bty323 lectures 15, 16 enzymes in industry markets types scale values future examples
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BTY323 Lectures 15, 16
Enzymes in Industry
Markets Types Scale Values Future Examples
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
Enzymes in Industry
Distribution of enzymes by substrate
Protein hydrolysing 59%
Carbohydrate hydrolysing 28%
Lipid hydrolysing 3%
Speciality (analytical, pharma, research) 10%
Enzymes in Industry
Textile processing
Grain processing
Food processing
Cleaning
Feed enzymes
Diagnostic/pharma
Waste management
Other
Textile processing 10Grain processing 12Food processing 18Cleaning 44Feed enzymes 4Diagnostic/pharma 4Waste management 4Other 4
Process % by value
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 enzymesFine chemicals manufactureChiral separation
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
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, -glucosidase) and diagnostic enzymes (DNA polymerase)
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
Examples of Industrial Enzyme Processes
Starch conversions and the production of High Fructose Syrups
Aspartame biosynthesis
Nitrile conversions• Acrylamide
• Nicotinamide
Corn starch processing 1Maize grain
Endosperm
Starch
Corn syrups
High fructose syrupsEthanol
Food additives
Corn steep liquor
Edible oilOil meal
Hulls
Gluten
Germ
Industrial andfood uses
Short chain dextrins (foods)
Maltose syrups
Corn starch processing 2.
Starch slurry40% wt., pH 6.5
* Add Termamyl®* Inject steam* Incubate at 105oC, 5-7 min
Maltose
Adjust pH to 4.5Reduce temperature to 95oCAdd amyloglucosidase
Glucose
High fructose syrup
Reduce temperature to 60-70oCAdd xylose (glucose) isomerase
Enzyme step 1: Action of Termamyl® on starch granules
Termamyl® is an -amylase (cleaves -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!)
0 10(minutes)
Amylase activity
Maltose concentration
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
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.
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)
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
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
Product out
Immobilised thermolysin
Product yield
Substrates in out-flow0%
100%
Column recharge
Time (weeks or months)
Nitrile biotransformations1. 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
Production of acrylamideAcrylonitrile 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.
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
nicotinamide
Rhodococcus whole cell biocatalyst
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)