Overview Of Dyadic InternationalDyadic uses a cutting edge patented C1 technology (Chrysosporium lucknowense) to create products for multi-billion dollar markets in industrial enzymes, biofuels, and biotherapeutics. Biopharma Agriculture Chemicals Enzymes Biofuels Diagnostics Health & Future Beauty Applications
Overview Of Dyadic InternationalProducts created through Dyadic’s patented C1 technology include: • Food Enzymes • Brewing Enzymes • Pulp and Paper Enzymes • Textile Enzymes • Enzymes used to create biofuels • Methods to facilitate the the discovery and development of human antibodies and therapeutic proteins
Dyadic International’s Market Niche• Dyadic International market capitalization is $17.7 million• Market capitalization up +293.3% YTD over the following industry peers:
Textile Food Brewing Animal Starch/ Pulp & Ethanol Food Alcohol PaperRocksoft® FoodCel Brewzyme Dyadic® Celustar, Fibrezyme® AlternaFueFamily of Family of Family of Xylanase, Alphasta Family of lEnzymes Enzymes Enzymes Beta r Enzymes Family of Glucanase, Family of Enzymes and Enzymes Cellulase Family of Products
Dyadic New Markets - BiofuelsHydrolysis of hemicelluloseand cellulose to monomersugars is one of thesignificant steps inbioethanol productionwhich can be accomplishedby using cellulases.
Dyadic Patented C1 Technology PlatformC1 is a fungal host production organism with superior genetic and fermentation characteristics that Dyadic originally used to manufacture enzymes for the textile industry.
Dyadic Patented C1 TechnologyDyadic International’s C1 technology is based on a system of protein production based on the patented Chrysosporiumlucknowense fungus.
Gene DiscoveryThe Fungal HTRS System from Dyadic, enables rapid genediscovery from all biodiversity (including eukaryotes) usingthe C1 host organism.Host OptimizationThe C1 platform allows for optimization of the hostproduction organism and manufacturing process.Proteins expressed using the C1 Expression System providethe foundation for proper glycosylation.Large Scale ManufacturingThe C1 organism has been used for biomolecule productionin 150,000 liter fermenters since 1996.
The C1 Advantage• Dyadic has innovated the only high throughput robotic gene discovery system that utilizes a non-yeast, eukaryotic host organism, ideal for rapidly discovering the full spectrum of nature’s gene sources that until now have been inaccessible.
Why Are Enzymes Important?Enzyme Group Industrial ApplicationsCarbohydrases Laundry and dishwashing detergents, industrial pipe/tank cleaners, textiles, pulp and paper, fermentation ethanolProteases Brewing, baking goods, protein processing, distilled spirits, laundry and dishwashing detergents, lens cleaners, leather and fur, chemicalsPeptidases Food and animal feed; Chiral resolution of chemical compounds or pharmaceuticalsLipases and Esterases Cleaners, leather and fur, dairy, chemicalsOxireductases Chemicals, detergent bleaches, pulp bleachingLyases Brewing industry; CosmeticsTransferases Manufacture of cyclodextrins from starch
EnzymesEnzymes are essential proteins that are found in all living cells. They have two main functions: 1) they act as catalysts ( a catalyst is a chemical which speeds up a chemical reaction ) 2) they control all the chemical reactions going on inside the cell.
A Word About EnzymesEvery cell has many different enzymes. Each enzyme isspecific : it only affects one particular chemical. Thischemical that the enzyme alters is called the substrate.An enzyme can either break apart one substrate intotwo different products, or it can join two substratesinto a single product. One example is shown below :
Assay Example - CMCaseCellulose• 33% of all plant matter is cellulose• Cellulose rich plant biomass is one of the foreseeable and sustainable source of fuel, animal feed, and feed stock for chemical synthsesis.
CelluloseCellulose is an organic compound consisting of a linear chain of several hundred to ten thousand linked p-glucose units • Component of primary cell wall in green plants • Most common organic compound on earth
QA/QC LaboratoryQA is short for Quality Assurance, which is defined asthe overall program that ensures that the finalresults reported by the lab are correct. QC is short for Quality Control, which refers to the measures that must be included during each assay run to verify that that the test is working properly.
Why Is A QA/QC Lab Important?• Determining the quality of the product• Translates to sales and profitability• Development of new productsFor example: – Enzymes that decrease in activity, may not meet client specifications. Those enzymes may than be sold to another client at a reduced price.
Dyadic Internship ProjectTest enzyme producedby Dyadic and itscompetitors to validateenzyme activity to meetrequired specifications.
Assays Performed• CMCase – DNS This assay measures the release of reducing sugars by the action of a cellulase on a cellulosic substrate• Xylanase – DNS This assay measures the release of reducing sugars by the action of a Xylanase on an Arabinoxylan substrate• Beta-Glucanase This assay measures the release of reducing sugars by the action of a Beta-Glucanase on a Barley Beta-Glucan substrate
Assays PerformedProtein Quantification WithBCA (bicinchoninic acid) SDS Page, used to To separate proteins according to electrophoretic mobility
Assay Example - CMCaseQuestion: Do current stores of Brewzyme BGX CONC (D12160810, Lot# MXEB02810) meet activity requirements of client X, who use it to break down the cellulose component of a particular product line?Answer: Perform CMCase Assay
Assay Example - CMCase• CMCase – DNS Assay Essential Components• Enzyme samples• Carboxymethylcellulose sodium salt• DNS (3,5 dinitrosalicylic acid)• .015 M Sodium acetate buffer, pH 4.8• UV spectrophotmeter
Assay Example - CMCaseCellulose• The conversion of cellulosic mass to fermentable sugars through biocatylist cellulase durived from cellulolytic organisms is at the core of Dyadic International’s business.
Assay Example - CMCase• CMCase – DNS • This assay measures the release of reducing sugars by the action of a cellulase on a cellulosic substrate • One unit of activity liberates 1 micromole of reducing sugar (expressed as glucose equivalents) in one minute. The sample activity is then related to a standard with a stated CMCase activity.
Assay Example - CMCase• Enzyme samples Samples are prepared using serial dilutions . Serial dilutions A serial dilution is the stepwise dilution of a substance in a solution.
Assay Example - CMCase • Performing a stability study using CMCase will tell us if enzymes meet stability standards. • 2 year stability studies • Room temperature, 4 C, and 40 C • pH & Appearance1510 5 Series 3 0 Series 2 Series 1
Assay Example - CMCase• Serial dilutions are used to establish a dynamic range, which is used to describe the ratio between the smallest and largest values of a changeable quality, such as light.
Assay Example - CMCase• In addition, glucose samples, standards, controls, and blanks are prepared.• Glucose samples – allows us to compare a known sugar to sugar produced.• Standards – a definitive method that produces a known result• Negative control – does not contain enzyme extract• Blanks – A sample that does not contain material that can absorb light at the level we want.
Buffer and pH• Buffers are used to dilute the sample and to maintain pH, a critical factor in enzyme reactions.
Assay SubstrateA Carboxymethylcellulose sodium salt substrate is prepared. • A substrate a molecule upon which an enzyme acts.
Assay Protocol• The substrate is heated in a boiling bath for 10 minutes, at which point the diluted sample is added. After another 10 minutes, DNS is added and placed in boiling water for 15 minutes.
Advice to Biotechnology Students• Pay close attention and take notes during lab, as well as lecture - you will be using these concepts and lab techniques in the lab and will need them as reference.• Take any opportunity to perfect your labs skills ( continuing education classes, volunteer opportunities). Knowing how to automatically perform calculations and basic set ups are expected!
Advice to Biotechnology Students• Ask lots of questions!• Research areas of interest so you have an idea of the kind of biotech work you would like to do.• Take responsibility for the direction of your education and career.• Stretch yourself – it’s OK to fail. It’s not OK to leave talent unused!
• Continue to work at Dyadic part-time while pursuing my biotechnology degree at PBCC.• Improve organic chemistry, biochemistry knowledge.• Learn more about enzyme kinetics.• Learn how to apply my business background to bringing enzyme based products to market.
• Dr. Libby Handel• Mark Emalfarb• Richard Jundzil• Loretta Tucker• Mickol Andrews• Rashida Thorpe• Karen Hubbard• Manuel Kavekos• Dr. Alexandra Gorgevska