Investigation of cellulase reaction mechanism

1
Investigation of cellulase reaction mechanism
using pure cellulosic susbstrate
Rajesh Gupta Y Y Lee Department of Chemical
Engineering Auburn University
Introduction Various forms of
pure cellulosic substrates were utilized to study
the reaction mechanism in cellulase reaction. The
substrates employed were micro-crystalline
cellulose (Avicel), a- cellulose, filter paper,
cotton, and non-crystalline cellulose (NCC).
These substrates were first characterized with
respect to Degree of polymerization (DP),
crystallinity, surface area and other physical
properties. NCC is a product of our laboratory.
It is highly amorphous cellulose with
crystallinity index less than 10. When
hydrolyzed with cellulase (Spezyme CP supplied
by Genencore Int.), it produces significant
amount of cello-oligosaccharides as reaction
intermediates, along with glucose and cellobiose.
Cello-oligosaccharides (COS) were categorized
into two separate fractions Low DP
cello-oligosaccharides (LD-COS) and high DP
cello-oligosaccharides (HD-COS). LD-COS, from DP
1-7, are detected by HPLC whereas HD-COS are
detected only after secondary hydrolysis. On the
basis of the profiles of these sugars during
enzymatic hydrolysis, individual actions of
Exo-glucanase (Exo-G), Endo-glucanase (Endo-G)
and ß-glucosidase (ß-G) the overall reaction
patterns are proposed. The major findings on
the function of individual cellulase components
are as follows (1) Exo-G and Endo-G do not
hydrolyze COS. (2) ß-G hydrolyzes cellobiose and
LD-COS. (3)Exo-G is responsible for LD-COS
production and Endo-G for HD-COS. Crystallinity
of substrates primarily affects the initial rate
of cellulose hydrolysis due to preference of
Endo-G towards the accessible amorphous region in
cellulose.
Proposed Mechanism of Cellulase action on NCC
HPLC Chromatograph of Cello-Oligosaccharide (COS
was prepared in our lab by acid Hydrolysis of
Cotton)
Comparison between Cystalline Cotton
and Non-Crystalline Cellulose (NCC)
(HPLC Column used Biorad Aminex P-Column)
Low DP Cello-oligosaccharides (LD-COS)
Low DP NCC
Endo-G
SOLID
LIQUID
HD-COS
Cellobiose
Glucose
14.1
LD-COS
NCC
CELLOBIOSE
Exo-G
Quantification of COS
ß-G
GLUCOSE
( Picture of Enzyme cartoon has been taken from
the Video on cellulase mechanism distributed by
NREL)

• Summary
• Enzymatic hydrolysis of NCC produces
  glucose(G1), cellobiose
• (G2) and cello-oligosaccharides (COS). This is
  in contrast to
• the crystalline cellulose, which produces only
  G1 and G2.
• In NCC reaction with very low enzyme loading, G2
  and LD-COS
• accumulate. Upon addition of external ß-G, both
  are hydrolyzed
• to glucose. This proves that ß-G not only works
  on cellobiose
• but also on LD-COS.
• In NCC reaction, formation of cellobiose is
  proportional to
• LD-COS, a proof that LD-COS is a product of
  Exo-G.
• When Exo-G hydrolyzes NCC, it produces
  cellobiose by sequential
• action. This process, however, ceases when DP
  goes below certain
• level, leaving unhydrolyzed LD-COS as one of
  the end-products.

Crystallinity of Pure Cellulosic Substrate
Actual COS concentration in solution 9 g/L COS
not shown in HPLC approx. 50 ------ HD-COS
Enzymatic Hydrolysis of NCC
Enzyme Loading High Loading 0.1 ml / g
Glucan Low Loading 0.005 ml / g Glucan
Degree of Polymerization (DP) of Pure Cellulosic
Susbtrate Calculated by measuring the absorbance
at _at_540nm after reaction of 50mg of substrate
with DNS reagent
Enzymatic Hydrolysis of Crystalline Substrate
Enzyme Loading 0.1 ml / g Glucan
Glucose
Glucose Cellobiose
Excess amount of ß-G added after 72hrs
Cellobiose
HD-COS

• Acknowledgement
• US Department of Energy for funding the project
• (US/DOE No. DE-PS36-00GO10482)
• Members of CAFI-II team
• Genencor International Inc. for supplying enzyme