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 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 enzymes Fine chemicals manufacture Chiral 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, b-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 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

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

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)

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 Immobilised thermolysin Product out Column recharge 100% Product yield Substrates in out-flow 0% Time (weeks or months)

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

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.

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

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)