ORIGINAL ARTICLE

Identification of a g-hexachlorocyclohexane dehydrochlorinase (LinA)variant with improved expression and solubility properties

MARIO MENCIA1, ANA I. MARTINEZ-FERRI1, MIGUEL ALCALDE2, & VICTOR DE

LORENZO1

1Department of Microbial Biotechnology, Centro Nacional de Biotecnologıa, CSIC, Cantoblanco, 28049 Madrid, Spain,2Departament of Biocatalysis, Instituto de Catalisis, CSIC, Cantoblanco 28049 Madrid, Spain

(Received 22 July 2005; revised 24 November 2005)

AbstractUnder aerobic conditions, the enzyme g-hexachlorocyclohexane dechlorinase (LinA) from Sphingomonas paucimobilis UT26catalyzes the elimination of chlorine atoms from the molecule of g-hexachlorocyclohexane (g-HCH) or lindane, arecalcitrant pesticide that is still widely used. In its native metabolic context, LinA starts the biodegradation process oflindane by transforming g-HCH to 1,2,4 trichlorobenzene (TCB), a less persistent chemical. In an attempt to generate animproved version of this enzyme to be used in lindane bioremediation schemes, we have run an experimental evolutionprocedure on LinA, using Escherichia coli as the surrogate host. One round of random mutagenesis and subsequentscreening for improved dechlorination in vivo sufficed to yield one mutant enzyme (LinAT10), bearing a single substitutionC132R, that displayed a two-fold enhanced expression and three-fold enhanced solubility of the enzyme compared to thewild type protein. This resulted in a biological product with a six-fold increase in dechlorination ability when expressed in E.coli. The potential of this protein and its expression system for in situ bioremediation is discussed.

Keywords: Bioremediation, dechlorinase, in vitro evolution, LinA, lindane, protein expression

Introduction

The g isomer of hexachlorocyclohexane (g-HCH or

lindane) has been produced and used as an insecti-

cide across the world in the last decades. Due to its

persistence in soil and ground water, and its toxicity

in the food chain, lindane use has been prohibited in

most countries, although it is still being used in some

parts of the developing world. While the g congener

of HCH is the one with insecticidal properties, its

industrial production generates a number of iso-

mers, four of which (a, b, g and d ) are particularly

persistent in the environment (Johri et al. 1996). A

number of microorganisms, both aerobes and anae-

robes, have been shown to dechlorinate the lindane

molecule and so start the biodegradation of the

compound (Johri et al. 1996). Aerobic biodegrada-

tion of lindane is frequently found in Sphingomonas

strains isolated from soils with a history of pollution

by HCH (Boltner et al. 2005; Mohn et al. 2006a;

Neufield et al. 2006), and to a lesser extent, in other

bacteria as well (Sahu et al. 1990; Thomas et al.

1996).

One key enzyme in aerobic degradation of lindane is

the HCH-dehydrochlorinase (LinA), which was ori-

ginally cloned from the strain Sphingomonas paucimo-

bilis UT26. The linA gene product is the first enzyme

of a catabolic pathway that also includes the products

of genes linB, linC, linD and linE , (Nagata et al.

1999b; Kumari et al. 2002; Dogra et al. 2004). This

pathway fully dechlorinates g-HCH and makes the

carbon backbone utilizable in the general metabolism

of the bacteria. It has been proposed that LinA and

LinB show periplasmic localization in their native host

S. paucimobilis UT26 without molecular processing

(Nagata et al. 1999a). This seems reasonable as by

being in the periplasmic space, LinA and LinB,

which catalyse the first two steps in catabolism,

would convert the HCH molecule into the less toxic

compound 2,5-dichloro-2,5-cyclohexadiene-1,4-diol

Correspondence: Vıctor de Lorenzo, Centro Nacional de Biotecnologıa-CSIC, Campus de Cantoblanco, Madrid 28049, Spain. Tel: 34-91

585 45 36. Fax: 34-91 585 45 06. E-mail: vdlorenzo@cnb.uam.es

Biocatalysis and Biotransformation, May�June 2006; 24(3): 223�230

ISSN 1024-2422 print/ISSN 1029-2446 online # 2006 Informa UK Ltd

DOI: 10.1080/10242420600667809

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(Dogra et al. 2004) before it crosses the cell mem-

brane and proceeds into central metabolism. Variants

of linA have been cloned and sequenced from other

Sphingomonas strains, which show ]/ 88% identity in

their amino acid sequences with the archetypal LinA

of strain UT26 (Dogra et al. 2004). Apart from that,

LinA shows low sequence similarity with other

proteins in the databanks. Despite this, a structural

model for LinA has been proposed (Nagata et al.

2001). In this model LinA was grouped along with

scytalone dehydratase, and other enzymes in the

frame of a new structural superfamily, which displays

a central six-stranded b-sheet motif complemented by

several a-helixes to form a mixed barrel structure.

When the predicted catalytic residues D25, H73 and

R129, of the proposed active center of LinA were

altered (Nagata et al. 2001), the mutants turned out to

be inactive, while changes of more distant residues did

not affect the activity of the protein. These experi-

mental results were in good agreement with the

proposed model.

Although in its native context LinA forms part

of the larger catabolic pathway mentioned above,

when tested in isolation, this enzyme displays an

unique activity capable of converting g-HCH to

1,2,4-trichlorobenzene via g-pentachlorocyclohex-

ene (Imai et al. 1991; Figure 1). LinA can dechlor-

inate the a, g and d isomers of lindane, but cannot

utilize the b congener as substrate (Kumari et al.

2002). This is probably due to the non-axial

disposition of all the chloro substituents in the bisomer. Strains that degrade the b isomer should

therefore have an alternative early dechlorination

mechanism (Kumari et al. 2002). The dehydro-

chlorination properties of LinA to yield 1,2,4

trichlorobenzene makes this enzyme very interesting

for bioremediation applications involving the release

of biological materials into a target polluted site. One

increasingly attractive technology for cleanup of soil

contaminated with toxic chemicals is the amend-

ment of the afflicted spot with killed Escherichia coli

cells in which a detoxifying enzyme of interest has

been previously over-expressed (Strong et al. 2000).

In this way, E. coli cells act as a vehicle to deliver a

biologically encapsulated catalyst to a given niche.

This procedure has been successfully used to in-

crease degradation in soil heavily contaminated with

atrazine and could be extended to other scenarios

(Strong et al. 2000).

Since the LinA protein catalyses the critical first

step in the degradation route of many HCH isomers

without the need for extra cofactors, its expression in

E. coli offers an opportunity for using whole cells of

this bacterium as an environmental amendment. In

this context, we set out a simple experimental

evolution procedure to generate LinA variants opti-

mized for dehydrochlorination activity when ex-

pressed in E. coli. As explained below, one round

of mutagenesis, expression and screening for the best

performing clones was enough to single out one

enzyme variant with improved features for environ-

mental release in E. coli , such as activity, stability

and solubility.

Materials and methods

In vitro evolution of the linA gene

In order to perform the directed evolution procedure

on the LinA protein we started from plasmid

pUC18NotLinA, which had been shown to produce

a good yield of LinA protein. Construction of this

plasmid is reported elsewhere (Mohn et al., 2006b).

pUC18NotLinA bears a 1240 bp fragment contain-

ing the linA gene from S. paucimobilis UT26 cloned

under the Plac promoter. Mutagenic PCR was

Figure 1. Scheme of the reactions catalysed by LinA in the proposed degradation route of g-HCH in Sphingomonas paucimobilis (Nagata

et al. 1999b; Kumari et al. 2002). Compounds: 1, hexachlorocyclohexane; 2, pentachlorocyclohexene; 3, 1,3,4,6-tetrachloro-1,4-

cyclohexadiene; 4, 1,2,4-trichlorobenzene. Compound 3 is unstable and evolves spontaneously to chemical species 4.

224 M. Mencıa

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performed using the Genemorph kit (Stratagene)

according to the manufacturer’s instructions in order

to get an average of 1 to 3 mutations per Kb of

template DNA. The template was 1 ng of plasmid

per 50 mL reaction, which was primed with 24 mer-

forward and reverse M13 sequencing primers for

amplification. The PCR was set to 40 cycles. The

amplified and mutagenized fragment was 1 Kb in

length and comprised the linA ORF plus a 481 bp

upstream sequence, including the putative linA

promoter, and 281 bp of the downstream flanking

sequences. The PCR product was digested with the

enzymes PstI and SacI and ligated to the corre-

sponding sites of pUC18Not. After transformation

of the strain E. coli CC118 and spreading onto plates

containing LB plus 150 mg mL�1 ampicillin, 960

colonies were picked up using a QPix colony picker

robot (Genetix) and inoculated into 200 mL of LB

plus ampicillin in 96 well plates. These were shaken

overnight at 308C in a moisture chamber and 10 mL

of each of the cultures were then inoculated into

fresh microtiter plates containing 200 mL of LB

medium plus ampicillin as before and supplemented

with 0.5 mM IPTG to induce expression of the

protein. These plates were incubated at 308C for

24 h and the induced cultures were subject to an

automated LinA activity assay as described below

(Phillips et al. 2001). The results were recorded with

a Victor 2 (Perkin-Elmer) multiplate colorimetry

reader equipped with a 550 nm filter. We previously

checked that the coefficient of variance was below

10% by measuring the activity from a 96-well plate

inoculated with E. coli cells carrying pUC18No-

tLinA as reference. About 30 clones from the

mutagenized library, exibiting an activity clearly

above that of the wild type, were re-screened for

confirmation of activity. Plasmid was extracted from

14 out of the 30 clones and the parental strain E. coli

CC118 was retransformed and the processing re-

peated including the activity assay.

High-throughput assay of LinA activity

This was performed essentially as described by

Phillips et al. (2001). Multiwell plates containing

the induced cultures were centrifuged at 48C, at

3220 g for 20 min. The supernatant was discarded

and the pellets were resuspended, using a liquid

handling robot Biomek 2000 (Beckman & Coulter),

in 60 mL of lysis buffer (40 mM Tris-HCl pH 8,

150 mM NaCl, 5% glycerol, 1 mM EDTA pH 8, 1%

Triton X-100, 1 mM phenylmethanesulfonyl fluo-

ride PMSF, 0.1 mg mL�1 DNase I, 0.1 mg mL�1

RNase, 300 mg mL�1 lysozyme). Plates were incu-

bated on ice for 45 min for the lysis to proceed and

then centrifuged at 48C at 3220 g for 20 min. During

the incubation time, the assay buffer was freshly

prepared as follows. 1 mL of 0.5 mM HEPES

pH 8.2, 10 mM sodium sulphate and 0.5 mM

EDTA pH 8, was added to 20 mL of a solution of

1 mg mL�1 phenol red dissolved in 20% ethanol and

10 mL of 12 mg mL�1 g-lindane dissolved in ace-

tone. 195 mL aliquots of the resulting assay cocktail

were then distributed into multiwell plates. At that

point, 6 mL from the lysed cultures were then added

to the 195 mL of assay buffer preloaded in each well

of the microtiter plates. All the liquid handling with

the 96 well plates was performed using the Biomek

2000 robot. The assay was allowed to proceed for 1

to 6 h at room temperature and the activity was

determined based on the change of colour of the

assay buffer due to the pH change resulting from

the release of Cl� and H� ions from the lindane.

The colour change was measured as the decrease

in the absorbance 550 nm (A550) and the activity of

each clone was calculated from the A550 values as the

release of protons (nmols) per minute, taking into

account the change produced in the buffer by the

addition of an extract without LinA. To this end,

appropriate calibration curves were established for

matching the colour change with a known amount of

HCl added to the assay buffer. One unit of LinA

activity was defined as the amount of enzyme

required for the release of 1 mmol of chloride ion

per minute under the assay conditions (the number

of chloride ions released is equal to the number of

protons). Samples were measured at different dilu-

tions and the activity experiments were repeated at

least three times. The standard errors were within

10% of the values.

Fractionation of cells extracts

The procedure was performed essentially as described

(Thomas et al. 2001). Starter cultures (5 mL) were

allowed to grow overnight and used to inoculate

50 mL of LB, supplemented with 150 mg mL�1 of

ampicillin to a starting optical density (OD600) of

0.01. Cultures were then grown at 378C to mid-

exponential phase and induced with 0.5 mM IPTG.

The cells were allowed to grow for a further 4 h and

harvested by centrifugation at 4000 g for 10 min at

48C. The pellets were resuspended in 7.5 mL of a

buffer containing 10 mM Tris-HCl pH 7.5, 20% (w/

v) sucrose, 0.1 mM EDTA, and incubated at room

temperature for 10 min. Cells were harvested as

before, resuspended in 2 mL of ice-cold 5 mM

MgSO4 and incubated on ice for 20 min to generate

sphaeroplasts. These were then collected by centrifu-

gation, and the supernatant retained as the periplas-

mic fraction. Spheroplasts were resuspended in 2 mL

of ice-cold 5 mM MgSO4 and sonicated for 5 �/ 20 s

Expanding the properties of dehydrochlorinase LinA 225

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bursts interrupted with 20 s cooling periods in ice.

Cellular debris and intact spheroplasts were removed

by centrifugation at 9300 g for 5 min at 48C, and

the remaining pellet was retained as the insoluble

fraction. Membranes were collected from the super-

nantant by a higher speed centrifugation at 250000 g

for 30 min, and the supernatant was retained as the

cytoplasmic fraction. The pellet was resuspended in

1.5 mL of ice-cold 5 mM MgSO4. Tris (1 M pH 8.0)

was then added to all fractions to a final concentration

of 10 mM before storage at �/208C. Samples were

mixed with an equal amount of protein loading buffer,

heated at 1008C for 10 min and analyzed on 12%

SDS-PAGE gels. After staining with Coomasie blue,

protein concentrations were determined by gel densi-

tometry using appropriate standards.

Computer modeling of LinA

Alignment of LinA with its structural homo-

logues was taken from Nagata et al. (2001). The

three-dimensional threading model of the LinA

structure and its variants was generated using the

3D-PSSM feature of server http://www.sbg.bio.ic.ac.

uk/�/3dpssm. The substitution of Cys132 by Arg

was entered manually in the structure.

Results

Experimental evolution of the HCH dehydrochlorinase

After one round of mutagenesis of linA and screen-

ing 960 clones, we obtained the activity distribution

shown in Figure 2. Approximately 25% of the clones

showed a large decrease in activity with respect to

that of the wild type protein of S. paucimobilis UT26.

This is compatible with an average rate of 1

mutation per linA gene. Fourteen clones showing

activities clearly above the standard were selected

and re-screened by the same procedure. Out of

14 candidates, only one clone (LinAT10) unam-

biguously showed an activity above the parent type,

and this was selected for a second round of

mutagenesis/screening by the same procedure. How-

ever, no significant further improvement in the

enzyme was detectable after analyzing a similar

number of clones (data not shown), so we focused

on the characterization of LinAT10.

Expression, solubility and localization of LinA and

LinAT10

The insert of the plasmid bearing the mutant linA

endowing the E. coli cells with a superior dechlori-

nation ability was sequenced and only one mutation

was detected. This was a change of T to C at

position 927 nt downstream of the BamHI site of the

cloned DNA. This change causes a C132R mutation

in the primary sequence of the LinA protein.

Interestingly, this residue is close to one of the

most catalytically important residues of LinA,

R129. Yet, according to the structural prediction,

C132 faces towards the protein surface (Figure 3),

while R129 faces the core of the protein, where it is

believed to form the catalytic center (Nagata et al.

2001).

The increase in dechlorination activity of the

C132R mutant could be due to improvement in

expression levels, higher solubility of the recombi-

nant protein, lower susceptibility to degradation, or

a genuine increase of the specific activity of the

enzyme. In order to distinguish between these

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

number of clone

Act

ivit

y re

spec

t to

WT

0 240 480 720 960

Figure 2. Distribution of activities in clones obtained after one round of random mutagenesis. E. coli cells were transformed with plasmids

harbouring the linA gene after being subject to random, PCR-based mutagenesis. Individual clones were picked, incubated at 308C, lysed

and the activities of the extracts were quantified as described in Materials and methods. Activity data relative to wild type (as reflected in

changes in absorbance) are represented on the Y-axis. Clones are arranged in decreasing order according to their relative activities.

226 M. Mencıa

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possibilities we first examined the amount of protein

in the cleared lysates, the activities of which were

measured in the high-throughput assay (see above).

The corresponding protein gels showed that the cells

bearing the mutant LinA clone contained consider-

ably higher levels of the protein than those with the

wild type counterpart (Figure 4). This indicated that

at least part of the higher activity of the mutant clone

could be traced to an elevated level of LinAT10 in

the E. coli host.

Since it has been reported that the LinA protein

has a periplasmic localization in its native host, we

next addressed the cellular location of the mutant

protein in E. coli . To this end we separated and

analysed the different subcellular fractions of the

bacterial host expressing the wild type LinA protein

and the C132R mutant (Figure 5). As expected, the

lanes corresponding to the SDS-PAGE analysis of

whole induced cells show that expression of the

mutant protein is about two-fold higher than that of

the wild type. Interestingly expression of the mutant

protein was higher at 308C than at 378C (data not

shown). However only a minor fraction of the LinA

protein could be detected in the periplasmic frac-

tions of cells bearing either the wild type protein or

the mutant. Why LinA goes mostly to the periplasm

in its native host (Nagata et al. 1999a) while it

remains predominantly cytoplasmic in E. coli is an

open question. In any case, the LinA protein

produced in the surrogate host appeared to maintain

an excellent ability for dechlorinating g-HCH.

The cell fractionation results of Figure 5 show also

that a good portion of the LinA protein is present in

the insoluble fraction, associated with cell debris

both in the wild type and the mutant clones.

However, the gel densitometry analysis of the same

samples clearly revealed that the relative production

of soluble LinAT10 was at least six-fold higher than

the wild type. This increase cannot be explained only

by the two-fold difference in soluble expression

levels between the two proteins. Instead, the data

suggest that the mutant protein possesses an intrin-

sically higher solubility in the cytoplasmic milieu of

E. coli than the original enzyme. The membrane

fractions also contained a certain amount of LinA

and LinAT10 proteins, although it is likely that such

Figure 4. SDS-PAGE of cell lysates of E. coli CC118 bearing

LinA proteins. Aliquots of the lysates obtained from cultures of E.

coli cells expressing either LinA or the LinAT10 mutant were

analyzed by SDS-PAGE and Coomasie blue staining. A typical

result from three independent experiments is shown.

Figure 3. Prediction of LinA structures. Left, localization of the residue Cys 132 in the three-dimensional structure model for LinA. Right,

model of LinA with the substitution Cys 132 to Arg. Tertiary structure prediction was performed using the program 3D-PSSM.

Expanding the properties of dehydrochlorinase LinA 227

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an association was the result of the extraction

procedure and not a genuine location of LinA in

cell membranes. In fact, we detect overproduced

LinA and LinAT10 in all cell fractions of E. coli ,

although we only consider that the soluble cytoplas-

mic fractions account for the improvement of the g-

HCH dechlorination ability of the whole-cell catalyst

(see below).

Enzymatic activities of LinA and LinAT10

When the specific activity of the different protein

fractions was measured (Table I) we observed that

the two cytoplasmic extracts were the most active.

The specific activity of the cell debris and membrane

fractions was roughly similar in all samples and

much lower that the activity of the soluble cytoplas-

mic fractions. This was expected, as protein over-

production often yields insoluble material that is

misfolded resulting in aggregates that are recovered

in the cell debris and the membrane fractions. In

contrast, the activities of the periplasmic fractions

were significant, although they only reached approxi-

mately 35% of those found in the cytoplasmic

samples. Whether the increase in intracellular solu-

ble LinAT10 is due to facilitation of protein folding

in E. coli or resistance to cell proteases warrants

further investigation which is beyond the scope of

this article.

The C132R LinAT10 mutant showed about the

same specific activity as the wild type protein in all

fractions and in several dilutions tested. Minor

differences were within the experimental error of

the procedures used. This indicated that the C132R

mutation did not affect the active site of the enzyme

despite occurring very close to the catalytic residue

R129. Also the mutation C132R did not alter the

pattern of location of the LinA protein when

expressed in E. coli , as we observed the same

proportional distribution of the protein and equiva-

lent specific activities in both the wild type LinA and

the LinAT10 mutant (Figure 5 and Table I). It thus

appears that C132R mutation improves the level of

expression and the amount of enzyme in the soluble

fraction when the LinA protein is overexpressed in

E. coli , and this happens without changing the

specific activity or the subcellular location properties

of the enzyme.

Discussion

This work was set out in the context of developing

superior biological materials for remediation of soil

polluted by lindane. In particular, we were interested

in increasing the operative dechlorination ability of

E. coli cells in which the linA gene of P. paucimobilis

UT26 was overexpressed as an early step in

the formulation of a whole-cell catalyst for soil

amendment (Strong et al. 2000). We have exploited

the power of experimental evolution and high

throughput screening for improve this feature and

thus expand the range of environmental applications

of LinA.

Table I. LinA activity (units mg�1 protein) of the different

fractions from extracts prepared from E. coli cells expressing the

wild type LinA protein or the T10 mutants. Activities were

calculated as described in Materials and methods.

Periplasmic Cell debris Cytoplasmic Membranes

Wild type 18.1 0.5 59.5 1.8

T10 20.3 1.2 55.0 1.8

Figure 5. SDS-PAGE of the cell fractions of E. coli CC118 bearing LinA proteins. Aliquots of the different fractions obtained from cultures

of E. coli cells expressing either LinA or the LinAT10 mutant were analyzed by SDS-PAGE and Coomasie blue staining as explained in the

text. Total: whole-cell sample; periplasmic: periplasmic fraction; cell debris: insoluble fraction; cytoplasmic: soluble cytoplasmic fraction;

membranes: resuspended membrane fraction. The whole procedure was performed in triplicate. One typical result is shown.

228 M. Mencıa

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The LinA enzyme of P. paucimobilis UT26 and its

orthologues in other soil bacteria efficiently dehalo-

genate lindane in aerobic environments. After appli-

cation of our procedures above, protein expression

levels were improved two-fold when overproduced in

E. coli and solubility in the E. coli cytoplasm three-

fold. In practical terms, the overall performance of

the whole-cell catalyst has therefore been enhanced

six-fold. Surprisingly, this considerable phenotypic

change was the result of a single amino acid

substitution at a residue very close to the catalytically

essential R129 which forms part of the active site of

the enzyme. In the three-dimensional model of LinA

(Figure 3), the mutated residue C132 (sidechain),

faces the exterior of the protein and hence, is

probably exposed to the solvent. A change of Cys

to Arg at the 132 position would produce a bulkier

and more hydrophilic sidechain, as arginine is a

charged residue. This could improve the solubility of

the enzyme by increasing its solvation surface. In

addition, it is possible that the Arg132 could

facilitate folding, and so yield a more stable enzyme

or make it less susceptible to degradation by

proteases which attack unfolded molecules. When

we look at the alignment of LinA together with four

other structurally related enzymes (Figure 6), we

observe that the position equivalent to C132 in three

out of five enzymes is occupied by charged residues

and in one of them it is an arginine. This supports

the notion that a charged residue at position 132

may contribute positively to the characteristics of the

enzyme. E. coli cells expressing LinAT10 are good

candidates for the applications envisioned above, as

they embody significant improvements in both the

total amount of protein produced and also the

fraction of soluble and thus operative LinA protein.

As shown in the case of atrazine, this approach is

feasible because the dehalogenating activity of LinA

does not require any co-factor or co-substrate, so

killed E. coli cells can be used as a vehicle to deliver

such biologically encapsulated catalysts to the target,

polluted sites (Strong et al. 2000).

Acknowledgements

We thank Sofia Fraile for providing technical assis-

tance, Dr Luis Angel Fernandez Herrero and Teca

Galvao for helpful discussions. M M is a Ramon y

Cajal Program researcher. This work was supported

by European grants of the 5th and 6th Frame Work

Programmes.

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Figure 6. Alignment of the relevant region of structurally related proteins (Nagata et al. 2001). Numbers correspond to catalytically

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1,2-dioxygenase.

Expanding the properties of dehydrochlorinase LinA 229

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