The incorporation of several non-canonical amino acids into the Thermoanaerobacter thermohydrosulfuricus lipase confers not only activity enhancement upon treatment with organic solvents (by up to 450%) and surfactants (resp. 1630%), but also protective effects against protein reducing (resp. 140%), alkylating (resp. 160%), and denaturing (resp.190%) agents as well as inhibitors (resp. 40%). This approach offers novel chemically diversified biocatalysts for hostile environments.DFG, EXC 314, Unifying Concepts in Catalysi

Acevedo-Rocha, Carlos G.

Antranikian, Garabed

Budisa, Nediljko

Hoesl, Michael G.

Nehring, Sebastian

Royter, Marina

Wiltschi, Birgit

Wolschner, Christina

Catalysis Science & Technology

The incorporation of several non-canonical amino acids into the Thermoanaerobacter thermohydrosulfuricus lipase confers not only activity enhancement upon treatment with organic solvents (by up to 450%) and surfactants (resp. 1630%), but also protective effects against protein reducing (resp. 140%), alkylating (resp. 160%), and denaturing (resp.190%) agents as well as inhibitors (resp. 40%). This approach offers novel chemically diversified biocatalysts for hostile environments

Acevedo-Rocha, C.

Hoesl, M.

Nehring, S.

Royter, M.

Wolschner, C.

Wiltschi, B.

Antranikian, G.

Budisa, N.

MPG.PuRe

Non-canonical amino acids as a useful synthetic biological tool for lipase-catalysed reactions in hostile environments

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1198 Catal. Sci. Technol., 2013, 3, 1198--1201 This journal is c The Royal Society of Chemistry 2013Cite this: Catal. Sci. Technol.,2013,3, 1198Non-canonical amino acids as a useful syntheticbiological tool for lipase-catalysed reactions in hostileenvironments†Carlos G. Acevedo-Rocha,z*ab Michael G. Hoesl,zc Sebastian Nehring,zcMarina Royter,d Christina Wolschner,c Birgit Wiltschi,e Garabed Antranikiand andNediljko BudisacThe incorporation of several non-canonical amino acids into theThermoanaerobacter thermohydrosulfuricus lipase confers not onlyactivity enhancement upon treatment with organic solvents (by upto 450%) and surfactants (resp. 1630%), but also protective eﬀectsagainst protein reducing (resp. 140%), alkylating (resp. 160%), anddenaturing (resp.190%) agents as well as inhibitors (resp. 40%).This approach oﬀers novel chemically diversified biocatalysts forhostile environments.Lipases are the most important biocatalysts in organic chemistryand biotechnology, but often these do not display stabilitytowards hostile environments necessary for industrial transfor-mations.1–3 To solve this issue, several strategies include the useof additives,3 enzyme chemical modification/immobilization,2freeze-drying,3 or pretreatment with organic solvents.4 Directedevolution has also emerged as a reliable tool for proteinengineering,5–7 including random8 or structure-guided9approaches for improving stability of enzymes towards organicsolvents10 or other hostile environments.11 From the syntheticbiology point of view, however, it would be useful to introducealternative methods. For example, the global incorporation ofnon-canonical amino acids (ncAAs) into proteins could delivernovel interactions by modifying residue hydropathy, surfaceareas, polarities, ionisation states and covalent radii in a radicalmanner that the standard twenty canonical amino acids cannotalways achieve.12 These physical properties are important enzymaticactivity determinants in apolar solvents because at lower dielectricconstants proteins show stronger intra-protein electrostaticinteractions and more rigid structures.13 We recently reportedthat ncAAs with various chemical modifications dramaticallyaltered the functionality of a lipase from the thermostablebacterium Thermoanaerobacter thermohydrosulfuricus (TTL).14The observed eﬀects include among others enhanced hydrolyticactivity, activation eﬃciency, as well as substantial changes inoptimal temperature and pH values (Table S1, ESI†).In this communication, we report the influence of variousorganic solvents on the hydrolytic activity of TTL congeners(these proteins originate from the same parent gene sequence,but contain a fraction of ncAAs). In addition, we also exploredthe eﬀects of various metal cations, surfactants, protein reducing,alkylating and denaturing agents as well as inhibitors. Here weused the tryptophan (1) analogues 4-aminotryptophan (1a),4-fluorotryptophan (1b), and 7-azatryptophan (1c); and we alsoincluded the previous TTL congeners,14 containing the proline(2) analogues cis-4-fluoroproline (2a), trans-4-fluoroproline (2b),cis-4-hydroxyproline (2c), and trans-4-hydroxyproline (2d); thetyrosine (3) analogues ortho-fluorotyrosine (3a) andmeta-fluoro-tyrosine (3b); the methionine (4) analogues norleucine (4a) andazidohomoalanine (4b); and the phenylalanine (5) analoguesmeta-fluorophenylalanine (5a) and para-fluorophenylalanine(5b) (Scheme 1).The parent TTL and thirteen TTL congeners were expressedusing appropriate amino acid auxotrophic Escherichia coli strainsunder controlled culture conditions, followed by purification andanalytical characterization to assess ncAA incorporation eﬃciency(see ESI† and Table S1). As noted earlier,14 TTL is a lipase thatdisplays thermal activation, i.e., it requires heat to display a shift(Thermal Activation Factor or TAF) of 14-fold from its basal to itsmaximal activity (see Table S1, ESI†). Importantly, the enzymaticactivation most likely depends on the opening or closing of alid covering TTL’s active site (see Fig. S1 and discussion inaMax-Planck-Institut fu¨r Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mu¨lheim,Germany. E-mail: acevedor@kofo.mpg.deb Philipps-Universita¨t Marburg, Fachbereich Chemie, Hans-Meerwein-Straße,35032 Marburg, Germany. Tel: +49 (0)-6421-28-26082c Berlin Institute of Technology, Department of Chemistry, Biocatalysis Group,Franklinstrasse 29, 10587 Berlin, Germanyd Institute of Technical Microbiology, Hamburg University of Technology,Kasernenstrasse 12, 21073 Hamburg, Germanye ACIB, Petersgasse 14, 8010 Graz, Austria† Electronic supplementary information (ESI) available: TTL information and 3Dmodel, eﬀects of other additives, chemicals and strains, protein protocols,spectrophotometric assays, general and detailed properties of TTL[1a/1b/1c]congeners. See DOI: 10.1039/c3cy20712a‡ These authors contributed equally.Received 21st October 2012,Accepted 13th February 2013DOI: 10.1039/c3cy20712awww.rsc.org/catalysisCatalysisScience & TechnologyCOMMUNICATIONOpen Access Article. Published on 14 February 2013. Downloaded on 29/03/2016 13:54:36.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article OnlineView Journal  | View IssueThis journal is c The Royal Society of Chemistry 2013 Catal. Sci. Technol., 2013, 3, 1198--1201 1199Table S1, ESI†). Interestingly, while TTL[4a] is the only congenerthat does not require activation to be as active as parent TTL,other congeners such as TTL[1b/3a] require diﬀerent activationvalues (resp. 11/7-fold; Table S1, ESI†). Exceptionally, TTL[5a]combines a TAF of only 6-fold with a higher activity than parentTTL by 1.25-fold (Table S1, ESI†). These findings suggest that theincorporation of ncAAs may be a strategy to not only activatelipases, but also to confer them functional advantages. Whilecurrently we are trying to gain structural insight into theTTL congeners studied, we decided to gain more functionalknowledge by pre-treating these with organic solvents, metalions, surfactants, protein reducing, alkylating and denaturingagents as well as inhibitors. The congeners were thus incubatedwith the various substances for 1 hour at 25 1C, and the residualhydrolytic activity was then measured using p-nitrophenylpalmitate ester, as described in the ESI.†The treatment of lipases with organic solvents bothenhanced and impaired lipase activity (Table 1). In parentTTL, most solvents were deleterious (o0.8-fold), with pyridine,DMSO and toluol displaying the highest eﬀects (0.0-fold), incontrast to the slightly positive eﬀects of tert-butanol (1.1-fold)and hexadecane (1.3-fold; Table 1).In the case of the 1 (Trp) congeners, noteworthy are theminimal eﬀects on the activity of TTL[1a/1b/1c] after pyridinetreatment by 0.8 to 1.0-fold, in contrast to all congeners, exceptfor TTL[4b], also by 0.8-fold (Table 1). This suggests that thencAAs at the two unique Trp residues, which are located closeto the active site of TTL (Fig. S1, ESI†), may interact withpyridine to prevent lipase inactivation. These findings areimportant because pyridine is useful for the lipase-catalysedsynthesis of biodegradable polymers.15 Of interest is also thefinding that TTL[1c] can be more active than TTL[1a/1b] aftertert-butanol, isopropanol, acetone, DMF, methanol, acetonitrileand N-decyl-alcohol treatment (Table 1). Ethanol, heptane andisooctane did not significantly alter the activity of all Trpcongeners, but these were completely inhibited by DMSO andtoluol (Table 1). In contrast, TTL[1a] was slightly better thanTTL[1b/1c] only upon exposure to n-hexane and hexadecane,thoughminimally (Table 1). Interestingly, under normal conditions(without solvent treatment and taking account of TAF), TTL[1a] andTTL[1c], respectively, lost their maximal activities from 32 to 25and 10 mU mg1, whereas that of TTL[1b] was negligiblyaﬀected from 32 to 31 mU mg1 (Table S1, ESI†). These findingssuggest that 1b may facilitate the exposure of the active site forcatalysis, a clear advantage in aqueous environments, but notin non-aqueous ones. Although 1c/1a may trade-oﬀ the activityof TTL under standard conditions, the potential advantages ofusing these ncAAs in organic media are evident. It is thusunadvisable to heat-activate the congeners prior to organicsolvent exposure, since the likelihood of compromising theiractivity exists.Regarding 2 (Pro) congeners, the fluorinated ones, TTL[2a/2b],were generally more active than the hydroxylated ones, TTL[2c/2d],upon treatment with tert-butanol, ethanol, isopropanol, acetone,DMF, acetonitrile, n-hexane, hexadecane, isooctane and n-decyl-alcohol (Table 1). Like parent TTL, all Pro congeners practicallydid not resist the eﬀects of pyridine, DMSO, methanol andtoluol, as no residual lipase activity was detected (Table 1). Underaqueous conditions, however, the original lipase activity wasaﬀected after maximal heat activation from 32 to 8.3/6.3/6.6/10.4 mU mg1 in TTL[2a/2b/2c/2d], resp. (Table S1, ESI†).Although the Pro congeners were marginally active in water,the ncAAs conferred them a selective advantage upon organicmedia treatment, TTL[2b] being the one with the highest eﬀect byup to 4.5-fold in tert-butanol (Table 1), an important co-solvent forthe lipase-catalysed production of biodiesel.16 Another practicalexample is TTL[2a] that displays the highest activity after treat-ment with isooctane (1.6-fold), another important co-solvent forScheme 1 Canonical and non-canonical amino acids used.Table 1 Residual lipase activity of TTL congeners upon treatment with variouswater miscible and non-miscible organic solventsa TTL congeners were incubated for 1 hour at 25 1C with 90% of organicsolvent. b To correct for p-nitrophenol auto-hydrolysis due to chemicalagent exposure and buﬀer, samples without lipase and without solventwere, respectively, included. Lipase activity was measured in triplicatefor all cases at the optimal temperature and pH 8.0 as described in theESI. To assess the sole eﬀect of solvents, the values were normalized to1.0 for each TTL. c Negative eﬀects are displayed on a white background(less than 1.0), whereas neutral and positive eﬀects are shown on light(1.0) and dark (more than 1.0) grey backgrounds, resp. DMSO: dimethylsulfoxide; DMF: dimethylformamide; NDA: N-decyl-alcohol.Communication Catalysis Science & TechnologyOpen Access Article. Published on 14 February 2013. Downloaded on 29/03/2016 13:54:36.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Online1200 Catal. Sci. Technol., 2013, 3, 1198--1201 This journal is c The Royal Society of Chemistry 2013the lipase-based synthesis of biodiesel from waste oils asfeedstocks.16The most prominent feature of globally substituting theseven 3 (Tyr) residues (Fig. S1, ESI†) in TTL by fluorinatedanalogues is the dependence of activity on the position of thering substitution. Under aqueous conditions, the fluorinationat themeta-position (3b) markedly aﬀected the maximal activity(3.7 mU mg1), whereas fluorination at the ortho-position (3a)did not (30.3 mU mg1), the latter being very close to that ofparent TTL (32 mU mg1; Table S1, ESI†). The main functionaldiﬀerence, under standard conditions, between TTL andTTL[3a] is a TAF of 14 and 7, resp. (Table S1, ESI†). However,TTL[3b] displayed more activity than TTL and TTL[3a] afterpre-treatment with all organic solvents, except for methanol inthe latter case (Table 1). Remarkably, TTL[3a] reveals exactly thesame patterns as parent TTL after organic solvent treatment –activity enhancement with tert-butanol and hexadecane butvarious degrees of impairment with all the other solvents(Table 1). Curiously, parent TTL and TTL[3a] have the sameoptimal pH and temperature as well as similar maximal activityupon heat activation with diﬀerent TAF values, in contrast toTTL[3b] with a 20 1C shift in optimal temperature and lossof activity under aqueous conditions (Table S1, ESI†). ThatTTL and TTL[3a] have the same patterns in their functionalproperties, while TTL[3a] and TTL[3b] exhibit opposite ones inspite of the ‘‘subtle’’ structural diﬀerences between 3a and 3b(ring substitution of fluorine) deserves further attention.Regarding the 4 (Met) congeners, TTL[4a] does not requireheat activation under standard conditions to show maximalactivity, but TTL[4b] needs a much higher thermal activation toreach 1/3 of that of TTL[4a] and parent TTL (Table S1, ESI†).Based on these findings, and on the location of the three out ofthe eleven Met residues in the lid (Fig. S1, ESI†), we suggestedbefore that the hydrophobic character of 4a may have retainedthe TTL’s lid in an open conformation, whereas 4b, hydrophilicin nature, may have blocked lid opening.14 If this may beplausible, then TTL[4a] would have its active site more exposedand, thus, it would be more vulnerable to organic solventtreatment, in contrast to TTL[4b]. Interestingly, TTL[4a] andTTL[3b] display very low resistance to most organic solvents,similar to parent TTL, but TTL[4b] was less active in most ofthem, except after treatment with pyridine (Table 1). ThatTTL[4b] has such a low catalytic activity may be due to a completeblockage of the lid opening. However, further structural investiga-tions are needed, to clarify this issue.In the case of the 5 (Phe) congeners, upon maximal heatactivation and under standard conditions, TTL[5a] was themost active of all lipases, whereas TTL[5b] lost 2/3 activity(Table S1, ESI†). In contrast, TTL[5b] performed better thanTTL[5a] after tert-butanol, acetone, DMF, acetonitrile and hexa-decane treatment, but both were equal regarding all the othersolvents (Table 1). The high number of Phe residues (sixteen) inTTLmight indicate a functional importance, as many are located inand near the lid as well as the active site (Fig. S1, ESI†).All congeners were also pre-incubated with several metalions because it is known that the latter ones can influencelipase activity.17 Unfortunately, the activity of all congeners,including parent TTL, was impaired by the treatment withall tri- and divalent cations, as well as Ag+, but not by themono-cations K+ and Na+ (Table S2, ESI†). This could beexplained by the possible interaction of the tri- and divalentions with the C-terminal His-Tag, which can be inferred fromdivalent copper, known to have aﬃnity for histidine, since non-His-tagged TTL is indiﬀerent to CuCl2 pre-treatment,18 whereasTTL with a His-Tag lost its activity by 2/3 (Table S2, ESI†).We also examined lipase activity of the TTL congeners upontreatment with surfactants, protein denaturing, reducing andalkylating agents as well as inhibitors (Table S3, ESI†). Ofinterest is the surfactant CHAPS, which enhanced the activityof almost all lipases, TTL[2d] being the most benefited by up to16.3-fold vs. 1.7-fold of TTL. Similarly, upon treatment with thedenaturant guanidium chloride TTL[1a/5a] resisted 0.7-fold vs.no activity of TTL, while the activity of TTL[1c] increased by1.9-fold vs. 0.9-fold of TTL after urea exposure (Table S3, ESI†).Finally, the protein inhibitor pefabloc inactivated almost allcongeners, except for TTL[2a/3b], which displayed a residualactivity of 0.4-fold (Table S3, ESI†). Importantly, the ringsubstituent is critical to confer partial ‘‘immunity’’ as TTL[2b/3a]was completely inactivated.In summary, the incorporation of ncAAs into this particularlipase not only enhanced its hydrolytic residual activity upontreatment with organic solvents and surfactants, but also conferredit protective eﬀects against protein denaturing, alkylating,denaturing, and inhibitory substances. Our data not only showthe potential advantages of using ncAAs in lipase-catalyzedreactions in hostile environments, but also highlight thechallenges ahead to meet the required traits such as selectivityfor practical biotransformations.This work was supported by the UniCat Excellence Cluster inBerlin as well as by the BioFuture Program and the Biokatalyse2021 cluster, both of the German Federal Ministry for Educationand Research.Notes and references1 U. T. Bornscheuer and R. J. Kazlauskas, Hydrolases in OrganicSynthesis: Regio- and Stereoselective Biotransformations, Wiley-VCH, Weinheim, 2005.2 K. M. Polizzi, A. S. Bommarius, J. M. Broering andJ. F. Chaparro-Riggers, Curr. Opin. Chem. Biol., 2007, 11,220–225.3 A. L. Serdakowski and J. S. Dordick, Trends Biotechnol., 2008,26, 48–54.4 M. Matsumoto, K. Kida and K. Kondo, J. Chem. Technol.Biotechnol., 2001, 76, 1070–1073.5 N. J. Turner, Nat. Chem. Biol., 2009, 5, 567–573.6 M. T. Reetz, Angew. Chem., Int. Ed., 2011, 50, 138–174.7 U. T. Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz,J. C. Moore and K. Robins, Nature, 2012, 485, 185–194.8 J. C. Moore and F. H. Arnold, Nat. Biotechnol., 1996, 14, 458–467.9 M. T. Reetz, P. Soni, L. Fernandez, Y. Gumulya andJ. D. Carballeira, Chem. Commun., 2010, 46, 8657–8658.Catalysis Science & Technology CommunicationOpen Access Article. Published on 14 February 2013. Downloaded on 29/03/2016 13:54:36.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article OnlineThis journal is c The Royal Society of Chemistry 2013 Catal. Sci. Technol., 2013, 3, 1198--1201 120110 R. J. Kazlauskas and H. K. Weber, Curr. Opin. Chem. Biol.,1998, 2, 121–126.11 V. G. Eijsink, S. Gaseidnes, T. V. Borchert and B. van denBurg, Biomol. Eng., 2005, 22, 21–30.12 N. Budisa, Angew. Chem., Int. Ed., 2004, 43, 6426–6463.13 A. M. Klibanov, Nature, 2001, 409, 241–246.14 M. G. Hoesl, C. G. Acevedo-Rocha, S. Nehring, M. Royter,C. Wolschner, B. Wiltschi, N. Budisa and G. Antranikian,ChemCatChem, 2011, 3, 213–221.15 M. Kitagawa and Y. Tokiwa, Biotechnol. Lett., 1998, 20,627–630.16 Z. Jin, S. Y. Han, L. Zhang, S. P. Zheng, Y. Wang and Y. Lin,Bioresour. Technol., 2012, 130C, 102–109.17 R. Gupta, N. Gupta and P. Rathi, Appl. Microbiol. Biotechnol.,2004, 64, 763–781.18 M. Royter, M. Schmidt, C. Elend, H. Hobenreich, T. Schafer,U. T. Bornscheuer and G. Antranikian, Extremophiles, 2009,13, 769–783.Communication Catalysis Science & TechnologyOpen Access Article. Published on 14 February 2013. Downloaded on 29/03/2016 13:54:36.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Online

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Non-canonical amino acids as a useful synthetic biological tool for lipase-catalysed reactions in hostile environments

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