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The activity of dehalogenating enzymes enables microorganisms to utilize halogenated compounds as sources of carbon and energy. The concomitant release of chloride ions observed when soil isolates were growing on such halogenated substrates suggested the presence of one or more dehalogenating mechanisms.

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CHAPTER 4

DEHALOGENASE COMPLEMENT UNDER BATCH-CULTURE CONDITIONS

Considerable variation has been recorded in the catalytic- activity of several dehalogenases from a number of species of micro organisms (Davies & Evans, 1962; Little & Williams, 1971; Goldman

e t a l ., 1968; Berry e t a l ., 1979; Slater e t a l ., 1979). These enzymes demonstrate different substrate specificities; for example, Jensen

(1960) noted that the MCA-induced dehalogenase of Pseudomonas dehalogenans

attacked MCA and DCA, when the organism belonged to groups I or II, but if a group III organism, the substrate range was increased to include 22DCPA. Several investigations on different bacterial strains (Goldman e t a l . , 1968; Weightman e t a l ., 1979; Weightman & Slater, 1980) have suggested that more than one dehalogenase was present. This was also shown for the fungu*iH ahoderm a v ir id e (Jensen, 1960).

Although a number of dehalogenase systems have been described, dehalogenase variation has not been systematically studied. The dehalogenase activities of soil bacteria, isolated by batch-enrichment, have been investigated and the enzyme profiles studied using poly acrylamide gel electrophoresis.

4.1 RELATIVE DEHALOGENASE ACTIVITIES

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activity in cell-free e tracts Drepared from Pseudomonas putida strain

PP3. Evidence for dehalogenase activity in cell-free extracts from the twenty soil isolates was obtained by detectinq chloride ion release using a chlor-O-counter (Section 2.3.1). The extracts were studied for activity towards the four substrates on which the organisms had been originally isolated, MCA, DCA, 2MCPA and 22DCPA. All the cell- free extracts possessed the ability to dehalogenate all the substrates, although activity towards each substrate differed with respect to the rate of dechlorination.

The varying abilities of the different cell-free extracts to dehalogenate the four substrates made it possible to group sixteen of these isolates according to their relative activities. The dehalogenation rates towards DCA, 2MCPA and 22DCPA were standardized to the MCA

dehalogenation rate, and the rates achieved by each isolate compared (Table 4.1). The determination of the rate of chloride release accounted for the presence of two chloride atoms per molecule in the disubstituted compounds.

The isolates were placed into five groups in accordance with their relative dehalogenase activities.

Group A organisms were segregated on the basis of the dominant MCA dehalogenase activity followed by DCA, 2MCPA and 22DCPA in order of decreasing activity. The rate of dehalogenation of DCA was in general 10 to 20" of the MCA rate. All of these organisms, with the exception of isolate El, were isolated by enrichment on MCA and it might be expected that the activity towards MCA would be the highest. Indeed, the exception, Isolate El, may not belong to this group as it exhibited

TABLE 4.1 Relative dehalogenase activity (standardized with respect to the MCA dehalogenating rate) in cell-free extracts of sixteen soil bacterial isolates. The errors quoted are standard deviations

Group Isolate Enrichment Relative Dehalogenase Activity substrate --- — ---

MCA

DCA

2MCPA

22DCPA

_El

_2MCPA

_{1 . 0}

_0.43-0.02

_0.28

_0.17

E12

MCA

1 . 0 0

.

1 2

,

.

1 1

,

0.07 E13

MCA

1 . 0

0.13x0.02

0.09x0.02

0.07x0.02

El 4

MCA

1 . 0

0.21j0.07

0.15x0.03

.

1 0

0 . 1 1

0.08 B

C

E2

2MCPA

1 . 0 0 . 2 0 0 . 2 1

0.03 D

E3

2MCPA

1 . 0 1 . 8 6

0.35

0 . 2 2

E

2MCPA

1 . 0

0.70-0.06

0.16-0.03

0.08-0.01

relatively greater DCA and 2MCPA activity than the other members of the group. Isolate E16 also showed atypical activity towards DCA demonstrating only 50% of the relative DCA activity exhibited by the other members of the group.

The correlation between enrichment substrate and maximal enzymatic activity towards the substrate did not hold for the other groups, all of which were isolated from 2MCPA enrichment cultures, yet showed greater activity towards MCA or DCA. That is, none showed highest activity towards 2MCPA.

Group B isolates demonstrated the highest DCA relative activities, which were 1.5 to 2.5 times greater than the MCA dehalogenation rates, and were 10 to 20 times higher than the relative DCA dehalogenation rate of group A organisms. In comparison with the group A organisms the group B isolates exhibited significantly higher relative 2MCPA and 22DCPA dehalogenating activities. Isolate El again represented an anomaly in group A as its 2MCPA activity was more comparable to the activity exhibited by group B organisms, as is also true for its 22DCPA relative activity. However, the DCA activity of isolate El precludes it from group B as the relative activity towards this substrate is 4 to 5 times lower than the relative activities exhibited by the group B isolates.

Isolate E2 [Pseudomonas sp. strain E2) was placed in a category of its own because of its significantly higher 2MCPA dehalogenating ability in comparison with group A isolates, and the 10-fold lower activity towards DCA than the group B organisms. The unique 22DCPA relative dehalogenase activity of this organism, being approximately 7

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times lower than the 2MCPA relative dehalogenation rate, contrasted with the 22DCPA relative activity of all the other isolates which demonstrated a 2-fold difference between 2MCPA and 22DCPA relative activities.

Group D was composed of isolate E3 (Paeudomonae sp. strain E3) as it possessed a greater 2MCPA relative activity and a high DCA dehalogenating ability.

Finally isolate E6 (Pseudomonas sp. strain E6) was placed into a separate group, group E, because of its intermediate DCA relative activity, which was 3 to 5 times higher than the same relative activities seen for group A organisms.

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