Contoh Cross Linking - Thesis Hydrolize of Cellulose by Immob Cellulase

ENZYMATIC HYDROLYSIS OF CELLULOSIC BIOMASS BY
USING IMMOBILIZED CELLULASE
by
SANGHA OH, B.S., M.S.
A THESIS
IN
CHEMICAL ENGINEERING
Submitted to the Graduate Faculty
of Texas Tech University in
Partial Fulfillment of
the Requirements for
the Degree of
MASTER OF SCIENCE
IN
CHEMICAL ENGINEERING
Approved
Acc&TTted
August 1982
900
'7 -'M
,-i^
• ^'?5îi-,.-^
ACKNOWLEDGMENTS
The author is deeply indebted to Dr. L. D. Clements for his timely
advice and continuous encouragements during the course of this work.
The author is profoundly indebted also to Dr. S. R. Beck for his
invaluable guidance in the field of immobilized enzyme technology.
Special thanks are given to Dr. H. R. Heichelheim for his helpful
criticism on the writing of this thesis.
The author expresses his heartful appreciation to Mrs. Sue W i H i s
for her skillful typing of this thesis.
Finally, the author gratefully acknowledges the Texas Energy and
Natural Resources Advisory Council for their financial support of this
work.
11
TABLE OF CONTENTS
PAGE
ACKNOWLEDGMENTS ii
LIST OF TABLES vi
LIST OF FIGURES vii

CHAPTER I INTRODUCTION 1
CHAPTER 11 LITERATURE REVIEW 5
Chemical Constituents of Cellulosic Biomass.. 5
Pretreatment of Cellulosic Biomass for

Enzymatic Hydrolysis 7
Properties and Mode of Action of Cellulase... 10
Production of Cellulase 12
Process of Enzymatic Hydrolysis of Cellulosic

Biomass 13
Immobilization of Enzymes 14
Carri ers 16
Immobi 1 ized Enzyme Reactors , 17

CHAPTER III MATERIALS 19
Determination of Amount of Reducing Sugars
by o-Toluidine Method 20
CHAPTER IV PREPARATION OF IMMOBILIZED CELLULASE THROUGH
COVALENT BONDINGS 22
1. Methods and Results. 22
A. S i l a n i z a t i ô n of FeÔ^ 22
B. Determination of Amount of Amino

Groups Bound on FeÔ. 22
C. Efficiency of Magnetic Recovery of

Silanized FeÔ^ from Buffer
Solution 24
iii
PAGE
D, Preparation of Immobilized Cellulase

Through Glutaraldehyde 25
E. Preparation of Immobilized Cellulase

Through Carbodiimide 31
2. Discussion 34
CHAPTER V EFFECT OF CROSS-LINKING ON ACTIVITY OF

IMMOBILIZED CELLULASE 38
1. Methods and Results 38
A. Activities of IMC's Cross-Linked With

Various Concentrations of
Glutaraldehyde 38
B. Retention of Activity of IMC Cross-

Linked Through 4.0% Glutaraldehyde 41
2. Discussion 50
CHAPTER VI LOADING OF VARIOUS AMOUNTS OF CELLULASE ON

SILANIZED FeÔ^ 52
A. Loading of Various Amounts of Cellulase

on Silanized FeÔ^ With Glutaraldehyde
as an Attaching Chemical 52
B. Loading of Various Amounts of Cellulase

on Silanized FeÔ^ With Carbodiimide as
an Attaching Chemical 54
2. Discussion 54
CHAPTER VII THE pH ACTIVITY PROFILES OF IMMOBILIZED AND

FREE CELLULASES 60
A. The pH Activity Profile of Immobilized

Cellulase Attached Through
Glutaraldehyde 60
B. The pH Activity Profile of Immobilized

Cellulase Attached Through
Carbodiimide 62
iv
PAGE
C. The pH Activity Profile of Free

Cellulase 65
2. Discussion 65
CHAPTERVIII EFFECTIVENESS OF GRINDING AS PRETREATMENT OF
COTTON GIN TRASH FOR ENZYMATIC HYDROLYSIS 69
1. Methods and Resul ts 69
2. Discussion 69
CHAPTER IX CONCLUSIONS AND RECOMMENDATIONS 73
Conclusions 73
Recommendations for Further Research 73
LITERATURE CITED 74
LIST OF TABLES
PAGE
Table 1. Content of Botanical Components in Cotton

Gin Trash (9) 8
Table 2. Retention of activity of IMC attached
through gl utaraldehyde 29
Table 3. Retention of activity of IMC attached
through carbodiimide 33
Table 4. Effect of cross-linking on the retention of
activity of IMC 40
Table 5. Glucose production by IMC cross-linked through
4.0% glutaraldehyde during six runs of
hydrolysis 46
Table 6. Retention of activity of IMC cross-linked

through 4.0% glutaraldehyde during six runs
of hydrolysi s 48
Table 7. Loading of various amount of cellulase on

silanized FeÔ^ with glutaraldehyde as an
attachi ng chemical 53
Table 8. Loading of various amounts of cellulase on

silanized FeÔ^ with carbodiimide as an
attaching chemical 56
Table 9. pH activity profile of immobilized cellulase

made with glutaraldehyde as attaching
chemical 61
Table 10. pH activity profile of immobilized cellulase

made with carbodiimide as attaching
chemical 64
Table 11. pH activity profile of free cellulase 66
Table 12. Effectiveness of grinding as pretreatment of

cotton gin trash for enzymatic hydrolysis 70
VI
LIST OF FIGURES
PAGE
Figure 1. Molecular structure of cellulose (6) 6
Figure 2. Chemical equation of silanization reaction 23
Figure 3. Chemical equation of treatment of the

silanized FeÔ^ with glutaraldehyde 26
Figure 4. Chemical equation of attachment of cellulase
on a carrier particle through glutaraldehyde..... 27
Figure 5. Retention of activity of IMC attached through
glutaraldehyde: Relative activity of IMC at
each run has been plotted in the middle of
the time of hydrolysis 30
Figure 6. Chemical equation of attachment of cellulase

on a carrier particle through glutaraldehyde 32
Figure 7. Retention of activity of IMC made with
carbodiimide as an attaching chemical:
Relative activity of IMC at each run has been
plotted in the middle of the time of
hydrolys i s 35
Figure 8. Chemical equation of cross-1inking of

immobilized cellulase through glutaraldehyde 39
Figure 9. Schematic diagrams of IMC. The shaded area
represents the FeÔ^ particle and the bars
represent covalent bonds. The small circles
represent immobilized cellulase molecules 42
Figure 10. Retention of activity of IMC's between Ist and

2nd runs of hydrolysis 43
Figure 11. Effect of cross-linking on the activity of IMC... 44
Figure 12. Accumulated glucose production by IMC's 47

Figure 13. Retention of activity of cross-linked IMC
during six runs of hydrolysis: the 4.0%
glutaraldehyde solution was used for
cross-linking 49
vn
PAGE
Figure 14. Loading of various amounts of cellulase on

silanized FeÔ^ with glutaraldehyde as an
Figure 15. Loading of various amounts of cellulase on

silanized FeÔ^ with carbodiimide as an
Figure 16. The pH activity profiles of immobilized and

free cellulase. The relative activities have
been adjusted to make each cellulase preparation
have relative activity 70 at its maximum
poi nt 63
Figure 17. Effectiveness of grinding as pretreatment of

cotton gin trash 71
VI 1 1
CHAPTER I
INTRODUCTION
Cellulose occurs abundantly in nature as the principal constituent
of the cell walls of most plants. It is usually present in close as-
sociation with other compounds, such as hemicellulose and lignin.
Impending shortages of petroleum force us to contemplate the vast
energy potential locked up in the currently unused and annually renew-
able sources of cellulosic biomass. The annual output of cellulosic
biomass in the U.S. exceeds one billion tons (1). In the South Plains
of Texas, there are 1.8 billion pounds of cotton-gin trash (CGT) pro-
duced annually (2).
The cellulose component of biomass can be hydrolyzed into glu-
cose which is then fermented to produce ethanol. Gasohol can be made
by using this ethanol.
Acids and enzymes have been used to hydrolyze cellulose to glu-
cose. The enzymatic hydrolysis is more specific and requires milder
reaction conditions than the acidic hydrolysis.
Enzymes are usually defined as proteins elaborated by living
cells which catalyze specific reactions necessary for the maintenance
of life. The enzymes can be isolated and used in industrial reactors
as catalysts. Unlike most inorganic catalysts, however, enzymes are
generally water-soluble. Thus, the enzymes can be used but once in
free solutions.
To overcome the problem of losing expensive enzymes in free solu-
tion, considerable world-wide interest has arisen in the use of

immobilized enzymes which are made to be water-soluble. Immobiliza-
tion refers to the modification of an enzyme so as to restrict its
gross movement and keep it in a relatively defined region of space.
This includes trapping in a gel, encapsulating in a membrane shell,
adsorption on a surface, covalent bonding to a solid, and possibly
other modifications. With an immobilized enzyme, one merely has to
recover the carrier to obtain the enzyme for reuse, thus overcoming
one of the economic drawbacks to the extensive use of enzymes as in-
dustrial catalysts.
From the standpoint of immobilized enzyme technology, the diffi-
culty in the hydrolysis of cellulosic biomass arises from the fact that
the cellulosic biomass is water-insoluble and some of the cellulosic
biomass is still present in the product stream as exhausted biomass.
The conventional techniques for recovery of the immobilized enzyme
from a product stream or retention in a reactor require that the
particle-size of solid carrier be substantially larger than that of
the exhausted biomass. A larger particle-size solid carrier, however,
causes problems because of its small surface area on which the enzymes
are to be bound and because of its slow movement which makes it diffi-
cult for the immobilized enzymes to approach the substrate particles.
Another approach is to use a solid carrier of small particle-size
which can be separated from the exhausted biomass of large particle-
size. This task may be carried out through a carrier which can be
separated magnetically from the exhausted biomass.
The cellulase enzyme system, which is secreted by fungi such as
Trichoderma viride, catalyzes the hydrolysis of cellulose to glucose.

The cellulase enzyme system is composed of three components which act
synergistically. Thus, the immobilization of the cellulase enzyme sys-
tem must be carried out by a method which does not deactivate any of
the three components.
Multi-step enzyme systems, such as the cellulase enzyme system,
immobilized on the same carrier may operate with high efficiency be-
cause the components of the system are present a \/ery short distance
from each other. The intermediate products made by one component are
available to the next component in the sequence at higher concentra-
tion than would be anticipated with a free multi-step enzyme system.
In nature, many of the enzymes carrying out a sequence of consecutive
reactions are either associated in more or less tight aggregates, are
jointly embedded in å cell membrane, or act in a 'gel-like surrounding
(3).
An important feature in enzyme catalysis is that the enzyme binds
to a substrate and the reaction proceeds in the confines of the enzyme-
substrate complex. An advantage of utilizing a non-porous carrier is
the fact that the enzyme is attached to an external surface and is in
immediate contact with the surrounding environment; therefore, diffu-
sion effects are minimal and a substrate of large particle-size like
cellulosic biomass can be easily reacted.
Enzymes can be more tightly bound to the carrier through covalent
bonding than through physical adsorption. The tight binding is re-
quired in the preparation of the immobilized cellulase because the
cellulase tends to leave the carrier to be adsorbed strongly on the
surface of the cellulosic biomass particle.

The overall goal of this thesis is to develop a method for immobi'
lization of cellulase enzyme while retaining reasonable enzyme acti-
vity. Specific tasks toward this goal are:
1) Preparation of active immobilized cellulase attached through
covalent bonding. To the author's knowledge, nobody has ever reported
the preparation of active immobilized cellulase attached through cova-
lent bonding. This preparation includes utilization of amino groups
and carboxyl groups of cellulase protein as linkage sites.
2) Investigation of activity retention of the immobilized cellu-
lases which are prepared with both methods.
3) Investigation of effect of cross-linking on activity and its
retention of the immobilized cellulases.
4) Investigation of loading of cellulase on non-porous magnetic
iron oxide with both methods of preparation.
5) Investigation of pH activity profiles of the immobilized
cellulases.
6) Investigation of effectiveness of grinding as pretreatment
of cotton gin trash for enzymatic hydrolysis.

CHAPTER II
LITERATURE REVIEW
Chemical Constituents of Cellulosic Biomass
The chemical constituents of cellulosic biomass include cellulose,
several hemicelluloses, lignin and a wide variety of extraneous mate-
rials.
Cellulose is a linear polymer of 3-D-glucopyranose units linked by
3(1^4)-glycosidic bonds. The only chemical difference between starch
and cellulose> both homopolysaccharides of D-glucose is that starch has
a(l-^4) linkage and cellulose has 3(1-^4) linkage (4). The number of glu-
cose units per cellulose molecule (degree of polymerization) ranges from
as few as 15 or less to as high as 10,000-14,000 (5). The molecular
structure of cellulose is shown in Figure 1 (6).
Hemicelluloses are mainly polymers of pentoses, coupled with a
small proportion of hexoses. Hemicelluloses can be hydrolyzed easily
by use of dilute acid or sometimes water alone, at elevated temperature
(7). Hemicelluloses are much lower in molecular weight than cellulose.
Their degree of polymerization seldom exceeds 200.
Lignin is a complex three-dimensional aromatic polymer, which is
very polydisperse but of high molecular weight. It is formed by the
polymerization of oxidatively formed radicals of p-hydroxycinnamyl al-
cohols and is consequently a polymer of phenylpropane units. The mol-
ecular structure of lignin has been reported by Freudenberg (8). The
extraneous materials in cellulosic biomass include proteins, waxes, fats, , ,,
essential oils, tannins, resin, terpenes, starch and inorganics.

5
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CT)
The cotton gin trash used in this research was harvested in Fall,
1980, in Lubbock County, Texas. The average composition of this cotton
gin trash is shown in Table 1. The cotton burr trash which is a kind
of cotton gin trash contains approximately 40% cellulose, 30% hemicellu-
lose and 25% lignin on a weight basis (10).
Pretreatment of Cellulosic Biomass for Enzymatic Hydrolysis
Native cellulose is water-insoluble and its structure is complex.
Thus, its susceptibility to attack by hydrolytic enzymes depends signi-
ficantly on its structural features. The major structural features of
cellulosic biomass that determine its susceptibility to enzymatic de-
gradation are the content of lignin and the degree of crystallinity.
Baker (11) reported that removal of lignin from hardwoods rapidly
increased the digestibility. He used sodium hydroxide and sodium sul-
fide to remove lignin. The fungus, Aspergillus fumigatus, can grow
well on hardwood pulps at lignin contents of 14% or less (12). Earlier
work on the fermentability of softwood pulps by thermophilic bacteria
showed that the lignin content had to be less than 1% for good utiliza-
tion (13).
Beck and Tuttle (14) reported that a caustic pretreatment increas-
ed the hydrolysis rate of cellulose of mesquite. This was partially
due to the fact that the mesquite was swollen during the caustic treat-
ment, which allowed the enzyme molecules to penetrate the structure
more easily.
Walseth (15) showed that cellulase readily degraded the more ac-
cessible amorphous portion of regenerated cellulose but was less able

8
Table 1. Content of Botanical Components in

Cotton Gin Trash (9).
Botanical Component Weight %
Bract 41.5
Leaf 4.7
Vein material 3.5
Petiole 1.6
Bark 3.3
Wood 1.1
Exo-mesocarp 1.8
Endocarp 3.8
Seedcoat 2.4
Cotyledon 0.3
Others 2.4
Weeds 33.7
to attack the less accessible, crystalline portion. Consequently, a
significant increase in crystallinity was observed during the hydrolysis
of cellulose with enzyme. As the cellulose becomes more crystalline,
it becomes more resistant to further hydrolysis (16).
The subdivision of cellulosic biomass to a wery fine particle-size
markedly enhances its susceptibility to acidic, enzymatic and biological
attack (17). Ghose and Kostick (18) found that excellent levels of
enzymatic hydrolysis could be obtained with newsprint which had been
ball-milled to -200 mesh. Pew and Weyna (19) obtained essentially com-
plete cellulase digestion of milled spruce and aspen after milling com-
pared to less than 10% digestion for the initial 60-80 mesh sawdust.
Vibratory ball-milling also enhanced cellulose digestibility of wood
and forages by rumen bacteria (17).
Millett, et al. (20) found tha, while vibratory ball-milling is
indeed an efficient pretreatment to improve ruminant utilization of
wood residues, the milling response is quite species selective. With
140 min. of milling, aspen and sweetgum showed 80% digestibility and
red alder showed only 20% digestibility. Softwoods were even less re-
sponsive than red alder; five different softwoods showed a maximum di-
gestion of only 18% after 120 min. of vibratory milling. Thus, this
selective species-response severely limits the broad application of
the milling as pretreatment.

10
Properties and Mode of Action of Cellulase
Cellulase is a multicomponent enzyme system. Through chromato-
graphic and electrophoretic techniques, the system has been resolved
into three components, C-,, C and 6-glucosidase (21).

I A
1) C-| component. The presence of C-, in the system is essential
if highly ordered substrates are to be attacked (21). The mode of ac-
tion of this component has been the subject of debate. According to
the hypothesis of Reese, et al. (22), C-, was believed to de-aggregate
the cellulose chains in preparation for attack by other components of
cellulase. Halliwell and Griffin (23) isolated the C-, component from
the cellulase system of Trichoderma koningii and found that the C-i
component released terminal cellobiose units from cellulose.
2) C component. This component can hydrolyze soluble deriva-
tives of cellulose or swollen and partially degraded celluloses (24).
Highly ordered substrates, however, are not attacked. Carboxymethyl
(CM) cellulose is normally used for the assay of C activity. The C
component attacks the cellulose at random (endo-3(l->'4)glucanase)
linkages. Cellobiose and cellotriose are the major products of endo-
3(l->-4) glucanase action.
3) 3-glucosidase. This component hydrolyzes cellobiose and short
chain celloolignosaccharides to glucose, but has no effect on cellu-
lose. The Trichoderma enzyme system has relatively low 3-glucosidase
activity (25), while the black Aspergilli are superior producers of S-
glucosidase (26).
n
The C-j component of one fungus acts synergistically with the C of
another. The molecular weights of C-, and C components lie in the re-
gion 45,000-75,000, with the exception of molecular weight 13,000 of
C^ which was removed from Trichoderma koningii cellulase on Sephadex
G-75 (27). The smallest C yet found had mol. wt. of 5,300 (28). The
3-glucosidase of Fusarium solani has mol. wt. of 400,000 (29). C-, and
C^ components are associated with various proportions of carbohydrate.
The carbohydrate may be covalently linked to the protein moiety in some
cases, while present as dissociable complex in others (30, 31).
The C-j and C^ components adsorb on the surface of cellulosic bio-
mass but the 3-glucosidase does not (32). A contact time of 15 minutes
was found to be sufficient for maximum adsorption to take place. After
15 minutes the enzymes started to return into the solution as a result
of hydrolysis.
Xylanase is the enzyme which hydrolyzes hemicellulose. This en-
zyme is excreted by Aspergillus wentii. Ghose and Bisaria (32) have
reported that enrichment of the Trichoderma cellulase with xylanase
has been shown to increase the hydrolysis rate of bagasse. Xylanase
enhances the hydrolysis of bagasse owing to the creation of more acces-
sible cellulosic regions that are readily reacted by the C-, and C
components. Hydrolysis of pure cellulose, however, was not affected
by the xylanase pretreatment because of absence of hemicellulose.
Furthermore, xylanase action is most effective when acting synergisti-
cally with other hydrolytic components, i.e. C-j and C components for
enzymatic degradation of bagasse.

12
Production of Cellulase
Thousands of microorganisms have the ability to grow on cellulose.
Although many of these grow quite rapidly, only a few produce extracel-
lular cellulases capable of converting crystalline cellulose to glucose
in vitro. Of these, culture filtrates from Trichoderma viride are the
most active. The cellulase activity in Trichoderma filtrates is stable
and may be stored for months under refrigeration or as lyophyllized or
acetone extract powders without any significant loss of activity.
Cellulase enzyme of Trichoderma is produced when cellulose is pre-
sent in the medium but not when substrates such as glucose are the sole
carbon source. For this reason, the cellulase is said to be an induc-
ible enzyme. The nature of inducer, is not certain.
Cellulase enzymes are repressible, which means that if the rate
of carbohydrate catabolism exceeds that required for cellular biosyn-
thesis, cellulase synthesis decreases or stops. By comparing the kin-
etics of cellulase repression by glucose with inhibition of RNA and
protein synthesis (by actinomycin D and puromycin, respectively),
Nisizawa, et al. (33) have inferred that cellulase synthesis is re-
pressed at the level of protein synthesis. Because the cellulases
are repressible, the addition of glucose, glycerol or any other rapid-
ly metabolized carbon source to a cellulose culture will interfere
with cellulase synthesis.
The biggest recent advance in improvement of cellulase yield has
been the generation and isolation of hyperproducing mutants (34). By
irradiating spores with a linear accelerator and screening survivors

13
for enhanced cellulase production, a strain (QM 9123) having twofold
increase in enzyme production was found. By taking this strain and
subjecting it to the same irradiation and screening procedure, strain
QM9414 was isolated and produces four times the amount of cellulase as
the original parent.
The cost of cellulase production occupies as high as 60% of the
total cost of enzymatic hydrolysis of cellulose (35). Therefore, every
effort to reuse the cellulase may be justified.
Process of Enzymatic Hydrolysis of Cellulosic Biomass
A tentative processing scheme for enzymatic hydrolysis of newsprint
has been presented by Wilke and Yang (35). This process has been formu-
lated on the basis of small-scale laboratory data for the key steps,
coupled with engineering analysis and assumptions as neede to complete
a preliminary process design and cost estimates for the conversion of
cellulosic materials to sugars. Such estimates enable one to ascertain
that the methods under development show at least some promise of econo-
mic feasibility. Such a study also serves to suggest priorities for
future research based on potential cost benefits.
In the process presented by Wilke and Yang, the feed is reduced to
approximately 20 mesh by means of moderate shredding and hammer-milling.
The cellulase is produced in a two-stage fermentation system, employ-
ing the fungus Trichoderma viride QM 9414. Hydrolysis is conducted
over 40 hrs. at 45°C at a solid/liquid ratio of 1/20 w/w based on in-
puts to the hydrolyzer. The hydrolyzer consists of five agitated
cylindrical concrete digestors. Cellulose conversion of 50:. is assumed.

14
The product sugar stream from the hydrolyzer is contacted countercur-
rently in three mixer-filter stages with feed solids for enzyme recov-
ery. Each mixer-filter stage consists of a mixing tank to provide 30
min. contact time and a horizontal belt vacuum filter to separate the
solid from the liquid.
Karube, et al. (36) have reported the immobilization of cellulase
inside of collagen beads and saccharification by employing a fluidized
bed reactor. Before hydrolyzing the cellulose, cellulase must adsorb
on the surface of insoluble partide of cellulosic materials. Most
of the immobilized cellulase prepared by their method, however was en-
trapped inside of collagen matrix so that it was hard for the enzymes
to contact the insoluble cellulose particles. Hydrolysis of cellulose
by the immobilized cellulase exceeds that by native cellulase only after
long incubation. The cellulose (Avicel AF) used in their experiment
was hydrolyzed quantitatively to glucose with the immobilized cellu-
lase. They infer that the cellulose particle may be adsorbed on cellu-
lases which are bound on the surface of collagen matrix and hydrolyzed
to low molecular-weight celluloses. Then, these low molecular-weight
celluloses may penetrate into the collagen fibril matrix and be hydro-
lyzed to glucose by cellulases which are immobilized inside.
Immobilization of Enzymes
One of the earliest reports of immobilized enzymes was that of
Nelson and Griffin in 1916 (37). These researchers reported the adsorp-
tion of invertase on charcoal and on alumina and demonstrated that
these immobilized enzymes retained their activity. Katchalski, et al.
(38) immobilized enzymes by covalent attachment to organic copolymers.

15
The work of the last several years has demonstrated that a wery
large number of different methods can be used to immobilize enzymes.
The general configurations used in enzyme immobilization are as follows
1) covalent bonding to a solid phase, 2) covalent bonding to soluble
polymers, 3) physical adsorption to a solid phase, 4) crosslinking at
solid surfaces, 5) crosslinking with difunctional reagents, 6) inclu-
sion in a gel phase, and 7) encapsulation. The largest amount of work
by far has been done on the covalent attachment to solid phases.
In general, the covalent attachment procedure involves the forma-
tion of an activated carrier, followed by the reaction of the activated
carrier with an enzyme to form a composite. A single reaction may be
involved, or several steps may be required in preparing the activated
carrier and coupling with an enzyme (39).
The most widely used technique for placing reactive organic groups
on inorganic surfaces is through reaction with silane coupling agents
(40). Silane coupling agents have dual functionality, with inorganic
functionality at one end and organic functionality at another. The in-
organic functional groups, which can be esters, halides or silanols,
condense with hydroxyl groups on inorganic surfaces (41).
A reactive aldehyde intermediate is readily prepared by reacting
glutaraldehyde with an amine group on surface of the carrier (42).
Aldehyde groups on an inorganic carrier react with primary amines to
form an imine coupling.
The amine groups on the carrier and the carboxyl groups on enzyme
react directly to produce amide linkages with help of carbodiimide (42)
A substituted urea is produced from each amide linkage as by-products.

16
In the last few years, attempts have been made to immobilize a
sequence of enzymes in close proximity to each other on the same car-
rier particle. Mattiasson, et al. (43) immobilized 3-glactosidase,
hexokinase and glucose-6-phosphate dehydrogenase together on the same
carrier particles to convert lactose to 6-phosphoglucono-lactone. It
was found that the efficiency of this solid-bound system of three en-
zymes was higher than that of the corresponding soluble system priot to
reaching steady-state.
Srere (44) reported efficient conversion of malate to citrate by
using malate dehydrogenase and citrate synthase immobilized on the same
carrier particles.
Monsan et al. (45) immobilized invertase covalently by cyanuric
chloride to bentonite. They mehtioned a possibility that cellulase
also could be immobilized using the same method.
Carriers
Many carriers have been reported in the literature during the past
20 years. The carriers can be made of organic or inorganic compounds.
The morphology and size of the carrier are extremely important with re-
spect to surface area and pore parameters, both of which in turn will
affect the loading of enzyme. A sample of the diversity and variety
of carriers currently available for immobilization includes: glass
particles (46), controlled-pore glass (47), alumina (48), controlled-
pore titania (49), nickel oxide (50), hydroxyapatite (51), iron oxide
(52), cellulose (53), agarose (54), colloidon (55), starch (56), poly-
arcrylamide (57), nylon (58), and collagen (59).

17
Recently, Heden (60) has commented on the potential use of magne-
tic materials as carriers. A wide range of magnetizable materials as
carriers, including iron, iron oxide, steels and ferrites are available
with properties which can be used to tailor materials for different re-
actor applications. Robinson, et al. (61) have reported immobilization
of two enzymes, a-chymotrypsin and 3-galactosidase, on two carrier mate-
rials, precipitated iron oxide activated with amino silane reagent and
a cellulose-iron oxide composite activated with cyanogen bromide.
Whiteside et al. (62, 63) have reported preparation of ferrimagnetic
catalyst carriers and magnetic filtration of small particles of ferro-,
ferri- and paramagnetic catalyst carrier.
mmobilized Enzyme Reactors
Immobilized enzyme reactors fall into several general categories
including batch reactors, continuous stirred tank reactors, fixed-bed
reactors, and fluidized-bed reactors.
Other variations of the basic reactor types include a stirred tank
reactor in which the immobilized enzyme is enclosed in mesh containers
attached to a stirrer to give adequate agitation with minimal attrition
of immobilized enzymes (64).
Closset et al. (65, 66) have analyzed and presented data for a
tubular membrane reactor for the hydrolysis of starch by a-amylase.
Enzyme and starch were contained in the membrane, which was permeable
to the produce, maltose, but not to the starch and enzyme. Venkatasub-
ramanian and Vieth (65) have used an arrangement consisting of alternate

18
collagen-enzyme membrane and backing layers wound around a feed distri-
butor.
A new approach to immobilize enzymes on magnetizable particles has
been carried out a step further by Gelf and Boudrant (68) who used a
fluidized-bed reactor containing papain bound to magnetic carriers.
The particulate immobilized enzymes were retained in the column by means
of a circular magnet encircling the upper part of the column.

CHAPTER I I I
MATERIALS
Cellulase of Trichoderma v i r i d e was purchased from ICN Co., Ohio,
and was i n a state of white f i n e power. I t was claimed by the company
that 1 mg of the cellulase would l i b e r a t e 4.4 y mole of glucose from
c e l l u l o s e i n one hour at pH 4.5 and temperature 40°C.
Magnetic iron oxide, FeÔ. whose p a r t i c l e size is less than 325
mesh, was purchased from Fisher S c i e n t i f i c Co., New Jersey. This chem-
i c a l is a combination of ferrous oxide, FeO, and f e r r i c oxide, FeÔ-.,
having a t h e o r e t i c a l formula of FeÔ..
Glutaraldehyde was purchased from Eastman Co., New York, and was
in a state of 50% water s o l u t i o n of density 1.09 g/ml.
The l-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene-
sulfonate was purchased from Sigma Chemical Co., Missouri. This chem-
i c a l w i l l be abbreviated as carbodiimide in the following parts of t h i s
thesis.
The 3-aminopropyltriethoxysilane was purchased from Aldrich Chem-
ical Co., Wisconsin.
The 2,4,6-trinitrobenzenesulfonic acid was purchased from Eastman
Kodak, Co., New York and was 99.9% pure. This chemical will be abbre-
viated as TNBS in the following parts of this thesis.
Toluene was purchased from MCB Co., Ohio and was A.C.S. reagent
grade.
Sodium tetraborate, Na^BÔ^ • 10 HÔ, was purchased from Sigma
Chemical Co., Missouri and was 99.9% pure.
19
20
Glycine was purchased from MCB Co., Ohio and was 99.5% pure.
The o-toluidine reagent was purchased from Sigma Chemical Co.,
Missouri (Stock No. 635-6) and was in a state of 6% (v/v) solution in
glacial acetic acid. This reagent had been specially manufactured by
the company for the purpose of glucose analysis.
All chemicals listed so far in this section were used as received
from companies without further purification.
Distilled water was prepared by using a metal still or a glass
still.
The origin and composition of cotton-gin trash (CGT) used for the
experiments of this thesis were described in the literature review.
The raw CGT was hammer-milled first and then pebble-milled for longer
than 3 days. After this period of milling, the CGT was in a state of
fine powder. Many batches of the CGT powder were collected in a large
container and mixed thoroughly by using a mechanical mixer. This pool
of CGT powder was used as substrate in all the experiments of this
thesis except the experiment which was carried out to study the effec-
tiveness of grinding as pretreatment of CGT for enzymatic hydrolysis.
The cotton gin trash powder w i U be abbreviated as CGT in the follow-
ing parts of this thesis.
Determination of Amount of Reducing Sugars

by o-Toluidine Method
A sample of 0.1 ml volume was taken fro-^ a hydrolysis nixture
whose amount of glucose was to be deternined. To this was aJded
5.0 ml of o-toluidine reanent.

21
A standard was prepared by mixing 0.1 ml of a standard glucose

solution (100 mg glucose/dl, Stock No. 635-100, Sigma Chemical Co.,
Missouri) and 5.0 ml of the o-toludine reagent. A blank was prepared
by mixing 0.1 ml of distilled water and 5.0 ml o-toluidine reagent.
Three test tubes which contained sample, standard and blank, re-
spectively, were shaken by using a lateral mixer and placed in a boil-
ing water bath for 10 min., after which they were cooled to room temp-
erature with tap water. The absorbances were then read at 620 nm on a
Bausch and Lomb Spectronic 21.
The amount of reducing sugars was reported in a form of correspond-
ing amount of glucose because molecular weights of the reducing sugars
were not fixed. There might be many kinds of reducing sugars in a
hydrolysis mixture, including cellobiose, cellotriose and oligosac-
charides as well as glucose. One sugar molecule of any molecular
weight was counted as one glucose molecule as long as the sugar mole-
cule had one reducing end.
The amount of reducing sugars present in the sample was calcu-
lated from the following equation.
„ 4. -
c j • í^9 qlucosex A sample - A blank
Amount of reducing sugars( Igg •. ) = T ^ ^ x -,p,p,
100
standard ' blank
where, A -, = Absorbance of sample at 620 nm

sample
AL-,
blanki = Absorbance of blank at 620 nm
A ^ ^ . = Absorbance of standard at 620 nm.
standard
CHAPTER IV
PREPARATION OF IMMOBILIZED CELLULASE THROUGH COVALENT BONDINGS
1. Methods and Results
A. Silanization of FepO.
The black fine power of FeÔ., 25.0 g, was dried at 200 ± 10°C
in an oven for 24 hours. The dried Fe-^P^ particles were silanized by
refluxing with 4.0 ml of 3-aminopropyltriethoxysilane in 196 ml of tol-
uene for 20 hours. This chemical reaction is described in Figure 2.
The silanized FeoO. particles were collected by using centrifugation
and washed thoroughly with distiUed water. The silanized FeÔ. par-
ticles were dried on boiling water bath.
B. Determination of Amount of Amino Groups Bound on FepO.
The colorimetric method developed by Snyder and Sobocinsi (69)
was employed in this thesis for this determination. The 3.0117 s sil-
anized FeÔ. was mixed with 20.0 ml of 0.766 mM TNBS solution made
with 0.1 M sodium tetraborate, pH 9.30, as buffer. The mixture was
agitated thoroughly for 30 min. by using a lateral mixer. During this
period of agitation, all the amino groups were assumed to react with
TNBS molecules which were present in excess amount. After the agita-
tion and reaction, some of the TNBS molecules were left unreacted.
The number of the unreacted TNBS molecules was determined by using
glycine which has an amino group as a part of it. The agitated mix-
ture of silanized FeÔ^ and TNBS was centrifuged. Excess glycine was
added to the supernatant to produce a colored product which has the
maximum absorption at 420 nm. Measurement of absorbance of light at
oo
23
ro
+
C
o
X u
o o (T3
OJ
rn s_
c
o
T3
<V
M
c c
13
(/) (/)
c
o
03
OJ OJ
c
fT3
03
z t/1
X OJ
-C
o
x-^ O)
C\J
oô"
OJ
>. s.
o
Q.
O
c
03
I
-I- cn
X>
u X
«_) '^
OJ
c ^
cn o
n ^
24
this wavelength made it possible to determine amount of the unreacted
TNBS. If we subtract this amount of TNBS from the amount of TNBS ori-
ginally used, we can find the amount of amino groups bound on FeÔ^
particles because amino groups react with TNBS molecules at 1:1 ratio.
A blank experiment was carried out by using non-silanized FeÔ. parti-
cles.
The results of these experiments show that 3.3 x 10" g moles of
amino groups are bound on 1.0 g of silanized FeoO^ particles.

C. Efficiency of Magnetic Recovery of Silanized FeÔ/. from
Buffer Solution
The recovery experiment began when the 0.5015 g silanized Fe-Ô^
was mixed with 50.0 ml of 0.05 M KAc buffer, pH 5.0. The magnetic re-
covery of silanized FeÔ. was performed by using two rings of doughnut-
shaped permanent magnets, 2.8 cm outer diameter. The magnets were placed
in a Ziploc sandwich bag (Dow Chemical Co., Indiana) and dipped into
the mix-ture. The silanized FeÔ. particles were attracted onto the
magnets during swirling. The recovered silanized FeÔ^ particles were
washed by swirling in 200 ml of 0.05 M KAc buffer, pH 5.10, while they
were still attached onto the magnets. The recovered silanized FeÔ^
particles were then released into 125 ml of 0.05 M KAc buffer, pH
5.10, by taking the magnets out of the sandwich bag. These procedures
of recovery were repeated for 15 times until no visible solid parti-
cles were present in the buffer solution.
The mixture of recovered silanized FeÔ^ and buffer was allowed
to settle for several hours until all the particles settled down on
bottom of the beaker. The buffer was then decanted. The moist
25
particles were dried on a boiling water bath. The dried particles were
placed in room temperature until there was no change in weight. The
weight of the dried particles was constant at 0.4945 g, which indicated
that 98.6% efficiency of recovery was achieved. Contribution of trace
amounts of KAc to the weight of dried FeoO^ was neglected.
D. Preparation of Immobilized Cellulase Attached Through

Glutaraldehyde
The 2.00 g silanized FeÔ^ was mixed with 20.0 ml of 2.5% glu-
taraldehyde which was prepared by diluting 50.0% glutaraldehyde solu-
tion with distilled water. The mixture was shaken for 1 hr. at room
temperature. The glutaraldehyde was to react with amino group bound
on silanized FeÔ. particles during the shaking (Figure 3 ) . The glu-
traldehyde-treated FeÔ. was centrifuged and then washed thoroughly
with 0.1 M NaPi buffer, pH 6.0. Then, the glutaraldehyde-treated
FeÔ. was mixed with 50.0 ml of 0.1 M NaPi buffer, pH 6.0, and 0.5201
g cellulase was added to this mixture. Immobilization of cellulase
was carried out through shaking of the mixture for 1 hr. at room temp-
erature (Figure 4 ) . The immobilized cellulase (IMC) was centrifuged
and washed thoroughly with distilled water. In the following parts of
this thesis, the weight of the IMC will beassumed to be the same as
that of silanized FeÔ^ which has been used to prepare the IMC because
weights of glutaraldehyde and cellulase actually bound on silanized
FeÔ. are not known at this stage of the research.
The 2.00 g IMC which had been prepared above served as a pool of
IMC for experiments throughout this thesis. A 0.500 g sample of IMC
was taken from the IMC pool and washed by employing centrifugation
26
O)
o^4
(V
-o
03
s_
03
cn
o
W
^n o
ro
N O)
o U
N
03
OJ
m >í (/)
-C
O) OJ
-a ri
03
03
X
+->
o O)
.o^ <v
03
OJ
C
o
03
cr
A OJ
03
OJ
ro
0)
s-
O)
27
©
03
c
o
O)
00
03
r—"
3
r—
•
(U
t ^
-o
OJ >«)
u _c
OJ
OJ <4- -o
03 o r—
03
-t-> i_
OJ
^ 03
gj -)->
c/) c ;3
O) 03
N CJ Û 1 —
CTí
03
-l-J - C
-.-' cn
CU .T3 3
U o
M- i -
"O O ^
OJ -t->
M
c
o O)
o H
O
•r—
-l->
r—
03 • r—
u
^
-y
.4-)
s_
QJ 03
1 —
^
í"
03
U OJ
•f— • r—
"" S-
Q
-C
J s-
03
CJ U
U
•^
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i_
3
\J
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28
with 0.05 M KAc buffer, pH 5.10. The supernatant was discarded. The
IMC was washed again with 100 ml of 0.05 M KAc buffer, pH 5.0 and then
centrifuged. Supernatant from this centrifugation was saved for the
preparation of sample and blank. A sample was prepared by mixing the
0.5005 g IMC, 5.00 g CGT and 50.0 ml supernatant. A blank was prepared
by mixing 5.00 g CGT and 50.0 ml supernatant. The sample and blank
were shaken at 46°C in an incubator-shaker for 12 hrs. The amounts
of reducing sugars produced during this period were determined by o-
toluidine method. The difference between the amounts of reducing
sugars produced in sample and blank was used to calculate activity of
the IMC. This was the first hydrolysis run. The activity of IMC at
the Ist run is listed in Table 2.
The'used IMC was recovered from the sample of the first run by
using two rings of permanent magnet which were placed in a sandwich
bag. The IMC was washed by swirling in 200.0 ml of 0.05 M KAc buffer,
pH 5.10, while the IMC was attached on the sandwich bag by magnets.
The IMC was released from the bag into 125.0 ml of 0.05 M KAc buffer,
pH 5.10, by removing the magnets from the inside of the bag. The IMC
was centrifuged and the supernatant was saved for preparation of a new
sample and blank for the second hydrolysis run. The preparation of the
new sample and blank followed the same procedure as the first run.
The activity of IMC at the second run is listed in Table 2.
The activities of IMC for four subsequent runs were determined by
repeating the same procedure as above. Figure 5 shows retention of
IMC activity.
29
OJ > í
C\J LO
> .(->
•^ •^
-M > o CD ro LO
03 •r- o <m
n— •+->
OJ OJ
CÍ:
u
<:
T3
>> ^^ s
-C
OJ OJ
oo
-o
o
CJ
> i
s_ -(-> 3
03 •1— r— S-
-»-> > CD J C
C\l CM
• r— LO CVJ
-(-> OJ C_J «^ <r)
cn CJ n— CM co
03
s: U3 cn c\j CM CSJ C\J
o 1—t
C7) C_) CT
O
s_
dJ
on
O 03
-o CU
OJ O
13 on
-C
O
CJ
(J
03
-(-> CSJ
co cn C\J LO o
-M
03 -o cr> o o o o
cn CsJ
(_J
•ão i
-
s_ :3.
O Q. —
>í cu


OJ on
C\J
00 OJ «r <0 o
U3
>> -— CNJ
Osl ,— oo I
O s- I
OJ S_ -C o «:3- LO 00 o
(U -o —' CVJ CvJ «:r
>í
03
00 S-
•r- OJ
OO -û
>, E -a XJ
+->
,— Z3 00 c s-
O C OJ ro «^ LO U3
s_
-o c
>. ^
-rr s_
30
100 -
90 -
80 -
70 -
>i
•M
60 -
CJ
<

50 -
•^
03
(U 40
Csl
30 -
20 -
10
0
0 12 24 36 48 60 72
Time of Hydrolysis (Hrs)
Figure 5. Retention of activity of IMC attached through

glutaraldehyde: Relative activity of IMC at each
run has been plotted in the middle of the time of
hydrolysis.
31
E. Preparation of Immobilized Cellulase Attached Through

Carbodiimide
The 0.1318 g cellulase, 0.5021 g silanized FeÔ^ and 0.1285 g car-
bodiimide were mixed in 50.0 ml distilled water. The pH of this mix-
ture was adjusted to 3.6 by adding 6 M HCl. The mixture was shaken for
2-1/2 hrs. at room temperature. The cellulase was to be attached on
the silanized êJd. during this shaking (Figure 6 ) . The IMC was cen-
trifuged and then washed thoroughly with distilled water. The IMC was
washed again with 200 ml of 0.05 M KAc buffer, pH 5.10, and centrifuged,
The supernatant of this centrifugation was saved for preparation of
sample and blank. The sample was prepared by mixing the 0.5021 g IMC,
5.00 g CGT and 50.0 ml supernatant. The blank was prepared by mixing
5.00 g CGT and 50.0 ml supernatant. The sample and blank were shaken
for 48 hrs. at 46°C in an incubator-shaker. After the shaking, amount
of reducing sugars produced by the IMC was determined by employing o-
toluidine method described previously in this thesis. Activity of the
IMC at this Ist run was calculated and listed in Table 3.
The used IMC was recovered from the sample by using two rings of
permanent magnet which were placed in a Ziploc sandwich bag. The IMC
was washed by swirling in 200.0 ml of 0.05 M KAc buffer, pH 5.10, while
it was attached on the sandwich bag by the magnets. The IMC was then
released from the bag into 125.0 ml of 0.05 M KAc buffer, pH 5.10, by
removing the magnets from inside of the bag. The IMC was centrifuged
and the supernatant was saved for preparation of new samples and blank
for the 2nd run. The sample was prepared by mixing all of the recov-
ered IMC, and CGT, 5.00 g, and supernatant, 50.0 ml. A blank was
32
03
•f • o
+ cOJ Jo3
f^ í.
- C 03
U
u
03
•M - C
C^ 0=0 -M CJ)
03 Z3
 U
o 03 • 1 —
X) CU 3 -(_>
s- U O"
03 s_
03
U -o u
N •
(U
s_
C7)
A
o
PO
^
> .(_> ro co
•(->
03 -r-
>
o CVJ
LO LD
OsJ
O
^ O
J2 u
•I- 3
s_ > 1—
03
U •I- ai C_) LO CO LO
u cu CTi oo CNJ
03 1—
O o cn
S- c—) E
"O cu
 o
-M cn u
03 o
CJ -o Ol co co o
^
-M
cu
 E
(U s_
û:: Q.
00
CT) 00
>> ^ 00 Cvj
> E -o -o
I— Z3 -(->
o c 00 c s_
s_ C\l ro
-o c
>í o
z: s_
34
prepared by mixing CGT, 5.00 g, supernatant, 50.0 ml. The shaking of
sample and blank and determination of amount of reducing sugars pro-
duced by the IMC was carried out with the same procedure as the Ist run.
Activity of the IMC at this 2nd run was calculated and listed in Table 3
The activities of IMC at subsequent two runs were determined by
repeating the same procedure as above. Figure 7 shows activity of IMC
decreases even after long time of hydrolysis, which is in a contrast
to Figure 5.
2. Discussion
Table 2 shows retention of activity of IMC attached through glu-
taraldehyde. Six hydrolysis runs have been carried out lasting for 12
hrs. Relative activities have been calculated by employing the acti-
vity of the IMC in the first run as a basis and plotted in Figure 5.
Activity of IMC decreases as time of hydrolysis increases. The decrease
of activity between the first and second runs is the most drastic of
any of the reuses. This implies that most of the immobilized cellu-
lase molecules are denatured during the first hydrolysis. The decrease
continues in a reduced degree in subsequent runs. Finally, the acti-
vity remains almost constant after the fourth run. The hydrolysis runs
done before the fifth run can be considered as a series of washing
steps. The CGT substrate has been used for this washing to destroy
unstable IMC molecules. Size of a cellulase molecule is so big that
there are many functional groups available for immobilization. Some
of them are essential to stability of the enzyme and some of them are
not. If one of the functional groups essential to the stability is used

35
100
80
>>
-i-j
>
u 60
03
>
03
O)
40
0
0
20
0
48 96 144 192
Time of hydrolysis (Hrs)
Figure 7 . Retention of activity of IMC made with carcoc'^m-ce as a^

attaching chemical: Relative activity cf :MC 5t eacn r-^-
has been plotted in the middle of the time of nycrclys^s.
36
for immobilization, there results an unstable IMC molecule which will
be destroyed easily in the vigorous environment of the hydrolysis reac-
tion. Ninety-eight percent of the activity of IMC at the fifth run is
reusable. If the fact that efficiency of magnetic separation is 98.6%
is considered, the 99.6% IMC is calculated to be reusable. The acti-
vity of this 99.6% reusable IMC is 22.8 (y mole glucose/g IMC • hr).
This result is quite significant, because nobody has ever reported pre-
paration of reusable IMC attached through covalent bondings.
Table 3 shows retention of activity of IMC attached through carbo-
diimide. Four runs of hydrolysis have been carried out and each run
lasted 48 hrs. Relative activities have been caTculated by employing
the activity of IMC at the first run as a basis and plotted against time
of hydrolysis in Figure 7. Activity of the IMC decreases as time of
hydrolysis increases. The decrease of activity between the first and
second runs is most drastic comparing the decreases between other runs.
This implies that most of IMC molecules are denatured during the Ist
run of hydrolysis. The decrease continues at the third and fourth run.
There is 9.5% decrease in relative activity between the third and
fourth runs. This is a big decrease comparing that of IMC attached
through glutaraldehyde. Attachment through carbodiimide is, therefore,
not so good as attachment through glutaraldehyde. This is even more
clear when the final activities of IMC's made with both methods are
compared. The final activity of IMC attached through carbodiimide is
1.6 (y mole glucose/g IMC • hr) and is still decreasing in contrast that
the final activity of IMC attached through glutaraldehyde is 22.8
(y mole glucose/g IMC • hr) and is almost constant. This implies that
37
the carbodiimide modifies cellulase molecules in such a way that every
IMC molecule is unstable and denatured in the vigorous environment of
hydrolysis.
CHAPTER V
EFFECT OF CROSS-LINKING ON ACTIVITY OF IMMOBILIZED CELLULASE
A. Activities of IMC's Cross-Linked lîth Various Concentrations

of Glutaraldehyde
The 0.1923 g IMC was taken from IMC pool made previously with
2.5% glutaraldehyde by the method described in section ID of this thesis.
The IMC was mixed with 20 ml of 10% glutaraldehyde solution which was
prepared by diluting 50% glutaraldehyde with distilled water. The mix-
ture was shaken for 1 hr. at room temperature to cross-link the im-
mobilized cellulase. A chemical equation of this cross-linking is
shown in Figure 8. The cross-linked IMC v/as centrifuged and then wash-
ed thoroughly with 200 ml of 0.05 M KAc buffer, pH 5.10. The cross-
linked IMC was washed again with 100 ml of 0.05 M KAc buffer, pH 5.10
and then centrifuged. The supernatant of this centrifugation was saved
to prepare the following sample and blank for the first run.
A sample was prepared by mixing all of the cross-linked IMC, 2.50
g CGT and 50.0 ml supernatant. A blank was prepared by mixing 2.50 g
CGT and 50.0 ml supernatant. The sample and blank were shaken for 12
hrs. at 46°C in an incubator-shaker. Amount of reducing sugars pro-
duced by the cross-linked IMC at this Ist run was determined by employ-
ing o-toluidine method described previously in this thesis. Activity
of the cross-linked IMC which had been prepared with lO:- glutaraldehyde
was calculated and listed in Table 4.
38
39
-Î<CH4N=CH(CH,)3CH=N
+ 0HC(CH2),CH0
=CH(CHACH=
q
-SÍ(CH4N=CH(CHZ)5CH=
o
> FeÔ^
\
o
O-5Í(CHAN=CH(CHZ)3CH=
p
+ 2.H10
Figure 8 . Chemical equation of cross-1inking of immobilized
c e l l u l a s e through glutaraldehyde.
40
o c on
s: 
o Xcu
J
-oc
C C^vJ OvJ <Æ C\l <T> o
O >í •
• • .
> í •r- -»-> - O ^ on <J^ c£> C\J «=^
-(-> -f- c ^ LO ci3 f^ • CO co
C > 03
cu •<-
-(-> 4-) -t->
 , S- 00 S- o on cr> r>-» ro
-(_) -(-> >, c o o o m <^ C\l C\l CVI
s_ •r- OsJ S_ E S I
'<v -t-> "O 1—1
-C U -(->>, ^
+-> <: 03 - C O)
c
o
o , S- 1/1 S- <T> r^v U3 <X) ro
I •-(-> > , 1 1 . U3 v^ r—
oo co
CO PO
• ^
> 00 O O O •^ ro ro
O •r- r - S_ E 21
s_ •4_) -O 1—.
u U -M > , a
e i ; 03 - C CT)
M-
O 1
u CU 00
O) C - O 00
o >> o
•.- - C s _ ^ - -
- M 5
03 - O -—
S_ .— S_
-(_> 03 O CJ) LO O O O
C S_ M- C • • • •
 -o -:«í .— í—
c o <u c
03 O .— on -r-
O CD Z3 1—
41
The used cross-linked IMC was recovered by using magnets and wash-
ed with 200 ml of 0.05 M KAc buffer, pH 5.10. This IMC was released
into 125 ml of 0.05 M KAc buffer, pH 5.10. The IMC was centrifuged and
the supernatant was saved for preparation of sample and blank for the
second run of hydrolysis. A sample was prepared by mixing all of the
IMC, 2.50 g CGT and 50.0 ml supernatant. A blank was prepared by mix-
ing 2.50 g CGT and 50.0 ml supernatant. The sample and blank were
shaken for 12 hrs. at 46°C in an incubator-shaker. Amount of reducing
sugars produced by the IMC in this second run was determined by employ-
o-toluidine method described previously in this thesis. Activity of
the IMC was calculated and listed in Table 4. Figure 9 shows schematic
diagram of IMC's.
Activities of IMC's which had been prepared with glutaraldehyde of
different concentrations than 10% were determined by employing the same
procedure as above except that different concentrations of glutaral-
dehyde were used. Figure 10 shows cross-linking increases retention
of activity. Figure 11, however, shows cross-linking decreases the
activity.
B. Retention of Activity of IMC Cross-Linked Through 4.0%

Glutaraldehyde
The 0.50 g IMC was prepared by attachment through 2.5% glutaralde-
hyde solution and cross-linking through 4.0% glutraldehyde solution.
The procedures for this preparation were the same as described pre-
viously in this thesis. The IMC was washed thoroughly with distilled
water. The IMC was washed again and then centrifuged with 100 ml of
0.05 M KAc buffer, pH 5.10. The supernatant of this centrifugation was
saved for preparation of sample and blank for the Ist run.
42
Cross section Top view
Uncross-
linked
Cross-
linked
Figure 9. Schematic diagrams of IMC. The shaded area represents the

Fe304 particle and the bars represent covalent bonds. The
small circles represent immobilized celljlase molecules.
43
O
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•D

0) c
O -D •f—
> i ^
• co c
"ai •^
-D ^—
^— (
00
<o (/)
s_
03
o
4~>
s_
o
^—
u
o• O) u_
" V£5 14-
o
u
<v
S_
-M
£1
<U
o u
«3- c CJ
o
o
CVJ
o
C\J
i rilUîÍLA,) 3N ] ô Aq.LAL;^V
âsoDntb d[o\h r'
45
A sample was prepared by mixing the 0.50 g IMC, 5.00 g CGT and
50.0 ml supernatant. A blank was prepared by mixing 5.00 g CGT and
50.0 ml supernatant. The sample and blank were shaken for 12 hrs. at
46°C in the incubator-shaker. The amount of reducing sugars produced
by the IMC was determined by employing the o-toluidine method described
previously in this thesis. Activity of the IMC at this first run was
calculated and listed in Table 5.
The used IMC was recovered by using permanent magnets. The recov-
ered IMC was washed with 200 ml of 0.05 M KAc buffer, pH 5.10 and re-
leased into 125 ml of 0.05 M KAc buffer, pH 5.10. The IMC was centri-
fuged and the supernatant was saved for preparation of new sample and
blank for the second run. The sample was prepared by mixing all of the
IMC, 5.00 g CGT and 50.0 ml supernatant. The blank was prepared by
mixing 5.00 g CGT and 50.0 ml supernatant. The sample and blank were
shaken for 12 hrs. at 46°C in the incubator-shaker. Amount of reducing
sugars produced by the IMC was determined by employing the o-toluidine
method described previously. Activity of the IMC at this second run
was calculated and listed in Table 5. Figure 12 shows accumulated glu-
cost production.
Recoveries of the IMC and preparations of new samples and blanks
were carried out by repeating the same procedures as above to obtain
activities of IMC at subsequent runs of hydrolysis. Table 6 and Figure
13 show retention of activity of IMC cross-linked through 4.0% glutral-
dehyde.
46
-o
 3 cn u
"D \
03 cu
O
S- </í "O
3 O
E CL cu
o 3 í
4-> ^— c
O 3
•c
s_
cu -o
-^ >.
•D
cu
c
•r— 00 c
r— 0.- <r> CT» <o CO
s-
( o c co
00
00
o I LO LD CT)
oo C^J 00
o C 00
u c
u s_ 3 c
"O s_
o u
s_
_-, c/) C- u <zn - c
fO cu
> a^ o 00 ro o o c\i O CT)
 cn u
-o .^
o OJ c
s_
Q.-C
r—

00
<:3 CT) M- -1- OsJ «3- 00 o cvj
O 00 ro <sD
CVJ

03
00 S_
•f- > E
•— 3 -o -D
O C 00 c S. •i-> x:
s_ OsJ ro Ln 4->
T3 C
>> 3
IC S_
47
Od
r>.
O
LO
n by I M C ' s .
00 o
•^ •r—
-—^ .«_)
oo u
S- 2
^ 13
^-^ o
oo s.
o.
•^
00
> </)
<•£> o
ro o u
s_ 3
r—
-o
>^ CT)
sz "D
TD ^-
CU cu
.(_>
JÍÍ: "D o o:
c CU ^_
•r— ^
cu
c= 3
r—
1
c
•r- «:r •f— c
Accu
C\l 1—
OO p—
00 (
o co
00
s.
u o
c
3
s.
u OsJ
o o C\J

o o o O O O O o o o
o Ln O Ln O LO
o Ln o Ln
on «;r ^ ro ro CNJ OsJ r— r—
(3WI 6/asoon[6 6«u) uoLaonpo-id 9S0Dn[5 pa:;p[nLun30\/

Activity Relative
Hydrolysis Time of
, |i m o l e g l u c o s e x Activity
run number hydrolysis (hrs) g IMC-hr,
Ist 0 - 12 35.8 100.0
2nd 12 - 24 26.8 74.9
3rd 24 - 36 26.2 73.2
4th 36 - 48 22.6 63.1
5th 48 - 60 20.7 57.8

O
on
<£)
6th 60 - 72
•
18.1
48
49
OsJ
r^
C during si runs of
ution was u ed for
o
LO
X 00
00 2: r-
«^ «—>. •— 0
00 00
s. -D
x: ,
00 •1- x:
•^ r— cu
00 1 -a
>) 00 (—
r— 00 n
LO o 0 s-
ro s_ S_ 03
-D U 4-)
>> 3
-C
0 cn
<+-
o
4.) 0

•r-
^
•^
^ f— 4-J <U
Retention of ac
hydrolysis: th
C\J
cross-linking.
CVJ
ro
s_
3
cn
0 0 0 0 0 0 0 0 0
0 C3^ 00 r^ LO on •^r 00 OsJ
/C:;LAL:ôe a A L ^ e ^ a y
50
2. Discussion
Table 4 shows how the cross-linking affects activity and retention
of activity of IMC. The higher the concentration of glutaraldehyde
that is employed, the higher the degree of cross-linking is expected.
The higher degree of cross-linking makes IMC molecules more tightly
bound to each other. This phenomenon is illustrated in Figure 9. Cross-
linking is formed when two aldehyde groups of a glutaraldehyde molecule
are reacted with amino groups of adjacent two cellulase molecules. Re-
tention of IMC activities between first and second runs is high when
highly cross-linked IMC is employed as expected. Figure 10 shows, how-
ever, the retention of IMC activity increases mildly when concentration
of glutaraldehyde increases. Figure 11 shows that the activities of
IMC's at first run decrease sharply at early stage when the concentra-
tion of glutaraldehyde increases. The activities of IMC's at second
run, however, decrease mildly. These results show only the effects of
cross-linking on activity of IMC but do not tell whether the cross-
linking is beneficial to overall glucose production or not.
Table 5 shows glucose production by IMC cross-linked through 4.0%
glutaraldehyde during six runs of hydrolysis. The IMC has been prepared
by 2.5% glutaraldehyde for attachment before the cross-1inking. The
4.0% concentration of glutaraldehyde for cross-linking has been chosen
arbitrarily. Table 6 shows retention of activity of the IMC cross-linked
through 4.0% glutaraldehyde during six hydrolysis runs. Tables 5 and 6
are originated from the same experiment. Figure 13 has been plotted
from the data which are shown in Table 6. Activity of IMC decreases
continuously but does not show any region of constant activity as in

51
the case of uncross-linked IMC. This continuous decrease implies con-
tinuous denaturation of the cross-linked IMC during hydrolysis runs.
The chemical reaction of cross-linking may damage the IMC and make it
unstable. The unstable IMC is denatured easily in a vigorous environ-
ment of hydrolysis reaction.
At this point, we have to decide whether the cross-linking is
beneficial for production of glucose during many runs of hydrolysis.
This can be achieved by comparing the accumulated amount of glucose
produced by the cross-linked IMC during the many runs of hydrolysis with
that produced by the uncross-1inked IMC. The data of Table 5 have been •
calculated from the data of Table 2 and Table 6. The accumulated amounts
of glucose produced by uncross-1inked IMC and cross-linked IMC have been
plotted in Figure 12. This figure shows that the accumulated amount of
glucose produced by the uncross-linked IMC is always higher than that
produced by the cross-linked IMC. Therefore, we can conclude that the
cross-linking of IMC is not beneficial on overall glucose production.

CHAPTER VI
LOADING OF VARIOUS AMOUNTS OF CELLULASE ON SILANIZED FeÔ^
A. Loadinq of Various Amounts of Cellulase on Silanized FepO/.

With Glutaraldehyde as an Attaching Chemical
The 2.00 g s i l a n i z e d FeÔ^ was mixed with 20.0 ml of 2.5% g l u t a r -
aldehyde s o l u t i o n . The mixture was shaken f o r 1 hr. at room tempera-
ture. The glutaraldehyde-treated FeÔ^ was centrifuged and then washed
thoroughly w i t h 0.1 M NaPi b u f f e r , pH 6.0. This FeÔ^ served as a pool
i n f o l l o w i n g experiments of t h i s section.
The 0.20 g a l i q u o t of FeÔ. was taken from the FeÔ^ pool prepared
above and mixed w i t h 0.1057 g cellulase and 20.0 ml of 0.1 M NaPi buf-
f e r , pH 6 . 0 . The mixture was shaken f o r 1 hr. at room temperature to
immobilize c e l l u l a s e on the c a r r i e r . The IMC was centrifuged and then
washed thoroughly with d i s t i l l e d water.
A sample was prepared by mixing a l l of the IMC made above, 5.00 g
CGT and 50.0 ml of 0.05 M KAc b u f f e r , pH 4.00. A blank was prepared by
mixing 5.00 g CGT and 50.0 ml of 0.05 M KAc b u f f e r , pH 4.00. The sample
and blank were shaken f o r 48 hrs. at 46°C in the incubator-shaker. The
amount o f reducing sugars produced by the IMC was determined by employ-
ing o - t o l u i d i n e method described previously. A c t i v i t y of the IMC was
c a l c u l a t e d and l i s t e d i n Table 7. The r a t i o (g c e l l u l a s e / g s i l . FeÔ^)
employed i n t h i s experiment was 0.529.
A c t i v i t i e s of IMC's with other r a t i o s than 0.529 were determined
by employing the same procedure as above except that d i f f e r e n t amounts

52
4c
 ,
> 4-J
•1— T -
4J > o UD oo o
03 • r - .
o ' — 4-> o on
. •
o
ro CU U
^
1 —
00
CT^
co CT>
,!—
4 J </) ^ j O ro ^— r^
<o 03 •»— r— Ln
00
Ln
>^
00 ro 4J
M 1— > 1—
•r- ^ • - - ICU
• . . •
o 00 OsJ o o

rc •r- 4-> o j 03 i c n U
^^ C O u l E 03
3 cu
1 — -C ^—^
GJ
1 —
u ^^,_^

u
fO
>^ ^'r^ .:^
4") 1 ; 03
c 4-í •r- CDi 4_>
3 4.) > 1 r--« r^ CD Ln
O 03 •
•r- CU J O • . . >.
E 4-^ >— fc^ o O on o — J 3
03 c
03
u o p m Ln vO <r>
uo
<C E "D
3 OO jo. 
O 03 03
•^ r—
s- CU
,,^ 3
03 - D U
> > í "^ f —
-C -D o 03
M- cu CU ro U
O r—
-D oo o <o N
03 3 •r— r— '^ 00 <Ti CT) ro 3 c r__ CJ^ C\l ro >
1 cn 03 r— 03 o o Ln 00 o:
CÍ; <— • • • . JZ
cu »r— o o o o O
u 00 00 o
r-^ CU .
cnicn •^ o

cn
•r—
-Q > oo
•r- 03
03
4-1
u o
fo • r -
<+- 
O oo S_
4-i ' — •r—
• • • •
C 3 — o ro CTi
r—
Ln LO 4_> ro
3 r— - D cn o
r—
oû 03 ro
O
E
-— cu
<U 00
E
—
1— 1 — C3
<: u 3
<U .
Q : o
54
of cellulase were used. Figure 14 shows relative activity of IMC vs.

ratio.
B. Loadinq of Various Amounts of Cellulase on Silanized FepO.

With Carbodiimide as an Attaching Chemica!
The 0.25 g cellulase, 0.50 g silanized FeÔ. and 0.25 g carbodi-
imide were mixed in 50.0 ml distilled water. The pH of the mixture was
adjusted to 3.60 by adding 6 M HCl. The mixture was shaken for 2-1/2
hrs. at room temperature to produce IMC. The IMC was washed thoroughly
with distilled water. The IMC was washed again with 100 ml of 0.05 M
KAc buffer, pH 5.10, and then centrifuged. The supernatant of this
centrifugation was saved for preparation of sample and blank. The
sample was prepared by mixing all of the IMC, 5.00 g CGT and 50.0 ml
supernatant. The sample and blank were shaken for 48 hrs. at 46°C in
an incubator-shaker. The amount of reducing sugars produced by the
IMC was determined by employing o-toluidine method described previously.
Activity of the IMC was calculated and listed in Table 8. The ratio
(g cellulase/g sil. FeÔ.) employed in this experiment was 0.500. Fig-
ure 15 shows relative activity of IMC vs. ratio.
2. Discussion
Table 7 shows the loading of various amounts of cellulase on silan-
ized Fe 0. with glutaraldehyde as an attaching chemical. The relative
activities of this table have been plotted against ratio in Figure 14.
This figure shows sudden increase in relative activities at early stage
and then no increase later. This phenomenon implies that cellulase can
not be loaded any more on the surface of the carrier once the surface
has been saturated by the enzyme molecules. The saturation ratio is

00
cr^
i
00
<V
cu
N
<Æ
•^ (/) 1 —
o o
ro c u

^_ C 4->
3 03
^ rI—
— oo
,— —J c
,
- ro 4-1 O J=
03 • ^ CU
Cí s_ -c
03 - —
> 03
S_
Oi_ 03
O ^
^ M '
oo cn^
c cn
•f—
-a ^
03 —J
O -r-
-^ s
sû CVJ
3WI jô /^ LAL ê aAL;e[oy

56
o
CT>
oo
03
activity*
4_>
Relative
03
S_
65.7
87.2
86.5
90.0
88.4
35.1
o
O o
on
O
ro
cu
-o
cu <4-
N O -—
icu >í—
0.060
0.080
0.147
0.156
0.109
0.149
oo 4-> 00 4-> : 3
<C fO •r— 1 <_)
N r— > j
• 1 - 3 T- \<V
O 03 O
r— r— 4-> i U
U •r- r - O ,
CU - 1 - 4-> CU 03 I C D
00 E
03 CU o u ;E >>
•— ^ -l_>
3 U
mole glucosev
>— c n

U •(- S- U
03
M- U
Activity
O 03 
19.5
19.3
19.1
14.5
4->
19.9
OO 4_> 00
4_> 03 •
C
C_) O r— cn
3 C
O 03
E 03
03 00 cn
/
03
oo
3 CU
>,
J3
O T3
Z E o -o
^
03 - 1 - ro CU
> •t- cu 4->
-a M_ 03
^ o
-o
( g cellulase
O J2
U
Ratio
s- 
03
CU
oo
J3

177.5
50.0
130.0
97.5
o >
(mg)
used
u
03
cu
>
03
— -C
•r— 3 U
OO —
r- cn
cn OJ c
""^ U '^

3 00 —J
- ro ,—
(— -_J .-13
<V ^ :;z
u O 'O
c:
C71 03 O!
03
oo
o 3 •r—
-f—
03 'i_ E
. cvi Q: 03 - r -
> -r-
-a
M- O
O J2I
>-
CT) 03
C U
•r—
-D -C
03 4-)
C •'-
_l 5
on
<u
Ô)
o o o o o
00 <vO OvJ
o X' LAL Oe 9AL B[ay
58
about 0.26. If larger ratio than the saturation ratio is employed, only
a small number of cellulase molecules will be used in immobilization
and the rest of them will be wasted. If smaller ratio than the satura-
tion ratio is employed, the carrier will be underloaded so that acti-
vity of the IMC will be low.
Table 8 shows the loading of various amounts of cellulase on sil-
anized FeÔ^ with carbodiimide as an attaching chemical. The relative
activities of this table have been plotted against the cellulase to car-
rier ratio in Figure 15. The results shown on the table and figure can
be interpreted in the same way as above. The saturation ratio is about
0.26 which is the same value as above. The plots of Figures 14 and 15
are known as adsorption isotherms since they have been obtained at a
fixed temperature. These plots show a Langmuir-type adsorption, and
are characterized by a monotonic approach to a limit, which presumably
corresponds to formation of a monolayer of enzyme. This type of be-
havior is that expected for chemisorption. In addition, the type of
plots indicates that the FeÔ. particles have no porous structures.
The IMC activity is listed in the second column of Tables 7 and 8.
These data were obtained using IMC's at their optimum pH's. These data
show that IMC's made with glutaraldehyde are 300 times more active than
the IMC's made with carbodiimide. The reason for this phenomenon may
be sought in denaturation of cellulase. The carbodiimide apparently
modifies carboxyl groups which are essential to activity of the enzyme.
This modification leads to denaturation which results in loss of acti-
vity. In other words, amounts of enzyme loaded on surface of carrier

59
are the same in both preparations but most of the IMC molecules made
with carbodiimide are inactive compared to IMC molecules made with
glutaraldehyde.
Activities of IMC's with other ratios than 0.500 were determined
by employing the same procedure as above except that different amounts
of cellulase were used. Amounts of the carbodiimide were the same as
the amounts of cellulase in ewery preparation.

CHAPTER VII
THE pH ACTIVITY PROFILES OF IMMOBILIZED AND FREE CELLULASES
A. The pH A c t i v i t y P r o f i l e of Immobilized Cellulase Attached

Throuqh Glutaraldehyde
The 1.03 g IMC was prepared with 2.5% glutaraldehyde s o l u t i o n as an
attaching chemical by repeating the same procedure as described previous
ly in this thesis. The IMC was mixed with 10.06 g CGT and 100.0 ml of
0.05 M KAc b u f f e r , pH 5.10. The mixture was shaken for 6 days at 46°C
i n an incubator-shaker. During t h i s long period of shaking, unstable
IMC molecules were to be completely denatured so that only stable IMC
molecules were to be remained a c t i v e . These active IMC molecules were
almost 100% reusable IMC according to Table 2 and Figure 5. After t h i s
period of shaking, the IMC was recovered magnetically and washed
thoroughly w i t h d i s t i l l e d water. This IMC would serve as a pool of IMC
i n the f o l l o w i n g experiments.
The 0.193 g a l i q u o t of IMC was taken from the pool of IMC. The
IMC was washed with 100 ml of 0.05 M KAc b u f f e r , pH 7.63 and then cen-
trifuged. The supernatant was saved f o r preparation of sample and
blank. The sample was prepared by mixing the 0.193 g IMC, 5.00 g CGT
and 50.0 ml supernatant. The sample and blank were shaken f o r 48 hrs.
at 46°C i n an incubator shaker. The amount of reducing sugars pro-
duced by the IMC was determined by employing o - t o l u i d i n e method. Acti-
v i t y of the IMC at pH 7.63 was calculated and l i s t e d in Table 9.
60
61
,
> 4_>
cu •r- "^ ro ro O r— r--
oo 4_J > • • • . •
03 03 -1- *^ v£) on co •^
1— 4-> <o <x> ro OsJ
<v u
Û: <C
cu •
u (—
03
-o U
cu •f—
N E
•r- cu
r— ^
• I —
u
J2
O CD > u
u 4_> 3 s_
<+- 03 •r— ^— JZ ro o 00 r^
o 4-) > CJ)
o• • • • •
+J •r— « 1 — Ln Ln 00 on
03 4-» — 2:
• f— oo <: o 1—1
M- 03 E
O _, CD
s_ >
>>x:
4_> r—
•r— 03
4_> S_
u 03
03 4_)
3
C2. CT)
VO <D Ln 00 <r)
oz ro • r «^ <T «a-
c^ • • • • •
CT) Osl ro ^ Ln r^
cu
03
62
Activities of IMC at other pH's than 7.63 were determined by re-
peating the same procedures as above except employing 0.05 M KAc buf-
fers whose pH's had been adjusted to other pH's than 7.63 by adding
various amounts of glacial HAc. The pH activity profiles are plotted

in Figure 16.
B. The pH Activity Profile of Immobilized Cellulase Attached

Throuqh Carbodiimide
The 0.6540 g cellulase, 2.5030 g silanized FeÔ., 0.6540 g carbodi-
imide were mixed in 250 ml distilled water. The pH of the mixture was
adjusted to 3.40 by adding 6 M HCl. This mixture was shaken for 2-1/2
hrs. at 46°C in an incubator-shaker. The IMC produced during this shak-
ing was centrifuged and then washed thoroughly with distilled water.
This IMC would serve as a pool of IMC in the following experiments.
The 0.2503 g aliquot of IMC was taken from the pool of IMC and
washed with 100 ml of 0.05 M KAc buffer, pH 7.65. The IMC was washed
again with 100 ml of 0.05 M KAc buffer, pH 7.65, and centrifuged. The
supernatant of this centrifugation was saved for preparation of sample
and blank. The sample was prepared by mixing the 0.2503 g IMC, 2.50 g
CGT and 50.0 ml supernatant. The blank was prepared by mixing 2.50 g
CGT and 50.0 ml supernatant. The sample and blank were shaken for 48
hrs. at 46°C in an incubator-shaker. The amount of reducing sugars
produced by the IMC was determined by o-toluidine method. Activity of
the IMC at pH 7.65 was calculated and listed in Table 10.
Activities of IMC at other pH's than 7.65 were determined by re-
peating the same procedures as above except employing 0.05 M KAc buf-
fers whose pH's had been adjusted to other pH's than 7.65 by adding
63
70 -
60
50 -
>,
-i->
•T—
> 40 -
u
03

-l_>
03
;2 30
<u
cc
20 -
10 -
0
8
PH
Figure 16. The pH activity profiles of immobilized and free cellulase

The relative activities have been adjusted to make each
cellulase preparation have relative activity 70 at its
maximum point.
64
4-)
•r—
 >,
> 4_>
CU Osl on 00 C\J
oo
+-> >
03 -r-
03 LO r»v Ln
C\J «•0 <X5 on
CU u
cc
<:
, o
4_) u
Ê cn •I— 3
•<- c Ln o <ô
> 1 —
•I—
cn Ln Ln Ln ro
o u -l-i
03 U cu o
 o
•f- 03 ;=
M-
O 00 cn
S_ 03
Q.
^-o
4_> -r-
•<— E
> •!-
•r— T—
4-> - D
U O
03 J 2
S_
z : 03 CT o on Ln
CJ. u LD on LD
•
Ln <>o
<u
03
65
various amounts of glacial HAc. The pH activity profiles are plotted

in Figure 16.
C. The pH Activity Profile of Free Cellulase
A sample was prepared by mixing 5.00 g CGT, 0.2528 g cellulase and
50.0 ml of 0.05 M KAc buffer whose pH had been adjusted to 7.47 by add-
ing glacial HAc. A blank was prepared by mixing 5.00 g CGT and 50.0 ml
of 0.05 M KAc buffer whose pH also had been adjusted to 7.47 by adding
glacial HAc. The sample and blank were shaken for 6 hrs. at 46°C in an
incubator-shaker. The amount of reducing sugars produced by the free
cellulase during this shaking was determined by employing o-toluidine
method. Activity of free cellulase at pH 7.47 was calculated and list-
ed in Table 11. The pH activity profiles are plotted in Figure 16.
Activities of free cellulase at other pH's than 7.47 were deter-
mined by repeating the same procedures as above except employing 0.05
M KAc buffers whose pH's had been adjusted to other pH's than 7.47 by
adding various amounts of glacial HAc.
2. Discussion
Table 9 shows the pH activity profile of immobilized cellulase at-
tached through glutaraldehyde as an attaching chemical. The relative
activities have been plotted against pH's of buffer solution in Figure
16. This figure shows that the immobilized cellulase attached through
glutaraldehyde has the optimum pH 4.00 at 46°C.
Table 10 shows the pH activity profile of immobilized cellulast at-
tached through carbodiimide as an attaching chemical. The relative ac-
tivities have been plotted against pH's of buffer solution in Figure 16.
66
>
CU > 4_)
oo •r- •T—
on ro
03 4-> >
03 •r- ro on r^
r— 4-> <X) Ln ro
cu U
CíL <=a:
, o •
4-> u ^— 1 03
CL •f—
cn 13
— Ln CT> «r on
4-) Ln O. c\i ro
>, U cu 1 — o Ln
4_) < r—
(—
o CD
u
03
cu
o r— CT LO
JD Ln •vr
03 Ln Ln «
c\j ro Ln
67
This figure shows that the immobilized cellulase attached through carbo-
diimide has the optimum pH 5.10 at 46°C.
Table 11 shows the pH activity profile of free cellulase. The re-
lative activities have been plotted against pH's of buffer solution in
Figure 16. This figure shows that the free cellulase optimum pH is
4.10 at 46°C.
The optimum pH of free cellulase found in this work is 0.7 unit
more acidic than the reported value of 4.80 (70). This difference may
be explained by difference in kinds of substrate used. In this experi-
ment, cotton gin trash has been used instead of pure cellulose which
had been used to obtain the reported pH value of 4.80.
The optimum pH's of immobilized cellulases are different from that
of free cellulase. This difference may be explained by two effects,
chemical modification effect and microenvironmental effect. The chemi-
cal modification of enzyme proteins by attaching chemicals may alter
the structures of the enzyme proteins so that the optimum pH's are
changed. The microenvironmental effect also plays a role in changing
the optimum pH of immobilized cellulase. The microenvironment around
the immobilized cellulase molecules is different from macroenvironment
which is a state of bulk solution. If the cellulase molecules were not
immobilized, they might be in the macroenvironment. The charged sur-
face of FeÔ« makes a microenvironment different from a macroenviron-
ment by unequal partition of hydrogen ions between the two environments.
The behavior of IMC attached through glutaraldehyde is similar with
that of free cellulase. The optimum pH's and shapes of curves are
very similar in two graphs. This phenomenon may indicate that the
68
carbodiimide changes the structure of active site of cellulase mole-

cules by modifying carboxyl groups which are present near the active
s i te.
CHAPTER VIII
EFFECTIVENESS OF GRINDING AS PRETREATMENT OF COTTON GIN
TRASH FOR ENZYMATIC HYDROLYSIS
A batch of hammer-milled CGT was prepared by using a hammer-mill
(Serial No. 74, J5124, Type SH, MikroPul Division, U.S. Filter Corpora-
tion). Then a jar of pebble-mill (Paul 0. Abbe, I n c , New Jersey) was
filled with this hammer-milled CGT. After 17 hrs. milling, about 1/50
of the total CGT in the jar was taken out.
The 1.3503 g CGT was taken from the 17 hrs.-milled CGT made above
and mixed with 0.1736 g free cellulase and 50.0 ml of 0.05 M KAc buffer,
pH 4.8. This mixture was shaken for 69 hrs. at 45°C in the incubator-
shaker. Amount of reducing sugars produced during this shaking was de-
termined by o-toluidine method and listed in Table 12.
Other data of Table 12 were obtained by taking the CGT out from
the jar of pebble-mill at various milling times and by repeating the
same procedures as above.

Figure 17 shows effectiveness of grinding as pretreatment of CGT.
2. Discussion
Table 12 shows the effectiveness of grinding as pretreatment of
cotton gin trash for enzymatic hydrolysis. The glucose production from
each size of cotton gin trash are plotted against milling times in Fig-
ure 17. There is a drastic increase in susceptibility of the CGT en-
zymatic hydrolysis along with an increase of milling time. The
69
70
Table 12. Effectiveness of grinding as pretreatment of cotton

gin trash for enzymatic hydrolysis.
Milling time Glucose production

(hrs) /y mole glucose
g cellulase-hr
0 2.9
17 11.4
26 13.7
39 16.1
46 17.8
62 19.7
71
20.0
cu
c/î
03
-^ 15.0 -
cu
u
cn
cu
oo
o
u
3
cn
10.0 -

U
3
"D
O
5.0 -
S-
CL
<v
</l
o
u
3
0
0 10 20 30 40 50 60 70
M i l l i n g time (Hrs)
Figure 17. Effectiveness of grinding as pretreatment of cotton

gin trash.
72
susceptibility increases more than 400% during 60 hrs. milling period.
This phenomenon may be explained by two effects, a size effect and a
crystallinity effect. The milling may produce small-sized particles so
that a large surface area of CGT is exposed to enzymatic attack. The
milling may also decrease the crystallinity of CGT so that loosened
structure of CGT invites the enzymatic attack. Between these two ef-
fects, the size effect may play more role than the crystallinity effect
does because the CGT appears to be more brittle than many other ligno-
cellulosic materials such as mesquite sawdust. The CGT will be broken
to small pieces rather than flattened out to flakes during the grinding.
CHAPTER IX
CONCLUSIONS AND RECOMMENDATIONS
Conclusions
1. Immobilized cellulase can be prepared through covalent bondings.
2. Immobilized cellulase covalently attached on FeÔ, through glu-

taraldehyde is 99.6% reusable and has activity of 23.0 (y mole glucose/
g IMC • hr) with cotton gin trash powder as substrate at temperature
46°C, pH 5.0 (Table 2 ) .
3. Glutaraldehyde is a better chemical than carbodiimide for co-
valent attachment of cellulase on Fe.^0-.
4.. Cross-linking of immobilized cellulase molecules through gluta-
raldehyde is not beneficial to long period hydrolysis of cotton gin trash,
5. The optimum loading ratio of cellulase on silanized FeÔ. is
0.26 when any glutaraldehyde or carbodiimide is used for attachment.
6. The optimum pH of the 99.6% reusable immobilized cellulase' at-
tached through glutaraldehyde is 4.00 at 46°C.
7. Grinding is an effective pretreatment of cotton gin trash for
enzymatic hydrolysis.
Recommendations for Further Research
1. Investigate the activity of immobilized cellulase made from cel-
lulase which is supplemented by 6-glucosidase.
2. Investigate the kinetics of immobilized cellulase.
3. Design immobilized cellulase enzyme reactors.
73
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ENZYMATIC HYDROLYSIS OF CELLULOSIC BIOMASS BY

USING IMMOBILIZED CELLULASE

SANGHA OH, B.S., M.S.

The author is deeply indebted to Dr. L. D. Clements for his timely

advice and continuous encouragements during the course of this work.

The author is profoundly indebted also to Dr. S. R. Beck for his

invaluable guidance in the field of immobilized enzyme technology.

Special thanks are given to Dr. H. R. Heichelheim for his helpful

criticism on the writing of this thesis.

The author expresses his heartful appreciation to Mrs. Sue W i H i s

for her skillful typing of this thesis.

Finally, the author gratefully acknowledges the Texas Energy and

Natural Resources Advisory Council for their financial support of this

LIST OF FIGURES vii

CHAPTER 11 LITERATURE REVIEW 5

Chemical Constituents of Cellulosic Biomass.. 5

Pretreatment of Cellulosic Biomass for

Properties and Mode of Action of Cellulase... 10

Process of Enzymatic Hydrolysis of Cellulosic

Immobi 1 ized Enzyme Reactors , 17

1. Methods and Results. 22

B. Determination of Amount of Amino

C. Efficiency of Magnetic Recovery of

D, Preparation of Immobilized Cellulase

E. Preparation of Immobilized Cellulase

CHAPTER V EFFECT OF CROSS-LINKING ON ACTIVITY OF

1. Methods and Results 38

A. Activities of IMC's Cross-Linked With

B. Retention of Activity of IMC Cross-

CHAPTER VI LOADING OF VARIOUS AMOUNTS OF CELLULASE ON

1. Methods and Results 52

A. Loading of Various Amounts of Cellulase

B. Loading of Various Amounts of Cellulase

CHAPTER VII THE pH ACTIVITY PROFILES OF IMMOBILIZED AND

1. Methods and Results 60

A. The pH Activity Profile of Immobilized

B. The pH Activity Profile of Immobilized

C. The pH Activity Profile of Free

CHAPTERVIII EFFECTIVENESS OF GRINDING AS PRETREATMENT OF

COTTON GIN TRASH FOR ENZYMATIC HYDROLYSIS 69

1. Methods and Resul ts 69

CHAPTER IX CONCLUSIONS AND RECOMMENDATIONS 73

Recommendations for Further Research 73

Table 1. Content of Botanical Components in Cotton

Table 6. Retention of activity of IMC cross-linked

Table 7. Loading of various amount of cellulase on

Table 8. Loading of various amounts of cellulase on

Table 9. pH activity profile of immobilized cellulase

Table 10. pH activity profile of immobilized cellulase

Table 11. pH activity profile of free cellulase 66

Table 12. Effectiveness of grinding as pretreatment of

Figure 1. Molecular structure of cellulose (6) 6

Figure 2. Chemical equation of silanization reaction 23

Figure 3. Chemical equation of treatment of the

Figure 6. Chemical equation of attachment of cellulase

Figure 8. Chemical equation of cross-1inking of

Figure 10. Retention of activity of IMC's between Ist and

Figure 11. Effect of cross-linking on the activity of IMC... 44

Figure 12. Accumulated glucose production by IMC's 47

Figure 14. Loading of various amounts of cellulase on

Figure 15. Loading of various amounts of cellulase on

Figure 16. The pH activity profiles of immobilized and