[38] PRODUCTION--APPLICATION OF IMMOBILIZED LACTASE 411

Production of a cacao butterlike fat using 1,3-specific lipases is very

interesting to the oils and fats industry. An illustration of an enzyme-
catalyzed reaction in a semiscale reactor is presented in Fig. 1. The en-
zyme reaction is carried out in a stirred tank fermenter, equipped with
temperature recording and control at 30-40 °, under agitation for 24-72 hr.
The reaction mixture is agitated at a speed giving a uniform dispersion.
After the enzyme reaction, the Celite particles are separated from the
reaction mixture, and the solvent is removed by evaporation. From this
oils and fats fraction, the interesterified triglycerides (or cacao butterlike
fat fraction) is concentrated and purified, using ethanol in an ordinary
fractionation procedure, accompanied by differential scanning calorimet-
ric analyses.
In order to make this process practical from an industrial point of
view, repeated use of adsorbed lipase as well as utilization of cheap raw
material (like the midfraction of palm oil) and efficient fractionation of the
products are indispensable.

[38] L a r g e - S c a l e P r o d u c t i o n a n d A p p l i c a t i o n o f
Immobilized Lactase
By J. L. BARET

Whey is a major by-product of the dairy industry. Most of it is spray-

dried or processed and used in a variety of applications as a food, a feed,
or a fermentation substrate. Ultrafiltration techniques have also been de-
veloping for the past decade and are now becoming a well-established
technology to recover whey proteins and also to process milk in cheese
making. Secondary by-products from the ultrafiltration, known as whey
or milk permeates, are now produced in a significant amount. A continu-
ous effort for better utilization of wheys and permeates is being made by
the dairy industry. The hydrolysis of lactose into glucose and galactose
appears to be an interesting approach to widen the profitable uses of
wheys and permeates.
Hydrolyzed lactose is sweeter and more soluble than lactose; it also
presents several additional advantages which allow producers to obtain
new attractive products. This report presents some aspects of the immo-

Copyright © 1987by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 136 All rights of reproduction in any form reserved.
412 ENZYME ENGINEERING (ENZYME TECHNOLOGY) [38]

bilized lactase processes developed by Corning for the hydrolysis of lac-

tose in permeates and wheys.

Lactose Hydrolysis Reaction and Applications

Lactose in solution can be hydrolyzed. The reaction is slightly exo-
thermic and can be catalyzed with acids, cationic resins in H ÷ form, or
enzymes. It is not strictly quantitative because side products may form
depending on the nature of the catalyst and on the pH and temperature of
the reaction.
These aspects were studied with demineralized whey permeates which
are fairly purified lactose solutions.l The studies showed that the lactose
hydrolyzed products which were obtained by acid or H ÷ resin hydrolysis
were less pure than those obtained by enzymatic hydrolysis. With more
complex lactose-containing feedstocks available on a large scale such as
milk, wheys, and their permeates, the acid and H ÷ resin hydrolysis routes
are not industrially attractive because of the complex side reactions oc-
curring during such processes.
Lactose hydrolysis by enzymatic routes presents a high selectivity. It
can be carried out batchwise using soluble fl-galactosidase, also called
lactase. This approach is justified in applications marginally sensitive to
the cost of enzyme such as for dietetics purposes or for lactose intoler-
ants. Immobilization of lactases was considered as a means to decrease
the cost of enzymatic hydrolysis. The production of hydrolyzed lactose
products at low costs is essential for applications as sweeteners or as
intermediate food products such as protein-sweetener mixes.
Immobilized lactase composites were developed using either Aspergil-
lus niger or Aspergillus oryzae acid lactases. Immobilized lactase systems
were designed and operated under industrial conditions to carry out the
lactose hydrolysis reaction in lactic acid wheys, acidified sweet wheys,
permeates, and demineralized permeates. Lactose hydrolysis can be con-
sidered to be a new unit operation which can be integrated in the transfor-
mation of a wide range of lactose-containing feedstocks. Starting from
wheys, "lactolyzed" whey syrups are obtained which can be used as food
ingredients or feed specialities. Depending on the level of demineraliza-
tion, hydrolyzed permeates can be utilized as fermentation substrates or
feed ingredients or elaborated into sweeteners or high DE syrup. (DE
stands for "dextrose equivalent," defined as total reducing sugars in the
syrup calculated as dextrose and expressed as a percentage of the total
dry substance.)

G. Coton, Address to the International Dairy Federation, Geneva, September (1979).

[38] PRODUCTION-APPLICATION OF IMMOBILIZED LACTASE 413

Immobilized Lactase Composites

Preparation of Immobilized Lactase Composites

fl-Galactosidases from A. niger and A. oryzae (also called acid fungal
lactases) are immobilized on a porous silica carrier based on the proce-
dures defined by Messing and Weetall. 2 This carrier is a controlled-pore
SiO2 ceramic of 30/45 US mesh size (or about 0.50 mm mean particle size)
and 350/k average pore diameter. The specific area is 45 m2/g, for a total
pore volume of 0.6 cm3/g. This carrier is morphologically and chemically
stable in a wide range of temperatures, it has good mechanical properties,
and it is not biodegradable. Particle size and pore dimension are opti-
mized to reduce external diffusion limitations to mass transfer, to limit
pressure drops, and to maximize the enzymatic activity.
The lactase is covalently bound to the controlled-pore silica cartier
using the silane-glutaraldehyde immobilization procedure already de-
scribed in the literature. 2 This technique can be briefly summarized as
follows. The silanol groups on the silica surface react with 7-aminopro-
pyltriethoxysilane to give an alkylamine-silica derivative. The free avail-
able amino groups are then activated by glutaraldehyde. The resulting
activated carrier contacts the enzyme in order to obtain the immobilized
lactase composite. The coupling efficiency decreases when higher lactase
loading is used. This immobilization process was optimized and scaled up
from the laboratory procedure to an industrial operation.

Properties of Immobilized Lactases

Regarding kinetic behavior, the pH profiles of immobilized lactase
composites are fairly similar to the soluble lactase with a shift to the acidic
side in the range of 0.5-1 pH units. Immobilized A. niger lactase exhibits
an activity of about 500 U/g at 50° at optimal pH in the range 3.5-3.8
(Table I). The Michaelis constant (Km) and the inhibition constant (Ki)
were determined as Km= 0.053 M and Ki = 0.005 M. The activation
energy calculated from the Arrhenius relationship of reaction rate to tem-
perature is 12 kcal/mol. The deactivation energy determined from the
variation of half-life (ti/2) as a function of temperature is 40 kcal/mol. 3
Immobilized A. oryzae lactase has an activity of 400 U/g at 40° at optimal
pH in the range 4-4.5. Values of gm = 0.05 M and Ki in the range 0.02-
0.05 M were reported 4 as well as an activation energy of 6 kcal/mol and a
2 R. A. Messing and H. H. Weetall, U.S. Patent 3,519,538.
3 j. R. Ford and W. H. Pitcher, Conf. Whey Prod. Chicago, Sept, (1974).
4 H. Hirohara, H. Yamamoto, E. Kawano, and T. Nagase, Int. Enzyme Eng. Conf., 6th
(1981).
414 ENZYME ENGINEERING (ENZYME TECHNOLOGY) [38]

TABLE I
MAIN CHARACTERISTICS OF LACTASES AND IMMOBILIZED LACTASES

Soluble lactases Immobilized lactases

pH pH Temperature pH
Source Status a optimum stability optimum optimum Activity b

A. niger GRAS 3.5-4 3-8 55-60 3-3.5 500 U/g (50°)

A. oryzae GRAS 4.5-5 3.5-8 50-55 4-4.5 400 U/g (40°)

GRAS, Generally recognized as safe.

b Units are micromoles of lactose hydrolyzed per minute at optimum pH and defined
temperature.

thermal deactivation energy of 70 kcal/mol. The operational characteris-

tics of A. oryzae lactase appeared to be more favorable to processing
whey and permeates because of its optimum pH (4.5) and less inhibition
by galactose.

Engineering Considerations
The development of immobilized lactase processes is basically depen-
dent on several parameters: the operational characteristics of the immobi-
lized lactase composite, the nature of the substrate feedstocks, the design
of the reactor system, and the operating strategy. The overall perfor-
mance of the system is the result of the interaction between these differ-
ent parameters and determines the economy of the process. The key
objective is to maximize the amount of hydrolyzed lactose which is pro-
cessed per unit weight of catalyst. A long operational life is necessary to
reach this objective. The deactivation of the immobilized lactase is mainly
influenced by the operating temperature and pH, the nature of the feed,
and the development of microbial contaminations.
In industrial practice, several processes based on immobilized enzyme
reactors are currently being used. The reactors have two points in com-
mon. First, a substrate feedstock of controlled purity is processed, and
second, the development of microbial contaminations can be controlled
because the substrate media are deficient and/or a selective environment
can be used (temperature, pH, substrate concentration, microbial inhibi-
tots). The situation using wheys and permeates is very different. These
media are nutritionally rich and thus are excellent growth media for mi-
croorganisms. The possibility of controlling the development of microbial
contaminations at low pH (below pH 3.5) and at temperatures above 35 °6
5 H. H. Weetall, N. B. Havewala, W. H. Pitcher, C. C. Detar, W. P. Vann, and S. Yaver-
baum, Biotechnol. Biogen. 16, 295 (1974).
[38] PRODUCTION--APPLICATION OF IMMOBILIZED LACTASE 415

is restricted for several reasons. Generally the addition of microbial inhib-

itors in the feedstock is industrially impossible because the presence of
those substances must be avoided in the end product. The control of
microbial contaminations in the reactor appeared as a critical technical
problem to be solved in order for this technology to be developed.

Feed Substrate
Different lactose-containing feedstocks have been evaluated. The
main characteristics of these feeds are reported in Table II. The complex-
ity of the feed increases as a function of the presence and concentrations
of salts (defined as ashes), nitrogenous matter (total nitrogen x 6.38), true
proteins (proteinaceous nitrogen × 6.38), and suspended solids.
The highly demineralized permeates are for practical purpose very
close to a pure lactose solution. However, their use may be limited be-
cause they are costly to produce by current ion-exchange techniques. In
order to widen the applications of the technology it was important to
develop processes that were adapted to the other different substrate
feeds. Processing of permeates and electrodialyzed permeates showed
that the presence of salts and different concentrations of cations and
anions normally found in these feeds had no adverse effect on the perfor-
mance and stability of the enzyme. The need for demineralization is there-
fore related to the application of the end product.
The level of suspended solids in wheys and the presence of the col-
loids in sweet whey were found to be of particular importance during
operations in a fixed bed reactor. Microbial contaminations in the feed
also has a critical impact as the main source of contamination.

Reactor Design
Reactors were designed in order to test the long-term stability perfor-
mance of the immobilized lactase and the economics of the processes
under industrial conditions. The design of the reactor aimed at maximiz-
ing the performances of the immobilized lactase reactor while minimizing
or controlling at the same time operational problems such as microbial
contaminations, pressure drops, and plugging. Three main types of reac-
tors were considered: fixed bed, fluidized bed, and stirred tank reactors
(Table III).
A perfect fixed bed reactor behaves ideally as a plug flow reactor. An
ideal continuous stirred tank reactor would behave as a perfect backmix
reactor. Some backmixing is observed in fluidized bed reactors and the
extent of bed expansion affects the performance. From a kinetic stand-

6 M. Harju, Nord. Med. Tidskr. 6, 155 (1977).

416 ENZYME ENGINEERING (ENZYME TECHNOLOGY) [38]

z
z O

b,
Z
0
O
,.= k~

o~

m
H

e~

o~

.r.,
©
[38] PRODUCTION-APPLICATION OF IMMOBILIZED LACTASE 417

TABLE III
COMPARATIVE PERFORMANCES OF CONTINUOUS STIRRED TANK AND
PLUG FLOW REACTORSa

Normalized residence
timeb (units/ml-hr)
Degree of Relative amount:
conversion Continuous continuous stirred
(%) stirred tank Plugf l o w tank/plug flow reactor

50 14 6.4 2.2
60 24.2 9.8 2.5
70 42.6 14.8 2.9
80 81.6 22.6 3.6
90 202.8 37.6 5.4

Aspergillus niger lactase, Ki = 0.0054 M; K m = 0.0528 M; lactose

5% (w/w).
b Normalized residence time (E/F). Plug flow reactor

(E/F)pF = ~ I_ Ki SoX + \ Ki +

Continuous stirred tank reactor

1 [SoX + XKm(1 + XSo/Ki) ]
(E/F)csTR = ~ 1 -- X J

where So is the initial lactose concentration (mol/liter); E, amount

of enzyme (units); F, volumetric flow rate (ml/hr).

point a plug flow reactor appears to be the most efficient at minimizing the
immobilized lactase requirement and the volume of the reactor. H o w e v e r ,
fixed bed reactors are k n o w n to be sensitive to the presence of suspended
solids which m a y be present in feeds. This aspect was found to be of
i m p o r t a n c e with w h e y feeds, so p r e t r e a t m e n t of w h e y feeds b e c a m e nec-
essary to run satisfactory operations in a fixed bed mode.

Microbial Contaminations

Sources of Contamination
T h e r e are three main sources of microbial contamination that m a y
affect the operations at industrial scale: microorganisms normally present
in the feed substrate, microorganisms present in the reactor or adsorbed
on the carrier, and accidental contamination occurring during the han-
dling of the lactase c o m p o s i t e or other operations.
418 ENZYME ENGINEERING(ENZYME TECHNOLOGY) [38]

It is not realistic to try to operate sterile reactors. However, the con-

trol of microbial contaminations to safe levels is an absolute necessity to
monitor the performances of the reactor and also to guarantee the quality
of the hydrolyzed products.
In practice, the level of microbial contaminants in the feed substrate
can be controlled by conventional heat treatment processes such as pas-
teurization which are well known in the dairy industry. The level of mi-
crobial contaminants in the product stream leaving the reactor is directly
related to the level of contaminants in the feed, and it is also a function of
the level of microbial contaminants which are present in the reactor.
Because of the short residence time in the reactor, it was found that the
increase of contaminants in the products is marginally related to the con-
taminants in the feed. The relative output/input ratios of microbial con-
taminants in the product and feed streams were good indicators in estab-
lishing the nature and trends of the microbial contaminations in the
reactor.

Approach to Immobilized Lactase Sanitation

The development in the reactor of microbial contaminants, mainly
yeasts and bacteria, can be critical. It was apparent that any commercially
viable immobilized lactase process must incorporate a sanitizing or disin-
fecting procedure. This procedure must efficiently destroy the contami-
nating microorganisms without any appreciable effect on the immobilized
lactase. Furthermore, when the product is intended for use in the food
industry, the disinfecting agent often must meet governmental regulatory
requirements.
Methods for disinfecting immobilized enzyme reactors were evalu-
ated. The different sanitizers or bacteriostatic agents were tested. Of
them, acetic acid, which is commonly used as a dilute aqueous solution in
laboratory studies, gives only limited results in industrial conditions. 7
Other known disinfectants, such as halogen derivatives, quaternary am-
monium, and biguanidine polymers, were unsuitable because of partial or
complete inactivation of the enzyme.
Substituted diethylenetriamines of the following general formula were
Ri R4
x /
/NCH2CH2NCHzCH2N \
R2 R3 R5

7 j. L. Baret, Brevet Franfais 2,471,192.

[38] PRODUCTION--APPLICATION OF IMMOBILIZED LACTASE 419

found useful at laboratory scale to disinfect the immobilized lactase. Syn-

ergistic mixtures of dioctyldiethylenetriamine and trioctyldiethylene-
triamine are commercially available from Th. Goldschmidt AG under the
name Tego-Diocto BS. Disinfection processes based on the use of this
product were scaled to industrial operations. 7

Operating Strategy

Industrial operations with immobilized enzyme reactors are depen-

dent on two critical factors, activity and stability. The activity has to be
maintained above some minimum value for an adequate period of time in
order to guarantee satisfactory operations. When no particular care is
taken, a rapid decrease in the activity is observed as a result of various
phenomena such as the deposition of material within the bed, the forma-
tion of a coating around the particles, the development of microbial con-
taminations, channeling, or other problems. The thermal deactivation of
the enzyme is not the controlling factor, as the apparent loss in activity
was found to be reversible when cleaning-sanitation operations were
operated on "dirty" or contaminated immobilized lactase (Table IV).
Depending also on the nature and the quality of the feed, some pretreat-
ment may be required to keep the activity constant during a period of time
adequate for continuous production (Table V).

T A B L E IV
HYDROLYSIS OF RAW WHEY AT p H 3.5 AND 50 °

Parameters Test A" Test B b

Flow rate (ml/hr) 102 103

Immobilized lactase (g) 4.68 4.68
Lactose (%) 4.26 4.26
t = 2hr
Glucose (g/liter) 17 16.5
Degree of conversion (%) 78 76
t = 6hr
Glucose (g/liter) 11.8 11.4
Degree of conversion (%) 54 53

" Test A was performed with freshly prepared immobi-

lized lactase.
b Test B was performed subsequently to test A, after
cleaning the immobilized lactase for 20 min with 1%
aqueous acetic acid solution in a fluidized bed mode.
420 ENZYME ENGINEERING (ENZYME TECHNOLOGY) [38]

TABLE V
APPARENT DEACTIVATION DURING HYDROLYSIS OF CLARIFIED AND
DEMINERALIZED WHEY WITH IMMOBILIZED LACTASEa

Operating temperature Apparent half-life

Whey (°C) (hr)

Without heat treatment 35 7

With heat treatment 35 1980
Without heat treatment 50 4
With heat treatment 50 124

a Whey was demineralized to 50% by electrodialysis (Ionics stack-pack)

and acidified to pH 3.5 with HC1. It was then clarified by centrifuga-
tion on a Alfa Laval LAPX 202. Heat treatment was 1 hr up to 80°.

Hydrolysis and Cleaning-Sanitation Cycles

Reactors were operated for long periods of time under semiindustrial
conditions on a cyclic mode including a continuous production phase at
constant temperature and cleaning-sanitation operations. 8 A first plant
was operated at the Milk Marketing Board (MMB) technical division at
Crudgington, United Kingdom, on a continuous basis 5 days a week with
permeates demineralized by ion exchange. It processed about 350 liters/
hr, achieving 80% hydrolysis for 15-20 hr per day. The immobilized lac-
tase was fluidized every day, when necessary with a dilute acetic acid
solution (1%, volume basis). More than 100 operating cycles were carried
out, during which the microbial contaminants in the hydrolyzed products
were controlled in the range of 100-2000 total counts per milliliter. The
conversion was maintained practically constant at the specified value
during the operations at constant flow rate.

Impact of Cleaning-Sanitation Procedures on Stability

Cleaning-sanitation procedures were developed to improve the opera-
tional stability for processing whey f e e d s . 9 In laboratory experiments,
crude whey was received from a cheese factory and clarified with an Alfa
Laval separator LAPX 202. The clarified whey was demineralized to 50%
in an electrodialysis module (Ionics). It was then acidified to pH 3.5 with
concentrated HC1 and stored at 2-4 °. The whey was heat-treated up to

s L. A. Dohan, J. L. Baret, S. Pain, and P. Delalande, Int. Enzyme Eng. Conf. 5th (1979).
9 j. L. Baret and L. A. Dohan, Brevet Franfais 2,483,748.
[38] PRODUCTION-APPLICATION OF IMMOBILIZED LACTASE 421

80 ° , centrifuged on the same separator, and cooled down to 4 ° . The whey

was subsequently used as the feed for three columns, each column con-
taining about 5 g of immobilized lactase, with the whey flow rate at 100-
110 ml/hr. Hydrolysis was conducted continuously for about 18 hr per
day at 35 ° . At the end of the hydrolysis phase, columns were rinsed with
water. In column 1, a solution of Alcalase 0.6L, at pH 7.5, 6 Anson units/
liter, from Novo was used to clean the immobilized lactase. It was then
disinfected with a 0.1% solution of Tego-Diocto BS (Th. Goldschmidt
AG). In column 2, only acetic acid was used at pH 3 and in column 3, only
Tego-Diocto BS 0.1% in solution was used.
These cleaning-sanitation procedures have a significant impact on the
performance as seen from Table VI. This was confirmed during two
semiindustrial projects using cottage cheese whey and acidified sweet
whey, cleaning-sanitation procedures were optimized to maximize the
productivity of the immobilized lactase.

Temperature Program
The knowledge of both the activation energy and the thermal deactiva-
tion energy allows an estimation of the theoretical life of the enzyme as a

TABLE VI
ACTIVITY AND STABILITY OF THE IMMOBILIZED LACTASE DURING
HYDROLYSIS OF WHEY IN RELATION TO DIFFERENT CLEANING AND/OR
DISINFECTING PROCEDURES

Column 1:
protease and Column 2: Column 3:
Day Tego-Diocto BS acetic acid Tego-Diocto BS

Activity (units/g)
1 235 258 174
5 296 202 143
10 233 163 125
15 220 117 122
17 223 36 (stop) 107
20 213 104
26 203 98
Half-life(days) 55 9 35
Stability (%)0 96 60 90

Stability (%) is the average statistical ratio of the activity after 17 hr to

the activity after 2 hr hydrolysis over all the days of operations.
422 ENZYME ENGINEERING(ENZYME TECHNOLOGY) [38]

function of the initial operating temperature. The theoretical life will in-
crease when the initial operating temperature decreases, so initial low
temperature operation is necessary to maximize the productivity. How-
ever, in practice, a pure thermal deactivation of the enzyme may not be
the only controlling phenomenon at low temperature. The initial produc-
tivities of the reactors were maintained constant at specified values by
raising the temperature when it was necessary. Actual gains in activities
were about 5-10% per degree centigrade in the range of operating tem-
peratures which were considered (20-45 °) with various feeds.
This operating temperature approach in conjunction with an adequate
cleaning-sanitation process made it possible to keep the performances of
the reactors constant over periods of time sufficient to demonstrate the
economic feasibility of the process.

Industrial Developments
These semiindustrial operations increased our confidence in the tech-
nology for hydrolyzing wheys and permeates. The technology was trans-
ferred to full-scale operations. Corning established joint ventures with
major partners in the food industry: the Specialist Dairy Ingredient com-
pany (SDI) with the Milk Marketing Board in England, the Nutrisearch
Company with the Kroger Company in the United States, and Corvire
with Union Laiti~re Normande (ULN) in France.
A plant processing 20,000 liters/day of sweet whey is operated by SDI
at Aston (Cheshire). The hydrolysis reactor can process 1000 liters/hr of
nondemineralized acidified sweet whey with a load of about 40 kg of
lactase composite. Lactolyzed whey products are obtained, and are for-
mulated as "sweet-protein" syrups which can be used as ingredients in
different sectors of the food industries. The SDI production was used to
develop the market for this new product in confectionary, ice cream, and
baked products. Capacity expansion is under way.
The most advanced operation is the Nutrisearch plant in Winchester,
Kentucky, which combines the immobilized lactase technology of Corn-
ing and continuous fermentation technology of Kroger Co. Investments
for that plant were 15 million dollars for a nominal capacity of 100,000
gallons of raw cottage cheese whey per day. Whey is processed by ultra-
filtration to obtain a protein-rich retentate. This stream is then formulated
as a dried whey protein concentrate. The permeate stream is pumped into
two hydrolysis columns. These columns are 3-ft-diameter and 15-ft-high
vessels filled with about 1500 kg of lactase composite. The lactolyzed
permeate stream can then be fermented by a selected Saccharomyces
cerevisae strain in a continuous fermenter to produce baker's yeast.
[39] UHT STERILIZED MILK TREATMENT USING SOX 423

A semiindustrial unit (10,000 1/day) is operated by Corvire in the U L N

plant at Conde-sur-Vire. H o w e v e r , the industrial development was
slower there because of the specific regulatory constraints in France.
L a c t o s e hydrolysis with immobilized lactase is now an industrial real-
ity. A subsequent expansion in industry is expected in the coming years.

[39] Continuous Treatment of Ultrahigh-Temperature

Sterilized Milk Using Immobilized Sulfhydryl Oxidase 1
B y HAROLD E. SWAISGOOD, VIOLETA G. JANOLINO, and
PAUL J. SKUDDER

Thiols are often responsible for undesirable flavors in foods because

of their extremely low organoleptic threshold concentrations. The
" c o o k e d " flavor of ultrahigh-temperature (UHT) 2 sterilized milk is a fa-
miliar example of such " o f f flavors." Aseptic packaging of U H T milk
allows the product to be merchandized and stored at ambient tempera-
ture. If the flavor could be acceptable by a larger fraction o f the popula-
tion, a potential energy saving could be realized. Consequently, discovery
that an e n z y m e indigenous to unheated milk, and thus aesthetically ac-
ceptable as a processing aid for milk, could be used to eliminate the
c o o k e d flavor 3,4 has important practical value.

Isolation of Protein Fractions Having Increased Sulfhydryl

Oxidase Activity
Sulfhydryl oxidase (SOX) is an iron-containing, glycomembrane en-
zyme existing primarily in the membrane vesicle fraction of skim m i l k ) -~
This work is Paper No. 9349 of the Journal Series of the North Carolina Agricultural
Research Service. The use of trade names in this publication does not imply endorsement
by the North Carolina Agricultural Research Service of the products.
2 Abbreviations used: UHT, ultrahigh-temperature; CPG, controlled-pore glass; SOX, sulf-
hydryl oxidase; DTNB, 5,5'-dithio-bis(2-nitrobenzoicacid); GSH, reduced glutathione.
3 H. E. Swaisgood, U.S. Patent 4,053,644 (1977).
4 H. E. Swaisgood, U.S. Patent 4,086,328 0978).
5 V. G. Janolino and H. E. Swaisgood, J. Biol. Chem. 250, 2532 (1975).
6 M. B. Sliwkowski, M. X. Sliwkowski, H. E. Swaisgood, and H. R. Horton, Arch. Bio-
chem. Biophys. 211, 731 (1981).
7 M. B. Sliwkowski, H. E. Swaisgood, and H. R. Horton, J. Dairy Sci. 65, 1681 (1982).
8 M. X. Sliwkowski, M. B. Sliwkowski, H. R. Horton, and H. E. Swaisgood, Biochem. J.
209, 731 (1983).

Copyright © 1987 by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 136 All rights of reproduction in any form reserved.