1. BTY323 Lectures 15, 16 Enzymes in Industry
Markets
Types
Scale
Values
Future
Examples

2. Industrial Enzyme Classes
Commodity enzymes
High volume (tonnes p.a)
Low purity (but not necessarily so)
Low cost (e.g. $5-40 per kg)
Low profit margins
Speciality enzymes
Low volume (g – kg)
High purity
High cost ($5 – 10,000 per g)
High profit margins

3. Enzymes in Industry Distribution of enzymes by substrate
Protein hydrolysing 59%
Carbohydrate hydrolysing 28%
Lipid hydrolysing %
Speciality (analytical, pharma, research) 10%

4. Enzymes in Industry
Process % by value

5. Industrial enzymes: Market trends
Increasing 10-15% annually by volume
Increasing 4-5% annually by value
Decreased margins for commodity enzymes
Increased use of speciality enzymes
Diagnostic enzymes
Fine chemicals manufacture
Chiral separation

6. Industrial enzymes Food processing Textiles Grain processing
Amylases in bread-making
Lipases in flavour development
Proteases in cheese making
Pectinases in clarifying fruit juices
Cellulases in treating denim to generate ‘stone-washed’ texture/appearance
Conversion of corn starch to high fructose syrups

7. Industrial enzymes Feed enzymes Waste management Diagnostic enzymes
Enzymes to assist in the digestibility of animal feeds (cellulase, xylanase, phytase)
Lipases as drain-cleaning agents
Reporter enzymes (alkaline phosphatase, glucose oxidase, b-glucosidase) and diagnostic enzymes (DNA polymerase)

8. Industrial enzymes Speciality Biotransformations
Lipases, esterases and oxidoreductases for chiral separations
Glucotransferases in synthesis of oligosaccharides
Thermolysin in aspartame synthesis
Nitrile hydratases in acrylamide and nicotinamide synthesis
Proteases in peptide synthesis
Penicillin acylase for manufacture of semisynthetic penicillins
Aspartase in the manufacture of L-aspartate

9. Examples of Industrial Enzyme Processes
Starch conversions and the production of High Fructose Syrups
Aspartame biosynthesis
Nitrile conversions
Acrylamide
Nicotinamide

10. Corn starch processing 1
Maize grain
Corn steep liquor
Germ
Edible oil
Endosperm
Oil meal
Hulls
Gluten
Starch
Industrial and
food uses
Short chain
dextrins (foods)
Maltose syrups
Corn syrups
Food additives
Ethanol
High fructose syrups

11. Corn starch processing 2.
Starch slurry
40% wt., pH 6.5
* Add Termamyl®
* Inject steam
* Incubate at 105oC, 5-7 min
Maltose
Adjust pH to 4.5
Reduce temperature to 95oC
Add amyloglucosidase
Glucose
Reduce temperature to 60-70oC
Add xylose (glucose) isomerase
High fructose syrup

12. Enzyme step 1: Action of Termamyl® on starch granules
Termamyl® is an a-amylase (cleaves a-1-4 glucosidic bonds in starch)
High temperature expands starch granules, making amylose and amylopectin chains more accessible
Termamyl is sufficiently stable at high temperatures if short reaction times are used
Starch hydrolysis is a batch process (the enzyme is not reused!)
Maltose concentration
Amylase activity 10
(minutes)

13. Enzyme step 2: Conversion of maltose to glucose
Amyloglucosidase is not as thermostable as Termamyl (temperature must be reduced)
Amyloglucosidase has a pH optimum of 6.5 (Termamyl® operates optimally at 8.5): pH must be reduced
Reaction kinetics are slower
Long incubations result in caramelisation of the saccharides - resulting in product loss and increase in impurities

14. Enzyme step 3: Conversion of glucose to fructose
Fructose is much sweeter than glucose; it can be used as a sweetening agent in foodstuffs, and is more profitable than glucose
The enzyme xylose isomerase will convert glucose to fructose, in an equilibrium reaction
Glucose  Fructose
A 50:50 mixture of glucose:fructose is sold as high fructose syrup (HFS)
Xylose (glucose) isomerase is much less thermostable, and inhibited by Ca ions.

15. Aspartame biosynthesis
Aspartame (L-phenylalanyl-L-aspartyl-methyl ester) is a low-calorie artificial sweetener used widely in soft drinks and confectionary products (e.g., Diet Coke). It can be synthesised chemically, or biocatalytically by peptide synthesis using a thermostable protease – Thermolysin® from the facultative thermophile, Bacillus thermoproteolyticus.
CBZ-L-Phenylalanine + L-Aspartyl-OMe
Immobilised Thermolysin in low-water content organic solvent system: 50oC
CBZ-L-Phe-L-Asp-OMe
Chemical removal of CBZ group (deblocking)
L-Phe-L-Asp-OMe
(Aspartame)

16. Key process characteristics
Immobilised enzyme allows continuous process and enzyme reuse
Proteases normally hydrolyse peptide bonds: a low water activity solvent system (organic solvent based) is necessary to reverse the normal equilibrium
Organic solvents often promote enzyme denaturation: Thermolysin® as a stable thermophilic protease
Product recovery is easy – the CBZ-L-Phe-L-Asp-OMe intermediate crystallizes out in the reaction media

17. Column-based biosynthesis of Aspartame®
Thermolysin® is used in a column format
Reaction will be run continuously until substrate ‘breakthrough’ is observed
This indicates that the enzyme efficiency is dropping (inhibition or denaturation)
Several columns may be operated in series to achieve maximum conversion efficiencies
Substrates in
Immobilised
thermolysin
Product out
Column recharge
100%
Product yield
Substrates in out-flow 0%
Time (weeks or months)

18. Nitrile biotransformations
Synthesis of acrylamide. A small proportion of the worlds supply of acrylamide (about 45kT p.a.) is synthesised biologically, using a whole cell catalyst. The catalyst is an engineered Rhodococcus strain containing high levels of the enzyme nitrile hydratase (NHase). This catalyst has been through three ‘generations’ of application:
1. Use of the native organism
2. Use of a recombinant Rhodococcus where the NHase gene was cloned and re-expressed at high levels in the parent organism.
3. Use of a recombinant Rhodococcus where the NHase gene was engineered to increase stability, and to reduce substrate and product inhibition, then re-expressed at high levels in the parent organism

19. Production of acrylamide
Acrylonitrile CH2=CH-CN
NHase-containing Rhodococcus cells in stirred tank bioreactor
Acrylamide CH2=CH-CONH2 + NH3
Acrylamide is widely used in industry as a precursor for the formation of acrylic polymers, in the construction, paint, and household products industries, and for laboratory use.
The biological production of acrylamide has advantages over the chemical synthesis because of the absence of side-reactions, and the simpler recovery of the reaction product.

20. Production of nicotinamide
Nicotinamide is an essential vitamin, and is widely used in the health-food and animal food-and-feed industries. Biological production, using the same Rhodococcus biocatalyst as for acrylamide production, operates at about 5kT p.a.
3-cyanopyridine
Rhodococcus whole cell biocatalyst
nicotinamide

21. Other large-scale industrial enzyme processes
Penicillin acylase
Penicillin (produced at very high yields by industrial-strain Streptomyces fermentations) is converted enzymatically to 6-aminopenicillanic acid
6-Aminopenicillanic acid is a substrate for chemical or microbial conversion to valuable commercial antibiotics (e.g. Ampicillin)