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)