Báo cáo khoa học: Characterization of recombinant prolidase from Lactococcus lactis – changes in substrate specificity by metal cations, and allosteric behavior of the peptidase pdf

Various culture conditions were examined using a pKK223-3–prolidase clone as described in Experimental procedures, and it was found that low temperatures with vigorous aeration yielded r

Lactococcus lactis – changes in substrate specificity by metal cations, and allosteric behavior of the peptidase

Soo I Yang and Takuji Tanaka

Department of Food and Bioproduct Sciences, College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon, Canada

Fermented foods have significant nutritional value and

are receiving growing attention from health-conscious

consumers During fermentation, microbial activity

changes the chemical, physical and nutritional

attri-butes of the food materials One of the main changes

during fermentation is the production of peptides and

amino acids via proteolysis These compounds are

major factors contributing to the flavor of fermented

foods Of these amino acids and peptides, hydrophobic

peptides exhibit undesirable bitterness in the fermented

foods [1] For example, in Cheddar cheese,

hydropho-bic peptides ranging from 2–23 residues were found to

be responsible for bitterness [2]

Hydrophobic peptides produced during

fermenta-tion undergo further hydrolysis through general

pepti-dase reactions, which result in reduced bitterness

However, peptides that contain proline behave

differ-ently from other peptides during general proteolysis

As proline is structurally and chemically unique among the 20 naturally occurring amino acids due to its imine structure, proline-containing peptides are much less susceptible to further enzymatic hydrolysis [3,4] Thus, hydrolysis of peptides during fermentation can ultimately produce proline-containing dipeptides, such as Xaa-Pro and Pro-Xaa Ishibashi et al [5] reported two interesting observations regarding pro-line-containing dipeptides: most of these dipeptides are bitter, and Xaa-Pro is generally more bitter than Pro-Xaa Proline-containing dipeptides tend to be accumulated as a result of the low susceptibility to enzymatic hydrolysis, and these dipeptides are bitter; therefore, peptidases specific for proline-containing dipeptides could control the bitterness of fermented foods

Keywords

bitterness; metallopeptidase;

overexpression; PepQ; proline

Correspondence

T Tanaka, Department of Food and

Bioproduct Sciences, College of Agriculture

and Bioresources, University of

Saskatchewan, 51 Campus Drive,

Saskatoon, Saskatchewan S7N 5A8, Canada

Fax: +1 306 966 8898

Tel: +1 306 966 1697

E-mail: takuji.tanaka@usask.ca

(Received 1 August 2007, revised 15

October 2007, accepted 16 November

2007)

doi:10.1111/j.1742-4658.2007.06197.x

The Lactococcus lactis NRRL B-1821 prolidase gene was cloned and over-expressed in Escherichia coli Under suboptimum growth conditions, recombinant soluble and active prolidase was produced; in contrast, inclu-sion bodies were formed under conditions preferred for cell growth Recombinant prolidase retained more than half its full activity between 30 and 60C, and was completely inactivated after 30 min at 70 C CD anal-ysis confirmed that prolidase was inactivated at 67C The enzyme was active under weak alkali to weak acidic conditions, and showed maximum activity at pH 7.0 Although these characteristics are similar to those for other reported prolidases, this prolidase was distinctive for two kinetic characteristics Firstly, different substrate specificity was observed for its two preferred metal cations, zinc and manganese: Leu-Pro was preferred with zinc, whereas Arg-Pro was preferred with manganese Secondly, the enzyme showed an allosteric response to changes in substrate concentra-tions, with Hill constants of 1.53 for Leu-Pro and 1.57 for Arg-Pro Mole-cular modeling of this prolidase suggests that these unique characteristics may be attributed to a loop structure near the active site

Abbreviations

IPTG, isopropyl thio-b- D -galactoside; LAB, lactic acid bacteria.

Lactic acid bacteria (LAB) are widely used to

pro-duce fermented foods LAB are nutritionally fastidious

and require amino acids as exogenous nutrients [6,7]

Required amino acids are assimilated in the form of

peptides that are produced from proteins by LAB

extracellular proteinase [8] The assimilated peptides

are further hydrolyzed by peptidases in LAB in order

to supply free amino acids for metabolism LAB may

have as many as 18 peptidases for efficient hydrolysis

of the imported peptides [9] Of these peptidases, four

are proline-specific: proline iminopeptidase, prolinase,

X-prolyl dipeptidyl aminopeptidase and prolidase [9]

Prolidase (EC 3.4.13.9) is specific to Xaa-Pro

dipep-tides, which can only be hydrolyzed by this peptidase

[8] As mentioned above, Xaa-Pro dipeptides are bitter

Therefore, reduction of the dipeptides via prolidase

may lead to the reduction of bitterness in fermented

food products

Prolidases have been reported from some microbial

sources, such as Lactobacillus delbrueckii subsp

bulgar-icus CNRZ 397 [4,10], Lactobacillus casei subsp casei

IFPL 731 [11], Pyrococcus furiosus [12] and Lb

helveti-cus [13] These prolidases have preferential activity on

Xaa-Pro dipeptides that have a hydrophobic amino

acid as the N-terminal residue The prolidases do not

hydrolyze Pro-Pro or Gly-Pro, and have little activity

on hydrophilic Xaa-Pro peptides Previous research

has shown that, in the absence of prolidase, LAB

growth is retarded by 13% [14] Most previous

research has concentrated on kinetic characterization

of LAB physiological activity As a consequence, there

is little information on the expression of recombinant

prolidases, the functionality of each residue or protein

engineering of this enzyme

In the present study, the prolidase-coding gene, pepQ,

was isolated from Lactococcus lactis NRRL B-1821 and

cloned Characterization of the recombinant protein

revealed some interesting characteristics of this

proli-dase Moreover, this research provides the means to

investigate the structure–function relationships of

proli-dase, hence providing a greater understanding of the

characteristics of this peptidase, which would be of

industrial use in the debittering of fermented foods

Results and Discussion

Cloning and expression of Lc lactis prolidase

The prolidase gene of Lc lactis NRRL B-1821 was

successfully isolated using PCR The DNA sequence of

the isolated gene (GenBank accession number

EU216565) was virtually identical to that reported

for Lc lactis Il1403 (GenBank accession numbers

NC_002662 and AE006395) The sequence of the iso-lated gene has a base difference compared with Lc lac-tis Il1403, and this substitution results in an amino acid change from Tyr67 to His67 in the putative amino acid sequence Attempts were made to express the gene using the tac promoter on pKK223-3, and the expres-sion system produced a large amount of recombinant protein under the preferred growth conditions (37C

in LB broth) of the Escherichia coli TOP 10 F’ hosts The amount of recombinant protein reached about 50% of total cell extracts, as determined from SDS– PAGE gel densitometry (data not shown) However, the growth under these conditions yielded recombinant prolidase as inclusion bodies In theory, inclusion bodies can be refolded into an active form; however, it

is uncertain whether the refolded proteins have an identical fold to that of the native proteins Therefore, the above expression system was refined in order to optimize the conditions to produce soluble protein without inclusion body formation

Optimization of expression and purification

of recombinant Lc lactis prolidase The overexpression of recombinant proteins can dis-turb host cell metabolism through their activities To avoid these negative effects, host cells often produce recombinant proteins as inclusion bodies [15] As pro-lidases are highly specific for Xaa-Pro, which does not have any specific roles in metabolism of the host cells, activity of prolidases would not have harmful effects

on the host cells Therefore, the rapid and ample expression itself would have caused sufficient stress to the host metabolism Growth conditions were exam-ined in order to decrease host stress by altering the conditions for expression Two possibilities for decreasing the stress were postulated: (a) that unfavor-able conditions for E coli growth (resulting in slower growth) would retard expression of the recombinant prolidase, and (b) that conditions allowing the host more resources for themselves, i.e rich media or weak induction of expression, could compensate for the con-sumption of energy and substances diverted to produce the recombinant prolidase Various culture conditions were examined using a pKK223-3–prolidase clone as described in Experimental procedures, and it was found that low temperatures with vigorous aeration yielded recombinant prolidase as a soluble protein Optimum results were achieved using a low concentra-tion of chloramphenicol (1 lgÆmL)1), induction with

1 mm isopropyl thio-b-d-galactoside (IPTG) at

A600= 0.5, and vigorous aeration (200 r.p.m with a low volume of medium in a large vessel) at 16 C

Under these conditions, 40 h cultures produced 20–

40% of soluble proteins as recombinant prolidase, as

determined by densitometry of the SDS–PAGE gel

Extension of the culture beyond 40 h did not increase

prolidase production

Purification of recombinant prolidase was achieved

using a two-step process: ammonium sulfate

precipita-tion and anion-exchange column chromatography

(Fig 1) Crude extracts were found to contain a

sub-stantial amount of recombinant prolidase (40 kDa;

lane C), compared with non-induced cell extracts (lane

B) Ammonium sulfate precipitation removed most of

the contaminating proteins from the crude extracts

(lane D) A final purification step using a DEAE–

Sephacel column resulted in a single prolidase band, as

evidenced by SDS–PAGE (lane E) A 900 mL culture

yielded 18.2 mg of purified prolidase, with a

purifica-tion factor of 11.8 and 89% recovery of activity from

the crude extracts (Table 1) The purified prolidase

had 197.2 unitsÆmg)1 (where one unit is as defined in

Experimental procedures) of specific activity using

2 mm Leu-Pro dipeptide as the substrate in 20 mm sodium citrate buffer (pH 6.5)⁄ 1 mm ZnCl2at 50C

Characterization of recombinant Lc lactis prolidase

The molecular mass of recombinant prolidase was esti-mated using mass spectrometry and gel filtration Based

on the gene sequence, an estimated molecular mass for the prolidase monomer of 40 kDa (39 970 Da) was determined The estimated molecular mass (40 164 Da) determined by mass spectroscopy of purified prolidase confirmed this value This molecular mass may not reflect the native state as mass spectrometers dissociate the protein molecules during the analysis process The molecular mass of prolidase in the native state (active form) was roughly estimated using four size-exclusion columns Prolidase appeared in the void volume fraction

of the Bio-Gel P-60 column (exclusion limit of 60 kDa) and in later fractions using other columns: Sephadex G-100 (100 kDa) and G-150 (150 kDa), and Bio-Gel P-200 (200 kDa) These results indicate that prolidase is larger than 60 kDa, but smaller than 100 kDa Only a dimeric structure can have a molecular mass within this range, as the monomer of this prolidase is 40 kDa as shown by mass spectrometry, SDS–PAGE and gene sequence We therefore propose that Lc lactis prolidase forms a homodimeric structure with a molecular mass

of approximately 80 kDa This proposed dimeric struc-ture is in agreement with the X-ray crystal strucstruc-tures of

P furiosus(Protein Data Bank accession number 1PV9 [16]) and Pyrococcus horikoshii OT3 (Protein Data Bank accession number 1WY2) prolidases

Recombinant Lc lactis prolidase exhibited activity over a broad range of temperatures, showing similar activities between 35 and 55C (Fig 2) The reaction rate dropped to 67% at 60C, and no activity was observed above 70C This range is broader than that for Lb delbrueckii prolidase, which has its highest activities between 40 and 50C [4] Figure 2 also shows the temperature stability The enzyme retained more than 60% of its activity after 30 min incubation below 50C; however, incubation at 60 C decreased

Fig 1 SDS–PAGE gel showing the final purification of recombinant

Lactococcus lactis prolidase Samples from each step of purification

process were compared by SDS–PAGE Lane B, whole cell extracts

of non-induced culture; lane C, crude extracts of induced culture;

lane D, after 60% ammonium sulfate precipitation; lane E, after

DEAE–Sephacel chromatography purification The arrow indicates a

molecular mass of 40 kDa Lane A shows the molecular mass

markers.

Table 1 Purification of Lactococcus lactis recombinant prolidase.

Purification process

Total protein (mg)

Total activity (units a )

Specific activity (unitsÆmg)1)

Yield (% activity)

Purification (fold)

a One unit of prolidase activity is defined as hydrolysis of 1 lmol of peptide in 1 min.

the residual activity to 22% Little activity was

observed after incubation at 70C The thermal

stability is comparable to that of Lb casei prolidase

[11] and higher than that of Lb delbrueckii

proli-dase [4]

This loss of activity in the recombinant Lc lactic

prolidase was most likely due to denaturation between

60 and 70C In order to confirm this speculation, CD

analysis was employed The CD signal started to

decline at 60C and reached a minimum at 71 C

(Fig 3), with the denaturation temperature estimated

as 67C This observation indicated that the enzyme

began to lose structure at 60C and was completely denatured at around 70C, thereby supporting the speculation that the loss of prolidase activity results from denaturation of the enzyme

Enzyme activity was measured between pH 4 and 10 (Fig 4) Activity was detected between pH 6.0 and 8.0 and reached a maximum at pH 7 for both Leu-Pro and Arg-Pro The optimum pH was consistent with values reported for Lb delbrueckii (pH 6.0) [4],

P furiosus (pH 7) [12], Lb casei (pH 6.5–7.5) [11] and partially purified Lc lactis subsp cremoris AM2 pro-lidases (pH 7.35 and 8.25) [17], although these enzymes worked in narrower pH ranges for Leu-Pro than that for Lc lactis prolidase

The reported prolidases vary in their metal require-ments, e.g Lb delbrueckii prolidase requires zinc [4],

P furiosus prolidase prefers cobalt and manganese [12], and Lb casei enzyme can utilize magnesium, manganese and cobalt [11] The recombinant Lc lactis prolidase showed its highest activity for Leu-Pro with zinc, but the activity with manganese was 21.5% of that with zinc (Table 2) Activity was not detected with other divalent cations, i.e cobalt, magnesium, nickel, copper and calcium

Substrate specificity of recombinant Lc lactis prolidase

To date, all known prolidases are dipeptide-specific, and cannot hydrolyze larger peptides Similar results were observed for the prolidase examined in this study (Table 2) Recombinant Lc lactis prolidase exhibited activity for Leu-Pro, Val-Pro, Phe-Pro, Arg-Pro and Lys-Pro Based on the peptide hydrolysis assay employed in this study, i.e., quantification of free

0

25

50

75

100

Temperature (°C)

Relative activity ( : 40°C = 100%) Residual activity ( : 20°C = 100%)

Fig 2 Effect of temperature on recombinant Lactococcus lactis

prolidase activity Open circles represent the observed activity of

fresh prolidase at each temperature The activities are expressed

as activities relative to the activity at 40 C Closed triangles

repre-sent the residual activity of Lc lactis prolidase after 30 min

treat-ment at each temperature The residual activities are expressed as

activities relative to the activity after 20 C incubation.

Temperatue (°C)

0

20

40

60

80

100

Fig 3 Thermal denaturation observed by CD The observed CD

signal at 222 nm is plotted against temperature The signal intensity

is expressed relative to the value at 20 C The determined

dena-turing temperature, 67 C, is indicated by a vertical dashed line.

0 20 40 60 80 100

pH

Fig 4 pH dependency of recombinant Lactococcus lactis proli-dase The activity of Lc lactis prolidase was measured using two dipeptides over a range of pH values The observed activities are expressed as the activity relative to that at pH 7.0 Open squares and closed circles represent Leu-Pro and Arg-Pro, respectively.

proline, no hydrolysis was observed for Gly-Pro,

Glu-Pro, Asp-Pro or the two tripeptides Leu-Leu-Pro and

Leu-Val-Pro Interestingly, substrate specificity was

dependent on the catalytic metal cation No activity

towards Pro-Pro was seen in the presence of zinc, but

low activity was seen in the presence of manganese Moreover, the preference for dipeptide changed from Leu-Pro to Arg-Pro in the presence of manganese

A comparison of the crystal structure of P furiosus prolidase and the sequence-based model of Lc lactis prolidase (Fig 5) indicated that the S1 sites are composed mainly of hydrophobic residues (Phe190, Leu193 and Ile308 of Lc lactis prolidase), suggesting

a preference towards hydrophobic residues at the N-terminus of the dipeptides In fact, the Xaa-Pro dipeptides preferred by Lc lactis prolidase were mostly hydrophobic peptides, as shown in Table 2 However, the preference for Arg-Pro, and the metal-dependent substrate specificity cannot be explained by the nature

of the S1 site residues described A notable difference between P furiosus and Lc lactis prolidases in the active site area is the length of the loop structure that

is contributed by the other subunit and covers the S1

site (yellow ribbon for P furiosus and cyan ribbon for

Lc lactis in Fig 5) The crystal structure of P furiosus suggests that this loop forms part of the S1 site The loop is longer in Lc lactis by four residues, and the middle of the loop is composed of charged residues (Asp36, His38, Glu39 and Arg40), whereas the

Arg295

Ser307

Arg295

Ser307

Fig 5 Active site superposition of Lactococcus lactis and Pyrococcus furiosus prolidases The residues in the active sites of a Lc lactis prolidase subunit are indicated by thick lines The S1site (Phe190, Leu193 and Val302; blue), S1‘ site (His292, Tyr329 and Arg337; green), and substrate size-limiting residues (Pro306, Ser307 and Ile308; orange) and metal-chelating residues (Asp221, Asp232, His296, Glu325 and Glu339; cyan) are shown Corresponding residues in P furiosus are indicated by thin lines The size-limiting arginine, Arg295, in P furiosus prolidase [16], and the corresponding residue, Ser307, in Lc lactis prolidase are labeled Ribbon models show the loop contributed from the other subunit The Lc lactis and P furiosus prolidase loop structures are in cyan and yellow, respectively Leu37B of P furiosus prolidase and Asp36B of Lc lactis prolidase are shown in the line model on the ribbons The illustration was generated using the VMD molecular modelling program [27].

Table 2 The relative activities of recombinant Lactococcus lactis

prolidase in the presence of zinc or manganese for various peptide

substrates Activity was measured with 2 m M peptides in 20 m M

sodium citrate (pH 6.5) and 1 m M metal (zinc or manganese)

chlo-ride; activities are expressed relative to the activity for Leu-Pro in

the zinc reaction mixture.

P furiosus loop has two hydrophilic residues (Thr34

and Ser35) It is speculated that this loop structure

contributes to the preference for the Arg-Pro

dipep-tide, i.e methylene groups (b, c, d–carbons) are

accommodated in the S1 site, and the amino group of

the side chain is associated with the negatively charged

residues (Asp 36 and Glu39) on the loop structure

Allosteric behavior of recombinant Lc lactis

prolidase

The relationships between substrate concentrations and

reaction rates were examined in order to determine

kinetic parameters Plots of substrate concentration

against observed catalytic rate showed sigmoidal

curves for both Leu-Pro and Arg-Pro (Fig 6) The

allosteric behavior indicated by the sigmoidal curves

was analyzed using the Hill plot, and Hill coefficients

of 1.53 and 1.57, respectively, were obtained for

Leu-Pro and Arg-Leu-Pro (Fig 6) Although allosteric behavior

is not common among proteinases⁄ peptidases, it has

been reported in several proteinases, e.g cathepsin C

[18] and Helicobacter pylori leucyl aminopeptidase [19]

Interestingly, the latter enzymes share characteristics

with Lc lactis prolidase: they hydrolyze peptides with

leucine at the N-terminus, they are metallopeptidases,

and their 3D structures share similar domain

struc-tures (based on the bovine leucyl aminopeptidase

structure, 1LAM [20]) Their similarities in 3D

struc-ture include (a) two distinctive domains that fold in

a⁄ b structures, (b) an active site located at the center

of the C-terminal domain, and (c) an active site that

faces another subunit

The Lineweaver–Burk plot using the Hill coefficient

(1⁄ sH against 1⁄ v plot) gave a Michaelis constant for

Leu-Pro of 3.7 mm and a rate constant of 247.9 s)1

The constants for known prolidases are: Lb del-brueckii, 2.2 mm and 225.9 s)1; Lb casei, 0.2 mm and 55.1 s)1 [11]; P furiosus, 3.0 mm and 271 s)1 [12] Similar to known prolidases [4,12,17], this Lc lactis prolidase exhibited substrate inhibition above 5 mm Leu-Pro; at 8 mm, the observed activity was 47% of that at 5 mm

Modelling of Lc lactis prolidase Molecular modeling provides insight regarding the allosteric nature of this enzyme A molecular model of

Lc lactis prolidase was successfully constructed and used to evaluate the structure–function relationship of this prolidase The Lc lactis model was superposed on the P furiosus model by comparison of their a-car-bons, yielding a root mean square deviation of 1.59 A˚ Figure 5 shows models of P furiosus (1PV9) and

Lc lactis prolidases around the active site zinc ions The P furiosus enzyme, which did not exhibit alloste-ric behavior, had a smaller loop structure over the active site (shown as a yellow ribbon in Fig 5) Maher

et al [16] discussed the contribution of this loop struc-ture as part of the substrate binding site, and it was suggested that Leu37B (B indicates the contribution from the other subunit) formed the hydrophobic S1 site in cooperation with Phe178, Ile181 and Ile 290 The comparable residues in the Lc lactis prolidase are Asp36B, Phe190, Leu193 and Ile308, respectively The positions and characteristics of the latter three residues are comparable to those of P furiosus prolidase How-ever, unlike Leu37B of P furiosus prolidase, Asp36B is

a hydrophilic charged residue, and is located on the longer loop structure (cyan ribbon in Fig 5) that was discussed in the substrate specificity section In some enzymes, the loop structures have been shown to con-tribute to the activity of enzymes by changing their shape [21,22] This suggests that the structure of this loop could take a different shape in the event of sub-strate binding, thus the residue comparable to Leu37B

of P furiosus might not be Asp36B but instead another residue on the longer loop This flexibility may mediate changes in the overall structure of the enzyme via subunit–subunit interaction Such changes may trigger the allosteric behavior of Lc lactis prolidase Another possibility is that Ser307 works as a key resi-due in the allosteric behaviour of this enzyme This residue is located close to the substrate-binding site, and is replaced by Arg295 in P furiosus (Fig 5) Maher et al [16] suggested that this residue limited the substrate to dipeptides We suggest that this residue can cooperate with the loop and contribute allosteric behavior to the peptidase These suggestions, i.e the

0

0.001

0.002

0.003

0.004

0.005

0.006

Substrate concentration (m M )

–1 )

–4 –2 0 2

–3 –2 –1 0 1 2

Ln (substrate)

Vmax –v)]

Fig 6 Allosteric behavior of Lactococcus lactis prolidase The plots

show the relationship between the observed activity and the

con-centration of Leu-Pro (open squares) and Arg-Pro (closed circles).

The inset shows the Hill plot of the assay with Leu-Pro.

contributions of the loop and Ser307, are being

exam-ined by our group

Conclusion

In this study, we have produced recombinant prolidase

in a soluble, active form The techniques use to achieve

solubilization could be used for other

difficult-to-express proteins Recombinant Lc lactis prolidase

exhibited characteristics similar to other prolidases,

but possessed distinctive properties of allosteric

behav-ior and metal-dependent substrate specificity Further

structure–function relationship studies will provide

insights into the behaviour of prolidase, thus

contrib-uting to applications of prolidase in fermented food

processing

Experimental procedures

Enzymes and chemicals

Enzymes for genetic engineering were purchased from

Fer-mentas (Burlington, Canada) and Invitrogen (Burlington,

Canada) All chemicals used in this study were

commer-cially available ACS grade, and were purchased from VWR

International (Edmonton, Canada)

Cultivation of Lactococcus lactis and genomic

DNA isolation

Lactococcus lactis NRRL B-1821 (Agricultural Research

Service culture collection, Peoria, IL, USA) was cultivated

in 100 mL of Lactobacillus MRS medium (BD-Difco,

Franklin Lakes, NJ, USA) for 24 h at 37C without

shak-ing The culture was harvested by centrifugation at 4000 g

for 5 min at 4C Harvested cells were treated with

pro-teinase K (1 mgÆmL)1 in 50 mm Tris–HCl pH 8.0, 50 mm

EDTA, 100 mm NaCl, 0.5% SDS; Roche Diagnostics,

Montreal, Canada) at 50C for 1 h, then disrupted using

phenol Extracted nucleic acids were collected, and RNA

was removed by RNase (Fermentas) treatment Genomic

DNA was purified by ethanol precipitation from the

reac-tion mixture

Isolation and cloning of the gene

A pair of primers (5¢-GGAGAATTCATGAGCAAAA

TTGAACGTATT-3¢; 5¢-ATTCTGCAGTTAGAAAATT

AATAAGTCATG-3¢) for PCR was designed based on the

sequence of the Lc lactis spp Il1403 prolidase coding gene

(GenBank accession numbers NC_002662 and AE006395)

and custom-synthesized (Integrated DNA Technologies

Inc., Coralville, IA, USA) The primers possessed EcoRI

(N-terminus) and PstI (C-terminus) restriction enzyme sites

(indicated by underlining) that flanked the ends of the open reading frame The PCR reaction mixture contained geno-mic DNA (20 lg), primers (20 pmol each), dNTPs (40 lm each) and Pfu DNA polymerase (0.5 units; Fermentas) in

100 lL of the buffer recommended by the manufacturer Each PCR reaction cycle consisted of 94C for 1 min,

55C for 1 min, and 68 C for 3 min (5 s was added to the

68C step for each cycle), and was repeated 30 times The amplified PCR fragments were hydrolyzed using EcoRI and PstI, and were then introduced into EcoRI–PstI-digested pUC18 plasmids The recombinant DNA was transformed into E coli TOP10F’, and positive clones were verified by DNA sequencing First, the sequenced gene was isolated using EcoRI–PstI restriction enzymes, and subcloned into the same sites of the pKK223-3 vector [23] Then, the con-structed recombinant DNA was transformed into E coli TOP10F’ The expression was examined under a variety of conditions in order to obtain recombinant prolidase in a soluble form The various conditions studied were: (a) cul-ture medium (LB or 2YT medium), (b) the concentration

of the inducing agent for the tac promoter (0.1, 1 or 10 mm IPTG), (c) protein synthesis inhibition using sublethal con-centrations of chloramphenicol (0.1 or 1 lgÆmL)1), (d) media with higher osmotic pressures (0.5 or 2% w⁄ v NaCl), (e) schedules of the induction (induced at A600=0.4, 0.5, 0.8 or 1.2), (f) the pH of the medium (pH 5.5 or 7.5), (g) the aeration conditions (100 or 200 r.p.m.), (h) the culture temperature (16, 18, 22, 29, 30, 33 or 37C), and (i) the duration of the culture (16, 40, 72 or 96 h) These condi-tions were tried individually or concurrently

Purification of the recombinant Lc lactis prolidase

The recombinant E coli was cultured in LB broth (pH 5.5)

at 16C The culture was carried out in 18 500 mL flasks with 50 mL medium in each (total 900 mL) Expression was induced by addition of 1 mm IPTG and chlorampheni-col (1 lgÆmL)1) when the A600reached 0.5 The culture was vigorously shaken at 200 r.p.m for 40 h before harvesting The harvested cells were resuspended in a lysis buffer solu-tion (20 mm sodium citrate buffer, pH 6.0, 1 mm zinc sulfate, 100 mm sodium chloride, 8 lgÆmL)1 RNase and 0.2 mgÆmL)1 lysozyme), and disrupted using ultrasonica-tion After removal of some proteins from the crude extracts by 40% saturated ammonium sulfate precipitation, the prolidase fraction was recovered using 60% saturated ammonium sulfate precipitation The recovered prolidase in the precipitate was dissolved in 20 mm sodium citrate (pH 6.0)⁄ 1 mm zinc sulfate and dialyzed against 2 L of the same buffer twice The dialyzed sample was applied to a DEAE–Sephacel anion exchange column (3 diameter·

15 cm; GE Healthcare, Chalfont St Giles, Buckingham-shire, UK), and prolidase was eluted using a 600 mL linear gradient from 0 to 0.5 m NaCl in 20 mm sodium citrate

(pH 6.0)⁄ 1 mm zinc sulfate The prolidase fractions were

concentrated and desalted using an YM30 Amicon Ultracell

filtration system (Millipore, Billerica, MA, USA) The

pur-ity of the prolidase was densitometrically estimated by

SDS–PAGE using Coomassie Brilliant Blue G250 and NIH

imagesoftware (developed at the US National Institutes of

Health and available at http://rsb.info.nih.gov/nih-image/)

The purified sample in 20 mm sodium citrate

(pH 6.0)⁄ 1 mm zinc sulfate was mixed with the same

vol-ume of glycerol and stored at)20 C until use

Enzyme activity assay

The amount of proline liberated from the peptide substrates

was determined using the ninhydrin method [24]

Dipep-tides were hydrolyzed in 20 mm sodium citrate buffer

(pH 6.5)⁄ 1 mm zinc chloride The reaction was initiated by

the addition of enzyme solution At 1 min intervals, an

aliquot (20 lL) was withdrawn and mixed with 50 lL of

glacial acetic acid and 50 lL of ninhydrin reagent (3% w⁄ v

ninhydrin, 60% v⁄ v glacial acetic acid, 40% v ⁄ v phosphoric

acid) The mixture was boiled for 10 min to develop the

color, and then cooled on ice The resulting chromophore

was quantified using 515 nm absorption All measurements

were carried out at least in triplicate One unit of prolidase

activity is defined as hydrolysis of 1 lmol of peptide in

1 min

Measurement of substrate specificity

The peptide substrates examined were Leu-Pro, Val-Pro,

Phe-Pro, Gly-Pro, Arg-Pro, Lys-Pro, Pro-Pro, Asp-Pro,

Glu-Pro, Leu-Val-Pro and Leu-Leu-Pro The peptides

(2 mm) were hydrolyzed in 20 mm sodium citrate

(pH 6.5)⁄ 1 mm zinc chloride or manganese chloride at

50C

pH dependency

The pH dependency of prolidase was examined using the

following buffer solutions in place of the sodium citrate

buffer in the method described above: 20 mm sodium

cit-rate (pH 4–5.5), 20 mm MES (pH 6.0–7.0), 20 mm Tris–

HCl (pH 7.5–9) and 20 mm sodium borate (pH 10) The

activity was analyzed using 2 mm Leu-Pro or Arg-Pro and

1 mm manganese chloride at 50C

Thermal stability and dependency

Recombinant prolidase was incubated in 20 mm sodium

cit-rate buffer (pH 6.5)⁄ 1 mm zinc chloride at the designated

temperature (20, 30, 40, 50, 60 or 70C) for 30 min The

residual activity was determined in order to evaluate the

stability of prolidase The temperature dependency was

separately examined in reactions using fresh enzyme at vari-ous temperatures (20, 30, 35, 40, 45, 50, 55, 60 and 70C) In both experiments, 2 mm Leu-Pro was used as the substrate

Metallic ion dependency

A variety of metal cations were tested for their effects on the prolidase activities Metal salts of zinc chloride, nickel chloride, cobalt nitrate, copper sulfate, manganese chloride, magnesium chloride and calcium chloride were used The activities were measured in 20 mm sodium citrate (pH 6.5)⁄ 1 mm solutions of each metal salt with 2 mm Leu-Pro at pH 6.5

Thermal denaturation temperature measurement for recombinant prolidase

The CD spectrum of purified prolidase was analyzed in

20 mm sodium phosphate buffer (pH 6.0) using a

PiStar-180 spectroscope (Applied Photophysics Ltd, Leatherhead, Surrey, UK) at the Saskatchewan Structural Sciences Cen-tre (University of Saskatchewan, Saskatoon, Canada) The thermal denaturation temperature was determined from the change in the CD spectrum at 222 nm over a temperature range from 25 to 90C The denaturation temperature was determined as the temperature at which the rate of CD spectrum change reached its maximum [15]

Mass spectrometry

The molecular mass of the recombinant prolidase molecule was determined using a mass spectrometer (API Q-star XL hybrid MS system; Applied Biosystems, Foster City, CA, USA) using the electrospray ionization method at the Sas-katchewan Structural Sciences Centre; measurements were carried out on the desalted sample in deionized water

Molecular mass estimation using gel filtration

The purified prolidase protein (0.8 lg in 10 lL) was loaded onto gel filtration columns: Sephadex G-100 and G-150 (GE Healthcare) and Bio-Gel P-60 and P-200 (Bio-Rad Laboratories, Hercules, CA, USA) The size of the columns was 0.5 diameter· 10 cm, and 20 mm sodium citrate buffer (pH 6.5)⁄ 1 mm ZnCl2 was used as the eluant The eluate was fractionated, and the activities of the fractions were qualitatively checked using Leu-Pro in order to determine the prolidase fractions

Computational molecular modelling

The protein sequence, deduced from the DNA sequence of the prolidase gene, was submitted to the 3d-jigsaw server

(http://www.bmm.icnet.uk/servers/3djigsaw/) [25] in order to

create an initial molecular model of Lc lactis prolidase The

initial model was derived based on the crystal structure of

P furiosus(Protein Data Bank accession number 1PV9 [16])

using the default parameters of the server This model was

then energy-minimized using the namd molecular modelling

program [26] The calculation used topology force field data

provided with the program, and was carried out in a

water-filled box The cut-off distance was set to 15 A˚ and the

calcu-lation run for 5000 iterations The minimized model was

compared with the P furiosus model (Protein Data Bank

accession number 1PV9 [16]) using the vmd software package

[27] on a Macintosh G4 computer

Acknowledgements

This research was supported by a grant from the

Natu-ral Sciences and Engineering Research Council of

Canada The authors appreciate the assistance of Lili

Liu and Guodong Zhang with the laboratory work

George Khachatourians, Rickey Yada (Guelph,

Canada), Nicholas Low, Michael Nickerson and Sylvia

Yada (Guelph, Canada) are acknowledged for their

helpful suggestions in the preparation of the

manu-script

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