231
FEMS Microbiology Letters 6 (1979) 231-234
© Copyright Federation of European Microbiological Societies
Published by Elsevier/North-Holland Biomedical Press
THE PARTIAL PURIFICATION OF TWO DEHALOGENASES FROM PSEUDOMONAS
PP3
PUTIDA
ANDREW J. WEIGHTMAN, J. HOWARD SLATER * and ALLAN T. BULL **
• Department of Environmental Sciences, University of Warwick, Coventry CV4 7AL, and ** Department of Applied Biology,
University of Wales Institute of Science and Technology, King Edward Vll Avenue, Cardiff CF1 3NU, U.K.
Received 12 June 1979
1. Introduction
Microbial growth on halogenated carbon compounds requires, at some stage of catabolism, the
cleavage of the carbon-halogen bond. In many cases,
particularly for halogenated aromatic compounds
[1,2], halogen removal may be fortuitous but in the
case of the halogenated alkanoic acids specific
dehalogenases [3,4] or halidohydrolases [1,5] have
been recognised. Many microorganisms have been
isolated capable of utilising halogenated alkanoic
acids as the sole carbon and energy source for growth
[6,7] but only a few have been examined in detail for
dehalogenase activity. Furthermore the studies of
Goldman [5,8,9 ] and Little and Williams [ 10,11 ]
provide the only instances of partial dehalogenase
purification and characterisation, although other
studies have examined the basic properties of other
dehalogenases in cell-free extracts [7,12,13 ]. This
communication describes the purification of two
dehalogenases from Pseudomonas putida PP3 which
was isolated from a microbial community growing on
2,2'-dichloropropionic acid [14] and some of the
results reported here have been the subject of preliminary communications [ 15,16].
2. Materials and Methods
The organism used in this study, Pseudomonas
putida strain PP3 (formerly described as P. putida P3)
was maintained and grown as previously described
* To whom reprint requests should be addressed.
[6,14] .,Drganisms for enzyme purification were
grown in 10 1 aspirators in a minimal medium [6]
supplemented with 2-monochloropropionic acid
(2MCPA), 0.6 g C 1-1 ; sodium acetate, 0.25 g C 1-1
and yeast extract, 0.5 g 1-1 . The culture was harvested
once 80-90% of the 2MCPA had been dechlorinated
and provided approx. 5 g wet weight of organisms.
Dehalogenase activity was assayed as previously
described [6], except that a Tris-sulphate buffer was
used instead of the phosphate buffer. Samples were
chosen for assay such that the maximum activity per
assay was 1.0 unit m1-1 , where one unit of activity is
defined as the dehalogenation of 1 ~mol substrate
min -1. Protein was estimated by the biuret method
with bovine serum albumin as the standard [17].
The dehalogenases were partially purified from
cell-free extracts prepared by disrupting the organisms by two passages through a French pressure cell
at 8.3 • 107 Pa (12 000 lbfin-2). D,L-Dithiothreitol
was routinely added to give a final concentration of
1 mM and the following operations were carried out
at 0-5°C. Pulverised ammonium sulphate was added
slowly with stirring to the crude extracts and the fraction precipitating between 30 and 70% saturation was
recovered by centrifugation at 45 000 × g for 20 min.
The precipitate was dissolved in 50 mM Tris-sulphate
pH 7.0 and desalted by gel filtration on a Sephadex
G-25 fine column (2.2 × 50 cm). The protein containing fractions were pooled and adsorbed onto a DEAESephadex A50 gel column (3.2 × 50 cm) which had
previously been equilibrated with 50 mM Trissulphate buffer pH 7.9 containing 50 mM ammonium
sulphate. The ion-exchange column was developed
with a linear concentration gradientfrom 50 mM to
232
500 mM ammonium sulphate in 50 mM Tris-sulphate
buffer pH 7.9. Pooled fractions of the separated
dehalogenase activities were concentrated separately,
approx. 10-fold by ultrafiltration in an Amicon UM
10 ultrafilter with a molecular weight retention of
greater than 10 000.
All the chemicals used were of the highest purity,
with the purity of the chlorinated alkanoic acids
checked by 13C.NMR.
3. Results
Initially the purification o f dehalogenase activity
from Pseudomonas putida PP3 was hindered by the
rapid loss of enzyme activity. The dehalogenase
activity for all the chlorinated alkanoic acids tested
[6], including the four major substrates, monochloracetic acid (MCA), dichloroacetic acid (DCA),
2MCPA and 2,2'-dichloropropionic acid (22DCPA),
were shown to be labile in either stored, concentrated
cell suspension or frozen crude extracts. In particular,
dehalogenase activity against DCA was unstable with
greater than 70% of the activity lost within 48 h of
storage of crude extracts at 4°C. MCA (Table 1),
2MCPA and 22DCPA dehalogenase activities were less
unstable. Four of five reagents tested (Table 1)
stabilised the loss o f DCA dehalogenase activity, but
only dithiothreitol also stabilised the loss of activity
of the other three major substrates.
Fig. 1 shows the separation of two dehalogenases,
after dithiothreitol stabilisation, by elution through a
DEAE-Sephadex A50 gel column with the elution of
fractions I and II occurring at 1300 ml and 1900 ml,
;/
500
I000
1500
Volume (m[)
20~00
Fig. l. The separation of the two dehalogenases on DEAESephadex (A-50) column. Dehalogenase activity against MCA
(o), DCA (o), 2MCPA (o) and 22DCPA (m). The total protein
(A) was estimated by measuring the absorbance at 280 nm.
respectively. Over 95% of the DCA activity was
contained in fraction II whilst the activity for 2MCPA
and 22DCPA was mainly located in fraction 1.
Dehalogenase activity against MCA was distributed
evenly between the two fractions. Table 2 shows a
typical partial purification scheme quantifying the
TABLE 1
The stability of MCA and DCA dehalogenase activity in crude extracts stored at 4°C and the effect of various reagents on improving enzyme stability
Time
(days)
Remaining activity as a percentage of the original activity
Control
0.5
6.0
5%(w/v)
ethanol
10%(w/v)
glycerol
lmM
EDTA
lmM
glutathione
lmM
dithiothreitol
MCA
DCA
MCA
DCA
MCA
DCA
MCA
DCA
MCA
DCA
MCA
DCA
96.7
62.1
67.9
11.5
94.2
61.6
67.3
29.6
97.7
64.9
71.2
47.1
87.2
67.9
77.5
35.6
96.5
63.3
74.4
10.9
97.7
91.4
100
74.8
233
TABLE 2
The partial purification scheme for the two dehal.ogenases
The figures in brackets are the dehalogenase activities for each substrate standardised with the MCA activity as unity.
Step
Crude extract
30-70% (NH4)2SO4
fraction
DEAE-Sephadex A-50
Fraction I
Fraction II
Concentration by ultrafiltration
Fraction I
Fraction II
Volume
(ml)
Protein
concentration
(mg m1-1)
Specific activity
(unit (mg protein)-I )
MCA
DCA
2MCPA
22DCPA
100
84
23.30
14.70
0.45 (1)
0.48 (1)
0.63 (1.40)
0.62 (1.29)
0.15 (0.33)
0.20 (0.42)
0.08 (0.18)
0.11 (0.23)
425
381
0.90
0.65
0.65 (1)
1.15 (1)
0.08 (0.12)
2.54 (2.21)
0.39 (0.60)
0.31 (0.27)
0.19 (0.29)
0.15 (0.13)
39
33
9.70
7.50
0.62 (1)
1.05 (1)
0.07 (0.11)
1.83 (1.74)
0.40 (0.65)
0.26 (0.25)
0.19 (0.31)
0.13 (0.12)
distribution of activities shown in Fig. 1. The activity
ratio for fraction I, after the ultrafiltration stage, was
shown to be the same as at the gel filtration stage
whereas the activity ratio in fraction II showed a
significant decrease in DCA dehalogenase activity.
Dehalogenase activity against two other substrates,
2-monochlorobutyric acid (2MCBA) and trichloroacetic acid (TCA) [6], was also interesting since all
the TCA activity was confined to the fraction II
dehalogenase and 80% of the 2MCBA activity was
associated with the fraction I dehalogenase.
Discussion
Some of the evidence previously presented [6],
particularly thermal denaturation data, suggested that
Pseudomonas putida PP3 contained two different
dehalogenases and the data reported in this paper
confirm the earlier observations. Furthermore the
separation of two dehalogenase fractions indicates
that one of the dehalogenases (found in fraction I)
has evolved to catalyse the dechlorination of chlorinated propionic (and, possibly, butyric) acids whilst
the second dehalogenase (found in fraction II) has a
far greater specificity towards chlorinated acetic
acids. However, the common MCA dehalogenase
activity suggests a close relationship between the
two dehalogenases and this is currently under further
examination. The fraction I dehalogenase showed a
remarkably consistent activity ratio between 2MCPA
and 22DCPA throughout the purification procedures,
providing further support for the suggestion that a
single enzyme catalyses the dechlorination of both
substrates [6]. The fraction II dehalogenase, however,
did not show such a constant ratio between the MCA
and DCA activities; for example, there was nearly a
25% decrease in the DCA activity relative to the MCA
during the concentration stage suggesting that possibly fraction II contains two enzymes which have not
been separated by these procedures. Alternatively, a
single enzyme with different susceptibility to the loss
of MCA and DCA dehalogenase capabilities could also
account for the observed variations in the activity
ratio for these two substrates.
The purification and characterisation of two
dehalogenase proteins in one micro-organism has been
described by Goldman [5,8,9] but in his case the two
enzymes were separately induced, one by MCA and
the other by DCA. Compared with previous reports
[5,7,8,11,13] the enzymes ofPseudomonas putida
PP3 differed markedly in their substrate specificities.
For example, the pseudomonad studied by Goldman
had no dehalogenase activity against 22DCPA and
other organisms showed different activity profiles.
Thus, a detailed examination of different dehalogenases could provide some interesting information
on the evolution of different catalytic activities
within a group of enzymes with the same basic
capability.
234
Acknowledgements
AJW thanks the Science Research Council for a
Research Studentship. We thank Mrs. D. Sanders for
valuable technical assistance.
References
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