Immobilization of Yeast Invertase in Sol Gel Beads

1
Immobilization of Yeast Invertase in Sol Gel Beads

• By
• Catherine V. Angley
• Earlham College
• Richmond, Indiana
• with
• Dr. Jonathan Woodward
• Dr. Hugh ONeill
• Chemical Technology Division
• Oak Ridge National Laboratory

2
Introduction

• The generation of hydrogen gas from sugars
  represents a novel method for the efficient
  production of a fuel.
• Invertase is an enzyme that hydrolyzes sucrose to
  glucose and fructose.
• Immobilization of invertase would allow
  continuous sucrose hydrolysis to glucose and
  fructose that are further oxidized in the
  hydrogen producing process (Woodward and Orr,
  1998).
• Candida utilis invertase has been used as a model
  for the immobilization of enzymes in sol gel
  beads.
• Characterization of the properties of the beads
  are currently under investigation.

3
Sol Gel Chemistry

• The sol gel process (Ellerby et al., 1992)
  consists of the following steps
• The sol gel solution is formed by the sonication
  of tetra-methylorthosilicate (TMOS) with water
  and the acidic catalyst, HCl (Fig. 1).
• Hydrolysis leads to the formation of silanol
  (Si-OH) and methanol groups. The silanol and
  methoxy groups react further, condensing, to form
  siloxane (Si-O-Si) with methanol and water as
  additional reaction products.
• The process of condensation to form siloxane
  continues during aging (2 weeks).
  Macromolecules such as enzymes can be immobilized
  within the pores of the covalent gel network as
  it forms.
• Gel drying involves the careful removal of the
  solvent phase.

4
Fig.1 The Mechanism for the Preparation of Sol
Gel
Acid Catalyzed Hydrolysis
Base Catalyzed Condensation
5
Materials and Methods

• Yeast invertase (Candida utilis) E.C. 3.2.1.26
  was purchased from Sigma.
• The activity of invertase was determined by
  measuring, spectrophotometrically, the amount of
  glucose produced by invertase in the presence of
  sucrose.
• The sol gel was formed by the procedure
  previously described.
• Immobilization procedure The enzyme stock was
  diluted in 10mM NaPhosphate buffer, pH7.2
  (300?L). To this, sol gel was added (200?L).
  This solution was mixed and aliquotted in to
  multiple 50?L beads onto parafilm. Gelation
  occurred within appoximately 3 minutes.
• After gelation occurred, the beads were placed in
  50mM NaAcetate buffer, pH 5.0 for aging.
• Properties of sol gel entrapped invertase were
  investigated.

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Results

• Invertase was immobilized by entrapment in sol
  gels. The amount of enzyme activity recovered
  was 30.
• Steps Taken to Increase Activity Recovered and
  the Results
• Different polymers were added to the enzyme
  solution before gelation. It was found that
  these conferred a substantial amount of
  additional protection to the enzyme against
  denaturation (Fig. 2).
• The polymer blue dextran yielded the most
  significant amount of activity at 60 recovered
  (Fig. 3).
• Over time, it became apparent that invertase
  leaks out of the beads. Evidence for such can be
  seen by the substantial decrease in activity
  after 3 days of being in a continuous flow system
  at 50C (Figs. 4 5 ).

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Fig. 2 Percent activity retained with various
Polymers
8
Fig. 3 Rate Comparison between Beads made with
and without Blue Dextran
70
60
Blue Dextran 60 activity
50
40
nmol Glucose/0.1 mL
30
20
w/o Blue Dextran 30 activity
10
0
5
10
15
20
Time (min)
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Fig. 4 Continuous Flow System with Immobilized
Invertase
50C Thermal Heating Loop
Product Glucose Fructose (collection vessel)
Substrate 10 mM Sucrose in 50 mM NaAc Buffer, pH
5.0
Sol Gel Beads with immobilized invertase
Peristaltic Pump Flow rate 0.25 ml/min
10
Fig. 5 Evidence for Leakage of Invertase from
Beads
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Conclusions

• Invertase can successfully be immobilized by
  entrapment within sol gel beads with 30 of its
  activity retained.
• A significantly greater amount of activity (60)
  is recovered when the polymer blue dextran is
  present during the gelation process.
• Invertase leaks out of the pores in the sol gel
  beads, given enough time.

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Future Work

• The prevention of enzyme leakage from the sol gel
  beads.
• Characterization of the properties of the
  immobilized invertase.
• The use of a continuous flow system whereby the
  activity of the beads is maintained as is the
  conversion rate of sucrose to glucose and
  fructose over an extended period of time.
• The effect of drying on the retained activity of
  the beads and any effect drying may have on
  enzyme leakage.
• Scanning electron microscopy of the beads at
  various stages in the sol gel process to
  investigate structural features and morphology of
  the beads at different phases in the process.

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References

• Ellerby, L. M. Nishida, C. R. Nishida, Fumito
  Yamanaka, S. A. Dunn, B. Valentine, J. S.
  Zink, Jeffrey I. Encapsulation of Proteins in
  Transparent Porous Silicate Glasses Prepared by
  the Sol-Gel Method. Science. 1992, 255,
  1113-1115.
• Woodward, Jonathan and Orr, Mark. Enzymatic
  Conversion of Sucrose to Hydrogen. Biotechnol.
  Prog.. 1998, 14, 897-902.
• Acknowledgements
• I would like to thank Dr. Jonathan Woodward and
  Dr. Hugh ONeill for all of their help, support
  and contributions to my research. I would also
  like to thank Dr. Sheng Dai for the useful
  technical discussions and John Getty for the help
  he provided in preparing this presentation. This
  research experience was made possible by the
  Great Lakes Colleges Association/ Associated
  Colleges of the Midwest.