Immobilized Enzymes for Food Processing

226 Pages·1980·20.142 MB·\226

by  Pitcher (Author)

Immobilized Enzymes
for
Food Processing
Editor
Wayne H. Pitcher, Jr., Sc.D.
Engineering Supervisor
Corning Glass Works
Corning, New York
Boca Raton London New York
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Main entry under title:
Immobilized enzymes for food processing.
Bibliography: p.
Includes index.
1. Immobilized enzymes—Industrial applications.
2. Food industry and trade. I. Pitcher, Wayne H.
TP456.E58I45 664’ .01 79-25738
ISBN 0-8493-5345-9
A Library of Congress record exists under LC control number: 79025738
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FOREWORD
Although enzymes have been used in the food processing industry for a number of
years, enzyme immobilization, allowing continuous processing, has been applied to
food processing only in the past decade. Much has been written about immobilized
enzymes during this period of time. So much, in fact, that it can become difficult even
for those involved in developing new enzymatic food processing operations to bridge
the gap between the field of immobilized enzymes and their specific requirements. It
is the purpose of this book to assist those engaged in this difficult task. Perhaps an
equally important goal is to bring to the researcher in enzyme immobilization an ap-
preciation for the requirements of the food processing industry. After all, most of the
commercial applications of immobilized enzymes, have been in the area of food proc-
essing.
The organization of the book was designed to give the reader background in enzyme
immobilization, engineering and economic factors, and the unique requirements of the
food processing industry in the first three chapters as a prelude to the consideration
of specific applications. Chapters dealing with applications include potential as well
as already commercialized processes. In so young a field the potential applications still
far outnumber those that have reached practical use.
As editor, I am extremely pleased that such a well-qualified group from industry
and the academic community consented to contribute to this work. My deepest thanks
go to them. Thanks should also go to the editors of CRC Press for their patience and
assistance.
THE EDITOR
Wayne H. Pitcher, Jr. received his B.S. in chemical engineering from the California
Institute of Technology in 1966. He continued his education in the field of chemical
engineering at the Massachusetts Institute of Technology where he received his S.M.
degree in 1968 and his Sc.D. in 1972.
Since joining Corning Glass Works in 1972, Dr. Pitcher has been primarily involved
in developing immobilized enzyme systems for industrial applications. Among other
responsibilities he has headed projects to develop immobilized glucose isomerase and
immobilized lactase systems.
Dr. Pitcher is a member of various professional organizations including the Ameri-
can Institute of Chemical Engineers and the American Chemical Society. He has
chaired sessions at several national and international technical conferences, authored
numerous articles, and holds several patents.
CONTRIBUTORS
Marvin Charles, Ph.D. Peter J. Reilly, Ph.D.
Department of Chemical Engineering Professor of Chemical Engineering
Lehigh University Department of Chemical Engineering
Bethlehem, Pennsylvania Iowa State University
Ames, Iowa
Robert W. Coughlin, Ph.D., P.E.
Professor of Chemical Engineering JohnF. Roland, B.S.
University of Connecticut Senior Group Leader
Storrs, Connecticut Kraft, Inc.
Research and Development
Robert V. MacAllister, Ph.D. Glenview, Illinois
Director
Scientific and Technological Evaluation Bhavender P. Sharma, Ph.D.
Clinton Corn Processing Company Senior Project Engineer-Chemical
Clinton, Iowa Technical Staffs Division
Corning Glass Works
Ralph A. Messing, M.S. Corning, New York
Senior Research Associate
Head Howard H. Weetall, M.S.
Department of Fundamental Life Manager
Sciences Biomedical Research
Corning Glass Works Corning Glass Works
Corning, New York Corning, New York
TABLE OF CONTENTS
Chapter 1
Introduction to Immobilized Enzymes 1
Wayne H. Pitcher, Jr.
Chapter 2
Immobilized Enzyme Engineering 15
Wayne H. Pitcher, Jr.
Chapter 3
Requirements Unique to the Food and Beverage Industry 55
J. F. Roland
Chapter 4
Manufacture of High Fructose Corn Syrup Using Immobilized Glucose Isomerase . .81
R. V. MacAllister
Chapter 5
Potential and Use of Immobilized Carbohydrases 113
P. J. Reilly
Chapter 6
Applications of Lactose and Immobilized Lactase 153
R. W. Coughlin and Marvin Charles
Chapter 7
Immobilized Proteases — Potential Applications 175
H. H. Weetall
Chapter 8
Application and Potential of Other Enzymes in Food Processing: Aminoacylase, As-
partase, Fumarase, Glucose Oxidase-Catalase 185
B. P. Sharma and R. A. Messing
Index 211
]
Chapter 1
INTRODUCTION TO IMMOBILIZED ENZYMES
Wayne H. Pitcher, Jr.
TABLE OF CONTENTS
I. Introduction 2
II. Adsorption 2
III. Covalent Bonding 6
IV. Entrapment 10
References 13
2 Immobilized Enzymes for Food Processing
I. INTRODUCTION
The purpose of this chapter on enzyme immobilization is to introduce the reader to
the diversity of immobilization techniques available and some of the variables that
affect the actual immobilization procedures. No effort has been made to judge the
suitability of these methods for immobilizing enzymes for food processing. These con-
siderations are discussed in Chapter 3. It is not intended that this section necessarily
be comprehensive or contain detailed descriptions of immobilization procedures. Since
enzyme immobilization can be accomplished in numerous ways under so many sets of
conditions, these details would, at best, only provide a starting point. What is impor-
tant is that the reader understand the various options known to be available in order
that he may be able to select a reasonable approach to his particular problem. Thus,
the emphasis in this chapter is on the attributes of the various methods of immobili-
zation and the ways in which workers in this field have attempted to vary them.
Although there are hundreds of immobilization procedures that have been catego-
rized in various ways, for the purposes of this treatment of the subject they are placed
into three general groups. These three types of immobilization are adsorption, covalent
bonding, and entrapment. Such a classification is convenient, if somewhat arbitrary.
There are cases where two methods are combined, a common example being adsorp-
tion and cross-linking (a form of covalent bonding). For more detailed information
on enzyme immobilization, other references should be consulted.12 Some additional
examples are also given in Chapter 4.
II. ADSORPTION
Adsorption is the oldest of the techniques used to immobilize enzymes, dating back
to 1916 when Nelson and Griffin3 used both charcoal and aluminum hydroxide to
adsorb invertase. Since that time a wide range of organic and inorganic substances
have been utilized as supports for adsorbed enzymes. Both organic materials such as
charcoal, various cellulose derivatives, and ion exchange resins and inorganic materials
including silica, alumina, titania, glass, and various naturally occurring minerals have
been used.
Although adsorption has had a dubious reputation in the past, probably as a result
of problems with desorption and inactivation upon adsorption, commercially it has
seen relatively frequent usage. Clinton Corn Processing Company has reported using
DEAE-cellulose to adsorb glucose isomerase.4 Tanabe Seiyaku Company has been im-
mobilizing aminoacylase on DEAE-sephadex and other ion exchange resins for use in
a process to racemize mixtures of D- and L-isomers of amino acids.5 CPC-International
is adsorbing glucose isomerase to porous alumina beads via a process developed by
Corning Glass Works.
Development of a useful adsorbed enzyme derivative depends on many factors. Per-
haps the most important, or at least the first to be encountered, is the interaction
between the enzyme and the surface of the carrier.
The same enzyme will be adsorbed on different carriers to varying degrees and ex-
hibit different levels of activity as a function of the support material properties. Simi-
larly, a carrier which is effective for one enzyme may be totally useless for another.
Several striking examples of the effect of carrier composition on adsorbed enzyme
activity have been reported. Pitcher and Ford6 adsorbed ^-galactosidase (lactase) onto
various porous ceramic beads with widely varying results as shown in Table 1. Stanley
and Palter7 reported adsorbing this same enzyme on a wide range of phenol resins.
They found that several materials including Duolite® A-l (Diamond Shamrock Co.),
3
Table 1
LACTASE ADSORPTION
Carrier Ave. pore Pretreatment Percent activity at pH
composition diameter (A) and 4.5 relative to lactase
adsorption pH chemically bound to
silica
Si02 370 3 35
SiO 370 7 24
2
Al,0 230 3 53
3
AU03 230 7 32
Ti02 380 3 117
Ti02 380 7 127
Note: Enzyme: Wallerstein Co. Lactase LP, 0.1 g enzyme per gram carrier
offered. Carriers: 30/45 mesh, porous particles.
From Pitcher, W. H., Jr. and Ford, J. R., Enzyme Engineering, Vol. 3, Pye,
E. K. and Weetall, H. H., Eds., Plenum Press, New York, 1978.
charcoal, p-hydroxybenzaldehyde, salicylaldehyde, and ocresol-phenol-formaldehyde
polymer failed to retain any enzyme activity. Oxidized catechol, humic acid, phloro-
glucinol-formaldehyde polymer, and catechol-formaldehyde polymer exhibited good
initial activity but exhibited significantly lower activity after treatment with salt solu-
tion. Duolite® S-30 (Diamond Shamrock Co.), resorcinol-formaldehyde polymer, and
Duolite® S-30 formylated with DMF-POC1 showed relatively high activity retention
3
even after soaking in 2MNaCl or 4Murea for several hours. The authors did eventu-
ally resort to glutaraldehyde cross-linking to stabilize the composite after adsorbing
the enzyme to the S-30 resin.
Caldwell et al.8 reported affecting the activity of a /?-amylase-Sepharose 6B deriva-
tive by varying the hexyl-group substitution levels in the sepharose. Maximum activity
was observed at a hexyl to galactose residue molar ratio of 0.5.
A patent granted to Eaton and Messing9 describes the effect of varying amounts of
magnesia in a porous alumina carrier on the activity of adsorbed glucose isomerase.
Addition of magnesia to the alumina support composition affects pH which may, in
turn, influence enzyme activity. From Table 2, it can be seen that enzyme activity falls
off at low and high MgO levels. Other data in the patent indicates the optimal magnesia
level to be in the 1 to 4% range.
Some of the effects of carrier composition in this case may be attributable to pH
shifts. Examples of pH effects on enzyme adsorption are well known. Kennedy and
Kay10 reported optimal adsorption of dextranase on porous titanium oxide spheres at
pH 5.0. This derivative was evidently at least fairly stable when used in a column
reactor. However, when the pH of the dextran solution feed was raised from 5.0 to
7.3, enzyme was rapidly eluted from the titania bed.
Boudrant and Cheftel11 reported on the stability of invertase adsorbed to several
macroreticular ion exchange resins. Enzyme desorption was more pronounced at pH
5.9 to 6.3 than at lower pH (2.4 to 3). However, other conditions such as temperature
and ionic strength more strongly influenced enzyme desorption.
High ionic strength solutions of electrolytes do tend to cause the desorption of ad-
sorbed proteins. The extent of this problem depends on the specific enzyme and sup-
port material involved, pH, temperature, and perhaps other variables. Boudrant and
Cheftel,11 and Stanley and Palter,17 and Baratti et al.12 all reported the effect of ionic
concentration on the adsorption of enzymes as had many others before them. Each of