English for biotechnology

◆ Enzymes don’t make reactions happen ◆ Enzymes don’t get used up, they can be reused ◆ Enzyme can transform ~ 10,000 substrate molecules into product per second Enzymes speed up the bio

Trinh Thi Phi Ly

English for Biotechnology

INDUSTRIAL ENZYMES

Enzymes are biological catalysts in

the form of proteins that catalyze

chemical reactions in the cells of

living organisms

What are enzymes?

◆ Enzymes don’t make reactions happen

◆ Enzymes don’t get used up, they can be reused

◆ Enzyme can transform ~ 10,000 substrate molecules into product per second

Enzymes speed up the biochemical

reactions without being used in the

reaction itself About 1300 enzymes

were found in human cells

What are enzymes?

Some enzymes help break large molecules into

smaller pieces that are more easily absorbed by the body Other enzymes help bind two molecules

together to produce a new molecule

Enzymes are highly selective catalysts, meaning that each enzyme only speeds up a specific

reaction .

Enzymology is the study of enzymes, their kinetics, structure, and function, as well as their relation to

each other

History of industrial enzymes

✔ Fermentation processes for brewing, baking,

and the production of alcohol, cheese have

been used since prehistoric times

✔ 1833 Payen and Persoz isolated an amylase

complex from germinating barley, this product

converted starch into sugars

✔ 1926 James Sumner discovered enzymes are

a type of protein when identifying urease as a

protein after purification and crystallization

History of industrial enzymes

✔ 1950 Novozyme launched the first fermented

enzyme, a bacterial alpha-amylase

✔ The first commercialized enzyme

expressed in a genetically modified organism

was a lipase for detergents

✔ Recombinant DNA technology has brought a

revolution in the development of new

enzymes

https://www.transparencymarketresearch.com/enzymes-market.html

Enzyme structure

❑ Active site (interactive site): the region of an enzyme where

substrate molecules bind and undergo a chemical reaction

❑ The active site consists of amino acid residues that form

temporary bonds with the substrate

❑ Substrate: a molecule upon which an enzyme acts

Enzyme

Active site

Substrate

Enzyme-substrate complex

◆ A cofactor is a non-protein chemical compound that is required for

the protein's biological activity Cofactors are able to execute chemical reactions that cannot be performed by amino acids

◆ The cofactor can be a metal ion, an organic molecule (coenzyme), or

a combination of both

Enzyme structure

Enzymes are rigid and

specific for one

particular substrate

Model of Enzyme-Substrate interaction

Enzymes are flexible, the active

site continually reshapes by its

interactions with the substrate until

the time the substrate is completely

bound to it

How Enzyme Work

During a chemical reaction, substrates reach a transition state

before they are transformed into products The activation energy

is the energy required to reach the transition state (start a reaction)

Enzymes accelerate the rate of chemical reaction by lowering the activation energy

How Enzyme Work

❑ Enzymes bring substrates together, bind substrate tightly and specifically

at the active site

❑ Substrates don’t have to expend energy for moving and binding

The characteristics of enzymes

✔ Enzymes possess great catalytic power, transforming

about 100 to 10,000 substrate molecules into product per second and proceeding > 100 times faster than

the uncatalyzed reaction.

✔ The active site of an enzyme has a strong affinity

for a specific substrate

✔ Enzymes remain unchanged during the reaction itself

While the enzyme's amino acid residues may break or form covalent bonds with the substrate, it will typically

reform those bonds, enabling the enzyme to react with more substrates.

3 Linkage specificity – the enzyme will act on a particular type

of chemical bond regardless of the rest of the molecular

structure.

4 Stereochemical specificity – the enzyme will act on a

particular steric or optical isomer

The active sites of enzymes have

some common features

◆ The active site is a three-dimensional cleft or crevice

◆ The active site takes up a small part of the total volume

of an enzyme

◆ Substrates are bound to enzymes by multiple weak

attractions

◆ The specificity of binding depends on the precisely

defined arrangement of atoms in an active site

Factors affecting enzyme activity

Enzyme inhibitors

◆ An enzyme inhibitor is a molecule that binds

to an enzyme and decreases its activity

◆ Enzyme inhibition can be either irreversible

or reversible

◆ An Irreversible inhibitor inactivates

enzyme by bonding covalently to a particular

group at the active site The inhibitor

dissociates very slowly from it target enzyme

◆ Reversible inhibitors inactivate an enzyme through a

non-covalent, more easily reversed interactions

◆ A reversible inhibitor can dissociate from enzyme

Allosteric regulation

❑ The regulation of an enzyme by binding an effector molecule

(regulator/modulator) at allosteric site.

❑ Allosteric enzymes are enzymes which have an additional site for an effector to bind to

Negative allosteric effector

Positive allosteric effector

Enzyme inhibitors in medicine

Major producers operating in the global

enzymes market

EC 2

Transferases

Catalyze the transfer of groups or atoms from one molecule

to another or from one position in a molecule to other positions in the same molecule

Glucosyltran sferase

Amylase Cellulase Lipase, pectinase, protease, phytase,…

EC 4 Lyases Catalyze the addition of groups to double bonds or the

formation of double bonds though the removal of groups

Pectate lyase

Catalase (1.11.1.6)

✔ Found in nearly living organisms exposed to oxygen

✔ H2O2 is a harmful byproduct, it must be quickly converted

into others

✔ 1 catalase can convert millions of H2O2 in 1s

✔ Protecting the cell from oxidative damages

Used in food or textile processing to remove H2O2 that is

used for sterilization or bleaching

Glucose oxidase (1.1.3.4)

• Catalyze the oxidation of glucose

• Synthesized in several species of fungi ( Aspergillus

which in turn kills bacteria.

◆ Used for determination of free glucose in blood fluids

◆ Found in honey and acts as a natural preservative

◆ Used as an additive due to its oxidizing effects; helps to remove O2from food packaging

◆ Control glucose level in fermentations/bioreactors and food

products

β-Amylase (E.C 3.2.1.2) hydrolyzes 1,4-α-D-glycosidic linkages, removing

successive maltose units from the nonreducing of the polysaccharide chain.

Pullulanase (limit dextrinase E.C 3.2.1.41) is a starch-debranching enzyme and hydrolyzes the 1,6-α-D-glycosidic bonds in amylopectin

Application of amylases

A group of enzymes that hydrolyze β-1,4 linkages in cellulose chains

Global market size:

US$ 1500 million (2019) forecast US$ 2320 million

by 2024 with 7.5% CAGR in terms of revenue

✔ Peptidases catalyze the breakdown of proteins into smaller

polypeptides or single amino acids

✔ Endopeptidases attack an internal peptide bond, as in trypsin,

chymotrypsin, pepsin, elastase and papain.

✔ Exopeptidases remove the terminal amino acids from the

protein chain Exopeptidases are divided into carboxypeptidases

or aminopeptidases depending on whether they digest proteins

from the carboxy- or amino-terminus

Proteases are

well-established ingredients

in most household laundry

detergents

Most of the commercial

detergent proteases are

subtilisins (extracellular serine endo-peptidases)

Lipases (E.C 3.1.1.3)

Hydrolyze triglyceride substrates in fats and oils

Improve the removal of fatty stains when used in powder and liquid detergents, special pre-soakers, or other cleaning agents

Lipolase was the first commercially

available lipase for

detergents and the first detergent

Enzymes for laundry detergents

✔ The most widely used detergent enzymes are

hydrolases, which remove soils consisting of

proteins, lipids, and polysaccharides To help

remove these stains, a number of different

hydrolases are added to detergents

✔ The major classes are proteases, lipases,

amylases, mannanases, cellulases and

pectinases

✔ Proteases were the first of these to be used

extensively in laundering for increasing the

effectiveness of detergents

Enzymes for laundry detergents

✔ Some lipases can act as alternatives to current

surfactant technology targeting greasy

lipid-based stains, and lipases are thus an

essential part of enzyme solutions used to replace

surfactants.

✔ Multienzyme systems may replace up to 25 % of

a laundry detergent’s surfactant system without

compromising the cleaning effect

✔ Mannases and pectinases are used for

hard-to-remove stains of salad dressing,

ketchup, mayonnaise, ice cream, frozen desserts,

milkshakes, body lotions, and toothpaste

Enzymes in dishwashing

✔ Phosphates have been used in dishwashing

detergents to get dishes clean Phosphates are

low toxicity, but they cause nutrient pollution and

feed the algae This leads to eutrophication and

harmful algal bloom

✔ For removal of starch soils, amylases are used;

and proteases are used for removal of protein

soils

Enzymes for cleaning membrane in the food industry

✔ Various enzymes are used in the

dairy and brewing industries for

cleaning microfiltration and

ultrafiltration membranes, as well

as for cleaning membranes used in

fruit juice processing

✔ As most proteinaceous stains or

soils are complexes of proteins,

fats, and carbohydrates, beneficial

synergistic effects can be

obtained in some cases by

combining different hydrolytic

enzymes

Enzymes for animal feed

• Many feed ingredients are not fully digested by livestock Adding

enzymes to feed, the digestibility of the components can be

enhanced

• Enzymes are added to the feed either directly or as a premix together with vitamins, minerals, and other feed additives

• In premixes, the coating of the enzyme granulate protects the

enzyme from deactivation by other feed additives or to protect the

enzyme from the heat treatments used to destroy Salmonella and

others in feed

• Enzymes for animal feed are now available to degrade substances

such as phytate, beta-glucan, starch, protein, and polysaccharides

Phytase for animal feed

✔ Around 50–80 % of the total phosphorus in

pig and poultry diets is present in the

form of phytate or phytic acid

✔ Phytate is capable of forming complexes

with proteins and inorganic cations such

as Ca, Mg, Fe, and Zn The use of phytase

not only releases the bound phosphorus

but also these other essential nutrients

✔ Use of phytase reduces the harmful

environmental impact of phosphorus from

animal manure in areas with intensive

livestock production

Enzymes for Biofuels

US $Bn 57.65

US $Bn 81.12

5

%

✔ Hydrolytic enzymes for

starch-based raw materials: alpha-amylases, glucoamylases

and related enzymes

✔ Producing fuel ethanol from cereals

such as wheat, barley, and rye presents a quite challenge

✔ Non-starch polysaccharides such

as beta-glucans and arabinoxylans create high viscosity, which reduce the efficiency of separation,

evaporation, and heat exchange

Cellulosic ethanol (2 nd generation)

The complex structure of biomass

is however more difficult to

convert into ethanol than

traditional starch substrates

Enzymes are vital in the

conversion of biomass to ethanol

Enzymes in food industry

• The first saccharification enzyme (glucoamylase) for the food

industry

• Glucose production switched from acid hydrolysis to enzymatic

hydrolysis because of the clear product benefits of greater yields, a

higher degree of purity and easier crystallization

• In 1973 with the development of immobilized glucose isomerase, which made the industrial production of high fructose syrup feasible

Enzyme applications in formula milk

• Proteases have been used for more than 50 years to produce infant milk formulas from cow’s milk,

converting milk proteins into peptides and free amino acids

• Endo- proteases with a preference for degrading

peptide bonds between amino acids in the highly

hydrophilic regions of a protein molecule are used for producing low-allergenic infant formulas

Enzyme applications for brewing

• The traditional source of enzymes used

for the conversion of cereals into beer is

barley malt, but too little enzyme activity

is present in the mash, the fermentation

process will be too slow; too little alcohol

will be produced

• As heat-stable amylases are much more

stable than malt amylases, simpler

liquefaction, shorter process times,

increased productivity

Enzymes for fruit juice processing

• Pectinolytic enzymes are a prerequisite for obtaining clear and

stable juices, good yields, and high-quality concentrates

• Pectins are composed of galacturonic acid units In the past, it was believed that the application of pectinesterase, polygalacturonase, and pectin lyase was sufficient to decompose pectic substances But, other sugars (e.g., rhamnose, xylose, galactose, arabinose) are incorporated into the pectin molecule

• Various other pectinolytic enzymes and hemicellulases are

needed

https://www.creative-enzymes.com/service/Industrial-Enzyme-Production_380.html

Fermentation process

Fermentation is a metabolic process that produces chemical changes

in organic substrates through the action of enzymes

The original definition of fermentation is the anaerobic conversion of

sugars to ethanol and carbon dioxide by yeast

❑ Production of cellular biomass

❑ Production of extracellular metabolites

Fermentation process

Submerged fermentation is a process involving the development of microorganisms in a liquid broth

SUBMERGED

Industrial enzyme production

✔ Both fed-batch and continuous fermentation processes are

common

✔ In the fed-batch process, sterilized nutrients are added to the

fermentor during the growth of the biomass

✔ In the continuous process, sterilized liquid nutrients are fed into

the fermentor at the same flow rate as the fermentation broth leaving the system, thereby achieving steady-state production

✔ Temperature, pH, feed rate, oxygen consumption, and carbon

dioxide formation are usually measured and carefully controlled to optimize the fermentation process

Enzyme harvesting

✔ Separate insoluble products (microbial cells) by

centrifugation or microfiltration.

✔ As most industrial enzymes are extracellular – secreted by

cells into the external environment – they remain in the fermented broth after the biomass has been removed

✔ The enzymes in the remaining broth are then concentrated

by evaporation, membrane filtration or crystallization depending on their intended application

✔ If pure enzyme preparations are required, they are usually

isolated by chromatography and/or by electrophoresis

Enzyme formulation

✔ Certain applications require solid enzyme products, so the crude

enzyme is processed into a granulate

✔ Other customers prefer liquid formulations because they are easier

to handle and dose along with other liquid ingredients

✔ Glucose isomerase used in the starch industry to convert glucose

into fructose is immobilized, typically on the surfaces of particles of

an inert carrier material held in reaction columns or towers This is done to prolong their working life; such immobilized enzymes may

go on working for over a year