Enzymes: Advances in Sustainable Industrial Processes and Bioenergy CDMA Fall Meeting Philadelphia, PA 29 September, 2005 Joel R. Cherry, Ph.D. Director, Bioenergy/Biotechnology Novozymes, Inc. Unlocking the magic of natureoverview of the versatility and the environmental advantages of enzyme technology.
Novozymes - Biotech-based world leader in enzymes & microorganismsGrowth by innovation - new products, applications, markets to drive sales growth of 8-9% Business Technical Food enzymes Feed enzymes Microorganisms Total area enzymes Main Detergent, starch, Baking, brewing, Animal feed Cleaning, plant Industrial markets textile, fuel wine care, waste biotech ethanol, juice, food treatment pulp&paper, specialities leather, personal care, fats&oils Market 45-50% 30-35% 45-50% 50%+ Approx. share (combined) 44% Novozymes, 2004 New growth areas: Pharma • Revenues: >$1 billion, world-wide sales discovery and production: • ~3,000 employees • Antimicrobial peptides • Major sites: • Denmark (Headquarters) • mAb production • US (N.C., CA, NY) • allergy vaccines • China, Japan, Brazil, France, Switzerland
Novozymes created the enzyme market 6 Novozymes sales 5 ons 4 li catiDKK w app f nebillion 3 tio no o duc 2 Intr 1 0 1987 1969 1978 1981 1990 1996 1999 1984 1963 1966 1972 1975 1993 Examples: 1974 Esperase First extremophile enzyme 1984 Maltogenase First recombinant enzyme, starch 1988 Lipolase First commercial lipase, detergent 1997 Kannase First low temperature and soft water protease, detergent 2000 Mannaway New enzyme class for stain removal, detergent
Novozymes’ Vision “We imagine a future where our biological solutions create the necessary balance between better business, cleaner environment and better lives”Intentionally left blank
The Novo Family, circa 1925, Denmark The Novo Nordisk Foundation Holding Company Pharmaceuticals Industrial Biotechnology
Towards sustainability The Industrial Revolution Conventional industry • Economic growth • Great costs to Nature threatening to growth for future generations The Industrial Evolution Biotechnology Unlocking the Magic of Nature Using our knowledge about living systems to create innovative solutions • Economic growth • Less costs to Nature enabling growth for future generationsWe think of ourselves as a business to change things – for the better. We want to play a role in the development towards sustainability. The change boils down to pushing ”the industrial evolution”concept – to move from the industrial revolution to the industrial evolution. For more than a century, global industry has looked to machines to revolutionise industry. This has been extremelysuccesful – it has created economic growth but it has been achieved at great cost to nature and people everywhere. It has been disturbing natures recycling of resources to a level which isthreatening to the opportunities for growth for future generations. At Novozymes we believe the time has come to evolve this traditional way of thinking and to use modern biotechnology to drivethis evolution of global industry forward. Thus The industrial evolution is about biotechnology. At Novozymes we are talking about unlocking the Magic of Nature. Because biotechnology is aboutusing and developing the knowledge we have about living systems in Nature to create innovative solutions - and that will enable economic growth – but do so at less costs to Nature than theconventional technology – so that instead of threatening the opportunities for further growth we are actually strengthening these opportunities.This all means that the development towards industrial evolution is a development towards sustainability
The Use of Enzymes Contribute to Sustainable DevelopmentEnzymes:• Are biological catalysts in the form of proteins that drive chemical reactions in the cells of living organisms• Are very speciﬁc catalytic properties• Work at mild conditions and only small quantities are required• Are fully biodegradable• Are made from renewable resources • e.g., glucose, starch, soy, protein• Excess biomass is used as soil conditioners and fertilizers e.g., cellulase• Improve product quality• Save water, energy, chemicals and waste• Speed up production processes• Enable novel products
Our enzyme technology and production Native enzyme Improved Leading-edge biotechnology alkaline activity expertise • Enzymes sourced from nature • World’s largest strain collection Altered • Bacteria, Fungi speciﬁcity • Recombinant production • Diversity generation: • Strong screening tools/capabilities Improved • Engineering thermostabilit • Shuffling y è We have the ability to ﬁnd the right enzyme to do the job ! Activity, Performance, Stability, Compatibility, Speciﬁcity, State-of-the-art production Temperature, pH, Pressure with continued optimisations Chelants, New activities, and economies of scale Expression• More than production sites globally • 5 strategic twenty commercialized protein engineered • Site DK is world’s largest plant for enzymesenzymes the ability to produce the è We have right enzyme in the amount needed !
Novozymes: A wealth of enzymesEndoproteases: Mutanases Catalases Subtilisin-proteases Dextranases Transglutaminases Trypsin-like proteases Alginate lyases Lactonohydrolases Acidic proteases Pectin esterase Phytases Pectin lyases GlutaminasesExoproteases: Pectate lyases Lactases Peptidases (amino-, Alpha-galactosidases carboxy-,di-, tri- Polygalacturonases Cyclodextrin peptidyl) Rhamnogalacturonase glycosyl transferasesAmylases s Alpha-acetolactateGlucoamylases Arabinanases decarboxylasesPullulanases Galactanases Disulphide isomerasesIsoamylases Lipases NitrilasesEndo Cellulases Cutinases UreasesExo Cellulases Esterases …just to mention aBeta-glucanases Phospholipases few !Xyloglucanases LaccasesXylanases Peroxidases …ability to ﬁnd or Haloperoxidases create THE correct Oxidases enzyme activity for the application
The beneﬁts of using enzymesEnzymes in Sustainable Development
Enzymes for the Detergent Industry e.g., proteases, amylases, lipases, cellulases, mannanases • Lower temperature (from 60oC to max. 40oC) • Energy savings in Denmark (28,000 tons coal/yr) • Less phosphate and chemicals in the environment e.g., Stainzyme • Enzymes are biologically • Stainzyme is a carbohydrate degradable degrading enzyme, which allows efficient removal of stains at washing temperatures as low as 300C. • More gentle to fabrics • If the average washing temperature in Europe is decreased from 400C to 300C a saving corresponding to 1/3 of the electricity-consumption from household washing will be achievedElectricity consumption from household washing in Europe is around 50 TWh a year, or 1½ times the total electricity consumption in Denmark or 10 times the yearly electricity production ofBarsebäck.
Enzymes for the Textiles Industry •Decrease/eliminate use of pumice stones •Decrease/eliminate pumice dust •Decrease/eliminate fabric damage •Increased fabric tensile strength •Decrease waste effluent •Increased garment life Laccase •Decreased pills, fuzz •New fashion looks Cellulase •Decrease/eliminate chlorine and other bleaching agents •New fashion looks •Eliminate use of pumice stones Amylase•Eliminate use of oxidizing agents or sodium hydroxide•Eliminate pressure cooking•Decrease/eliminate fabricdamage
Enzymes for the Baked Goods Industry Without Novamyl • Fungal alpha-amylase: Increases loaf volume and improves eveness of crumb structure. • Maltogenic alpha-amylase: Increases shelf-life. • Glucose oxidase: Oxidizes free sulfhydryl groups to make weak doughs stronger and more elastic. • Lipase: Dough conditioning by producing more uniform, smaller crumb cells and silkier testure and whiter crumb color. • Xylanase: Dough conditioning and improved crumb structure. • Protease: Weakens the gluten to provide better dough for biscuits. With Novamyl
Textiles: Scourzyme application Scourzyme is a pectin degrading enzyme that removes impurities in natural cotton. Scourzyme • Substitutes harsh chemicals (NaOH, peroxide) • At lower temperatures • With less consumption of water as compared to the traditional chemical cleaning process
Life Cycle Assessment LCA is about comparing the environmental impacts of 2 or more alternative systems satisfying the same demand System 1 (enzymatic) LCA User Beneﬁt System 2 (chemical) Auxiliaries (chelator and soda ash) • LCA only considers the environmental impact Scourzyme L Substrate • LCA addresses the whole Fermentati Recovery Formulati LCA addresses a wide range of productionon on production system, from cradle environmental impacts: to grave; from production of raw materials to handling of waste Consumption of resources Cotton Knitting Scouring Bleaching/ Using the Chemicals, energy, water, production land use Colouring cloth Emissions with global impact potential: Global Warming Stratospheric Ozone Depletion Auxiliaries (NaOH, acetic acid and surfactant) Emissions with local/regional impact e.g., Flow diagram, potential: Energy (electricity and heat) Scourzyme Acidiﬁcation Nutrient Enrichment Summer smog Water Toxicityif we want to claim that enzyme technology contributes to a sustainable development we need to demonstrate that theenvironmental load of producing the enzymes is outbalanced by the environmental beneﬁts of using the enzymes.advantages which typically outweigh the load of producing the enzymes in the order of magnitude 10-100 timesAn LCA provides the bass for choosing the environmentally most attractive process from a number of alternative processesproviding the same service.Scourzyme is a pectate lyase which is used in the textile milling industry for the removel of the waxy impurities found in nativecotton.The overall objective is to evaluate the environmental impact of substituting the conventional chemical process for scouring of cotton with a process based on application of ScourzymeKey assumptions: the type of scouring process does not inﬂuence the down stream processing of the fabric (bleaching and dyeing)and the use phase of the fabric. The type of scouring process does not inﬂuence the amount of raw cotton produced.
Life Cycle Assessment LCA is about comparing the environmental impacts of 2 or more alternative systems satisfying the same demand System 1 (enzymatic) LCA User Beneﬁt System 2 (chemical) • LCA: from cradle to grave LCA addresses a wide range of environmental impacts: Emission to the Environmental impacts: Consumption of resources environment: Chemicals, energy, water, land use CO2 Global warming potential Emissions with global impact potential: CO Acidiﬁcation potential Global Warming NOx Stratospheric Ozone Depletion SOx Smog formation potential Emissions with local/regional impact VOC potential: CH4 Nutrient enrichment Acidiﬁcation potential Nutrient Enrichment NO3 Summer smog PO4
Scourzyme application - the environmental impact Scouring of knitted cotton ﬁbre Source: Öeco-Institute e.V. March 2003 Based on case study in the Chinese market.
Examples of environmental beneﬁts incl. contribution from enzyme production Normalised data (expressed as person equivalents)Using enzyme technology has advantages which typically outweigh the load of producing the enzymes in the order of magnitude 10-100 timesNovamyl:person equivalents:Each time we use 1 kg Novamyl (extra) we save CO2 emission corresponding to 3870 kg CO2 equivalents.Each time we use 1000 kg Novamyl (extra) we save CO2 emission corrersponding to 3870 t CO2 equivalents (1000 kg is chosen arbitrarily)1 average world citizen causes an emission of approximately 8 t CO2 equivalents annuallyThus the saving of 3870 t CO2 correspond to the CO2 emission from approximately 470 people.Thus for each time we are using 1000 kg Novamyl (extra) and save 3870 t CO2 equivalents - we could be 470 more people on this earth living a life like the rest of us - without any inﬂuence at all on the global warming.Sustainability is about allowing more people a life at a higher living standard - without increasing the environmental load. It is about diminishing the environmental load of our activities to allow more people have such activities. The savings of bread produced is the single most important contributor to the result. Thus the reduced wheat production (incl. use of fertilizer) can explain 49 % of the reduction of the global warming potential, 49% of theacidiﬁcation potential and almost 100% of the nutrient enrichment potential. However, for the effects on energy the savings on crude oil (for transport) and heat for baking becomes important. 40% of the energy saving is related tocrude oil. The savings of polyethylene packaging material contributes 12% to the energy savings. The creation of summer smog is almost exclusively related to the emission of ethanol from the bakeries.Pulp: smog – disadvantage using enzyme. The smog formation in the thermo mechanical pulp application is highlighted because this is a negative impact - the only case among those listed here where we actually have a disadvantageof using enzymes. The explanantion that we get this negative contribution is the following:When we save energy in the pulping process we obtain less heat for other purposes. Thus we need to generate some heat. We do this by combustion of wood chips - which gives rise to smog formation. However, if you take a look atslide 3 - the normalised data (that is expressed as person equivalents) - the importance of this negative effect is very low. The importance is almost 1000 times less than the positive effect on global warming.Enzymes diminish the pollution of the environmentAn LCA study has demonstrated that 1 kg of Scourzyme can reduce the emission of nutrient salts to the aquatic environment with 2,3 kg phosphate equivalents. If all European cotton (incl. Turkish) was pretreated the savings wouldcorrespond to the emission of nutrient salt pollutants from approximately 400.000 people.Enzymes save energyNZ51054 is an endo-mannanase product used for thermomechanical pulping. 1 kg enzyme product can process 13 t of pulp and reduce the energy consumption with 24.400 MJ. By treatment of 1 million t pulp energy correspondingto the annual consumption of 12.000 people will be saved.Enzymes increase yieldLecitase Ultra is a phospholipase used for removal of the phospholipids from vegetable oil. The use of Lecitase Ultra increases the yield of oil leading to savings in the agricultural production of oil seeds. Savings in agriculturalproduction typically have great environmental beneﬁts –including the savings originating from the environmental load of for instance fertilizers, pesticides and transportation.The LCA study has been build on a speciﬁc soybean oil plant in the US. For each day this plant is in operation the savings in CO2-emission correspond to the annual emission from 30 people or from driving 61.000 miles.Enzymes save non-renewable resourcesPhytase is used in animal feed as a replacement of inorganic phosphate. Phosphate is a limited resource, which are used by all living organisms. By using 1 kg of Ronozyme P5000 CT 30 kg of phosphate is saved. The average annualper capita consumption is estimated to be 22 kg phosphate. The savings originating from providing phytase to the 23 million pigs in Denmark represent phosphate savings corresponding to the yearly consumption of phosphate forabout 1 million people.Enzymes substitute harsh chemicals
Enzymes can reduce the Enzymes can save Enzymes can save agricultural rawpollution of the aquatic energy materialsenvironment Detergents Fats&OilsPulp & paper • Enzymes in detergents for •Phospholipase for removal of• Lipase for stickies control the phospholipids from low temperature washing• Protease for bioﬁlm removal vegetable oil Pulp&Paper• Cellulase/amylase for de- • Lipase for interesteriﬁcation •Lipase for pitch control in of fatinking the paper industry (reduction ofLeather industry down time) Juice• Lipase for degreasing Enzymes can save money! for juice-production Textile •Pectinase• Protease for deharing •Pectinase for pretreatment Feed of cottonFood industry •Cell-wall degrading enzymes Juice for animal feed• Lipase for interesteriﬁcationof fat • Pectinase for juice Food manufacturing• Protease/peptidase for •Protease for treatment ofhydrolysis of vegetable proteins Brewing meat • Hemicellulase for brewing • Lactose oxidase for up- (viscosity reduction) grading of whey Textiles •Pectinase for pretreatment of cotton
Just as oil declines, population peaks… Source: United Nations, World Population Prospects, The 1998 Revision; and estimates by the Population Reference Bureau. Oil will never run out, it will just become prohibitively expensive… …in our lifetimes, or our children’s lifetimes.
Historical and projected development of the grain-based fuel ethanol production a. p. h 30 Europe wt o gr China % Billion litres/year 25 20 e ag er 20 Av 15 USA 10 5 0 Year: 81 83 85 87 89 91 93 95 97 99 1 3 5 7 9 0 0 `0 `0 `0 Novozymes’ 2004 estimateDette er den historiske udvikling af hele det amerikanske ethanol marked gennem de seneste 20 år med en gennemsnitsvækst på ca. 5% frem til 2001.Denne vækst er sket under betingelser hvor der ikke har været særlig bevågenhed og efterspørgsel. Vores estimater peger på en langt højere vækst 20% p.a. for de næste 5 årvil være realistisk.Det kan vi sige ret præcist da vores estimater bygger primært på den stærkt forøgede aktivitet vi ser for planlægningen og bygning af nye tør formalings fabrikker I USA. Manerkender allerede dette I stigningen fra 00 til 01.
Ability to signiﬁcantly impact petroleum consumptiondepends on biomass utilization 150.0 112.5 Billion gallons / yr 75.0 37.5 0 Corn (today) Corn (max) MTBE replacement (max) + Stover Corn Total Biomass Baseline gasoline demand
Issues: BioethanolEnvironmental • Biofuel is a sustainable and almost CO2-neutral energy source • Fuel ethanol can replace MTBE as octane booster in gasoline 10 US states have successfully banned MTBE (ex. California, New York and Connecticut), creating a 1.4 billion gallon per year market • Fuel ethanol - as a ﬂuid energy source for the transport sector - is the only alternative to gasoline (except from biodiesel and gas) • Already two type blends of ethanol/gasoline on the market • blends of 10% ethanol and 90% gasoline = E10 • blends of 85% ethanol and 15% gasoline = E85
Novozymes Biotech, Inc.National Renewable Energy Labs US Dept of Energy BioEnergy Project $17.8 million/4 years Unlocking the magic of nature
Converting biomass to fuel ethanol Biomass 38% Cellulose Pre-treatment Acid pretreated corn stover 56% Cellulose Costs 10X too much! 32% Hemicellulose 5% Hemicellulose 28% Lignin 17% Lignin 13% Other 13% Other Processed biomass Enzyme hydrolysis (cellulose) (cellulases) Starch Glucose Currently: Fermentation Starch to ethanol Enzyme hydrolysis (alpha-amylase) Ethanol (glucoamylase) 72% Starch 10% Cellulose/Hemicellulose 9% Protein 4% Oil 4% OtherFacts: (for background information only)•Grant in January 2001 from NREL / US DoE worth up to USD 14.8 million over three years (with milestones) to develop more efficient cellulases for degradation of cellulose from biomass into fermentable sugars. About 27 scientists in the US, but also groups inDK and China. Novozymes to keep all IPRs due to 20% self-contribution.Fuel ethanol is a sustainable and CO2 neutral fuel source and can replace MTBE as oxygenate in gasoline (MTBE originally replaced lead, but is now polluting the ground water reservoirs). Fuel ethanol is also able to supplement normal gasoline as basic fuel for thetransportation sector•Several US states are phasing out the use of MTBE; California from 1 January 2003 (>2 million litres in excess demand), New York from 2004. In addition, the following states either have implemented or will implement a reduction in use of or a phase out ofMTBE: Arizona, Colorado, Connecticut, Illinois, Iowa, Kansas, Maine, Michigan, Minnesota, Nebraska, South Dakota and Washington. Currently, ethanol is subsidized from government side for use as fuel, but when replacing MTBE, the costs of using ethanol iseither on or below current MTBE costs•Current business, from starch to ethanol: Enzyme competitors are Genencor, EBS, Alltech. US fuel ethanol output per year around 2 billion gallons (6.8 billion litres), growing >20 per cent per year•Worldwide market around 20 billion litres from sugarcane or starch (in the form of corn, wheat, barley, cassava, etc.)•Future business, if successful, from cellulose to ethanol (enzyme hydrolysis; cellulose to starch, then liquefaction and sacchariﬁcation to fermentable sugar). The result is lower production price and higher competitiveness also when compared to gasoline(without subsidy)•Markets: US fuel ethanol to grow >20 per cent per year. EU to implement goals (for bio fuels, not only ethanol), 2% of all transportation fuels in 2005, 6% in 2010 and 20% in 2020. China introduced fuel ethanol as gasoline replacement in 2001 and is set toincrease capacity signiﬁcantly, largest ethanol plant; >700 million litres in capacity at one single plant - has large excess capacity in some agricultural grains like corn•Enzyme market potential: 2005; USD 60 million, 2010; USD 200 million (both are conservative estimates)
How can Novozymes make enzymes less expensive? New tools for an old industryReduce enzyme production costs by:• Reduced cost feedstocks• Reduced enzyme recovery• On-site production• Increased fermentation yieldIncrease enzyme activity on a per gram basis by:• More thermostable enzymes• Higher specific activity• Optimization of cellulase enzyme mix
Bioenergy Project Organization Microarray •Novel mRNA’s Fungal Expression Proteomics •Expression of new genes Fungal Expression •Novel secreted proteins •Fermentation •Fungal host development •Fermentation optimization Bioinformatics •New fungal cellulases •Homologue analysis Biochemical Characterization •Protein purification •Substrate specificity •Thermostability •Synergy Directed Evolution •Rational design •Regio-specific mutagenesis •Random mutagenesis •Shuffling •Screening!!!A multidisciplinary approach was taken on the project using many different NZ biotech tools (read slide).Microarray, proteomics and bioinformatics fed novel genes into both expression work, to test their biochemical characteristics on converting biomass to glucose, as well as these genes being fed into the directed evolution technology – which was used to make them more active or with higher temperature stability.In the end, all wild-type and/or protein engineered enzymes were put back into the recombinant host, Trichoderma reesei, for co-expression and ﬁnal testing on biomass conversion to glucose (which is then fermented to ethanol by yeast). The process is either a hybrid or simultaneous sacchariﬁaction/fermentation.Using microarray analysis, over 728 biomass-induced clones were identiﬁed in a cDNA library and sequenced. Sequence analysis revealed 170 non-redundant genes, many of which were recognizable carbohydrate-active enzymes.Sequence analysis revealed 170 non-redundant genes, many of which were recognizable carbohydrate-active enzymes. In addition, genes encoding hemicellulases, lignin-active enzymes, carbohydrates transporters, and others were identiﬁed. 24 genes without signiﬁcant homology to previously cloned geneswere found that could not be assigned a function. These proteins are of signiﬁcant interest as they may represent previously uncharacterized enzymes involved in efficient lignocellulose breakdown.Proteomics was used on the same T. reesei samples used in microarray experiments to help pinpoint those proteins that are both secreted and present in large abundance celluloytic fungi grow on cellulosic substrates. This method relies on the separation of proteins on a two-dimensional electrophoretic gelSeveral proteins were identiﬁed with increased expression on PCS in T. reesei and multiple proteins were a number of fungi that were chosen due to their ability to degrade PCS.Directed Evolution:Natural Diversity: Identiﬁed, cloned and expressed cellobiohydrolases, endoglucanases, and β-glucosidases Bioinformatics, Proteomics, Microarrays, Fungal Expression, Protein Chemistry : Discovery of naturally occurring enzymes with improved thermal stabilityCreating Diversity in the Laboratory: Directed Evolution Screen for improved residual activity following thermal denaturation: β-glucosidase Screen for improved enzyme activity at higher temperature: CBHI
Signiﬁcant biochemical improvements incellulase efficiency have been made over the Expression of four recombinant proteins in production host > 6X
Cost comparison after recent achievements:Grain vs. biomass in USD/gallon ethanol $7.00 3-D Column 1 3-D Column 2 Biomass USD/gallon EtOH produced 3-D Column 4 $5.25 Enzymes Depreciation of capital Improvements: 3-D Column 7 Mix of: Total • enzyme engineering Gasoline • novel enzymes $3.50 gasoline, $/gal • process changes 3-D Column 12 $1.75 $0 StarchCellulose 1999 Cellulose 2004
Enzymes In Action:Contributing to Sustainable Development Goodbye to… Hello to… • Solvents • Reduction in aquatic • Acids environment pollution • Oxidizing Agents • Reduction in agricultural • Chlorine production burden • Food Chemicals • Reduction in energy • Sulphides needs • Pumice • Reduction in petroleum • High Temperatures dependancy • High Energy Requirements
Biotechnology - Added value to the chemicalindustry Impact of industrial Sustainable biotechnology development Cost reduction • Reduction in aquatic • Raw materials environment pollution • Process costs • Reduction in agricultural production • Investments burden Additional revenues • Reduction in energy • New products needs • Value-added • Reduction in processes petroleum dependancy
Microorganisms, the art of our business sustainable developmentEnzymes are unlocking the Magic of Natures Own technologyThe Magic of Nature is the magic of the natural ecosystems operating to maintain balance and proper recycling of the resources of the Earth. The industrial revolution has inﬂuenced this naturalbalance signiﬁcantly - to an extent which requires new technological platforms for continued economic growth.Unlocking The Magic of Nature is about biotechnology, about using and developing our knowledge about living organisms and systems to create solutions, which are more acceptable to theenvironment than current production technology. Enzyme technology is an example of modern biotechnology. This article demonstrates that enzyme technology represents a technology platformwhich can enrich many different industries with new and more sustainable technologies. The platform both offers a variety of potential products as well as advanced production-technology, whichenables quick development of competitive products.The potential of enzyme technology in a sustainability perspective builds on this broad applicability. There are already developed so many enzyme applications that alone a higher penetration ofthese will contribute signiﬁcantly to a better environment. However, hopefully a broader knowledge about the potential of enzymes will also foster the development of completely new and moreradical solutions in support of our need for continuous economic growth.To Novozymes Unlocking the Magic of Natures own Technology is about expanding the use of enzymes to established and completely new industries. It is about transforming the traditionalstandards of industry to the beneﬁt of nature and mankind and it is about dialogue with potential customers, scientists and regulatory authorities who can help us unlock the full potential of ourtechnology. We imagine a future where the industrial revolution is replaced by an industrial evolution building on the multiple opportunities offered by enzyme technology.