UNDERSTANDING THE DOSE RESPONSE OF CELLULASE

ENZYME COCKTAILS UNDER VARIABLE TEMPERATURE, pH AND

IONIC LIQUID CONDITIONS

Davis Hu1, Vimalier Reyes-Oritz2, Kenneth Sale2, Steve Singer2, Blake Simmons2

1Maryville College, Maryville, TN 37804 · 2Lawrence Berkeley National Laboratory

ABSTRACT

ACKNOWLEDGMENTSThis work was supported and made possible by the Center of Science and

Engineering Education at Lawrence Berkeley National Laboratory, U.S.

Department of Energy, Office of Science, and The Joint BioEnergy Institute. I would

like to thank my wonderful supporting mentors Vimalier Reyes-Oritz and Kenneth

Sale. I would also like to thank my safety work lead supervisor Steve Singer. Lastly,

I would also like to thank Vice President Blake Simmons who made my internship

possible by his wise selection of me for my participation in this program. This work

conducted by the Joint BioEnergy Institute was supported by the Office of Science,

Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.

BACKGROUND

RESEARCH QUESTION

HOW CELLULASES WORK

EXPERIMENTAL/DATA FIGURES

MATERIALS/METHODS

Production of biofuels from sugars in lignocellulosic biomass is a promising

alternative to liquid fossil fuels, but there are barriers to overcome to maximize

production of fuels from these resources. Due to its composition of a complex

array of cellulose, hemicellulose and ligin polymers, lignocellulosic biomass is

extremely recalcitrant to degradation to glucose. The experiment proposed

consists of finding a mixture of enzymes that maximize the release of glucose at

an optimized temperature and pH and to study the dose response of the enzyme

mixture. Initial experiments were run on the individual enzymes over a range of

temperatures and pH to determine their optimal enzymatic conditions. This set of

enzymes was then mixed in various ratios to determine the combination of

cellulases that maximize glucose yields from ionic liquid pretreated switchgrass.

With these preliminary results, we will be able to seek future development of

inexpensive cellulosic biofuels at high yield production which would limit the

dependence on fossil based fuels which causes many significant adverse effects

to the environment and society.

INSTRUMENTAL ANALYSIS

• A major proportion (~85% in US) of energy

consumed is derived from oil, coal and natural gas.

• Concerns over depleting oil reserves and global

warming are fueling the development of alternative

sources of liquid transportation fuels to meet

increasing demands for generations and beyond.

• Lignocellulosic biomass is a potential feedstock for

renewable transportation fuels.

• Mixtures of enzymes compatible with the biomass

pretreatment process are required for production of

fermentable sugars.

• Thermophilic and Ionic Liquid Tolerant Enzymes

• Endo-Cellulase (Endo) – Cel_9A & Cel_5A

• Cellobiohydralase (CBH) – Csac

• β-Glucoside (BG) – βG

• Reaction Conditions

• Temperature – 50°C-80°C – Thermophilic to prevent contamination

and increase rate of reaction

• Ionic Liquid Content – 0%-20% Concentration

• Enzyme Load – 20 mg of enzyme/g of glucon

• Materials and Supplies

• Enzymes – E. Coli Grow Lysate Purify Buffer Exchange –

3-4 Days

• Substrate – Weigh into analytical 1.7 mL tubes

• Characterization Equipment

• High Performance Liquid Chromatography (HPLC)

• 2950 Biochemistry Analyzer (YSI)

• SpectraMax M2 Instrument for Dinitrosalycylic Acid Colorimetric

Assay

• Preparation and Running a DNS assay for Temperature Variance with enzymes Cel_9A and

Cel_5A AND for Temperature and pH Variance with enzymes Endo-cellulase,

Cellobiohydrase and β-Glucosidase

• Preparation and Running a DNS assay for an Enzyme Cocktail Solution

Figure 6. Typical wet-lab space at the Joint BioEnergy Institute

Figure 1. Cellulases work synergistically to catalyze the conversion

of cellulose to glucose by catalyzing the cleavage of the β(1-4)

bonds between the glucose units.

Figure 7. YSI 2950 Biochemistry Analyzer Instrument

Description/Diagram.

Figure 8. DNS colorimetric progression from yellow to red as a function

of increasing temperature.

Figure 9. SpectraMax M2 Instrument.

Figure 10. Schematic Diagram of a Spectrophotometer in SpectraMax M2.

0.340 0.345

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

35 45 55 65 75 85 95

Ab

so

rba

nc

e

Temperature(°C)

Temperature Profiles of Enzymes Cel_9A & Cel_5A

Cel_9A

Cel_5A

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

0.450

0.500

25 35 45 55 65 75 85

Ab

so

rba

nc

e

Temperature(°C)

Temperature Profiles of Enzymes ENDO, CBH and βG at pH 5, 6, 7

ENDO pH 5

ENDO pH 6

ENDO pH 7

CBH pH 5

CBH pH 6

CBH pH 7

βG pH 5

βG pH 6

βG pH 7

DISCUSSION/CONCLUSION

• Cel_9A had maximum activity 65°C and Cel_5A had maximum

activity at 85°C.

• The optimal (Temperature, pH) for each enzyme tested was (40 °C, 5)

for the endocellulase, (45°C, 5) for the cellobiohydralse and (75 °C,

7) for the β-glucosidase.

• Glucose yields increase steadily as a function of total enzyme dose.

- With ionic liquid pretreated switch grass (ILSG) as the substrate,

glucose yields increase rapidly between 100 and 400 nM befoe

beginning to level off.

- To the contrary, glucose yields from avicel reached a plateau at

200 nM and no further increase was observed even at a higher

enzyme doses.

- The approximate 3X greater glucose yields produced from ILSG

are likely due it having a higher content of amorphous cellulose

compared to the highly crystalline avicel substrate.

• The study could be extended to producing an optimized multi-

component enzyme mixture that maximizes glucose yields at a specific

temperature and pH.

Figure 3. Example of the colorimetric DNS cellulase assay.

Figure 4. Veriti 96 Thermal Cycler Heater for heating enzyme samples.

What is the proportion of each enzyme in a

mixture of enzymes that maximizes release

of glucose? What is the optimal enzyme

dose and the optimal temperature and pH for the saccharification reaction?

Figure 5. Eppendorf Thermomixer Apparatus for heating and mixing of

enzyme/substrate samples

Figure 2. Schematic diagram example of enzymes Cel_9A and Cel_5A

arrangement in 96-well plate along with specified temperatures.

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

0 25 50 100 200 400

Ab

sorb

ance

Enzyme Concentration [nM]

Absorbance vs Enzyme Concentration of ILSG and Avicel

ILSG IL-Avicel

0

1

2

3

4

5

6

7

8

0 50 100 150 200 250 300 350 400 450Glu

co

se

Co

nc

en

tra

tio

n (

nM

)

Enzyme Concentration (nM)

Glucose vs. Enzyme Concentration of ILSG and Avicel

GC ILSG

GC Avicel

Crystal

structure of a

b-glucosidase

Crystal

structure of an

endoglucanas

e

Crystal

structure

of a CBH

Plants Enzymes Microbes

feedstock

engineering

enzyme

engineeringfuels synthesis

pretreatment

LIGNOCELLULOSIC

BIOFUELS