering has improved the properties of detergent lipases
• Substrate specificity
• Thermostability,
• Laundry wash performance
• Protease stability.
• It has wash relevant properties like alkaline pH optimum, stable at wash
temperatures up to 60 degree C, stable in proteolytic wash solutions, oxidation
stability, stable towards several other detergent ingredients including surfactants.
 Case Study- Lipolase
Genetic Engineering Protein Engineering

Plasmid
Lipase gene from (expression vector)
Humicola sp

Aspergillus oryzae
(cell factory)

Recombinant Humicola lipase

 Enzyme Production Flow Sheet
Upstream processing
Enzyme secreted Downstream
from cells Processing

Enzymes inside
cells (intracellular) Cell Lysis
Cell Factories –Engineered
for the production of Separation of cells from
industrial enzyme fermentation media

Downstream Processing/Purification
Purified enzyme Liquid formulation

Solid
(lyophilisation/drying)

Chromatography Immobilisation
(High Resolution Purification)
https://www.youtube.com/watch?v=pq3veCjuI7s

https://www.youtube.com/watch?v=ghXR3ImYgvA

https://www.youtube.com/watch?v=FGKWmWeN5Kg