Báo cáo khoa học: Engineering thermal stability of L-asparaginase by in vitro directed evolution ppt

Analysis of the electrostatic potential of the wild-type enzyme showed that Asp133 is located at a neutral region on the enzyme surface and makes a significant and unfavourable electrosta

directed evolution

Georgia A Kotzia and Nikolaos E Labrou

Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Greece

Bacterial l-asparaginases (l-ASNases, EC 3.5.1.1)

have been used as therapeutic agents in the treatment

of lymphoblastic leukaemia [1,2] l-ASNase exerts its

antitumor activity by depleting the nonessential

amino acid l-Asn from human blood and other

extracellular fluids [3,4] Certain malignant cells,

unlike normal cells, are unable to synthesize l-Asn

due to the lack of l-asparagine synthetase activity

[5,6] These cells are dependent on extracellular

sources of l-Asn in order to complete protein

synthe-sis [1,7] Therefore, administration of l-ASNase

selec-tively destroys the neoplastic cells by starving them

of l-Asn [8,9]

To date, l-ASNases of Erwinia chrysanthemi and Escherichia coli are in clinical use, as effective drugs in the treatment of acute lymphoblastic leukaemia, Hodg-kin’s disease, acute myelocytic leukaemia, acute myelo-monocytic leukemia, lymphosarcoma, melanosarcoma, etc [10,11] The main restrictions to the therapeutic use of l-ASNase include its premature inactivation, thus necessitating frequent injections to maintain ther-apeutic levels, and several types of side reactions from mild allergies and the development of immune responses to dangerous anaphylactic shock [9,12–14] Over the years, many homologous l-ASNases have been cloned and characterized to find enzymes with

Keywords

directed evolution; enzyme engineering;

leukaemia; saturation mutagenesis; thermal

stability

Correspondence

N E Labrou, Laboratory of Enzyme

Technology, Department of Agricultural

Biotechnology, Agricultural University of

Athens, Iera Odos 75, 11855 Athens,

Greece

Fax: +30 210 5294308

Tel: +30 210 5294308

E-mail: lambrou@aua.gr

(Received 19 September 2008, revised 2

December 2008, accepted 19 January 2009)

doi:10.1111/j.1742-4658.2009.06910.x

l-Asparaginase (EC 3.5.1.1, l-ASNase) catalyses the hydrolysis of l-Asn, producing l-Asp and ammonia This enzyme is an anti-neoplastic agent; it

is used extensively in the chemotherapy of acute lymphoblastic leukaemia

In this study, we describe the use of in vitro directed evolution to create a new enzyme variant with improved thermal stability A library of enzyme variants was created by a staggered extension process using the genes that code for the l-ASNases from Erwinia chrysanthemi and Erwinia carotovora The amino acid sequences of the parental l-ASNases show 77% identity, but their half-inactivation temperature (Tm) differs by 10C A thermo-stable variant of the E chrysamthemi enzyme was identified that contained

a single point mutation (Asp133Val) The Tmof this variant was 55.8C, whereas the wild-type enzyme has a Tmof 46.4C At 50 C, the half-life values for the wild-type and mutant enzymes were 2.7 and 159.7 h, respec-tively Analysis of the electrostatic potential of the wild-type enzyme showed that Asp133 is located at a neutral region on the enzyme surface and makes a significant and unfavourable electrostatic contribution to overall stability Site-saturation mutagenesis at position 133 was used to further analyse the contribution of this position on thermostability Screen-ing of a library of random Asp133 mutants confirmed that this position is indeed involved in thermostability and showed that the Asp133Leu muta-tion confers optimal thermostability

Abbreviations

Eca L- ASNase, L- asparaginase from Erwinia carotovora; ErL - ASNase, L- asparaginase from Erwinia chrysanthemi 3937; L- ASNase,

L- asparaginase; StEP, staggered extension process; Tm,half-inactivation temperature.

less toxic effects [15,16] In addition, many attempts

have been made with a view to extending the half life

of l-ASNase in the blood circulation [17,18], with the

most promising approach being its covalent coupling

to poly(ethylene glycol) [19–21]

Unfortunately, naturally available enzymes are

usu-ally not optimusu-ally suited for therapeutic purposes

This incompatibility often relates to the stability of

the enzymes under body conditions In the case of

engineering proteins for thermostability, researchers

are in the enviable position of being able to choose

between three different, apparently equally successful,

strategies: rational design, directed evolution and the

construction of (semirational) synthetic consensus

genes Although there are many examples of enzymes

that have been stabilized by the introduction of only

one or two mutations [22] and despite many

success-ful efforts to understand the structural basis of

pro-tein stability, there is still no universal strategy to

stabilize ‘any’ protein by a limited number of

ratio-nally designed mutations Well-known and reasonably

successful types of rational engineering work

inc-lude rigidifying mutations (e.g Xxx fi Pro or

Gly fi Xxx or the introduction of disulfides),

work-ing primarily through their effect on the entropy of

the unfolded state, improvement of molecular packing

(e.g shortening of loops, improvement of interactions

in the hydrophobic core, for example, by the removal

of internal cavities), modification of surface charge

networks or reinforcement of a higher oligomerization

state [23] Because the structure–function relationship

is not known or fully understood for the majority of

proteins, directed evolution provides a powerful

approach for improving thermostability [24–27] This

method, in combination with high-throughput

screen-ing, can be used even in cases where no information

on the 3D structure exists, and a single experiment

may provide enough variants to obtain the best

thermostable mutant [28]

In this study, l-ASNases from Erwinia carotovora

(Ecal-ASNase) [15] and Erwinia chrysanthemi

(Erl-ASNase) [16] were subjected to directed evolution,

with a view to find variants with improved

thermosta-bility Enhancing the stability of l-ASNase by protein

engineering improves its body-residence time, and

thereby minimizes immunosuppressive effects by

lower-ing the therapeutic dose After one round of directed

evolution one variant with a point mutation

(Asp133Val) was found An extensive search for the

best-fit residue at position 133 was made using

satura-tion mutagenesis, which revealed that this site plays an

important role in the thermal stability of the enzyme

The present work represents the first experimental

approach for improving the thermal stability of this therapeutic important enzyme

Results and Discussion

Identification and kinetic characterization of a thermostable mutant

One of the most important goals of protein engineer-ing is to produce modified enzymes with improved thermostability However, the thermal stability of a protein is not readily predictable from its 3D structure Thus, directed evolution is by far the best way to alter this enzyme property [24–28]

In this study, we used in vitro directed evolution using two homologous l-ASNase sequences from

E carotovora and E chrysanthemi (77% sequence identity) The library of mutated genes was generated using the staggered extension process (StEP) and expressed in E coli Wild-type and mutant enzymes were expressed as non-tagged proteins The diversity

of the DNAs in the resulting library was examined by sequence analysis of 10 randomly picked clones, and the results showed a satisfactory variability in recombi-nations The thermal stability of the enzyme variants was evaluated at 55C Under these conditions, wild-type enzymes from E carotovora and E chrysanthemi displayed 19.9% and 37.2% remaining activity, respec-tively One clone that showed 87.3% remaining activity was identified and selected for further study This enzyme variant was sequenced and found to be a single point mutant of the E chrysanthemi enzyme The mutation was at position 133 of the amino acid

GAC fi GTC) Shis mutation was the result of an error introduced by the DNA polymerase

Following sequencing, the mutant enzyme was sub-jected to purification according to a purification proce-dure established for the wild-type enzyme using an S-Sepharose FF column [16] The results showed that the mutant enzyme hardly bound to the cation exchan-ger For example, the dynamic capacities for the wild-type and mutant enzymes were determined to be 8.9 UÆmL)1 adsorbent and 0.1 UÆmL)1 adsorbent, respectively Thus, a new purification protocol was developed using a two-step chromatographic procedure involving a negative purification step using DEAE– Sepharose CL6B followed by immobilized metal chelate-affinity chromatography on a Ni-NTA column (Fig 1) It is of particular importance to point out that the Asp133Val mutant enzyme showed unusual chromatographic behaviour on ion exchangers In particular, at pH 5.5, the enzyme did not bind to the

cation exchanger (S-Sepharose FF column) or to the

anion exchanger (DEAE–Sepharose CL6B), although

its theoretical isoelectric point is 8.57 This was the

first indication that the mutant enzyme exhibits

unu-sual electrostatic potential

The basis of thermal stability of the Asp133Val

mutant

The thermal stability of the purified Asp133Val mutant

enzyme was assessed by measuring its residual activity

after heat treatment for 7.5 min at various

tempera-tures (Fig 2) The half-inactivation temperature (Tm)

of the mutant enzyme was found to be 55.8C, which

is almost 9.4C higher than that of the wild-type

Erl-ASNase [16] Kinetic analysis of the thermal

inactiva-tion of the wild-type and Asp133Val mutant enzymes

at 50C gave linear plots (Fig 3) For both enzymes,

the inactivation process followed first-order kinetics

[29] The inactivation rate constant was calculated

using the following equation:

log(% remaining activity) = 2.303
kin
t

where kin is the inactivation constant kin for the

4.34· 10)3h)1, which is 59-fold lower than that of

wild-type Erl-ASNase The half-lives (t1⁄ 2) at 50C of the wild-type and mutant enzymes were determined to

be 2.7 and 159.7 h, respectively

To gain a deeper insight into the structural basis of thermal stability, a molecular model of the mutant was constructed (Fig 4) Analysis of the structure showed that Asp133 lies at the external surface of Erl-ASNase,

in particular at a loop region formed by residues Thr129–Lys134 between a4 and b4 (Fig 4A) It forms

a salt bridge and two hydrogen bonds with Arg169 The first hydrogen bond is formed between the OD1 atom of Asp133 (2.87 A˚) and the side chain NH2 of Arg169, whereas the OD2 atom forms a weak H-bond (3.47 A˚) with the side chain NH2 of Arg169 (Fig 4B)

In the Asp133Val model, these two hydrogen bonds are lost and the side chain of Val133 no longer inter-acts with Arg169 (Fig 4C)

Fig 2 Thermal inactivation curves The residual activities of the wild-type (h) and mutant Asp133Val ( ) enzymes were measured after heat treatment at various temperatures (30–70 C) for 7.5 min.

Fig 1 SDS ⁄ PAGE of Asp133Val mutant enzyme purification

Pro-tein bands were stained with Coomassie Brilliant Blue R-250 Lane

A, molecular mass markers; lane B, E coli BL21 (DE3)pLysS crude

extract after induction with 1 m M isopropyl thio-b- D -galactoside;

lane C, unbound fraction from DEAE-Sepharose CL6B, pH 7.5;

lane D, eluted Asp133Val mutant enzyme from the Ni-NTA affinity

adsorbent.

Fig 3 Kinetics of thermal inactivation of the wild-type and Asp133Val mutant enzyme The residual activities of the wild-type ( ) and mutant Asp133Val (¤) were measured at various times after incubation at 50 C The results are presented as plot of log(% remaining activity) versus time (h).

Thus, any attempt to understand the greater thermal

stability of the Asp133Val mutant enzyme in terms of

intramolecular interactions (e.g H-bonding, ionic

bonds) in the structure was unsuccessful Instead, the

explanation appears to lie in the much greater entropy

of activation in the case of the wild-type enzyme In

principle, the entropy of inactivation consists of two

parts: the increased configurational entropy due to

partial unfolding of the protein in the transition state

and the decrease in entropy of the solvent due to

expo-sure of hydrophobic side chains The latter effect is

likely to be small at elevated temperatures [30] and the

major contribution must then be the increase in

config-urational entropy Thus, the greater rate of thermal

inactivation of the wild-type enzyme than of the mutant is probably mainly due to greater flexibility or disordering of the transition state with some contribu-tion from a lower degree of exposure of hydrophobic residues To assess whether Asp133 contributes to structural flexibility, we analysed the plots of the crys-tallographic B-factors along the polypeptide chain of the enzyme structure This plot can give an indica-tion of the relative flexibility of porindica-tions of the protein [15,31] As shown in Fig 5, the structure dis-plays a well-defined flexibility pattern Several highly mobile regions throughout the entire sequence can be identified, and these are separated by a number of segments with low mobility Asp133 displays high

Fig 4 Structural representations of the

wild-type and Asp133Val mutant enzyme.

(A) Diagram of the modelled Asp133Val

mutant enzyme subunit with succinamic

acid bound to the active site The bound

ligand and the mutated residue (Val133) are

shown in a stick representation and are

labelled (B) Structural representation of the

mutation site 133 of the wild-type ErL -

ASN-ase (C) Structural representation of the

mutation site 133 of the Asp133Val mutant.

Hydrogen bonds are shown as dashed lines

and residues are labelled The model of the

mutated enzyme was constructed using

WHAT IF [51] (D) A closer view of the

electro-static potential of the mutation site 133.

Negative, positive and neutral values of

electrostatic potential are indicated by

shades of red, blue and white colour,

respectively (E) Superposition of the

Pois-son–Boltzmann electrostatic potential of the

mutant and the wild-type enzymes Asp113

is shown as a spacefill representation

(col-oured black) and labelled The colour code

utilized to represent the electrostatic

poten-tial along with the potenpoten-tial range is: red:

)1.8; white: 0; blue: 1.8 All figures were

created using PYMOL [54], except (E) which

was created using SWISS-PDB VIEWER [53].

crystallographic B-factors located at a region with high

mobility, indicating that this residue undergoes large

fluctuations This may cause local disordering with

increased flexibility, which may contribute to the

increase in configurational entropy

Erl-ASNase is composed of four identical subunits,

and the active enzyme is always a tetramer To

deter-mine whether the higher thermal stability observed for

the Asp133Val mutant enzyme is due to higher

stabil-ity of the quaternary or tertiary structure,

subunit-dissociation experiments were carried out Shifrin et al

[32] showed that guanidinium chloride dissociates the

enzyme tetramer in 50 mm phosphate buffer at pH 7.5

The dissociation is accompanied by the appearance of

an ultraviolet difference spectrum with a maximum at

288 nm The band at 288 nm in the difference spec-trum has been ascribed to tyrosyl residues [32] In this study, the rate of subunit dissociation in the wild-type and Asp133Val mutant enzymes was monitored by following the rate of appearance of the 288 nm band

in the ultraviolet difference spectrum As illustrated in Fig 6, the rate of dissociation of the tetramer was found to be approximately equal for both enzymes (first-order rate constants: 0.011 and 0.013 min)1 for the wild-type and Asp133Val mutant enzymes, respec-tively), indicating that the stabilizing effect of the mutation is not due to quaternary structure stabiliza-tion Instead, it is the result of stabilization of the tertiary structure

Taking into account the unusual chromatographic behaviour of the mutant enzyme on ion exchangers, and because in silico structural analysis of the interac-tions in the microenvironment of Val133 in the mutant enzyme did not provide adequate explanations for its higher thermostability, electrostatic potential analysis was performed Analysis of the wild-type enzyme showed that Asp133 is located at an uncharged (neu-tral) region of the enzyme (Fig 4D) Replacement of a surface charge (Asp) with a hydrophobic residue (Val) would not normally be expected to increase the stabil-ity of the protein, because for a hydrophobic side chain it is more favourable to be buried within the core of the protein than to be exposed to solvent It has been suggested, however, that in some cases there can be residues located on the surface of a protein that provide an unfavourable electrostatic contribution to the overall stability of the domain, due to the asymme-try of the electrostatic potential mapped on the surface

of the protein [33] In our case, because Asp133 lies in

a neutral environment, its charge is unfavourable and

it may therefore destabilize the overall structure Com-parison of the Poisson–Boltzmann electrostatic poten-tial of the mutant and the wild-type (Fig 4E) showed that greater symmetric positive potential was observed

in the mutant enzyme than in the wild-type enzyme This may cause less structural perturbations in the mutant enzyme

Effects of the Asp133Val mutation on kinetic parameters

The mutant enzyme was subjected to steady-state kinetic analysis using three different substrates: l-Asn, l-Gln and Na-acetyl-Asn (Table 1) The results showed that the kcat and Km values for l-Asn were increased

by 1.6- and 2.9-fold, respectively, compared with the wild-type enzyme By contrast, the Km values for the

Fig 5 The dynamics of ASNase A plot of the crystallographic

B-factors along the polypeptide chain obtained from the crystal

structure of E chrysanthemi ASNase, (PDB code 1O7J) The plot

was produced using WHAT IF software package [51] The height at

each residue position indicates the average B-factor of all atoms in

the residue B-factors are available in the PDB file 1O7J.

Fig 6 Guanidinium chloride induced subunit dissociation Rate of

appearance of the 288 nm band of the wild-type (s) and mutant

Asp133Val (d), as a function of time in 0.05 M phosphate buffer,

pH 7.5, containing 3 M guanidine chloride Changes in absorbance

at 288 nm were monitored for 5 min.

l-Gln and Na-acetyl-l-Asn were reduced and kcat

values were increased (Table 1) The increased Km

value for l-Asn, although undesirable, is still within

the range of acceptability for therapeutic applications

[35]

The results of the kinetic analysis indicate that

although the site of the mutation is distant from the

active site, it makes a significant contribution to

catalysis (kcat and Km), suggesting that long-range

effects play an important role Considering the

inter-action of the enzyme with the substrate, it is

reason-able to propose that there may be a long-range effect

in the active site In particular, the mutation at

posi-tion 133 may destabilize the crucial hydrogen bond

formed between the side chain of the substrate and

the main chain oxygen of Ala139 This interaction

has been shown to be responsible for the higher

mobility of the active-site loop and of the flexible

cat-alytic region 120–140, and it is believed to contribute

to substrate specificity and to the rate-limiting step

[15,16,36] These structural observations are consistent

with the results of the kinetic analysis (Table 1) and

presumably explain the effect of the mutation on

kinetic constants Similar long-range effects have been

found in the serine protease subtilisin BPN’ [37] For

example, charged residues on the surface of the

enzyme some 13–15 A˚ from the active site have been

found to modulate enzyme–substrate complex

forma-tion and catalysis Also, long-range interacforma-tions have

been found in the case of aminoacyl-tRNA synthetase

and 4-chlorobenzoyl-CoA dehalogenase catalysis [38,39]

Site-saturation mutagenesis at position 133

In vitrosite-directed evolution (saturation mutagenesis) can be used to advantage during protein engineering

to explore additional evolution pathways and enable rapid diversification in protein traits [40] This method makes possible the creation of a library of mutants containing all possible mutations at one or more pre-determined target positions, in order to determine the best-fit residue at that position It has been used suc-cessfully for rapid improvement of various protein functions [40,41]

In this study, site-saturation mutagenesis at posi-tion 133 was used to investigate in greater depth the contribution of this position to the thermostability A library of enzyme variants was created by overlap extension PCR using two degenerate synthetic oligo-nucleotides in which the mutation site (position 133) was diversified using a randomized NNN codon The library was subsequently screened for clones with improved stability at 60C for 5 min Under these conditions, the mutant Asp133Val shows 21.3% resid-ual activity Four clones that showed ‡ 45% residual activity (compared to the Asp133Val mutant) were selected and sequenced Three clones were found to have single point mutations at position 133: Asp133Leu, Asp133Ile and Asp133Thr In addition,

Table 1 Kinetic parameters of the wild-type and mutant enzymes Steady-state kinetic measurements were performed at 37 C in 0.1 M

Tris ⁄ HCl, pH 8 (or pH 8.2 for L -Gln) All initial velocities were determined in triplicate The kinetic parameters k cat and K m were calculated by nonlinear regression analysis of experimental steady-state data using the GRAFIT (Erythacus Software Ltd, Staines, UK) program [34].

)

kcatÆKm)1 (m M )1Æs)1)

(· 10 3

) Wild-type a

a Data for the wild-type enzyme were from Labrou & Kotzia [16] and are included for comparison.

one clone had a double mutation: Asp133Leu⁄

Asp103Thr The spontaneous Ala103Thr mutation

introduced by Pfu DNA polymerase The four most

thermostable clones have an uncharged substitution at

position 133 (Leu, Ile, Thr) This further supports the

finding that position 133 contributes significantly to

the electrostatic potential of the enzyme, and it

pro-vides evidence for the necessity of uncharged⁄

hydro-phobic residue at position 133 for attainment high

thermostability

The selected saturation variants were purified using

the same procedure that was used for the Asp133Val

mutant Subsequently, their thermal stabilities were

evaluated using heat-inactivation studies at 57.5C

(Fig 7) and the results are listed in Table 2 All

mutants showed reduced kin values (from 1.75- to

3.2-fold) compared with the Asp133Val mutant,

indi-cating further improvement in their thermal stability

The Asp133Leu mutant appeared to be the most

thermostable, with a kin of 0.040 min)1 The double

mutant Asp133Leu⁄ Ala103Thr showed a kin compa-rable with that of the single point mutant Asp133Leu

Structural analysis of 3D models of the mutant enzymes showed the formation of additional non-covalent interactions in the mutated enzymes (Fig 8) In particular, the side chain hydroxyl group

of the Asp133Thr mutant is involved in an H-bond with the side chain of Arg169, similarly to the wild-type Erl-ASNase (Fig 4C) By contrast, the Asp133Leu and Asp133Ile mutants appear to form additional van der Waals interactions The side chain

of Leu133 interacts (van der Waals contacts) with the side chain of Asn226, and Ile133 interacts with Arg169 and Gly170 All residues are parts of loops; therefore, the additional stabilizing effects of the mutations, compared with the Asp133Val enzyme, may be due either to restriction of the conforma-tional freedom of the protein or to the energetic contribution of the newly formed H-bond and van der Waals contacts

Variants bearing a single mutation (Asp133Leu, Asp133Ile, Asp133Thr) were subjected to kinetic analysis using l-Asn, l-Gln and Na-acetyl-l-Asn as substrates The results showed little to moderate effect

of the Leu, Ile and Thr substitutions on the Kmvalues compared with the Asp133Val mutant (Table 1) It is interesting to note that for the Asp133Thr mutant, the

Km value for l-Asn was reduced fourfold compared with the Asp133Val mutant and by 1.5-fold compared with the wild-type Erl-ASNase By contrast, the kcat values were significantly reduced although the enhanced specificity (kcatÆKm )1) towards l-Asn was maintained in all mutants

Conclusions

The results of this study provide new data on the structural basis of the thermal stability of E chry-santhemi l-ASNase, and provide a basis for the design

of new, improved forms of the enzyme for future ther-apeutic use It has been shown that the hydrophobic effect, hydrogen bonding and packing interactions between residues in the interior of the protein are dominant factors that define protein stability The role

of surface residues in protein stability has received much less attention The stability of the Asp113Val mutant enzyme is a particularly interesting rare case

in which replacement of a surface charge with a hydrophobic residue leads to an increase in the stabil-ity of the protein Whereas conventional chemical intuition would expect that salt bridges should contribute favourably to protein stability, recent

Fig 7 Kinetics of thermal inactivation of the mutant Asp133Val

and its saturation variants The residual activities of the mutant

Asp133Val (e) and of the saturation variants were measured at

var-ious times after incubation at 57.5 C The results are presented as

plot of log(% remaining activity) versus time (min) Asp133Leu ( ),

Asp133Ile (d) and Asp133Thr (D).

Table 2 First-order inactivation rate constants (kin, min)1) of the

mutant enzymes at 57.5 C.

computational and experimental evidence has shown

that salt bridges can be either stabilizing or

destabiliz-ing [41] For example, alleviation of unfavourable

sur-face charge can increase the stability of proteins [42] Many proteins contain clusters of positively or nega-tively charged residues, and optimization of the surface electrostatic potential may enhance protein stability For example, in recent studies of ribonucle-ase T1 and ubiquitin, it was shown that relieving surface charge through mutation increased protein stability [42–44]

During the development of a therapeutic protein, it

is important to improve its long-term stability Under-standing the stability parameters and the factors that affect them are critical steps The thermostable enzyme variants reported in here may be tested further on ani-mals and⁄ or humans in order to create a new drug for future therapeutic use

Experimental procedures

l-Asn and l-Gln were obtained from Serva (Heidelberg, Germany) a-Ketoglutaric acid and Sepharose CL6B from Sigma (St Louis, MO, USA) Na-Acetyl-l-Asn was obtained from Sigma-Aldrich, (Milwaukee, WI, USA) NADH (disodium salt, grade II,  98%) and crystalline bovine serum albumin (fraction V) were purchased from Boehringer Mannheim (Mannheim, Germany) Nessler’s reagent and glutamate dehydrogenase were obtained from Fluka (Taufkirchen, Germany) All primers were synthe-sized and purified by MWG-biotech AG (Ebersberg, Germany) TOPO cloning kit and all other molecular biology reagents were from Invitrogen (Carlsbad, CA, USA)

Directed evolution ofL-ASNase

Directed evolution of l-ASNase was carried out using the StEP [45] Plasmids (pCRT7⁄ CT-TOPO) containing the nucleotide sequences of l-ASNase from E carotovora (Ecal-ASNase; NCBI accession number: AY560097, the enzyme was cloned as a non-tagged protein) [15] and

E chrysanthemi 3937 (ErL-ASNase; NCBI accession num-ber AY560098, the enzyme was cloned as a non-tagged pro-tein) [16] were used as parental sequences in the PCR The forward primers used in the reaction were the 5¢-ATGGAACGATGGTTTAAATCTCTG-3¢ and 5¢-ATG TTTAACGCATTATTCGTTGTTGTTTTTG-3¢, and the reverse primers were the 5¢-TCAATAGGTGTGGAAATA GTCCTGG-3¢ and 5¢-TTAAGCTTTTAATAAGCGTGG AAGTAATCC-3¢ The PCR was carried out in a total vol-ume of 50 lL containing 25 ng of each primer, 10 ng of template plasmid DNA, 0.2 mm of each dNTP, 5 lL of 10· Taq buffer, 1.5 mm MgCl2buffer and 2.5 units of Taq DNA polymerase (Stratagene, La Jolla, CA, USA) The PCR procedure comprised 99 cycles of 30 s at 94C and

10 s at 46C

Fig 8 Structural representations of the mutant enzymes A closer

view of the mutation site of Asp133Thr, (A); Asp133Ile, (B); and

Asp133Leu, (C) Hydrogen bonds are shown as dashed lines and

residues are labelled The models of the mutated enzymes were

constructed using WHAT IF [51] The figure was created using PYMOL

[54].

Cloning, expression and screening for

thermostable mutants

Following completion of StEP, the resulting PCR amplicon

was treated with the restriction enzyme DpnI to eliminate

parental plasmid DNA and was TOPO ligated to

pCRT7⁄ CT-TOPO expression vector The presence of

the stop codon in the 5¢-end of the reverse primers allowed

the expression of non-tagged enzyme variants The resulting

expression constructs pT7Mut-ASNase were used to

trans-form competent BL21(DE3)pLysS E coli cells E coli cells,

harbouring plasmids pT7MutASNase, were grown at 37C

in 30 mL of Luria–Bertani medium containing 100 lgÆmL)1

ampicillin and 34 lgÆmL)1 chloramphenicol The synthesis

of l-ASNases was induced by the addition of 1 mm

isopro-pyl thio-b-d-galactoside when the absorbance at 600 nm

was 0.6–0.8 Five hours after induction, cells were harvested

by centrifugation at 4000 g and 4C for 20 min In order

to find thermotolerant mutants, cell-free extracts were

incu-bated at 55C for 7.5 min and were then used to measure

the residual activities using the coupled enzyme assay

method described below

Saturation mutagenesis, library creation and

screening

Saturation mutagenesis at amino acid position 133 was

per-formed by overlap extension using PCR [46] Mutations

were introduced using a set of degenerate synthetic

oligonu-cleotides, in which the mutation site was diversified using a

randomized NNN codon The pairs of oligonucleotide

primers used in the PCR for the saturation mutagenesis

were as follows: the first pair 5¢-ATGGAACGATG

GTTTAAATCTCTG-3¢ (P1) and 5¢-GTGAAAAGCNNN

AAGCCGGTAGTG-3¢ (P2), and the second pair 5¢-CACT

ACCGGCTTNNNGCTTTTCAC-3¢ (P3) and 5¢-TCAAT

AGGTGTGGAAATAGTCCTGG-3¢ (P4) Sites of

muta-tion are indicated in italics The expression construct

encod-ing the wild-type ErL-ASNase [16] was used as template

DNA After completion of the PCR (using primers P1, P2

and P3, P4), the PCR products were digested with DpnI to

eliminate parental DNA, and were then used in another

PCR as templates using P1 and P4 primers to amplify the

entire mutated gene The latter was TOPO ligated into a T7

expression vector (pEXP5-CT⁄ TOPO) and recombinant

plasmids were isolated and were used to transform

compe-tent BL21(DE3)pLysS E coli cells E coli cells were grown

at 37C in 30 mL of Luria–Bertani medium containing

100 lgÆmL)1 ampicillin and 34 lgÆmL)1 chloramphenicol

The synthesis of the mutated enzymes was induced by the

addition of 1 mm isopropyl thio-b-d-galactoside when the

absorbance at 600 nm was 0.6–0.8 Four hours after

induc-tion, cells were harvested by centrifugation at 4000 g and

4C for 20 min Hundreds of transformants of the library

were examined for their thermotolerance at 60C for

5 min, in order to identify promising mutants showing increased thermotolerance compared with the Asp133Val The thermotolerance was estimated by measuring the residual activities using the coupled enzyme assay method described below For the variants showing ‡ 45% residual activity, the mutations were determined by DNA sequencing

Purification of the wild-type and mutant enzymes

Purification of the wild-type enzymes was carried out according to published methods [15,16] The purification of mutants was accomplished by a two-step procedure, comprising a negative purification step using a DEAE– Sepharose CL6B, followed by an immobilized metal che-late-affinity chromatography on a Ni-NTA column This was carried out as follows: cell paste was suspended in potassium phosphate buffer (5 mm, pH 7.5), sonicated and centrifuged at 10 000 g for 5 min The supernatant was col-lected and applied to DEAE–Sepharose CL6B (1 mL, 0.5· 2 cm i.d.) column, previously equilibrated with potas-sium phosphate buffer (5 mm, pH 7.5) Nonadsorbed pro-tein was washed off with 3 mL equilibration buffer The flow-through and the first fraction (1 mL) of the washing step were mixed, adjusted to pH 8 (with the addition of 0.1 m potassium phosphate buffer, pH 8) and 0.1 m NaCl (by the addition of 5 m NaCl) before being applied to Ni-NTA (0.5 mL, 0.5· 1 cm i.d.) column The column was previously equilibrated with potassium phosphate buffer (50 mm, pH 8) containing 0.3 m NaCl Nonadsorbed protein was washed off with 8 mL equilibration buffer, followed by 8 mL potassium phosphate buffer (50 mm,

pH 7.5) containing 0.3 m NaCl, and 8 mL potassium phos-phate buffer (50 mm, pH 7) containing 0.3 m NaCl Bound l-ASNase was eluted with potassium phosphate buffer (50 mm, pH 6.2) containing 0.3 m NaCl Collected fractions (2 mL) were assayed for l-asparaginase activity and pro-tein Following purification, the wild-type enzyme as well

as the mutant enzymes were dialysed against 1000 vol of 0.1 m Tris⁄ HCl pH 8.0 (for kinetic analysis; see below) or against 10 mm KH2PO4buffer pH 7 (for thermal inactiva-tion studies; see below)

Assay of enzyme activity and protein

Enzyme assays were performed at 37C at a Hitachi U-2000 double beam UV⁄ Vis spectrophotometer carrying

a thermostated cell holder (10 mm path length) Activities were measured by determining the rate of ammonia forma-tion, by coupling with glutamate dehydrogenase, according

to Balcao et al [47] The final assay volume of 1 mL con-tained 71 mm Tris⁄ HCl buffer, pH 8.0, 1 mm Asn, 0.15 mm a-ketoglutaric acid, 0.15 mm NADH, 4 units glutamate dehydrogenase and sample containing l-ASNase activity Alternatively, the rate of ammonia formation was measured

at 37C using the Nessler’s reagent [48] One unit of

l-ASNase activity is defined as the amount of enzyme that

liberates 1 lmol of ammonia from l-Asn per min at 37C

Protein concentrations were determined at 25C using

the method of Bradford [49] using BSA (fraction V) as

standard

Kinetic analysis

Steady-state kinetic measurements were performed as

described previously [15,16,50] The kinetic parameters kcat

and Km were calculated by nonlinear regression analysis of

experimental steady-state data Turnover numbers were

cal-culated on the basis of one active site per subunit Kinetic

data were analysed using the computer program grafit

(Erythacus Software Ltd, Staines, UK) [34]

Molecular modelling and computational analysis

Molecular modelling for creating the model of E

chrysant-hemi l-ASNase was carried out as described by Kotzia &

Labrou [16] The molecular modelling program what if

[51] was used to predict the conformation of the mutant

enzymes Prediction of the conformation of the new side

chains was performed as described by Chinea et al [52]

Poisson–Boltzmann electrostatic potential analysis of the

mutant and the wild-type enzymes was carried out using

swiss-pdb viewer[53] The parameters utilized to calculate

the electrostatic potential were: dielectric constant (protein)

4, dielectric constant (solvent) 80, solvent ionic strength

0.00, partial charged ‘on’ Nonbonded interactions were

analysed by moltalk (http://i.mol.talk.org) The program

pymolwas used for inspection of models and crystal

struc-tures [54]

Thermal stability of the wild-type, Asp133Val and

saturation variants

Thermal inactivation of the wild-type and Asp133Val and

saturation variants was monitored by activity

measure-ments Samples of the enzymes, in 10 mm KH2PO4 buffer

pH 7, were incubated at a range of temperatures from 30

to 70C for 7.5 min Subsequently, the samples were

assayed for residual activity, using the coupled enzyme

assay method described above The Tm values were

deter-mined from the plots of relative inactivation (%) versus

temperature (C) The Tm value is the temperature at

which 50% of the initial enzyme activity is lost after heat

treatment The kinetics of thermal inactivation of the

wild-type and Asp133Val was monitored at 50C The

kinetics of thermal inactivation of saturation variants was

monitored at 57.5C The rates of inactivation were

followed by periodically removing samples for assay of

enzymatic activity Observed rates of inactivation (kin)

were deduced from plots of log (% remaining activity) versus time

Guanidinium chloride induced subunit dissociation

Guanidinium chloride induced dissociation of the wild-type and mutant Asp133Val enzymes was carried out according

to Shifrin et al [32] in 50 mm KH2PO4pH 7.5 Guanidinium chloride treatments were performed in the presence of 3 m guanidinium chloride and measurements were taken for

5 min The rate of dissociation was determined by the increase of the absorbance at 288 nm as described by Shifrin

et al.[32]

Electrophoresis

SDS⁄ PAGE was performed according to the method of Laemmli [55] on a slab gel containing 12.5% (w⁄ v) poly-acrylamide (running gel) and 2.5% (w⁄ v) stacking gel The protein bands were stained with Coomassie Brilliant Blue R-250

Acknowledgements

This work was financially supported by the Hellenic General Secretariat for Research and Technology: Operational Program for Competitiveness, Joint Research and Technology Program

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