[Reprinted from THE JOURNAL OF GENERAL PHYSIOLOGY, January 20,1941,
         Vol. 24, No. 3, pp. 377-3971

STUDIES ON THE LACTASE OF ESCHERICHIA COLI

BY H. P. KNOPFMACHER AND A. J. SALLE

(From the Departmtxd of Bacleriology, U&e&y of California, Berkeley)

(Received for publication, September 16,194O)

A yeast capable of fermenting lactose was first described by Adametz
(1889).  He found it in his studies on the microorganisms of cheeses and
gave it the name Saccharomyces lads.  In the same year Beijerinck work-
ing with two species of yeast, Saccharomyces kejir and S. tyrocola, succeeded
in demonstrating in the filtrate of his cultures a lactose-hydrolyzing enzyme,
which he named "lactase."

Following these investigations lactases were soon detected in many yeasts,
molds, bacteria, and in animal tissues.  In 1896 Fischer and Niebel voiced
the opinion that hydrolysis had always to precede the fermentative decom-
position of lactose.  From their study of the structure of carbohydrates
they concluded that the enzyme concerned must be specific for the alpha-
glucose-beta-galactoside linkage of milk sugar.  Due to more recent work,
however, the validity of these assumptions has become rather questionable.

Lactases are widely distributed in the plant and animal kingdoms.  Euler
(1922) in reviewing the literature on this subject points out that they are
always found in the intestinal tract of young mammals but decrease mark-
edly with age.  As to their occurrence in the pancreas there is no agreement
among the various authors.  More recently Cajori (1935) has reported a
lactase from the dog's liver.

Bierry and Rant (1909) found a lactase in the gastrointestinal tract of
the edible snail, Helix pomatia, and Wigglesworth (1927) reported it from
the midgut of the cockroach, Periplaneta americana.  It is, however, very
doubtful whether these lactases are identical with those of higher animals,
and the same holds for the lactases of higher plants, most frequently encoun-
tered in the family Rosaceae.  The best known example in this group is the
enzyme emulsin of bitter almonds, which can hydrolyze lactose as well as
beta-glucosides.

Various species of yeasts, molds, and bacteria are capable of fermenting
lactose and may contain lactases.  Such have been found in Aspergillus
niger and A. oryzae by Hofmann (1934 a), in Diplococczcs pneum&ae by
Fleming and Neill (1927 a), in CIostridium perfringens by the same authors
                        311


378             LACTASE OF ESCHERICHIA COLI

(19273), in Escher&&a GO& by Lowenstein, Fleming, and Neil1 (1929), and
in Escherichia coli mutabile by Hershey and Bronfenbrenner (1936) and
Deere, Dulaney, and Michelson (1936).  The presence of lactases in these
organisms, however, does not necessarily mean that hydrolysis of the lactose
into its constituent sugars has to precede fermentation.  The evidence ob-
tained by Willstatter and Oppenheimer (1922) for lactose yeast, by Wright
(1936) for Streptococcus thermopkailus, and more recently by Leibowitz and
Hestrin (1939) for maltose yeast points very strongly to the possibility of
direct fermentation of lactose and other disaccharides under certain
conditions.

Escherichia co& was selected for a general study of its lactase, special
emphasis being placed on the kinetics of enzyme action, heat inactivation,
and the behavior of the enzyme toward some reducing and oxidizing agents
and salts of heavy metals.

EXPERIMENTAL

1. Preparation of the Enzyme Solution

Fleming and Neil1 (1927) were successful in obtaining cell-free extracts of carbo-
hydrases from pneumococci by subjecting them to repeated freezing and thawing.
In this process zymases were destroyed, while the activity of the hydrolytic enzymes
was preserved. This method is very tedious and time-consuming and therefore was
not investigated further.

Hofmann (1934 b, c) obtained active lactase preparations from E. coli and B. del-
brukii by treating the bacteria with an alcohol-ether mixture and then drying them
at room temperature. This method was found to be unsatisfactory in our hands largely
because of the susceptibility of the enzyme itself to the solvents used.  The activity
of preparations of lactase so obtained was very low and decreased on prolonged contact
with alcohol, which sometimes was unavoidable.

To obtain appreciable amounts of enzyme, masses of E. `coli were grown on standard
meat extract agar in which 1.5 per cent of lactose had been incorporated.  After 48
hours incubation the organisms were washed off with a physiological salt solution, con-
taining 1 per cent of toluene, and subsequently centrifugated. This procedure was
repeated three times in order to reduce to a minimum the concentration of adhering
metabolic waste products. The resulting suspension contained 6 X 10" organisms
per ml. It was treated with an additional amount of toluene bringing the total con-
centration of the latter up to 5 per cent. Toluene serves three purposes: (1) It acts
as a preservative, (2) it inactivates the zymase complex without affecting the lactase
(Willstgtter and Oppenheimer, 1922), and (3) it destroys the semipermeability of the
cell walls, bringing about a gradual autolysis of the bacteria.

Several attempts were then made to obtain a cell-free enzyme preparation.  As was
mentioned above it was found that the lactase apparently was very susceptible to alcohol
and ether. It was also completely inactivated on dehydration with acetone.  When a
toluene-treated cell suspension was incubated overnight at 37oC. a dry gelatinous sub-
stance was obtained. This was removed and ground to a powder, the relative activity


H. P. KNOPFMACHER AND A. J. SALLE         379

of which, as determined by a method to be described later, was found to be 82 per cent
of that originally present in the bacterial suspension.
The dried powder, consisting of whole cells and cell fragments, was subjected to
more rigorous autolysis. Measured portions were suspended in M/U phosphate buffer
solutions of pH 7.0, 8.0, and 9.0 and incubated overnight at temperatures of 37oC.
and 46oC. They were then centrifugated and supematants and sediments tested for
lactase activity. The opaque supematant fluids were practically inactive, whereas
the precipitates still exhibited a marked activity though less than that of the dry powder,
probably because of the severity of the treatment to which they had been subjected.
A microscopical examination revealed that practically all bacterial cells were disinte-
grated, and only cell fragments were present. The enzyme, apparently, adhered to
these cell fragments.

These observations are contrary to reports by Karstrijm (1930), who obtained cell-
free lactase preparations from E. coli by suspending the dried organisms in phosphate
buffer solution of pH 7.0. They are, however, in agreement with results reported by
Hershey and Bronfenbrenner (1936), who were unable to separate the enzyme from the
bacterial cell and therefore concluded that it was an intracellular water insoluble enzyme.
In another experiment equivalent amounts of toluene-treated cell suspension were
exposed to the action of trypsin and papain.  In both instances lactase activity was
destroyed.

Finally, 120 ml. of bacterial suspension were ground for 18 hours in a ball mill devised
by Krueger (1933). But again lactase was inactivated.
In view of these experiences it was decided to use the original cell suspension in all
subsequent experiments, and it will be referred to in this report as "enzyme solution"
or "E. cuEi lactase" inasmuch as it was solely employed for hydrolyzing lactose.  This
preparation was stored in an icebox at PC. where its activity decreased only slightly
during the course of several months.

2. Materials and Methods

Standard sugar solutions: 1 gm. of lactose hydrate and glucose, respectively, were
dissolved in 100 ml. of distilled water and a few drops of toluene added.
Throughout the course of the experiments dilutions were prepared from these standard

solutions, 1 ml. of which contained 10 mg. of the respective sugar.
The Folin-Wu method (1920) for blood sugar determination was chosen as best fitted
for measuring the total amount of sugar present before and after hydrolysis by the
enzyme.
Experiments were conducted as follows: The desired dilution of the standard was
prepared by the use of M/U phosphate buffers of measured hydrogen ion concentration.
One-tenth ml. portions of enzyme preparation were added to 5 ml. of lactose solution
and the tubes shaken in a water bath at 36'C. for a certain length of time.  Thereupon,
they werecentrifugated for 30 minutes and the supernatant liquid used for sugar deter-
mination. 2 ml. were pipetted into Folin-Wu sugar tubes, 2 ml. of copper solution
added, and the tubes then placed in boiling water for 8 minutes.  After cooling 2 ml.
of color reagent (phosphomolybdic acid) were added, the tubes made up to a volume
of 25 ml. with water, and the resulting color compared with that of a standard.
In preliminary readings, employing glucose and lactose solutions of different concen-
trations, it was found that 1 mg. of lactose corresponded to 0.504 mg. of glucose.  In


380              LACTASE OF ESCHERICHIA COLI

all experiments, therefore, the values obtained have been expressed in terms of glucose
or total reducing sugar on the basis of the above empirical determination.
For example, if the initial concentration of lactose is l/40 of that of the standard
solution, i.e. 2 ml. contain 0.5 mg. of lactose, it will be read as 0.252 mg. of glucose or
total reducing sugar, a glucose `solution being always used as the standard for com-
parison.

For each experiment a parallel control had to be set up since most of the chemicals
whose effect on the enzyme was to be tested were oxidizing or reducing agents, and the
enzyme solution itself slightly reduced copper sulfate.  For this purpose corresponding
amounts of enzyme and chemical reagent were added to 5 ml. of phosphate buffer solu-
tion and the reducing values obtained then subtracted from the total.
Finally, a correction for volume had to be made to an extent dependent upon the
amount of enzyme solution and chemical reagent added.
It was impossible to maintain a perfectly uniform rate of hydrolysis for the duration
of the experiments. The values fluctuate between 51 and 59 per cent hydrolysis per
hour for a l/40 lactose solution. This circumstance, however, was not regarded as of
importance inasmuch as the problem selected concerned merely the comparative study
of rates of reaction as affected by hydrogen ion concentration, temperature, and chemicals.

RESULTS

      1. The Eject of Hydrogelz Ion Concentratiolz
Optimal conditions with regard to hydrogen ion concentration differ
for lactases from various sources (Oppenheimer, 1935).
To determine the effect of pH on the activity of E. coli lactase, experi-
ments were carried out as follows: M/PI phosphate buffer solutions of
different pH were prepared and their hydrogen ion concentration checked
by means of a glass <electrode.  They were then used to make up lactose
solution of a concentration of l/40 with respect to the standard (0.252 mg.
of total sugar per 2 ml.).

As described above, 5 ml. were then mixed with 0.1 ml. of enzyme prep-
aration and shaken in a water bath at 36'C. for 1 hour, and the reducing
sugar was determined.  The results are given in Table I.
The values are plotted in Fig. 1.
The results indicate that the activity of the enzyme is markedly reduced
by slight acidity but much less affected by alkalinity of the medium.  The
optimum pH for the time period and temperature given seems to extend
over the range between 7.0 and 7.5.  Consequently, all subsequent experi-
ments were carried out at a pH of 7.5.

2. The Mechanism of Enzyme Action

Michaelis and Menten (1913) worked out general rate laws for the action
of invertase on sucrose by assuming a chemical combination of the enzyme
with its substrate as the governing step in the hydrolysis of the sugar.


        H. P. KNOPFMACHER AND A. J. SALLE         381

The enzyme-substrate equilibrium can be represented by the equation:
         =, E (El (S)
                     as)

                        TABLE I
   Eject of pH on the Degree of Eydrolysis of Lactose by E. coli Lactase

PH        Amount of total sugar      Hydrolysis

5.0
6.0
6.5
7.0
7.5
8.0
9.0

FIG.

--









-

mg.psr.zd.           P@--
0.254            0.8
0.357           41.7
0.386           53.2
0.399           58.3
0.400           58.7
0.393           56.0
0.381           51.2

Ratio of activity to that
of maximum activity

0.01
0.71
0.91
0.99
1.00
0.95
0.87

          5 5        6 6  PH
               PH    7 7        8 8        9 9
1. Effect of pH on the rate of hydrolysis of lactose by E. coli lactase.
1. Effect of pH on the rate of hydrolysis of lactose by E. coli lactase.

where (E) and (ES) refer to the concentration of free and combined enzyme
respectively and (S) to the concentration of the substrate,
The constant K, could be determined by simple mathematical calculation,
leading to the equation

u -o&~(S)
vm  k. + (S)


382            LACTASE OF ESCHERICHTA COLI

in which v represents the initial velocity at the substrate concentration
(S), V, the maximum velocity, K,, therefore, being equivalent to the sub-
strate concentration at which half the limiting velocity is reached.
Lineweaver and Burk (1934) developed graphic methods for determining
dissociation constants of enzyme-substrate compounds. Since in some
cases one molecule of enzyme reacts with several molecules of substrate they
modified the Michaelis-Menten equation accordingly:

k _ m (S)"
`---La-

and

V    (SP
YE-
VU8  6)" + k.

The latter equation can then be written

                   1 k.
       -=-+f
                 v vmw   m'
in which V,,,, the maximum velocity, and K, are constant.
A plot of 1 against & must therefore give a straight line for some in-
          V

tegral value of 12. The intercept of this line on the 1 axis is f and its
                                             V         m
slope +.  In this fashion, then, the constants are easily determined.
Whe: the above equation is multiplied by (Q it assumes the form
(an k (S)*        6-v
-= v + 7.
v m m By plotting z, against (S)" a straight line is again

obtained.           (an
  The intercept on the T axis is $            1
                     and the slope is -.
                                        m          VW2

The latter plot is not only of importance in checking the values obtained
by the former but also in discovering any departure from a straight line
due to substrate inhibition.            (SP
        In such a case plots of 21 against (S)" give
curves that rise concavely with increasing substrate concentration.
The following solutions were prepared:
(a) A l/10 dilution of the standard (2 mg. of lactose per 2 ml. = 29.3 X
10%)

(b) A l/20 dilution of the standard (1 mg. of lactose per 2 ml. = 14.6
x lo-%d)


         II. P. KNOPFMACBER AND A. J. SALLE         383

(c) A l/40 dilution of the standard (0.5 mg. of lactose per 2 ml. = 7.3 X
10-%X)

(d) A l/60 dilution of the standard (0.33 mg. of lactose per 2 ml. = 4.9
x 10-k)

The results of hydrolysis after 30 and 60 minutes are given in Table II.
Upon plotting l/v against l/S and S/t, against S practically straight lines
were obtained.  (See Figs. 2 and 3.)  Consequently, it can be concluded
that one molecule of enzyme combines with one molecule of lactose as is
the case with all the other carbohydrases so far investigated.

TABLE II

Rate of Hydrolysis of Vary&x Concentrations of the Substrate

1. Substrate A
3olnin.
60 61
2. Substrate B
30 min.
60 "
3. Substrate C
30 min.
60 "
4. Substrate D
30 min.
60 "

--

Amount of total
  sugar

m*. )tv 2 d.

)n min.

1.164      0.0053
1.312      0.0051

0.628      0.0041
0.722      0.0036

0.332     0.0027
0.394      0.0024

0.233      0.0022
0.273      0.0018

-

Velocity

=

=

l/O (av.)

192

260

392

scul

w

0.5



1.0



2.0



3.0

--

-

s/v

384

260

1%

167

The intercept on the l/v axis is at 138, hence V,,, = l/138 = 0.0072 mg.
per 2 ml. per minute.

K. = V, X slope = 0.0072 X 132 - 0.95 mg. per 2 ml. = 13.9 X 10-.1x or 0.00139
V, and K,, as evaluated from the second plot, are somewhat higher.

4

The intercept on the S/v axis is at 132 = ha/V,,,.  l/V,,, = 130, hence
V, = 0.0077 and K, = 1.02 = 14.9 X W4aa, or 0.00149.
From these plots it may be inferred that the substrate has no inhibiting
effect on the rate of hydrolysis by the enzyme.
To determine the effect of different concentrations of E. COG lactase on
the rate of hydrolysis of lactose an experiment was set up in the ordinary
way, using a l/40 lactose solution but adding varying amounts of enzyme
preparation.  The results are recorded in Table III.
Plotting the figures of the third column against those of the first gives
practically a straight line (Fig. 4). This may be also expressed in a mathe-


384            LACTASE OF ESCHERICHIA COLI

FIG. 2.

          -5      IO  A3 % 2o w =O
Nature of the enzyme-substrate intermediate of E. coli lactase with

    .5          10          I5
FIG. 3. Test of enzyme-lactose intermediate.

lactose.


H. P. KNOPFMACHER AND A. J. SALLE         385

matical form by the equation: K = ?- where x represents the amount
                     Et'
hydrolyzed, E the enzyme concentration, and t the time, which in the
above experiment was constant; Z&L, 1 hour. It is at once evident that


                        TABLE III
         Hydrolysis by Varying Enzyme Concentrations

                                                  scllute constant
Amount of enzyme  Amount of total sugar Amount hydrolyzed (z)        I
                        ==El   KY&

     ml.        mg. pm 2 ml.
    0.05         0.319        0.067         1.34        0.30
    0.10         0.383         0.131         1.31        0.41
    0.15         0.452         0.200         1.33        0.52
    0.20         0.499        0.247         1.24        0.53

               005        010       0.15     Cl20
           EnTyme concentration InrmZJ
FIG. 4. Relation between enzyme concentration and the rate of hydrolysis.

the values for K fit the above data far better than those for K1, the so called
Schiitz constant (1885). Analogous results with yeast lactase were reported
by Willstatter and Oppenheimer (1922).  In view of the fact that, as has
been shown previously, a definite equilibrium between enzyme and sub-
strate is established the products of reaction apparently do not decrease
the rate of hydrolysis.


386              LACTASE OF ESCHERICHIA COLI

     3. Kinetics of Lactose Hydrolysis by E. coli Lactase
The hydrolysis of lactose by E. coli lactase follows a course between a
zero and first order reaction which is quite common for hydrolytic enzymes.
Michaelis and Menten (1913), confronted with such difficulty in the

  FIG. 5. Relation between substrate concentration and the rate of hydrolysis.
case of invertase, showed that its action could be expressed by a formula
that is actually a combination of zero and first order equations.

          Zero order: k - kd

a

First order: In - = kd,
          a-x

Michaelis-Menten equation: Vd = x -I- k. ln a&z

Essentially the same formula was derived by Van Slyke and Cullen
(1914) for the action of urease on urea.  Barendrecht (1913) showed that
it also held for lactase prepared from yeast.
As suggested in the original paper of Michaelis and Menten, the data of
Table II have been presented graphically in two ways. In Fig. 5 the
amount hydrolyzed (x) is plotted against time (t).  It is readily seen that


H. P. KNOPFMACHER AND A. J. SALLE         387

for the highest concentration a straight line is obtained indicating that the
zero order reaction holds in this case which is in agreement with Michaelis
and Menten's observations.

In Fig. 6, x + 2.3 R, log a; is plotted against time, and practically
straight lines result, at least for the 1st hour.  Only in the case of the l/40
lactose solution were additional data for the 2nd hour available (x = 0.185

1.3

Substrate

                     concentration

I       /V

                   IF z9.3xu' M

30         60         90        PO

FIG. 6. Kinetics of the hydrolysis of lactose by E. coli lactase.

after 1.5 hours and x = 0.214 after 2 hours), and these, too, follow prac-
tically a straight line.  Hence, one can draw the conclusion that hydrolysis
of lactose by E. coli lactase approximates the reaction course of the in-
tegrated Michaelis-Menten equation.

4. The Eject of Temperature

5 ml. of a l/40 lactose solution were incubated with 0.1 ml. of enzyme
preparation for 30 minutes at 26OC., 36OC., 46"C., and 56oC. after preheat-
ing the enzyme for 5 minutes at the respective temperature. Table IV
shows the results obtained.


388              LACTASE OF ESCHERICHIA COLI

The recorded drop of lactase activity between 36oC. and 56oC. may be
attributed most probably to a more rapid heat inactivation of the enzyme
at the higher temperatures.
To elucidate this point further a few experiments were set up designed to
determine the rate of enzyme destruction at different temperatures.  Test
tubes containing measured amounts of enzyme solution were immersed in
a water bath at the desired temperature which was closely controlled.

                     TABLE IV
EJ'ect of Temperature on the Degree of Hydrolysis of Lactose by E. coli Lactase

Temperature      Amount of total sugar

  "C.           mg. per 2 ml.
  26            0.295
  36            0.330
  46           0.379
  56            0.249

Hydrolysis       Ratio of activity

per ted
17.1 1
31.0            1.81

50.4 I         1.63

0             0

Temperature of
preheating

"C.
45

53

Rates of Heat Inactivation

Time of preheating

0
15
20
30


0
3
5
7

TABLE V

f E. coli Lactase at Different Temperatures

A
-

Lmount of total sugar

h-mint hydrolyzed  1 First order constant

i

mg. per 2 ml.
0.379
0.354
0.345
0.332

0.127
0.102
0.093
0.080

0.379        0.127
0.336        0.084
0.319        0.067
0.300         0.048

-

0.0146
0.0156
0.0154

0.138
0.128
0.139

After heating for varying times the tubes were placed in ice water to check
as quickly as possible further destruction of enzyme.  The residual lactase
activity was then determined in the ordinary way of mixing 0.1 ml. of en-
zyme preparation with 5 ml. of a l/40 lactose solution and shaking it in a
water bath of 36oC. for 1 hour.  The results of experiments carried out at
temperatures of 45oC. and 53oC. are given in Table V.

It was found that thermal inactivation of E. COG lactase followed the
equation of a simple first order reaction:
             2.3 log Ao/A = kt,


H. P. KNOPFMACHER AND A. J. SALLE         389

where A0 is the activity of the unheated enzyme solution, in other words,
the amount hydrolyzed under ordinary conditions, A the activity of the
enzyme heated for the time t, and R the constant of heat inactivation.
The average values for K are thus 0.0152 at 45'C., and 0.135 at 53oC.
They can be determined also by plotting log A against t as has been done in
Fig. 7.

It is at once evident that the rate constant for heat inactivation changes

3-






,I _







2-







I.3 _

5

FIG. 7. Rate of heat inactivation of E. coli lactase.

very considerably with a relatively small change in temperature.  Similar
observations have been made with all the enzymes so far investigated,
and they are in close agreement with those reported with regard to the de-
naturation of proteins.

Destruction rates are best considered in their relation to the correspond-
ing "heats of enzyme inactivation."  These latter values, known as "critical
thermal increments," can be calculated with aid of the van't Hoff-Arrhenius
equation

d In k AH
-=-
dt KT=


390              LACTASE OF ESCHERICHIA COLI

in which k is the reaction velocity constant, T the absolute temperature,
R the gas constant, and AH the "critical thermal increment."
Integrated between the limits Tz and T1, the above equation assumes the
following form :

Since k1 = 0.0152, kt = 0.135, T1 = 318", Tz = 326", and R = 1.99
calories, the value of AH is calculated as 56,400 calories per mol.

This thermal increment is of the same order of magnitude as those meas-
ured in protein denaturation and hence indicates the possibility that E. coli
lactase may be a protein.  Besides, previously cited experiments on de-
struction of lactase activity through the agency of trypsin and papain
constitute another strong evidence for the protein nature of the enzyme.

5. Activation and Inhibition by Chemicals

Inhibition phenomena have been most thoroughly investigated with re-
spect to the action of yeast invertase on cane sugar.

Among others, Euler and Svanberg (1920) and MyrbLck (1926) have
studied in great detail the inactivation of this enzyme by various chemical
reagents.  From the results of his experiments Myrback has derived some
tentative conclusions as to the nature of the groups that enable the enzyme
to decompose its substrate.  According to him, an acidic, a basic amino,
and an aldehydic group are parts of the invertase molecule concerned with
the hydrolysis of cane sugar.  He found no evidence that sulfhydryl was
an essential group.

Only recently, however, Manchester (1939) noted an acceleration of
invertase activity due to the addition of potassium cyanide which is in
close agreement with the results obtained for E. coli lactase.  As will be
discussed later this may point to the presence of SH groups in the enzyme
molecule.

Activation and inhibition of E. coli lactase is produced by a variety of
chemical agents (see Tables VI-XIII).  All experiments were carried out
at 36oC. and at a pH of 7.5 for a period of 1 hour.  The substrate, as usual,
was 5 ml. of a l/40 lactose solution.  One-tenth ml. of enzyme preparation
was at first treated with the chemical agent whose action on the rate of hy-
drolysis was to be determined, by incubation for about 15 minutes and then
added to the substrate.


H. P. KNOPFMACHER AND A. J. SALLE         391

6. Attemfded Reactivation

Von Euler and Svanberg (1920) succeeded in reactivating, by means of
hydrogen sulfide and sodium cyanide, yeast invertase poisoned by heavy
metal salts such as silver nitrate and mercuric chloride.  A similar reactiva-
tion of the protease papain is well known.  It was demonstrated first by
Vines (1902) and Mendel and Blood (1910) and later by many other in-
vestigators.

Only recently, however, were Hellerman, Perkins, and Clark (1933)

                      TABLE VI

Activation by Potassium Cyanide

Amount of KCN added    Amount of total *lag**      Hydrolysis

None
0.1 ml. 10%
0.1 ml. lo-aaa
0.1 ml. lo-f?d
0.1 ml. lO%d

w&g. per 2 d.          per cml
0.391           55.2
0.388           54.0
0.418           65.8
0.433           72.2
0.381           51.2

_









-

Ratio of activity to that
of untreated enzyme

1.00
0.98
1.19
1.31
0.93

    TABLE VII
Activation by Sodium Suljide

Amount of N&S added

Amount of total sugar

Hydrolysis

Ratio of activity to that
of untreated enzyme

None
0.1 ml. IO+
0.1 ml. lo-%I
0.1 ml. 10%

fug. pe.? t d.         pa CsrJ
0.380           51.2           1.00
0.382           51.6           1.01
0.422          67.4           1.32
0.412           63.5           1.25

and Hellerman and Perkins (1934) successful in restoring to normal the
activity of urease and papain by suitable reducing agents after a preceding
inactivation by salts of heavy metals and oxidants.
On the basis of these tindings analogous experiments were set up with
E. coZi lactase. They were carried out as usual, the enzyme, however,
being treated first with the inhibiting, then with the reducing agent, each
for about 15 minutes.

The results are given in Table XIV.
E. coZi lactase was irreversibly inactivated by most of the enzymic
poisons used, in contrast to yeast invertase, urease, and papain. (See
Table XIV.)


   TABLE VIII
Activation by Cysteine*

Amount of cyst&e added    Amolmt of tot81 sugar    Hydrolysis

Ratio of activity to
that of untreated
   ewe

                  ma. )W z:mt.         )e? cnl
None                0.389         54.4          1.00
0.1 ml. 1.8 X lO-%r        0.388         54.0          0.99
0.1 ml. 4.5 x 10%        0.416         65.1          1.20
0.1 ml. 9.0 X 10-ard        0.413          64.2          1.18
* Dilutions were prepared from Pfanstiehl's cysteine hydrochloride adjusted to

pH 7.0.

      TABLE m
Inhibition by Mercuric Chloride

Amount of H&h added

None
0.1 ml. 10%
0.1 ml. 2.0 X 10%
0.1 ml. 3.3 X 10-i
0.1 ml. 5.0 X 10%
0.1 ml. lo-%

=









-

Amount of total sugar

mg. per 2 d.
0.395
0.395
0.353
0.318
0.282
0.250

=









-

Hydrolysis

*n c.ml
56.7
56.7
40.0
26.0
11.9
0

Ratio of activity to
that of untreated
  enzyme

     TABLE X
Inhibition by Silver Nitrate

Amount of A.qNOr added    Amount of total sugar    Hydrolysis

Ratio of activity to
that of untreated
   enzyme

                  mg. )cI 2 Pd.        Qn cmU
None                0.398         57.9          1.00
0.1 ml. lo-%f            0.398         57.9          1.00
0.1 ml. 3.3 x IO+        0.311          23.4          0.43
0.1 ml. 5.0 x lo-54        0.281          11.5          0.20
0.1 ml. 10-%x          0.252 to 0.260       0 to 3.0        0 to 0.05


                    TABLE XI
          Inhibition by Copper Sdfaie

Amount of CuSOd added    Amount of tot81 sugar    Hydrolysis

None
0.1 ml. 10-k
0.1 ml. 2.0 X 10%
0.1 ml. 3.3 x lo-%a
0.1 ml. lO-a,

SUE. fwl2 mt.        per G&ml
  0.382         51.6
  0.382         51.6
  0.351         39.4
  0.333         32.1
0.262 to 0.270     4.0 to 7.1

Ratio of activity to
that of untreated
   enzyme

  1.00
  1.00
  0.76
  0.62
0.08.to 0.14

392


TABLE XII

     Inhibition by Iodine (Aqueous Solution in Potussiunt Iodide)
     .-___-.                      -~~ --__.-.--__ _____-.~..---...
-__- ____-__         ---.--.-.- -

  Amount of L added    Amount of total sugar      Hydrolysis     Ratio of activity to that
                                        of untreated enzyme

               mg. )CI 2 ml.          $ecr cent
None              0.392          55.6           1.00
0.1 ml. 10%         0.392          55.6           1.00
0.1 ml. lO+         0.324          28.6          0.53
0.1 ml. 10-2M         0.255            1.2           0.02

      TABLE XIII
Inhibitian by Hydrogen Peroxide*

Amount of I&Or added     Amount of total sugar     Hydrolysis

Ratio of activity to
that of untreated
   enzyme

None

0.1 ml. 2.0 x 10-a,
0.1 ml. lO+d
0.1 ml. 10%

mgg,per2td.        pn ted
0.382          51.6
0.383         52.0
0.364          44.3
0.361          43.3

1.00
1 .Ol
0.86
0.84

* Dilutions were made from 30 per cent hydrogen peroxide (Merck's Superoxol).

                     TABLE XIV

Attempted Reactivation of E. coli Lactare

Nature and amount of inhibitor

None

0.1 ml. W3 M HgClz
Same
Same
0.1 ml. BP8 Y AgNOs

Same
Same

0.1 ml. 3.3 X W4 Y AgN&
Same

0.1 ml. W3 rd Cd04
Same
Same
0.1 ml. 10-2 M 12
Same
Same
0.1 ml. 1W3 M 4
Same
0.1 ml. W2 M H&J
Same
Same

Nature and amount of reducing
       agent

None
None

0.1 ml. 1W2 M KCN
0.1 ml. 1(r2 Y Na2S
None

0.1 ml. 1t2 M KCN
0.1 ml. 1c2 M NatS
None

0.1 ml. 1W2 M Na2S
None

0.1 ml. W2 M KCN
0.1 ml. 1W2 M NazS
None

0.1 ml. 1CF2 M NazS
0.1 ml. 9 X 1W3 ht cysteine
None
0.1 ml. l&' M NazS
None
0.1 ml. 1(r2 Y Na&
0.1 ml. 2 X 1(r2 Y KCN

       393

-

A
to


-
Tl









0.

".A' 2
0.382
0.250
0.253
0.248
252 to
0.26(
0,256
0.251
0.311
0.311
0.262
0.331
0.305
0.255
0.258
0.262
0.324
0.324
0.364
0.377
0.388

`ydrolysi

pa cent

51.6
0
0
0
to 3.0

1.6
0
23.4
23.4
4.0
31.4
21.0
1.2
2.4
4.0
28.6
28.6
44.3
49.9
54.0

I
s









-

Ratio of
activity to
that of
untreated
aEyme

1.00
0
0
0
0 to 0.06

0.03
0
0.43
0.43
0.08
0.61
0.41
0.02
0.05
0.08
0.53
0.53
0.86
0.97
1.05


394              LACTASE OF ESCHERICHIA COLI

Similar observations with heavy metal salts have been reported recently
by Winnick, Davis, and Greenberg (1940) with regard to asclepain, a pro-
tease from the latex of the milkweed AscZe@s speciosa.  This enzyme is
even inhibited by the practically insoluble sulfides of silver and mercury.
In this respect E. coli lactase behaves differently. Equal amounts of
10-3~ solutions of mercuric chloride and silver nitrate and of a 10-2~
solution of sodium sulfide were mixed and then added to the enzyme.
Table XV gives the results obtained.

TABLE XV

Eject of Heavy Metal S&des on E. coli Lactase

None

0.1 ml. lO-" M AgN03
+O. 1 ml. 1W2 M Na2S
0.1 d. lo--' M HgC12
+O. 1 ml. 1W2 M NazS

Amount of total sugsr

mg. pt.? 2 ml.
0.387

0.402

0.386

Hydrolysis

pa cent
53.6

59.5

53.2

Ratio of activity to
that of untreated
   elK5yme

1.00


1.10


0.99

DISCUSSION

In its reaction course E. coli lactase obviously follows the general pattern
of carbohydrases as best exemplified by yeast invertase.

Studies of heat inactivation and destruction by proteases indicate its
protein nature, but beyond this little can be said about the active groups
of the enzyme molecule responsible for the decomposition of the substrate.

It is doubtful how far Hellerman, Perkins, and Clark's theory (1933)
concerning the oxidation-reduction state of urease may be applicable to
E. coli lactase.  The above investigators postulated sulfhydryl groups to
be part of the active enzyme molecule.  They contended that oxidation of
SH groups to the dithio-stage or the formation of mercaptides with heavy
metal ions led to an inhibition of the enzyme studied.

There seems to exist some analogy to their findings in the case of E. coli
lactase.  Its slight activation by reducing agents such as sulfide and
cysteine and by cyanide and its readily reversible inactivation by hydrogen
peroxide point to easily oxidizible and reducible radicals such as sulfhydryl
groups.  The same holds for yeast invertase.  But if they play any r6le
in this connection it is apparently of minor importance.  Maybe they are
protected in the lactase and invertase molecules or not as actively functional
as in proteolytic enzymes.  Groups other than sulfhydryl seem to be essen-


H. P. KNOPFMACHEB AND A. J. SALLE         395

tial for enzyme action.  Through them the enzyme molecule apparently
forms insoluble complexes with mercury and silver ions, whereas cupric
ions are bound in a looser combination.

As for the action of iodine one might speculate on the formation of addi-
tion compounds such as have been suggested by Herriott (1936) in the case
of pepsin.  He was able to isolate diiodo-tyrosine from pepsin that had
been inactivated previously by treatment with iodine.  On the other hand,
he noticed no appreciable oxidation of the enzyme by iodine.

SUMMARY

A "lactase solution" was prepared from Eschericltia coli.  The mechanism
of its action has been studied and changes in the rate of hydrolysis under
various conditions investigated.

The hydrolysis of lactose by the enzyme approximates the course of
reaction of the integrated Michaelis-Menten equation. One molecule of
enzyme combines with one molecule of substrate.
E. coli lactase is readily inactivated at pH 5.0, and its optimal activity
at 36oC. is reached between pH 7.0 and pH 7.5.
The optimal temperature for its action was found to be 46oC. when
determinations were carried out after an incubation period of 30 minutes.
Its inactivation by heat follows the course of a first order reaction, and
the critical thermal increment between the temperatures of 45oC. and 53oC.
was calculated to be 56,400 calories per mol.
The enzyme is activated by potassium cyanide, sodium sulfide, and cys-
teine, and irreversibly inactivated by mercuric chloride, silver nitrate, and
iodine.

After inactivation with copper sulfate partial reactivation is possible,
while the slight inhibition brought about by hydrogen peroxide is com-
pletely reversible.

The possible structure of the active groups of E. coZi lactase as compared
with other enzymes has been discussed.

BIBLIOGRAPHY

Adametz, L., Saccharomyces lactis, eine neue Milchzucker vergahrende Hefeart, Cen&.
B&t., 1889, 6, 116.

Barendrecht, H. P., Enzyme action, facts and theory, Btichem. J., London, 1913,7,549.
Beijerinck, M. W., Die Lactase, ein neues Enzym, Centr. B&t., 1889, 6, 44.
Bierry, H., and Rant, A., Dbdoublement du lactose et de ses d&iv& par les lactases
animales, Cotnpt. rend. SOG. biol., 1909, 66, 522.
Cajori, F. A., The lactase activity of the intestinal mucosa of the dog and some char-
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396               LACTASE OF ESCHERICHIA COLI

Deere, C. J., Dulaney, A. D., and Michelson, T. D., The utilization of lactose by Es-
cherichia coli-mutabile, J. Bact., 1936, 31, 625.
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Euler, H. v., and Svanberg, O., tuber Giftwirkungen bei Enzymreaktionen, Ferm.&-
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Fischer, E., and Niebel, W., tuber das Verhalten der Polysaccharide gegen einige thie-
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Herriott, R. M., Inactivation of pepsin by iodine and the isolation of diiodo-tyrosine
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Lowenstein, L., Fleming, W. L., and Neill, J. M., Studies on bacterial enzymes.  VII.
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H. P. KNOPFMACHER AND A. J. SALLE         397

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