ABSTRACT

1

Kinetic Investigation of Cellulase Enzyme Using
Non-Crystalline Cellulose and Cello-Oligosacchara
ides
Suma Peri and Y.Y.Lee Department of Chemical
Engineering, Auburn University,AL,USA.
ABSTRACT
Hydrolysis of cellulose by cellulase enzyme is a
solid-liquid heterogeneous reaction. As such the
reaction is strongly affected by the physical
resistances caused most notably by the
crystalline structure. Recently we have invented
a method to produce non-crystalline cellulose
(NCC) from a commercial a-cellulose or cotton by
a relatively simple process. This material is
drastically different from natural cellulose in
that the crystalline structure is disrupted. We
have used this material for the kinetic study of
cellulose hydrolysis. Among the major findings
is that a significant amount of oligomers was
found to accumulate throughout the reaction. It
appears that oligomers are inhibitory to
cellulase enzyme, especially the endo-glucanase.
We have also produced cello-oligosaccharides
(beta 1-4 glucan) from alpha-cellulose
and used as the substrate for cellulase. When
treated separately, the oligomers can be easily
hydrolyzed to glucose by sulfuric acid, but was
not hydrolyzed significantly cellulase enzyme.
Introduction
Acid and Enzymatic Hydrolysis of
Cello-oligosaccharides

• The crystallinity of cellulose is a major factor
  hindering the enzymatic reaction by cellulase.
  Under the influence of the crystallinity, it is
  difficult to obtain the intrinsic kinetic
  information.
• We have produced non-crystalline cellulose (NCC)
  from alpha cellulose by treatment with
  concentrated sulfuric acid.
• The main objective of this work is to investigate
  the kinetic behavior of enzymatic hydrolysis
  against NCC and seek additional information that
  may not be available from crystalline cellulose.

Glucose
Cellobiose
Materials and Methods
COS

• NonCrystallineCellulose(NCC), and
  Cello-Oligosaccharides(COS) were prepared in
  our laboratory.
• Cellulase enzyme Spezyme CP, Genencor, .Lot No.
  301-00348-25
• The conditions for acid hydrolysis of
  cello-oligosaccharides were 121C, 20 min.,
  4H2SO4.
• The enzymatic hydrolysis conditions 1(w/v)
  glucan concentration, pH 4.8, 50ºC and 150 rpm

Acid Hydrolysis
DP5
DP3
DP4
Cellobiose
DP6
Glucose
Glucose
Glucose
Product distribution from the enzymatic
hydrolysis of Avicel cellulose and NCC
Cellobiose
DP4
DP3
Enzymatic Hydrolysis, 15FPU/g-glucan
1 FPU/g- glucan (96 hours)
1 FPU/g- glucan (6 hour)
Glucose
Cellobiose

• Acid hydrolysis of COS resulted in 93 glucose
  yield in 20 min
• Enzymatic hydrolysis gave 17.7 of glucose yield

Cellobiose
Glucose
Avicel
Avicel
Glucose
Enzymatic Hydrolysis of COS and Avicel
Cellobiose
Glucose
Cellobiose
NCC
NCC
Cello-oligosaccharides are more difficult to
hydrolyze than Avicel.
oligomers
oligomers
Profiles of glucose, cellobiose, and oligomers in
hydrolysis of NCC
Summary

• Hydrolysis of NCC exhibits extremely high initial
  rate. The reaction then essentially ceases after
  10 hours.
• In the enzymatic hydrolysis of NCC, a significant
  amount of cellobiose and COS are formed as
  reaction intermediates.
• Cellobiose gradually disappeared whereas COS
  remained constant throughout the enzymatic
  hydrolysis.
• COS are easily hydrolyzed by sulfuric acid, but
  are not significantly hydrolyzed by cellulase.
• Hydrolysis of COS by Spezyme CP was slower than
  that of Avicel.
• COS appear to be inhibitory to endo-glucanase.

Oligomers were not degraded throughout the
reaction.