Problem-Based Learning Laboratories on

Chemicals from BiorenewablesBioseparations

C. Glatz, S. Mallapragada, B. Narasimhan, P. Reilly and J. Shanks

Department of Chemical Engineering

M. HubaEducational Leadership and Policy StudiesIowa State University, Ames, IA 50011-2230

Z. NikolovProdiGene, Inc. and TAMU, College Station TX

Vision We have developed four 1-credit open-ended,

multidisciplinary laboratory courses involving “Chemicals from Biorenewables”. These problem-based learning laboratories have been integrated with existing and new bioengineering-related ChE classes

Target audience: – undergraduate (seniors) and graduate students in

Chemical Engineering – undergraduate and graduate students in

Biochemistry and Biophysics, Biology and Food Science.

Motivation: Topic ChE evolving from a petrochemical-based to a

biorenewables-based discipline. Examples:Product Species used Company

Indigo Microbial Genencor

poly(lactic acid) Microbial Cargill/Dow

Biopol Microbial/plants Monsanto

1,3 propanediol Microbial DuPont

Current ChE curriculum does not reflect this trend Introduce new courses to cover this new technology

Motivation: Educational Problem-based learning

– Open-ended problems– Learning-based approach– Students direct learning of the topic– Problems provide motivation for learning

Multidisciplinary – Team-based approach

ABET criteria– Life-long learning

Curriculum Structure Four new 1-credit laboratories - each associated

with an existing or new ChE undergraduate/ graduate level biotechnology related theory course

Each laboratory course has one open-ended design project topic and list of desired outcomes

Students work in teams of three - each team has a student with a biology/biochemistry background

Opportunity for problem-based, student-directed, multidisciplinary team-based learning

Bioethics component

General Lab Course Outline First two weeks: Common component for all the lab

classes - Teach students statistics, bioethics, how to work in teams, literature searches, laboratory notebooks. Faculty member plays role of instructor with learning exercises in context of technical content of the course.

Next three weeks: Literature review, coming up with plan for solving the problem, team roles, some laboratory training. Faculty member plays role of coach.

Next nine weeks: Implementation of plan, experimental design. Faculty member plays role of coach

Last two weeks: Wrapping up, written and oral presentations

Description of Laboratory Courses Bioinformatics - (Spring 03: Reilly) - Development of

bioinformatic and virtual reality techniques for investigating and predicting enzyme structure and function.

Metabolic Engineering - (Spring 02: Shanks) - Combination of experimental methods with mathematical analysis of the metabolism of ethanol fermentation from yeast.

Bioseparations - (Fall 02: Glatz) - Development of a process for recovering a recombinant protein expressed in corn germ.

Tissue Engineering - (Fall 02: Mallapragada, Narasimhan) - Development of a bioreactor to cultivate bioartificial skin in vitro on suitable biodegradable polymer scaffolds

Acknowledgments

NSF Combined Research and Curriculum Development Grant EEC 0087696

Barry Lamphear and Susan??, Prodigene, Inc. for assistance with ELISA.

Nicolas Deak, Erin Denefe and Tom Mathews for their presentation.

Summer research crew of Danielle McConnell, Jim Kupferschmidt, Yandi Dharmadi, Zhengrong Gu, Maureen Griffin

Tutors Todd Menkhaus and Kevin Saunders

Brazzein Purification

ChE 562

12/06/02

Goal Objectives:• Develop a separation process to recover Brazzein

from transgenic corn • Purity must be > 80% of total protein content• Salt content in final product must be less than

0.01M

Starting material:

• Defatted transgenic corn germ meal with some endosperm contamination. Initial brazzein concentration 250 g per gram of meal

Brazzein molecular structure:

Brazzein Information

• Small molecule 6500 Da

• Thermo-stable 32-82 C

• Water Soluble pH= 3.6, 4, and 7

• pI=5.4

• Water solubility will be at least 50 mg/ml.• Two different types of brazzein. Type II twice

as sweet as Type I.

Experimental ProceduresTransgenic Corn Germ

Extraction Size Exclusion

Cation Exchange Chromatography

Size Exclusion

Purified Brazzein

Extraction Variables• Protein/Water Ratio = 1g/6 mL, temperature = 23 • pH= 4.0-5.5• Salt concentration =50 NaOAc: 100 mM NaCl• Mixing time of 45 minutes

Protein extraction vs pH

594.4690.7

1559.5

1357.9

0

200

400

600

800

1000

1200

1400

1600

1800

pH 4 pH 4.5 pH 5 pH 5.5

Pro

tein

co

ncen

trati

on

(u

g/m

L)

Size Exclusion HPLC• The objective for this test was to asses whether

simple membrane filtration was applicable in this case.

• The Brazzein rich extract (pH 4) is eluted in a Size Exclusion HPLC

• Elution conditions:– pH 4.0

– NaAc Buffer ( NaAc 20mM, NaCl 30 mM)

• A standard elution curve was run in parallel, using known MW markers.

HPLC Standard Curve

Molecular Weight = 147416 exp(-0.4751* Time)

R2 = 0.9404

0

250

500

750

10 12 14 16 18 20 22 24

Time (min)

Mol

ecul

ar W

eigh

t (k

D)

HPLC Results

Brazzein

6.5 kDa

21.1 min.

Contaminant

2.91 kDa

22.8 min.

HPLC Conclusions

• A direct membrane filtration of our Brazzein rich extract is not applicable in this case

• The overlapping contaminant protein peak is important enough to try some other means to purify our product

Cation Exchange Chromatography

• Goal: Separate brazzein from the 2.91 kDa contaminant

• Cation exchange successful removing brazzein from yeast cells (Irwin)

• Resin type: SP (sulphopropyl) Sepharose Fast Flow

• Elute with linear salt gradient 0-1 M NaCl

Cation Exchange Setup

• Column: SP Sepharose fast flow resin• Length = 8 cm• Diameter = 1 cm• Volume = 6.28 mL

• Buffers: • A = 20 mM NaOAc, pH 4.0• B = Buffer A + 1.0 M NaCl

• Operation: • Flowrate = 1.0 mL/min

Cation Exchange for Pure Brazzein

• Sample 40 g/mL pure brazzein in DI water

• Equilibration of column with Buffer A

• Load of 10 column volumes

• Gradient elution from 0%-100% Buffer B in 15 column volumes

• Isocratic flow of 100% buffer in 5 column volumes

Cation Exchange for Corn Germ

• Sample 20 g + 120 mL Buffer A• Mixed 40 min, centrifuged 30 min. (15000

rpm), and filtered (0.22m CA)• Equilibration of column with Buffer A• Load of 5 column volumes • Gradient elution from 0%-100% Buffer B in 15

column volumes • Isocratic flow of 100% buffer in 5 column

volumes

Cation Exchange Results

• Section of concern 27-43 minutes after elution begins (30-45% Salt gradient)

• Estimate fold of purification• assuming no nonprotein UV280 adsorbing

materials

• Perform experiment to determine molecular weight of material in this section

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

-3 -2 -1 0 1 2

Time (hr)

Ligh

t Abs

orba

nce

(UV

)

0

20

40

60

80

100

Sal

t Gra

dien

t (%

20

mM

NaO

Ac)Pure Brazzein

Nontransgenic Corn

Salt Gradient

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5

Time (hr)

Abs

orba

nce

Fra

ctio

n

0

20

40

60

80

100

Sal

t Gra

dien

t (%

20

mM

NaO

Ac)

Pure Brazzein

Transgenic Corn

Salt Gradient

Conclusions:

• Cationic exchange chromatography is more effective (72 %)

• This is not enough to comply with our specifications

• After analyzing SDS-PAGE electrophoresis results we decided to do a size exclusion

• Expected results will be within specifications

Questions?

Do you have any suitable problem?

Impact Make ChE education more relevant for our

undergraduate students Teach students

– problem-based learning techniques

– develop their metacognitive abilities

– life-long learning

Coupling these educational techniques with valued new technologies

Integrate some of these new experiments in a non open-ended manner into the required ChE undergraduate laboratories

Assessment

Self- and instructor-assessment using– Teamwork rubric– Design rubric– Written report rubric– Oral presentation rubric

Curriculum Structure

MetabolicEngineering

Microbial Engineering Lab

Product Development

Polymeric Biomaterials

Tissue Engineering Lab

Downstream Processing

Bio-separations

Bio-Separations Lab

UpstreamProcessing

Biochemical Engineering

Bioinformatics Lab