research article purification and characterization of

Hindawi Publishing CorporationEnzyme ResearchVolume 2013 Article ID 670702 6 pageshttpdxdoiorg1011552013670702

Research ArticlePurification and Characterization of Phenylalanine AmmoniaLyase from Trichosporon cutaneum

Andrea Goldson-Barnaby12 and Christine H Scaman1

1 Food Nutrition and Health University of British Columbia 2205 East Mall Vancouver BC Canada V6T 1Z42Department of Chemistry University of the West Indies Kingston Jamaica

Correspondence should be addressed to Christine H Scaman christinescamanubcca

Received 25 June 2013 Accepted 13 August 2013

Academic Editor Qi-Zhuang Ye

Copyright copy 2013 A Goldson-Barnaby and C H Scaman This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Trichosporon cutaneum phenylalanine ammonia lyase was selected as amodel to investigate the dual substrate activity of this familyof enzymes Sequencing of the PAL gene identified an extensive intron region at the N-terminus Five amino acid residues differingfrom a prior report were identified Highest Phe Tyr activities (16 plusmn 03 04 plusmn 01 120583molh g wet weight) were induced by Tyr Theenzyme has a temperature optimum of 32∘C and a pH optimum of 8ndash85 and shows no metal cofactor dependence Michaelis-Menten kinetics (Phe 119870

119898

50 plusmn 11mM) and positive allostery (Tyr 1198701015840 24 plusmn 06mM Hill coefficient 19 plusmn 05) were observedAnion exchange chromatography gave a purification fold of 50 with 20 yield The His-Gln motif (substrate selectivity switchregion) indicates the enzymersquos ability to act on both substrates

1 Introduction

Phenylalanine ammonia lyase (PAL EC 43124) catalyzesthe conversion of phenylalanine to trans-cinnamic acid asa step in the phenylpropanoid pathway of plants and in theformation of secondary products of metabolism in somemicroorganisms [1 2] In some instances the enzyme alsoconverts tyrosine to para-hydroxycinnamic acid These dualsubstrate enzymes are classified as phenylalaninetyrosineammonia lyases (EC 43125) Enzymes with a greater cat-alytic efficiency for tyrosine are known as tyrosine ammonialyase (TAL EC 43123) There are no known genes that codefor a lyase that has activity exclusively with tyrosine

Interest in PAL is from two perspectives First thestructural features of the enzyme responsible for its substratespecificity have not been fully elucidated Second a moreselective and efficient TAL is of interest for industrial appli-cations The specificity of PAL for phenylalanine relative totyrosine varies by over 106 between biological sources andtypically the efficiency of Phe turnover is higher than Tyr[3 4] An understanding of the basis for this astounding rangeof substrate preference is required to rationally engineer an

efficient tyrosine-specific enzyme for use in the synthesisof p-hydroxycinnamic acid for industrial applications [5]Themicrobial production of aromatic chemicals continues toincrease as it allows for the use of greener technologies andrenewable energy sources [6]

PAL has been extensively characterized from a widevariety of plant sources [7] but only a few microbial sourcesof the enzyme have been investigated [8 9] PAL fromthe yeast Trichosporon cutaneum (TcPAL) identified as anenzyme able to metabolize both Phe and Tyr and possessinga relatively high level of activity with tyrosine was selectedas a model to further investigate the dual substrate activityof this family of enzymes Several novel findings were notedand are reported herein The cloned gene was found to havea single intron region near the N-terminus of the enzymeand five amino acid residues that differed from a previousreport [6] A His-Gln motif was identified which appears tobe a characteristic feature of PAL enzymes displaying dualsubstrate activity with tyrosine and phenylalanine This isthe first reported characterization of the TcPAL enzyme withregard to its temperature and pH optimum as well as metaldependence

2 Enzyme Research

2 Materials and Methods

21 Cloning of the TcPAL Gene Escherichia coli bacterialstrains were cultured in Luria Bertani (LB Mediatech Hern-don VA) liquid medium Kanamycin (50120583gmL) was addedto LB media and plates when growing bacteria containingplasmids

The yeast strain Trichosporon cutaneum (ATCC 58094)was purchased from the American Type Culture Collection(Manassass VA) Shake cultures were grown aerobically inDagleyrsquos medium at 30∘C at 100 rpmwith addition of tyrosine(20mM) at 30∘C to an OD600 of 3 Cells were pelleted bycentrifugation (9000 xg 15min 4∘C) Genomic DNA wasextracted utilizing aQiagenDNApurification kit and utilizedas template for PCR amplification

Primer sequences were designed based on the DNAsequence of the T cutaneum PAL protein The PAL genewas PCR amplified using Phusion DNA polymerase (NewEngland Biolabs) from T cutaneum genomic DNA withthe 51015840 primer TcPAL- F 51015840-CGCGAATTCATGTTTATTGAGACC-31015840 as the forward primer (EcoRI site underlined)and 31015840 primer TcPAL- R 51015840-GAAGCTTTTAGAACATCTT-GCCAAC-31015840 as the reverse primer (HindIII site underlined)Control reactions were also performed The amplified PCRfragment was purified digested with HindIII and EcoRI andinserted in the pET30a vector to give plasmid pET-30a (+)TcPAL E coli DH5120572 competent cells were transformed withthe plasmid construct by use of heat shock treatment Positiveclones were identified by restriction analysis and plasmidDNA was submitted for DNA sequencing

Database comparison was performed with the BLASTsearch tools on the server of the National Center for Biotech-nology Information National Library of Medicine NIH(httpwwwncbinlmnihgov) Multiple sequence align-ments were performed using ClustalW Intron analysis wasperformed using GENSCAN

22 Cell Culture and Extraction Shake cultures of T cuta-neum were grown aerobically in Dagleyrsquos medium at 30∘Cat 100 rpm with addition of tyrosine (20mM) at 30∘C toan OD600 of 3 Cells were harvested by centrifugation at23500 xg for 15min at 4∘C The cell pellet was resuspendedin extraction buffer (50mM Tris-HCl pH 80 1mM EDTA10mM 2-mercaptoethanol protease inhibitor cocktail tabletsEDTA free and 1 tablet per 100mL) in a 1 4 ratio (wetweight buffer) and the cells were disrupted by vortexingwith glass beads for 6min with intermittent cooling on iceAfter centrifugation aliquots of supernatant were desaltedusing a PD-10 column (GE Healthcare) and used for enzymepurification Protein concentration was determined by theBradford assay [10]

23 Enzyme Assays The PALTAL activity of enzymeextracts was measured using a spectrophotometric assay [11]For preliminary induction studies the enzyme activity wasdetermined using an end point assay using Phe (40mM) orTyr (2mM) in 10mM Tris-HCl (pH 85) at a temperatureof 37∘C for 15min The reaction was terminated by the

addition of 25 trichloroacetic acid (200120583L) Samples werecentrifuged at 13000 xg for 15min and the absorbance wasmeasured All other experiments were conducted utilizinga continuous spectrophotometric assay at a temperature of30∘C for 2min Assays were initiated by addition of enzymeextract (200120583L) to substrate solution (800 120583L) preequili-brated at 30∘C For kinetic analysis solutions containing Phe(05mM to 100mM) or Tyr (05mM to 10mM) in 10mMTris-HCl (pH 80) were utilized The same enzyme extractwas used to compare the kinetic constants for PAL andTAL activities The absorbance of trans-cinnamic acid wasmeasured at 290 nm whereas para-hydroxycinnamic acidwas measured at 315 nm Kinetic data was analyzed usingGraphPad Statistical Software (Version 5 California) whichcombines biostatistics curve fitting (nonlinear regression)and scientific graphing into a comprehensive program

24 PAL Purification and Characterization All purificationsteps were carried out at 4∘C Crude cell free extract of Tcutaneum from PD-10 columns was applied to a Fast ProteinLiquidChromatography anion exchangeHiTrapQSepharosecolumn (25 times 10 cm) pre-equilibrated with Tris-HCl buffer(01M pH80)The adsorbed enzymewas elutedwith a lineargradient of KCl from 0 to 03M in the same buffer [12]Fractions with PAL activity were pooled and exchanged withextraction buffer using Millipore centrifugal filter units witha 30 kDa molecular cutoff

The temperature optimumof the enzymewas determinedby performing enzyme assays at 28∘C 32∘C 36∘C and 40∘Cwith Tris-HCl (50mM pH 80) as buffer The pH optimumwas determined by conducting enzyme assays at 32∘C withTris-HCl (50mM) adjusted to 73 76 82 86 and 89 Metaldependence studies were performed using the chloride saltsof sodium potassium magnesium and ferrous at a total saltconcentration of 100mM inTris-HCl buffer (50mM pH80)

3 Results and Discussion

31 Sequencing of the PAL Gene The PAL gene from Tcutaneum was amplified from genomic DNA using PCRA 2114 bp fragment size was generated cloned into thepET30a vector and submitted for sequence analysisThe genesequence showed high similarity with other PAL enzymesin the NCBI database Protein blast analysis revealed thatthe PAL shares identity with the yeast PALs Trichosporonasahii (70) Rhodosporidium toruloides (50) Rhodotorulamucilaginosa (49) and Rhodotorula glutinis (48)

Sequencing of the TcPAL gene from two independentclones revealed five amino acid residues that differ from thesequence previously reported by Breinig et al [6] namelyGln 74 Ala 274 Val 298 Pro 322 and Lys 486 A comparisonof our TcPAL sequence with the closely related Trichosporonashaii PAL revealed that the residues at positions 74 274and 486 were identical (Table 1) However the residue equiv-alent to 298 matched the Ala residue previously reportedrather than the Val in our sequence There was no obviousconsensus for the amino acid residue corresponding to Pro322 for the three proteins Analysis of the T cutaneum gene

Enzyme Research 3

Table 1 Amino acids reported for Trichosporon cutaneum PAL andequivalent residues identified in T ashaii PTALa

PTAL source Residueb

T cutaneum (current work) Q74 A274 V298 P322 K486

T cutaneum (ABA698981) R74 V274 A298 S322 R486

T ashaii (EKD033381) Q129 A327 A351 E375 K539aGenbank number in bracketsbBlue polar uncharged residues red positively charged green negativelycharged black polar purple proline

sequence byGENESCANutilizing the parametermatrices forArabidopsis and maize predicted the presence of an intronwithin the TcPAL gene sequence at position 121 through 1182(1062 bp) which was confirmed with the translated proteinsequence

In the substrate selectivity switch region of PAL TALenzymes a His-Leu motif is characteristic of TALs whereasPhe-Leu is characteristic of PALs [3] In TcPAL His-Leu wasreplaced by His-Gln This motif was also observed in othermicrobial PAL enzymes (Figure 1) Other PTAL enzymeswith the His-Gln motif possess dual substrate activity to Pheand Tyr with a 119870

119898PheTyr ratio greater than one Examplesinclude T cutaneum with ratios of 32 or 82 (current work[13]) R toruloides ratio 16 [14] R glutinis ratio 23 and 19[9 15] R mucilaginosa ratio 20 [13] and R graminis ratio29 [13] Activity with Tyr has not been previously reportedfor T asahii but based on this motif it is predicted to possessboth PAL and TAL activities In addition to the His-Glnselectivity switch region observed in the protein sequenceof these enzymes other residues surrounding this motifwere conserved specifically Glu His Gln Leu Cys Residuesforming the hydrophobic substrate binding pocket describedbyWatts et al [3] were also conserved (Leu 194 Leu 242 andVal 245)

32 PAL Purification and Characterization PAL purificationusually requires multiple steps to obtain a homogenouspreparation [16] Due to the enzymersquos relative instabilitya rapid purification procedure is desirable Purification ofTcPAL by acid precipitation aqueous two-phase partition-ing and anion exchange chromatography was investigated(Table 2) TcPAL was obtained with an approximate yield of20 and 50-fold purification from the HiTrap Q Sepharosecolumn (Figure 2) SDS PAGE of the eluted protein revealedthe presence of the desired band at 79 kDa confirmed byWestern blot (not shown) and two other unidentified bandsat 67 kDa and 31 kDa PAL is typically tetrameric with fouridenticalmonomers ranging from 77 to 83 kDa [17]This two-step procedure is rapid and efficient in comparison to thefour-step procedure reported by Vannelli et al [13] whichproduced a purification fold of 14 and an overall yield of6 Vannelli et al [13] used a quaternized polyethyleneimineanion exchange column resulting in a 4-fold purificationThetheoretical pI of TcPAL reported by Vannelli et al [13] was63 while the pI of the enzyme as determined from isoelectricfocusing analyses was 58 TcPAL from the present study has

lowast lowast

QKELIRFLNAGIFG 143QKELIRFLNAGVFG 141QKELIRFLNAGIFG 151QRELIRFLNAGAFG 140QRELIRFLNAGAFG 142QRELIRFLNAGAFG 139QRELIRFLNAGAFG 127QKELIRFLNAGVFG 154QTNLVWFLKTGAG- 113QTNLIWFLKSGAG- 113QKALLEHQLCGVLP 144QKALLEHQLCGVLP 226QKALLEHQLCGVLP 150QKALIEHQLCGVTP 150QKSLLEHQLCGVLP 40QLSLLEHQLCGFLP 175QISLLEHQLCGFLP 123QANLVHHLASGVGP 96GGSLISHLGTGQGA 94

PcPAL

NtPAL

AtPAL

ZmPAL

BoPAL

OsPAL

TaPAL

GbPAL

AvPAL

NpPAL

RtPAL

RgPAL

RmPAL

RrPAL

SsPAL

TsPAL

TcPAL

RsTAL

SeTAL

Figure 1 Substrate selectivity switch region (residues in bold)identified in PAL and TAL enzymes FL is indicative of PALsHQ for PALTALs and HL for TAL enzymes Abbreviations withUniprot or Genbank number Pc Petroselinum crispum (P45729)Nt Nicotiana tabacum (P25872) At Arabidopsis thaliana (P35510)Bo Bambusa oldhamii (C0LL35) Zm Zea mays (B6SWA0) OsOryza sativa (A2X7F7) Ta Triticum aestivum (Q43210) GbGinkgobiloba (A7UHB6) Av Anabaena variabilis (Q3M5Z3) Np Nostocpunctiforme (B2J528) Rt Rhodosporidium toruloides (P11544) RgRhodotorula glutinis (G0SVG1) Rm Rhodotorula mucilaginosa(P10248) Rr Rhodotorula graminis (CAD238281) Ss Sporidiobolussalmonicolor (E0YNE2) Ts Trichosporon asahii (EKD033381) TcTrichosporon cutaneum (ABA698981) Rs Rhodobacter sphaeroides(Q3IWB0) and Se Saccharothrix espanaensis (Q2EYY5)

a theoretical pI of 62 because of the sequence differenceswe obtained which may account for the difference observedin the behaviour of the enzyme during anion exchangechromatographyThe enzyme has a pH optimum in the rangeof 80ndash85 (Figure 3) and a temperature optimum of 32∘C(Figure 4) and shows no metal dependence on the chloridesalts of sodium potassium magnesium and ferrous

TcPAL exhibited the highest activity with Phe Tyr (16 plusmn03 04 plusmn 01 120583molh g wet weight) when induced by tyrosine(2mM) Control and phenylalanine (2mM) induced culturespossessed lower activities (Phe Tyr 09 plusmn 003 02 plusmn 01 and04 plusmn 02 03 plusmn 01 120583molh g wet weight resp) while noactivity was found when 2mM glucose was in the growthmedia consistent with previous reports [17]

Purified TcPAL displayed typical Michaelis-Mentenkinetics with Phe but atypical Michaelis-Menten kineticswere the best fit with Tyr (Figure 5) Vannelli et al [13] alsoreported typical Michaelis Menten kinetics with Phe (119870

119898

49 plusmn 09mM) and positive allostery with Tyr (1198701015840 06mMHill coefficient 26 plusmn 04) comparable to the findings ofthis present study Interestingly the yeast PALs from Rtoruloides R glutinis and R mucilaginosa with high proteinsequence identity to TcPAL appear to follow Michaelis-Menten kinetics with both substrates Positive allostery may

4 Enzyme Research

Table 2 Purification of the PAL enzyme from Trichosporon cutaneuma

Method Total activityb (U) Total protein (mg) Specific activity (Umg) Purification fold Yield ()Anion exchange

Crude 005 plusmn 001 038 plusmn 009 015 plusmn 000 mdash 100Post 001 plusmn 000 0001 plusmn 000 739 plusmn 073 496 20

Acid precipitationCrude 016 plusmn 003 104 plusmn 074 020 plusmn 010 mdash 100Post 015 plusmn 004 061 plusmn 028 026 plusmn 006 13 94

Aqueous two-phasec

Crude 007 plusmn 003 039 plusmn 014 021 plusmn 005 mdash 100PEGNa2SO4 002 plusmn 000 008 plusmn 000 033 plusmn 003 15 28PEG(NH4)2SO4 002 plusmn 000 010 plusmn 008 010 plusmn 004 05 28PEG Na2CO3 001 plusmn 000 031 plusmn 004 002 plusmn 001 01 14

aResults are the average plusmn range of two independent trials Crude samples are cell culture extracts obtained from a PD-10 column b1 U of activity = 1120583moL ofreleased tran-cinnamic acid per min cFor aqueous two-phase partitioning the same crude enzyme extraction was used for each of the PEGsalt systems

KCI elution gradient (0 to 03M)Relative absorbanceActivity

79kDa67kDa

31kDa

250130100

7055352515

10(kDa) A B C

0006000500040003000200010

Activ

ity (U

)

0 2 4 6 8 10 12 14 16Volume (mL)

435

325

215

105

0minus05

Rela

tive a

bsor

banc

e(280

nm)

1U of activity = 1120583moL of released trans-cinnamic acid per minute

Figure 2 Chromatographic profile illustrating the purification ofthe PAL enzyme from Trichosporon cutaneum extract by anionexchange chromatography Insert SDS PAGE of Trichosporon cuta-neum PAL enzyme Lane A protein ladder B crude extract Cpurified protein from anion exchange chromatography T cutaneumPAL at 79 kDa

be a characteristic feature of the Trichosporon species and itis possible that Trichosporon asahii PAL may share similarenzyme kinetic characteristics to that of TcPAL

Vannelli et al [13] reported a PALTAL ratio((119881max119870119898)Phe(119881max119870

1015840

)Tyr) of 08 while we observeda ratio of 22 (Table 3) This implies that the enzyme isintrinsically a PAL and not a TAL enzyme The His-Glnmotif observed in the substrate selectivity region of theprotein sequence further substantiates this claim

4 Conclusion

Five different amino acid residues not previously reportednamely Gln 74 Ala 274 Val 298 Pro 322 and Lys 486were identified from translation of the TcPAL gene sequence

15

1

05

0

Rela

tive a

ctiv

ity

PheTyr

73 76 79 82 85 88pH

Figure 3 pH dependence studies on the PAL enzyme from the yeastTrichosporon cutaneum A pH optimum of 82 was observed

15

1

05

0

Rela

tive a

ctiv

ity

PheTyr

28 32 36 40

Temperature (∘C)

Figure 4 Temperature dependence studies on the PAL enzymefrom the yeast Trichosporon cutaneum A temperature optimum of32∘C was observed

Enzyme Research 5

Table 3 Kinetic parameters for purified extracts of the PAL enzyme from Trichosporon cutaneum

Trial Phenylalanine Phenylalanine Phenylalanine Tyrosine Tyrosine Tyrosine PALTAL ratio119881max (120583Mminminus1) 119870

119898

(mM) 119881max119870119898 119881max (120583Mminminus1) 1198701015840 (mM) 119881max119870

1015840

1 13 plusmn 01 50 plusmn 11 03 02 plusmn 002 24 plusmn 06 01 302 47 plusmn 06 57 plusmn 30 08 10 plusmn 01 20 plusmn 04 05 163 72 plusmn 08 42 plusmn 20 17 08 plusmn 01 09 plusmn 04 09 19For each trial activity assays for Phe and Tyr were performed on the same enzyme extract Extracts were obtained from anion exchange chromatography

15

1

05

00 20 40 60 80 100

Phenylalanine (mM)

Velo

city

(120583M

min

minus1)

(a)

025

020

015

010

005

0000 2 4 6 8 10

Tyrosine (mM)

Velo

city

(120583M

min

minus1)

(b)

Figure 5 Purified Trichosporon cutaneum PAL kinetics (a) Phenylalanine119870119898

50 plusmn 11mM and 1198772 = 093 (b) Tyrosine1198701015840 = 24 plusmn 06mM119867 = 19 plusmn 05 1198772 = 099 Kinetic data was analyzed using GraphPad Statistical Software

The His-Gln motif identified in the substrate selectivityswitch region of TcPAL may potentially be utilized to assistin assigning other uncharacterizedmicrobial ammonia lyasesin the genomic database as PAL-specific enzymes possess-ing activity with tyrosine TcPAL exhibits greater catalyticefficiency for Phe over Tyr although the greatest inductionof enzyme activity occurred in the presence of Tyr Weconfirmed that the enzyme exhibits positive allostery withTyr in contrast toMichaelisMenton kineticswith PheA two-step purification of the enzyme was achieved by applying acrude cell extract on anion exchange chromatography (20yield 50-fold purification)

Acknowledgments

This work was supported by an NSERC Discovery grantto Christine H Scaman and a Canadian CommonwealthScholarship and Wilson Henderson Fellowship to AndreaGoldson-Barnaby

References

[1] K R Hanson and E A HavirThe Biochemistry of Plants vol 7Academic Press New York NY USA 1981

[2] J S Williams M Thomas and D J Clarke ldquoThe gene stlAencodes a phenylalanine ammonia-lyase that is involved inthe production of a stilbene antibiotic in Photorhabdus lumi-nescens TT01rdquo Microbiology vol 151 no 8 pp 2543ndash25502005

[3] K TWatts BNMijts P C Lee A JManning andC Schmidt-Dannert ldquoDiscovery of a substrate selectivity switch in tyrosineammonia-lyase a member of the aromatic amino acid lyasefamilyrdquo Chemistry and Biology vol 13 no 12 pp 1317ndash13262006

[4] M C Moffitt G V Louie M E Bowman J Pence J P Noeland B S Moore ldquoDiscovery of two cyanobacterial phenylala-nine ammonia lyases kinetic and structural characterizationrdquoBiochemistry vol 46 no 4 pp 1004ndash1012 2007

[5] W W Qi T Vannelli S Breinig et al ldquoFunctional expressionof prokaryotic and eukaryotic genes in Escherichia coli for con-version of glucose to p-hydroxystyrenerdquoMetabolic Engineeringvol 9 no 3 pp 268ndash276 2007

[6] S Breinig W W Qi F Sariaslani T Vannelli and Z XueldquoUnited States Patent US6951751rdquo 2005

[7] D H Jones ldquoPhenylalanine ammonia-lyase regulation of itsinduction and its role in plant developmentrdquo Phytochemistryvol 23 no 7 pp 1349ndash1359 1984

[8] M J MacDonald and G B DrsquoCunha ldquoA modern view ofphenylalanine ammonia lyaserdquo Biochemistry and Cell Biologyvol 85 no 3 pp 273ndash282 2007

[9] Z Xue M McCluskey K Cantera A Ben-Bassat F SSariaslani and L Huang ldquoImproved production of p-hydroxycinnamic acid from tyrosine using a novel thermostablephenylalaninetyrosine ammonia lyase enzymerdquo Enzyme andMicrobial Technology vol 42 no 1 pp 58ndash64 2007

[10] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

6 Enzyme Research

[11] C W Abell and R Shen ldquoPhenylalanine ammonia-lyase fromthe yeast rhodotorula glutinisrdquoMethods in Enzymology vol 142no C pp 242ndash248 1987

[12] R I Monge M Lara and A Lopez-Munguia ldquoPurification andstabilization of phenylalanine ammonia lyase from Sporidiobo-lus pararoseusrdquo Biotechnology Techniques vol 9 no 6 pp 423ndash428 1995

[13] T Vannelli Z Xue S Breinig W W Qi and F S SariaslanildquoFunctional expression in Escherichia coli of the tyrosine-inducible tyrosine ammonia-lyase enzyme from yeast Tri-chosporon cutaneum for production of p-hydroxycinnamicacidrdquo Enzyme and Microbial Technology vol 41 no 4 pp 413ndash422 2007

[14] M Ameyama E Shinagawa K Matsushita and O AdachildquoGrowth stimulation of microorganisms by pyrroloquinolinequinonerdquo Agricultural and Biological Chemistry vol 48 no 11pp 2909ndash2911 1984

[15] AAGatenby F S Sariaslani X TangWWQi andTVannellildquoUS Patent 6368837rdquo 2002

[16] R R Fritz D S Hodgins and C W Abell ldquoPhenylalanineammonia-lyase Induction and purification from yeast andclearance inmammalsrdquoThe Journal of Biological Chemistry vol251 no 15 pp 4646ndash4650 1976

[17] H J Gilbert and M Tully ldquoSynthesis and degradation ofphenylalanine ammonia-lyase of Rhodosporidium toruloidesrdquoJournal of Bacteriology vol 150 no 2 pp 498ndash505 1982

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Enzyme Research



Microbiology

2 Enzyme Research

2 Materials and Methods

21 Cloning of the TcPAL Gene Escherichia coli bacterialstrains were cultured in Luria Bertani (LB Mediatech Hern-don VA) liquid medium Kanamycin (50120583gmL) was addedto LB media and plates when growing bacteria containingplasmids

The yeast strain Trichosporon cutaneum (ATCC 58094)was purchased from the American Type Culture Collection(Manassass VA) Shake cultures were grown aerobically inDagleyrsquos medium at 30∘C at 100 rpmwith addition of tyrosine(20mM) at 30∘C to an OD600 of 3 Cells were pelleted bycentrifugation (9000 xg 15min 4∘C) Genomic DNA wasextracted utilizing aQiagenDNApurification kit and utilizedas template for PCR amplification

Primer sequences were designed based on the DNAsequence of the T cutaneum PAL protein The PAL genewas PCR amplified using Phusion DNA polymerase (NewEngland Biolabs) from T cutaneum genomic DNA withthe 51015840 primer TcPAL- F 51015840-CGCGAATTCATGTTTATTGAGACC-31015840 as the forward primer (EcoRI site underlined)and 31015840 primer TcPAL- R 51015840-GAAGCTTTTAGAACATCTT-GCCAAC-31015840 as the reverse primer (HindIII site underlined)Control reactions were also performed The amplified PCRfragment was purified digested with HindIII and EcoRI andinserted in the pET30a vector to give plasmid pET-30a (+)TcPAL E coli DH5120572 competent cells were transformed withthe plasmid construct by use of heat shock treatment Positiveclones were identified by restriction analysis and plasmidDNA was submitted for DNA sequencing

Database comparison was performed with the BLASTsearch tools on the server of the National Center for Biotech-nology Information National Library of Medicine NIH(httpwwwncbinlmnihgov) Multiple sequence align-ments were performed using ClustalW Intron analysis wasperformed using GENSCAN

22 Cell Culture and Extraction Shake cultures of T cuta-neum were grown aerobically in Dagleyrsquos medium at 30∘Cat 100 rpm with addition of tyrosine (20mM) at 30∘C toan OD600 of 3 Cells were harvested by centrifugation at23500 xg for 15min at 4∘C The cell pellet was resuspendedin extraction buffer (50mM Tris-HCl pH 80 1mM EDTA10mM 2-mercaptoethanol protease inhibitor cocktail tabletsEDTA free and 1 tablet per 100mL) in a 1 4 ratio (wetweight buffer) and the cells were disrupted by vortexingwith glass beads for 6min with intermittent cooling on iceAfter centrifugation aliquots of supernatant were desaltedusing a PD-10 column (GE Healthcare) and used for enzymepurification Protein concentration was determined by theBradford assay [10]

23 Enzyme Assays The PALTAL activity of enzymeextracts was measured using a spectrophotometric assay [11]For preliminary induction studies the enzyme activity wasdetermined using an end point assay using Phe (40mM) orTyr (2mM) in 10mM Tris-HCl (pH 85) at a temperatureof 37∘C for 15min The reaction was terminated by the

addition of 25 trichloroacetic acid (200120583L) Samples werecentrifuged at 13000 xg for 15min and the absorbance wasmeasured All other experiments were conducted utilizinga continuous spectrophotometric assay at a temperature of30∘C for 2min Assays were initiated by addition of enzymeextract (200120583L) to substrate solution (800 120583L) preequili-brated at 30∘C For kinetic analysis solutions containing Phe(05mM to 100mM) or Tyr (05mM to 10mM) in 10mMTris-HCl (pH 80) were utilized The same enzyme extractwas used to compare the kinetic constants for PAL andTAL activities The absorbance of trans-cinnamic acid wasmeasured at 290 nm whereas para-hydroxycinnamic acidwas measured at 315 nm Kinetic data was analyzed usingGraphPad Statistical Software (Version 5 California) whichcombines biostatistics curve fitting (nonlinear regression)and scientific graphing into a comprehensive program

24 PAL Purification and Characterization All purificationsteps were carried out at 4∘C Crude cell free extract of Tcutaneum from PD-10 columns was applied to a Fast ProteinLiquidChromatography anion exchangeHiTrapQSepharosecolumn (25 times 10 cm) pre-equilibrated with Tris-HCl buffer(01M pH80)The adsorbed enzymewas elutedwith a lineargradient of KCl from 0 to 03M in the same buffer [12]Fractions with PAL activity were pooled and exchanged withextraction buffer using Millipore centrifugal filter units witha 30 kDa molecular cutoff

The temperature optimumof the enzymewas determinedby performing enzyme assays at 28∘C 32∘C 36∘C and 40∘Cwith Tris-HCl (50mM pH 80) as buffer The pH optimumwas determined by conducting enzyme assays at 32∘C withTris-HCl (50mM) adjusted to 73 76 82 86 and 89 Metaldependence studies were performed using the chloride saltsof sodium potassium magnesium and ferrous at a total saltconcentration of 100mM inTris-HCl buffer (50mM pH80)

3 Results and Discussion

31 Sequencing of the PAL Gene The PAL gene from Tcutaneum was amplified from genomic DNA using PCRA 2114 bp fragment size was generated cloned into thepET30a vector and submitted for sequence analysisThe genesequence showed high similarity with other PAL enzymesin the NCBI database Protein blast analysis revealed thatthe PAL shares identity with the yeast PALs Trichosporonasahii (70) Rhodosporidium toruloides (50) Rhodotorulamucilaginosa (49) and Rhodotorula glutinis (48)

Sequencing of the TcPAL gene from two independentclones revealed five amino acid residues that differ from thesequence previously reported by Breinig et al [6] namelyGln 74 Ala 274 Val 298 Pro 322 and Lys 486 A comparisonof our TcPAL sequence with the closely related Trichosporonashaii PAL revealed that the residues at positions 74 274and 486 were identical (Table 1) However the residue equiv-alent to 298 matched the Ala residue previously reportedrather than the Val in our sequence There was no obviousconsensus for the amino acid residue corresponding to Pro322 for the three proteins Analysis of the T cutaneum gene

Enzyme Research 3










lowast lowast


PcPAL

NtPAL

AtPAL

ZmPAL

BoPAL

OsPAL

TaPAL

GbPAL

AvPAL

NpPAL

RtPAL

RgPAL

RmPAL

RrPAL

SsPAL

TsPAL

TcPAL

RsTAL

SeTAL





119898


4 Enzyme Research





Aqueous two-phasec




79kDa67kDa

31kDa

250130100

7055352515

10(kDa) A B C

0006000500040003000200010

Activ

ity (U

)

0 2 4 6 8 10 12 14 16Volume (mL)

435

325

215

105

0minus05

Rela

tive a

bsor

banc

e(280

nm)





1015840


4 Conclusion


15

1

05

0

Rela

tive a

ctiv

ity

PheTyr

73 76 79 82 85 88pH


15

1

05

0

Rela

tive a

ctiv

ity

PheTyr

28 32 36 40

Temperature (∘C)


Enzyme Research 5



119898


1015840


15

1

05

00 20 40 60 80 100

Phenylalanine (mM)

Velo

city

(120583M

min

minus1)

(a)

025

020

015

010

005

0000 2 4 6 8 10

Tyrosine (mM)

Velo

city

(120583M

min

minus1)

(b)




Acknowledgments


References











6 Enzyme Research















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Enzyme Research 3










lowast lowast


PcPAL

NtPAL

AtPAL

ZmPAL

BoPAL

OsPAL

TaPAL

GbPAL

AvPAL

NpPAL

RtPAL

RgPAL

RmPAL

RrPAL

SsPAL

TsPAL

TcPAL

RsTAL

SeTAL





119898


4 Enzyme Research





Aqueous two-phasec




79kDa67kDa

31kDa

250130100

7055352515

10(kDa) A B C

0006000500040003000200010

Activ

ity (U

)

0 2 4 6 8 10 12 14 16Volume (mL)

435

325

215

105

0minus05

Rela

tive a

bsor

banc

e(280

nm)





1015840


4 Conclusion


15

1

05

0

Rela

tive a

ctiv

ity

PheTyr

73 76 79 82 85 88pH


15

1

05

0

Rela

tive a

ctiv

ity

PheTyr

28 32 36 40

Temperature (∘C)


Enzyme Research 5



119898


1015840


15

1

05

00 20 40 60 80 100

Phenylalanine (mM)

Velo

city

(120583M

min

minus1)

(a)

025

020

015

010

005

0000 2 4 6 8 10

Tyrosine (mM)

Velo

city

(120583M

min

minus1)

(b)




Acknowledgments


References











6 Enzyme Research















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 Enzyme Research





Aqueous two-phasec




79kDa67kDa

31kDa

250130100

7055352515

10(kDa) A B C

0006000500040003000200010

Activ

ity (U

)

0 2 4 6 8 10 12 14 16Volume (mL)

435

325

215

105

0minus05

Rela

tive a

bsor

banc

e(280

nm)





1015840


4 Conclusion


15

1

05

0

Rela

tive a

ctiv

ity

PheTyr

73 76 79 82 85 88pH


15

1

05

0

Rela

tive a

ctiv

ity

PheTyr

28 32 36 40

Temperature (∘C)


Enzyme Research 5



119898


1015840


15

1

05

00 20 40 60 80 100

Phenylalanine (mM)

Velo

city

(120583M

min

minus1)

(a)

025

020

015

010

005

0000 2 4 6 8 10

Tyrosine (mM)

Velo

city

(120583M

min

minus1)

(b)




Acknowledgments


References











6 Enzyme Research















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Enzyme Research 5



119898


1015840


15

1

05

00 20 40 60 80 100

Phenylalanine (mM)

Velo

city

(120583M

min

minus1)

(a)

025

020

015

010

005

0000 2 4 6 8 10

Tyrosine (mM)

Velo

city

(120583M

min

minus1)

(b)




Acknowledgments


References











6 Enzyme Research















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 Enzyme Research















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article purification and characterization of

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