Biotechnology Program

Internship

John SchneiderDyadic International, Inc.

Presentation Overview

• Overview of Dyadic International• Products• The C1 Fungus Platform• Dyadic’s QC/QA Laboratory • Internship Project• Assays Performed• Conclusions• Future Plans

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 & Beauty

Future Applications

Overview Of Dyadic International Products 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:

Dyadic International’s Major Product Lines

Textile Food Brewing Animal Food

Starch/Alcohol

Pulp &Paper

Ethanol

Rocksoft®Family of Enzymes

FoodCelFamily of Enzymes

BrewzymeFamily of Enzymes

Dyadic®Xylanase, Beta Glucanase, and CellulaseFamily of Products

Celustar, AlphastarFamily of Enzymes

Fibrezyme®Family of Enzymes

AlternaFuelFamily of Enzymes

Dyadic New Markets - Biofuels

Hydrolysis of hemicellulose and cellulose to monomer sugars is one of the significant steps in bioethanol production which can be accomplished by using cellulases.

Dyadic Patented C1 Technology Platform

C1 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 Technology

Dyadic International’s C1 technology is based on a system of protein production based on the patented Chrysosporiumlucknowense fungus.

The C1 Advantage

Gene DiscoveryThe Fungal HTRS System from Dyadic, enables rapid gene discovery from all biodiversity (including eukaryotes) using the C1 host organism.

Host Optimization The C1 platform allows for optimization of the host production organism and manufacturing process. Proteins expressed using the C1 Expression System provide the foundation for proper glycosylation.

Large Scale ManufacturingThe C1 organism has been used for biomolecule production in 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 Applications

Carbohydrases Laundry and dishwashing detergents, industrial pipe/tank cleaners, textiles, pulp and paper, fermentation ethanol

Proteases Brewing, baking goods, protein processing, distilled spirits, laundry and dishwashing detergents, lens cleaners, leather and fur, chemicals

Peptidases Food and animal feed; Chiral resolution of chemical compounds or pharmaceuticals

Lipases and Esterases Cleaners, leather and fur, dairy, chemicals

Oxireductases Chemicals, detergent bleaches, pulp bleaching

Lyases Brewing industry; Cosmetics

Transferases Manufacture of cyclodextrins from starch

Enzymes

Enzymes 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 Enzymes Every cell has many different enzymes. Each enzyme is

specific : it only affects one particular chemical. This chemical that the enzyme alters is called the substrate. An enzyme can either break apart one substrate into two different products, or it can join two substrates into a single product. One example is shown below :

Assay Example - CMCase

Cellulose • 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.

Cellulose

Cellulose 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

Sample Reaction

QA/QC Laboratory

QA is short for Quality Assurance, which is defined as the overall program that ensures that the final results 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 Project

Test enzyme producedby Dyadic and itscompetitors to validateenzyme activity to meet required specifications.

Assays Performed

• CMCase – DNSThis assay measures the release of reducing sugars by the

action of a cellulase on a cellulosic substrate

• Xylanase – DNSThis assay measures the release of reducing sugars by the

action of a Xylanase on an Arabinoxylan substrate

• Beta-GlucanaseThis assay measures the release of reducing sugars by the

action of a Beta-Glucanase on a Barley Beta-Glucan substrate

Assays Performed

Protein Quantification With BCA (bicinchoninic acid)

SDS Page, used toTo separate proteinsaccording to electrophoreticmobility

Assay Example - CMCase

Question: 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 - CMCase

Cellulose• 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 & Appearance

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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.

Assay Colometric Reagent

3,5-dinitrosalicylic acid >> reduction>> 3-amino, 5 nitrosalicylic acid

3,5-dinitrosalicylic acid 3-amino. 5 nitrosalicilic acid

Assay Example - CMCase

Assay Example - CMCase

• Samples are removed, cooled, DNS bonds to the broken ends, showing color. Deeper color, more reaction. Samples are than read on a spectrophotometer.

Assay Example - CMCase

• Absorbance levels are measured through the use of a UV spectrophotometer

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Assay Example - CMCase

Enzyme + Buffer

Serial Dilutions

CMCase + Heat

DNS Enzyme Activity Level

Product Measured By

Spectrophotmeter

Assay Example - CMCase

Sample Data Analysis

Overview of Dyadic InternationalProductsThe C1 Fungus PlatformEnzymesQC/QA LaboratoryInternship Project Assays PerformedConclusionsFuture Plans

Presentation Summary

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!

• 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!

Advice to Biotechnology Students

Future Plans

• 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.

Acknowledgements• Dr. Libby Handel• Mark Emalfarb• Richard Jundzil• Loretta Tucker• Mickol Andrews• Rashida Thorpe• Karen Hubbard• Manuel Kavekos• Dr. Alexandra Gorgevska

Thank You