Haloalkane Dehalogenases: Biotechnological Applications*

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Biotechnol. J. 2012, 7 DOI 10.1002/biot.201100486 www.biotechnology-journal.com

Review Haloalkane dehalogenases: Biotechnological applications*

Tana Koudelakova1, Sarka Bidmanova1, Pavel Dvorak1, Antonin Pavelka1, Radka Chaloupkova1, Zbynek Prokop1,2 and Jiri Damborsky1,2

1 Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Masaryk University, Brno, Czech Republic 2 Enantis Ltd., Brno, Czech Republic

Haloalkane dehalogenases (EC 3.8.1.5, HLDs) are α/β-hydrolases which act to cleave carbon- Received 24 MAY 2012 halogen bonds. Due to their unique catalytic mechanism, broad substrate specificity and high Revised 30 JUN 2012 robustness, the members of this enzyme family have been employed in several practical applica- Accepted 20 JUL 2012 tions: (i) biocatalytic preparation of optically pure building-blocks for organic synthesis; (ii) recycling of by-products from chemical processes; (iii) bioremediation of toxic environmental pollutants; (iv) decontamination of warfare agents; (v) biosensing of environmental pollutants; and (vi) protein tagging for cell imaging and protein analysis. This review discusses the application of HLDs in the context of the biochemical properties of individual enzymes. Further extension of HLD uses within the field of biotechnology will require currently limiting factors – such as low expression, product inhibition, insufficient enzyme selectivity, low affinity and catalytic efficiency towards selected substrates, and instability in the presence of organic co-solvents – to be overcome. We propose that strategies based on protein engineering and isolation of novel HLDs from Supporting information available online extremophilic microorganisms may offer solutions.

Keywords: Biocatalysis · Biodegradation · Biosensors · Cell imaging · Protein analysis

1 Introduction reviews focused on soil-based discovery of novel dehalogenases [20] and hexachlorocyclohexane degradation Haloalkane dehalogenases (EC 3.8.1.5, HLDs) are α/β- [21–24] have also been published. The aims of the present hydrolases which convert halogenated compounds to cor- review are to summarize the properties of characterized responding alcohols, halides and protons [1]. Hydrolytic HLDs with regards to potential application and to criti- cleavage of a carbon-halogen bond proceeds by the SN2 cally discuss their possible practical use. mechanism (Fig. 1) followed by the addition of water [2, 3]. Water is the only co-factor required for catalysis. More- over, HLDs are relatively stable and easy to handle, which 2 Properties of HLDs makes them attractive for both academic research and practical applications. The importance of dehalogenating HLDs represent one of the best characterized enzyme enzymes is highlighted by the number of reviews that de- families that act on halogenated hydrocarbons and their scribe different biochemistry, genetics and applications, derivatives [25]. During the past twenty five years, 17 dif- mainly in the field of biodegradation [4–19]. Specialized ferent bacterial HLDs have been identified and at least partly biochemically characterized [26–38] (Prudnikova et al., in preparation). Characteristics of HLDs are summa- Correspondence: Prof. Jiri Damborsky, Loschmidt Laboratories, rized in Figure 2. The properties important for various Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno, practical applications, such as expression, substrate Czech Republic E-mail: [email protected]

Abbreviations: HLD, haloalkane dehalogenase; SSG, substrate specificity *We dedicate this paper to the founder of this field, our mentor and group friend, Professor Dick B. Janssen from the Groningen University.

Figure 1. Simplified scheme of the reaction mechanism of HLDs. (A) Formation of an alcohol catalyzed by a wild-type enzyme. (B) Irreversible formation of an alkyl-enzyme complex in the HaloTag, where the catalytic histidine is mutated into phenylalanine. The different products of reactions are in gray. Depicted according to Verschueren et al. [2] and Los et al. [105]. specificity, catalytic efficiency, enantioselectivity and expressed constitutively in and Xanthobacter autotroph- stability, are discussed in the following paragraphs. icus GJ10 and Sphingobium japonicum UT26, respectively [39, 40], the expression of DhaA in Rhodococcus 2.1 Expression rhodochrous NCIMB 13064 is regulated by a repressor responding to 1-chlorobutane and 1-haloalkane levels in the The expression of HLDs in their natural hosts was studied cellular environment [41–43]. However, strains with con- with DhaA, DhlA and LinB, which were isolated from stitutive expression of DhaA have also been described bacterial strains utilizing halogenated compounds as a [43]. Although the expression and function of other HLDs sole carbon source [26–28]. Whereas DhlA and LinB are in their original strains has not been identified, most

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Figure 2. Biochemical and structural properties of HLDs. (A) Summary of properties of DatA [35], DbeA [46] (Prudnikova et al., in preparation), DbjA [31, 45–47, 58], DhaA [27, 45–47, 49, 50, 55, 119, 120], DhlA [26, 46, 48, 50, 56, 121], DhmA [29, 32], DmbA [30, 46, 122], DmbB [30], DmbC [33, 46], DmlA [31, 47], DmmA [37], DpcA [38], DppA [36], DrbA [33, 46], LinB [45–47, 51, 123]. The isoelectric point (pI) and molecular weight (MW) were predicted by Expasy server [124]. The detailed description of the thermal denaturation measurement and the calculation of cavity properties is given in the Supporting information. Abbreviations: EC, Escherichia coli; MS, Mycobacterium smegmatis; RE, Rhodococcus erythropolis; ×, *, ** and ***, qualitative measures; ∞, mul- timer. (B) pH and temperature profile.

HLDs can be heterologously expressed in Escherichia coli 2.2 Substrate specificity (Fig. 2) [26–39, 42] (Prudnikova et al., in preparation). Pro- tein yields obtainable from shake flask cultivations under HLDs possess broad substrate specificity. Cleavage of the laboratory conditions range between 1–150 mg L–1, de- carbon-halogen bond by HLDs has been reported using pending on the particular heterologous enzyme ex- more than a hundred chlorinated, brominated and iodi- pressed and expression system used. Improved expres- nated aliphatic compounds containing a monohalogenat- sion may be achieved by optimized codon usage and the ed sp3 hybridized carbon as substrate (Fig. 3A, Support- use of a suitable expression system. ing information, Tables S1 and S2) [1, 44–47]. Compounds

Figure 3. Substrate specificity and enantioselectivity of HLDs. (A) Substrate specificity of HLDs. The enzymes are colored according to membership to individual substrate specificity groups: SSG-I (DbjA, DhaA, DhlA and LinB), SSG-II (DmbA), SSG-III (DrbA) and SSG-IV (DatA, DmbC and DpcA). DppA is unclassified. The columns of specific activities higher than 80 nmol s–1 mg–1 are trimmed for clarity. (B) Enantioselectivity of HLDs.

halogenated on sp2 hybridized carbon, multihalogenated Even though these numbers are far from the theoretical on a single carbon, fluorinated, or aromatic are not con- maximal value of 108-109 M–1 s–1 for diffusion-limited en- verted [1]. A recent study of substrate specificity divided zymes, they are close to the recently estimated median HLDs into four substrate specificity groups (SSGs) [46]. catalytic efficiency of an “average” enzyme: 105 M–1 s–1 HLDs that convert a number of resistant chlorinated sub- [54]. Catalytic efficiency achieved with anthropogenic strates were categorized as one group (SSG-I: DbjA from chlorinated substrates, such as 1,2,3-trichloropropane, is Bradyrhizobium japonicum USDA 110, DhaA, DhlA and significantly lower (40 M–1 s–1 for DhaA), making protein LinB). Another group preferring brominated and iodinat- engineering of the enzyme to improve catalytic efficien- ed compounds was identified (SSG-IV: DatA from cies essential to gain performance sufficient for industri- Agrobacterium tumefaciens C58, DbeA from Bradyrhizo- al applications [52]. The Michaelis-Menten constants and bium elkanii USDA94 and DmbC from Mycobacterium the rate constants reported for HLDs vary across sub- bovis 5033/66) (Fig. 2). SSG-II and SSG-III were each strates by five and four orders of magnitude, respectively

populated with only one enzyme (DmbA from Mycobac- (Km: 0.005 – 45 mM and kcat: 0.04 – 40 mM) [30, 31, 33, terium bovis 5033/66 and DrbA from Rhodopirellula balti- 35–38, 48–51]. ca SH1, respectively), reflecting the unique substrate preferences of these enzymes. The study also identified 2.4 Enantioselectivity the “universal” substrates, i.e. 1-bromobutane, 1-iodo - propane, 1-iodobutane, 1,2-dibromoethane and 4-bro- Initial study of DhaA and DhlA revealed only a weak mobutanenitrile and the “poor” substrates, i.e. 1,2-di - enantioselectivity of HLDs with selected chiral and chloropropane, 1,2,3-trichloropropane, chlorocyclohexane prochiral halogenated substrates (Supporting informa- and (bromomethyl)cyclohexane of analyzed HLDs. tion, Table S4) [55, 56]. While DhaA was assayed using 2- bromobutane, 2-bromopentane and 2-bromo-1-phenyl- 2.3 Catalytic efficiency propane by Janssen et al. [55], both DhaA and DhlA were tested with six short-chain dihalogenated alkanes, six HLDs exhibit catalytic efficiencies ranging from 104 to 105 terminally monohalogenated esters and four halogenated M–1 s–1 with their best (brominated) substrates [48–53]. prochiral propanes by Pieters et al. [56]. The subsequent

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studies by Prokop et al. [45, 57] revealed that DhaA, LinB hydroxyesters [45, 57], (S)-α-bromoamides and (S)-α-hy- and DbjA strongly discriminates between enantiomers of droxyamides [47], useful building blocks in the production several α-brominated esters (Fig. 3B, Supporting infor- of pharmaceuticals. For instance, (S)-2-pentanol is an in- mation, Table S3), and DbjA additionally exhibits high termediate in the synthesis of potential anti-Alzheimer’s enantioselectivity towards two β-brominated alkanes. disease drugs [59], ethyl (S)-2-hydroxypropionate is used The same three enzymes were successfully used for ki- for preparation of lofexidine [60], and optically pure netic resolution of α-bromoamides [47]. Most recently, amides are precursors of non-natural peptides, vitamins three novel HLDs enantioselective towards α-brominat- and antibiotics [61] (Fig. 5). ed esters were discovered [35, 38] (Prudnikova et al., in Enantioselectivity of an enzyme can be described preparation). The enzyme DatA also exhibits exception- either by enantiomeric excess (e.e., the excess of one al enantioselectivity towards two β-brominated alkanes enantiomer over the other) or the E value (E, the ratio of [35]. All enantioselective HLDs tested so far have exhib- specificity constants for the prefered or non-prefered ited the preference for (R)-brominated substrates [35, 38, enantiomer). Besides high enantioselectivity (e.e. > 99%, 45, 47]. E > 100), further requirements for the application of enzyme to a biocatalytic process have been proposed: (i) 2.5 Stability conversion of more than 98%; (ii) substrate loading higher than 100 g L–1; (iii) substrate/biocatalyst ratio higher Increasing reaction temperature to 35–50°C has a positive than 50 with maximal biocatalyst loading 5 g L–1; and (iv) effect on the catalysis of HLDs. Above these tempera- reaction time less than 24 h [62]. Due to the character of tures, a rapid drop in activity is observed [58]. The only kinetic resolution of racemic substrates, the theoretical exception is cold-adapted DpcA from Psychrobacter yield is limited to 50%, unless an additional chemical or cryohalolentis K5, which shows its highest activity at enzymatic process [61] or racemization of the substrate 25°C [38]. Although elevated temperature increases the [63] is used. Activation of the hydroxyl group of three dif- catalytic rate, HLD structures start to denature between ferent (S)-α-hydroxyamides by methanesulfonyl chloride 40–50°C (Fig. 2, Supporting information, Table S3) [30, 33, has enabled the enantioconvergent chemoenzymatic 35, 58]. Similarly, extremely acidic or alkalic conditions are synthesis of optically active N-alkyl (R)-α-azido amides, not suitable for HLDs, which generally perform catalysis N-alkyl (R)-α-benzylaminoamides, N-alkyl (R)-α-phenoxy - best at pH 8-10 (Fig. 2, Supporting information, Table S3) amides and N-alkyl (R)-α-ethylthioamides [61] (Fig. 6). [26–28, 30, 33, 35, 58]. The study of the behavior of DbjA, The compounds were prepared within two days to give a DhaA and LinB in the presence of 14 water-miscible or- 57-96% yield and e.e. of 28–98%. The first reported dy- ganic solvents showed that the resistance of individual namic kinetic resolution using a HLD employed polymer- enzymes to organic co-solvents differs (Stepankova et al., based phosphonium bromide as a racemization agent for submitted). While DbjA was stable in most of the co-sol- N-phenyl-2-bromopropionamide and incorporated an ex- vents tested up to concentrations of 20% (v/v), the activi- perimental setup with DbjA in a semipermeable mem- ty of LinB and DhaA gradually decreased at a co-solvent brane compartment [63]. A 63% yield and an e.e. of 95% concentration of 5% and 10%, respectively. were achieved using this process. The use of semipermeable membrane reactors and organic solvents could solve the issue of low substrate 3 Applications of HLDs loadings under aqueous reaction conditions, which are reported as approximately 0.3 g L–1 [45, 47]. In the case of HLDs have attracted considerable attention due to their (S)-2-pentanol preparation, such low substrate loading is unique catalytic mechanism, broad substrate specificity the biggest disadvantage compared to the alternative ki- and robustness. They have been used for biocatalytic netic resolution catalyzed by Candida antarctica lipase B preparation of optically pure building-blocks for organic which employs racemic 2-pentanol as both substrate and synthesis; recycling of by-products from chemical pro- solvent [59]. Nevertheless, processes catalyzed by HLDs cesses; bioremediation of toxic environmental pollutants; generally possess favorable substrate/biocatalyst ratios decontamination of chemical warfare agents; biosensing (>50). Reactions can be completed within 2 hour to 2 day environmental pollutants; and protein tagging for cell im- time frame [45, 47]. aging and protein analysis (Fig. 4). A further factor contributing to the economical feasi- bility of the process is the price of biocatalyst production. 3.1 Biocatalysis Enzyme production yields have a dramatic effect on production costs as they can vary from units of mg L–1 in non- The discovery of high enantioselectivity of HLDs (Sup- optimized systems up to 15 g L–1 and 25 g L–1 in optimized porting information, Table S3) has opened up the possi- intracellular and extracellular systems, respectively [64]. bility to utilize HLDs to prepare optically pure (S)-β-bro- The cost of enzyme should be less than 10% of total moalkanes, (S)-β-alcohols, (S)-α-brominated esters, (S)-α- process cost [65]. Therefore, the current expression yields

Figure 4. Biotechnological applications of HLDs. Summary of reported applications of DatA [35], DbeA (Prudnikova et al., in preparation), DbjA [45, 47, 57, 61, 63, 125], DhaA [14, 44, 45, 47, 57, 66, 78, 79, 83, 87, 89, 92, 94, 96, 105–115, 125], DhlA [75, 76, 93, 97], DmbA [44, 89], DmlA [47], DpcA [38] and LinB [28, 44, 45, 47, 57, 61, 89, 95]. Abbreviations: BSA, bovine serum albumin; CB, 1-chlorobutane, DCA; 1,2-dichloroethane; DKR, dynamic kinetic resolution; KR, kinetic resolution; POI, protein of interest; TCP, 1,2,3-trichloropropane; TEV, cleavage site for TEV protease.

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Figure 5. Examples of optically pure compounds obtained from kinetic resolutions employing HLDs.

of HLDs reaching 150 mg L–1 under laboratory conditions the presence of product inhibition (Supporting informa- require optimization. tion, Table S5). The limitations described above have motivated fol- 3.2 By-product recycling low-up DNA enrichment studies and protein engineering experiments [20, 52, 68–70]. The enrichment of specific 1,2,3-Trichloropropane, 1,2-dichloropropane, and 1,2- sequences from a soil sample using degenerated oligonu- dichlorobutane are by-products from the chemical syn- cleotides led to discovery of 19 new genes and the char- thesis of epichlorohydrin, propylene oxide, and butylene acterization of four novel enzymes active with 1,2,3- oxide, respectively [14, 20, 66] (Fig. 7). Recycling of the trichloropropane [20]. Three of them lacked product halogenated aliphatic compounds to halohydrins could inhibition, but their kinetic parameters were comparable save manufacturing costs; therefore the Dow Chemical with those reported for DhaA (Supporting information, Company attempted to develop an efficient catalyst to Table S5). drive halohydrin formation (Supporting information, Table The significant improvement of DhaA kinetic param- S5). The initial culture enrichment of various soil and wa- eters was reported in three protein engineering studies ter samples from Midland County, Michigan in medium [52, 68, 69]. A mutant possessing a four-fold higher cat- with a mixture of 1-chlorobutane, 2-chlorobutane, and alytic constant with 1,2,3-trichloropropane than the wild- 1-chloro-2-methylpropane led to isolation of an Rhodococ- type was obtained after one round of error-prone PCR by cus sp. TDTM003 possessing a variant of DhaA [20, 66]. Gray et al. [68]. Bosma et al. [69] constructed a variant M2 Application of an enzyme biocatalyst for large-scale using DNA shuffling and error-prone PCR that was nearly processing requires suitable specificity, but also favorable eight-times more efficient in conversion of 1,2,3-trichloro- kinetic properties and good stability [14, 67]. Although propane than DhaA. Computer modeling provided a wild type DhaA can convert 1,2,3-trichloropropane into deeper understanding of the improved conversion of required 2,3-dichloropropane-1-ol [66] and immobilized 1,2,3-trichloropropane by the variant M2 [71] and addi- enzyme kept 10% of its activity in neat substrate [67], the tional mutations were introduced to the access tunnels technology was not competitive due to poor kinetic pa- of DhaA by saturation mutagenesis [52, 72]. The result- –1 rameters of the biocatalyst (Km > 1 mM, kcat < 1 s ) and ant five-fold mutant DhaA31 exhibited a 26-times higher

Figure 6. Examples of optically pure compounds obtained from chemoenzymatic syntheses employing HLDs.

Figure 7. Examples of halogenated by-products from chemical processes (up) and the desired products (down) catalyzed by HLDs.

catalytic efficiency than the wild-type enzyme (with Km Agrobacterium radiobacter AD1, the strain reported to –1 close to 1 mM and kcat > 1 s ; Supporting information, possess a biochemical pathway for utilization of halo- Table S5). genated alcohols [78]. An engineered strain with an as- In addition to kinetic parameters, enantioselectivity sembled pathway can use 1,2,3-tribromopropane and 1,2- and stability of DhaA also have been optimized. Van dibromo-3-chloropropane as a sole carbon and energy Leeuwen et al. [73] used DhaA31 as a template for addi- source but its growth on 1,2,3-trichloropropane was re- tional five rounds of directed evolution and obtained ported to be poor [79]. In a follow-up study, the previously enantiocomplementary mutants r5-90R and r5-97S, described DhaA mutant M2 (Supporting information, which can be used for the preparation of high-value chi- Table S5) was used instead of wild-type DhaA [69]. How- ral epichlorohydrins. Gray et al. [68, 74] obtained a re- ever, even an eight-fold improvement of DhaA catalytic markably thermostable DhaA variant (designated Dhla8), efficiency was not sufficient to provide feasible biodegra- which exhibited a half-life of 138 min at 80°C by a combi- dation of 1,2,3-trichloropropane. nation of mutations from gene site saturation mutagene- Continuous solid-gas biofilters with dehydrated bac- sis. terial cells containing DhaA or DhlA have been exten- sively studied for removal of 1-chlorobutane from waste 3.3 Bioremediation gas [80–83]. Pre-treatment of the dehydrated bacterial cells of E. coli BL21(DE3) containing DhaA with ammonia Bioremediation is one of the most promising fields to ap- vapor improved the stability of the catalyst and retained ply HLDs because their natural substrates, chlorinated maximal reaction rates for 60 hours [83]. aliphatic and cyclic compounds, are prominent environ- Biodegradation of four hexachlorocyclohexane iso- mental pollutants. The compounds, such as 1,2-di - mers (α-, β-, γ- and δ-) was recently reviewed by Lal et al. chloroethane, 1-chlorobutane and hexachlorocyclohexa- [24]. Significant reductions in hexachlorocyclohexane lev- ne (Fig. 8), have been introduced into the environment els (30–100%) were achieved after the growth stimulation through their use as organic solvents or pesticides [14, 18, of hexachlorocyclohexane-degrading strains at four dif- 24, 52, 75]. ferent contaminated sites. Inoculation of hexachlorocy- The most thriving use of HLDs described to date is the clohexane-degrading strains into diverse samples of con- application of the 1,2-dichloroethane utilizing strain taminated soil led to successful biodegradation of hexa- X. autotrophicus GJ10 in a full-scale groundwater purifi- chlorocyclohexane as well. The two most recent papers cation plant in Lübeck, Germany [13, 76]. The plant oper- reported in vitro biotransformation of ε-hexachlorocyclo- ates at 8–12°C and treats water contaminated by 1,2-di - hexane, heptachlorocyclohexane [84] and isomers of chloroethane in concentrations from 2–15 mg L–1 to the fi- hexabromocyclododecane [85] (Fig. 8) into mono- and/or nal concentration 0.01 mg L–1 with a speed of 5–20 m3 h–1. dihydroxylated products by LinB from Sphingobium in- Previous attempts to utilize the same strain in air-lift and dicum B90A. extractive membrane bioreactors for removal of 1,2-di - Due to the restricted use of genetically modified bac- chloroethane from the contaminated water or gas has re- teria for environmental clean-up applications, the possi- mained at the level of laboratory studies [13, 77]. bility of phytoremediation has been investigated as well Several studies have been focused on microbial [75, 86–88]. Tobacco plants containing a haloalkane and a biodegradation of 1,2,3-trichloropropane. A continuous haloacid dehalogenase from X. autotrophicus GJ10 or an effort to enrich 1,2,3-trichloropropane-degrading organ- apoplast-targeted haloalkane dehalogenase from Rho - isms from environmental samples was not successful. dococcus sp. were recently engineered. These trans- Therefore, attempts to construct a synthetic biochemical genic plants could degrade 1,2-dichloroethane [75] and pathway for 1,2,3-trichloropropane utilization in bacteri- 1-chlorobutane [87], respectively, and represent a poten- um were made. Initially, the wild-type DhaA from tially cost-effective and efficient remediation strategy Rhodococcus sp. m15-3 was heterogously expressed in [75].

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Figure 8. Examples of the warfare agent sulfur mustard and common halogenated environmental pollutants catalyzed by HLDs.

3.4 Decontamination

The ability of DhaA, DmbA and LinB to degrade the warfare agent sulfur mustard (yperite, Fig. 8) has been reported [44, 89]. While spontaneous hydrolysis of sulfur mustard leads to formation of a sulfonium ion, an un- wanted reaction intermediate with adverse health ef- fects, dehalogenase hydrolysis of sulfur mustard proceeds via a non-toxic intermediate [44]. The application Figure 9. Examples of halogenated compounds detectable by biosensors of HLDs offers a safe and environmentally friendly alter- employing HLDs. native to chemical detoxification. The concept has been exploited in the enzyme-based decontamination mixture The subsequently constructed biosensors utilized Yperzyme Kit [90] developed by the company Enantis. cells immobilized directly on the sensing membrane of ion The major limiting factor of current technology is the poor selective electrode [92] or on the layer with a fluorescent water solubility of the target molecule. If sulfur mustard pH indicator applied on optical fiber [93, 94]. This concept is predissolved in an organic solvent miscible with water, has been commercialized by the company OptiEnz Sen- the agent readily dissolves in the mixture and rapid en- sors, which offers biosensors for the detection of 1,2- zymatic hydrolysis can occur [91]. However, the unsatis- dichloroethane, 1,2-dibromoethane, methylene chloride factory stability of the enzymes in the presence of organ- and other halogenated aliphatic hydrocarbons (Fig. 8 and ic co-solvents can limit such an application. Novel HLDs 9). These biosensors possess low detection limits and en- hydrolyzing sulfur mustard or the use of immobilized/en- able simultaneous detection of analytes using muti-chan- gineered enzymes with improved stability could elimi- nel optoelectronics. nate these limitations [44] (Prokop et al., patent applica- The first biosensor exploiting purified HLD was re- tion). ported recently [95]. The biosensor tip consists of a layer of the enzyme and the fluorescent pH indicator conjugat- 3.5 Biosensing ed to bovine serum albumin [98] (Bidmanova et al., unpublished data). The enzyme-based biosensor offers sig- Detection of chemicals using biosensors provides a sim- nificantly shorter measurement time compared to con- ple and rapid alternative to the traditional analytical tech- ventional methods, exhibits broad temperature and pH niques, such as gas and liquid chromatography, which working range and stability (Bidmanova et al., unpub- are time-consuming, expensive and not suitable for con- lished data). This sensing concept was exploited by tinuous measurements [92–95]. Initial attempts to con- the company Enantis in the biosensor EnviroPen, for struct biosensors for halogenated substrates (Supporting detection of common halogenated environ mental pollu- information, Table S6) led to a system with dehalogenat- tants, e.g. 1,2-dibromoethane, 3-chloro-2-(chloromethyl)- ing cells immobilized in polymer beads as a biological 1-propene and 1,2,3-trichloropropane (Figs. 7 and 9). component and ion selective electrodes as a transducer There is no natural form of HLD possessing sufficient ac- [96, 97]. tivity towards 1,2,3-trichloropropane, so the biosensor

suitable for the detection of this compound uses variant [102–105]. The covalent binding of a chlorinated ligand is DhaA31 (Supporting information, Table S6). enabled by the mutation of the catalytic base His272Phe, The performance of the biosensor is critically influ- which impairs the final hydrolysis step and leads to ir - enced by the selection of an appropriate immobilization reversible trapping of the alkyl-enzyme intermediate method for the biological component. The dehalogenat- (Fig. 1B) [104]. The synthetic ligands consist of a ing cells were encapsulated on the transducer [92–94, 96, chloroalkane linker attached to various fluorescent dyes, 97], while the purified enzyme was covalently bound [95]. affinity handles, or solid surfaces (Supporting information, Two previous reports on covalent immobilization of HLDs Table S7). described covalent binding of DhaA onto alumina support This innovative application of HLDs has been devel- impregnated by polyethyleneimine [67] and combination oped by the Promega Corporation, which has demon- of affinity and covalent binding of DhlA on iron oxide strated its successful application to fluorescence imaging nanoparticles [99]. In this case, cross-linking of LinB with [102–105]; protein expression and purification from E. coli glutaraldehyde was favorable due to the highest activity [107] and mammalian cells [108, 109]; protein expression retention and negligible leakage compared to other test- and immobilization from a cell-free extract [110]; and pro- ed methods (Bidmanova et al., submitted). tein-protein or protein-nucleic acid interaction studies Other critical characteristics of a biosensor are selec- (including arrays and chip technology) [111–115]. The tivity, sensitivity and functionality under operating HaloTag technology is currently protected by a number of conditions. Due to their wide substrate scope, individual patents, e.g. [106, 116–118] and has been already utilized HLDs are not specific to the particular substrate, but act in about 40 independent studies (see Supporting infor- within whole class of halogenated compounds [95]. mation). Limited selectivity of HLD-based biosensor can be im- To overcome a low affinity of the wild type DhaA to proved through protein engineering or using sensor long chlorinated ligands, saturation mutagenesis with arrays employing enzymes with different selectivity [94]. high-throughput fluorescence screening was employed Such a strategy is applicable for the detection of 1,2- [105, 106]. The beneficial mutations (Lys175Met, dichloroethane, which is converted only by DhlA [46]. Cys176Gly, and Tyr273Leu) were combined in a single The detection limits of current biosensors employing mutant, synergistically improving binding rate by four or- HLDs vary from 1.0 to 25 000 μg L–1 [92–95]. Although ders of magnitude, corresponding to the apparent sec- such detection limits can be sufficient for the monitoring ond-order rate constant 106 M–1 s–1 [105]. Good expression of contaminated sites, a lower limit of detection is re- of HaloTag fusion proteins in mammalian or E. coli cells quired for analysis of drinking water [94]. The limits of 1,2- was supported by removal of harmful sequence motifs dibromoethane, 1,2-dichloroethane, 1,2-dichloropropane, and optimization of codon usage according to human 1,2-dibromo-3-chloropropane and lindane in drinking codon bias, while avoiding low usage codons of E. coli water set by US Environmental Protection Agency range [118]. HaloTag was further engineered for maximal solu- from 0.05 μg L–1 to 5 μg L–1. To decrease the detection lim- ble expression of fusion proteins in E. coli using rational it, protein engineering needs to be applied. design and directed evolution [107]. This optimization While monitoring contaminants in the environment, yielded a fusion tag with increased stability called the temperature and pH under which the enzymatic re- HaloTag7. action proceeds strongly depends on properties of the application site, e.g. a normal groundwater system has a constant temperature derived from the average surface 4 Conclusions temperature and geothermal gradient, and pH in the range of 6.5 to 8 [100]. However, acidification caused by To date, 17 HLDs have been characterized and nine of industry can lead to pH values lower than 5.5 [101]. Novel them possess properties suitable for biotechnological ap- enzymes functional at low temperatures and pH would be plications, as described in section 2. HLDs have already an advantage for the preparation of biosensors suitable for been successfully used for laboratory-scale production of such environments. optically pure organocompounds; to neutralize sulphur mustard; for bioremediation of groundwater contaminat- 3.6 Cell imaging and protein analysis ed with 1,2-dichloroethane and biodegradation of hexachlorocyclohexane; in biosensors suitable for the detec- The wealth of knowledge on HLDs provided a theoretical tion of environmental pollutants; and for protein tagging. basis for the design of a versatile fusion tag (HaloTag) sys- Several other potential applications still require addition- tem, which can be used for both in vivo and in vitro analy- al research to solve limiting factors, such as low expres- sis of mammalian proteins [102–106]. The technology is sion, product inhibition, insufficient enzyme selectivity, based on irreversible covalent binding of miscellaneously low affinity and catalytic efficiency towards selected sub- labeled ligands by the mutant dehalogenase DhaA, which strates, or instability in the presence of organic co-sol- forms a genetically encoded fusion with a target protein vents. Recently introduced high-throughput methods for

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exploiting natural diversity and laboratory protein evolution have significantly contributed to the preparation of Jiri Damborsky is the Loschmidt Chair optimized HLDs. We propose this trend will continue in Professor at the Department of Experi- the foreseeable future and will further expand the practi- mental Biology and the Research cal usefulness of this fascinating enzyme family. Centre for Toxic Compounds in the En- vironment, Masaryk University, Brno, Czech Republic. He received his mas- The authors would like to thank T. Prudnikova, I. Kuta ter’s degree in 1993 and a PhD in Smatanova and P. Rezacova for providing the unpublished Micro biology in 1997 at Masaryk Uni- structure of DbeA for analysis of the enzyme’s active-site versity. His research interests lie in the parameters and to E. Chovancova and J. Brezovsky for field of protein engineering and synthetic biology. His research group their useful comments on the manuscript. The work was develops new concepts and software tools for the rational design of supported by the Grant Agency of the Czech Republic enzymes and bacteria with improved properties for biocatalysis, bio- (203/08/0114, P503/12/0572 and P207/12/0775), the Grant transformation and biosensing. He has published more than 100 origi- Agency of the Czech Academy of Sciences nal articles and co-authored two patents for the use of enzymes in de- (IAA401630901) and the European Regional Development contamination and biocatalysis. He is a co-founder of the biotechnolo- Fund (CZ.1.05/2.1.00/01.0001). The work of AP was sup- gy spin-off company Enantis and a holder of the EMBO/HHMI Scien- ported by Brno PhD Talent Scholarship provided by Brno tist award of the European Molecular Biology Organisation and the City Murlicipality. Howard Hughes Medical Institute. Zbynek Prokop and Jiri Damborsky are co-founders of Enantis. 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