Dyadic International uses its patented C1 fungus platform to produce industrial enzymes for markets like biofuels, chemicals, and biotherapeutics. The presentation overview included Dyadic's products, C1 platform, quality control laboratory, internship assays on cellulase activity, and future plans. The internship project involved testing Dyadic and competitor enzymes using assays like CMCase, xylanase, and beta-glucanase. These assays help ensure enzyme activities meet specifications. The presentation provided advice to biotechnology students and concluded by thanking Dyadic staff.

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

3. Overview Of Dyadic International
Dyadic 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

4. 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

5. Dyadic International’s Market Niche
• Dyadic International market capitalization is $17.7 million
• Market capitalization up +293.3% YTD over the following
industry peers:

6. Textile Food Brewing Animal Starch/ Pulp & Ethanol
Food Alcohol Paper
Rocksoft® FoodCel Brewzyme Dyadic® Celustar, Fibrezyme® AlternaFue
Family of Family of Family of Xylanase, Alphasta Family of l
Enzymes Enzymes Enzymes Beta r Enzymes Family of
Glucanase, Family of Enzymes
and Enzymes
Cellulase
Family of
Products

7. 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.

8. 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.

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

10. Gene Discovery
The 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 Manufacturing
The C1 organism has been used for biomolecule production
in 150,000 liter fermenters since 1996.

11. 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.

12. 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

13. 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.

14. 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 :

15. 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.

16. 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

17. Sample Reaction

18. 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.

19. Why Is A QA/QC Lab Important?
• Determining the quality of the product
• Translates to sales and profitability
• Development of new products
For example:
– Enzymes that decrease in activity, may not meet
client specifications. Those enzymes may than be
sold to another client at a reduced price.

20. Dyadic Internship Project
Test enzyme produced
by Dyadic and its
competitors to validate
enzyme activity to meet
required specifications.

21. 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

22. Assays Performed
Protein Quantification With
BCA (bicinchoninic acid)
SDS Page, used to
To separate proteins
according to
electrophoretic
mobility

23. 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

24. 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

25. 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.

26. 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.

27. 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.

28. 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
15
10
5 Series 3
0 Series 2
Series 1

29. 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.

30. 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.

31. Buffer and pH
• Buffers are used to
dilute the sample
and to maintain
pH, a critical factor
in enzyme reactions.

32. Assay Substrate
A Carboxymethylcellulose sodium salt substrate
is prepared.
• A substrate a molecule upon which an enzyme acts.

33. 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.

34. Assay Colometric Reagent
3,5-dinitrosalicylic acid >> reduction>> 3-
amino, 5 nitrosalicylic acid
3,5-dinitrosalicylic acid 3-amino. 5 nitrosalicilic acid

35. Assay Example - CMCase

36. 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.

37. Assay Example - CMCase
• Absorbance levels are measured through the use of a
UV spectrophotometer
14
12
10
8
6 Series 3
4
2 Series 2
0 Series 1

38. Assay Example - CMCase
Enzyme + Buffer Serial Dilutions CMCase + Heat
Product Measured
Enzyme Activity
By DNS
Level
Spectrophotmeter

39. Sample Data Analysis
Assay Example - CMCase

40. Presentation Summary
Overview of Dyadic International
Products
The C1 Fungus Platform
Enzymes
QC/QA Laboratory
Internship Project
Assays Performed
Conclusions
Future Plans

41. 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!

42. 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!

43. • 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.

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

45. Thank You