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Research ArticleReceived: 24 October 2012 Revised: 21 January 2013 Accepted article published: 25 January 2013 Published online in Wiley Online Library: 29 April 2013

(wileyonlinelibrary.com) DOI 10.1002/jctb.4050

Purification, characterization and comparisonof Penicillium purpurogenum β-glucuronidasesexpressed in Escherichia coli and Pichia pastorisShuping Zou,b† Shuyuan Guo,a† Imdad Kaleema and Chun Lia∗

Abstract

BACKGROUND: To investigate the effects of post-translational modifications in different recombinant expression systems onthe catalytic properties of recombinant β-glucuronidase. The β-glucuronidase (GUS) gene from Penicillium purpurogenum Li-3was cloned and successfully expressed in Escherichia coli BL21 and Pichia pastoris G115.

RESULTS: The recombinant E. coli produced a 15-fold increased level of β-glucuronidase while the recombinant P. pastoris strainproduced a 6.9-fold increased level of β-glucuronidase compared with their parent strains. The β-glucuronidases from recombi-nant E. coli (PGUS-E) and P. pastors (PGUS-P) were purified to 35.9- and 47.4-fold, respectively, through affinity, ion exchange andgel filtration chromatography. PGUS-E from E. coli was a non-glycosylated protein with an apparent molecular mass of 72.43 kDa,while PGUS-P from P. pastors was appropriately glycosylated with a molecular mass of 78.83 kDa measured by MALDI/TOF-MS.Although both recombinant β-glucuronidases exhibited similar pH optima, the glycosylated PGUS-P showed a significantlyhigher thermal stability and less sensitivity to metal ions compared with the non-glycosylated PGUS-E. The glycosylated PGUS-Palso displayed lower Km values, and higher kcat/Km ratios than the non-glycosylated enzyme towards glycyrrhizin.

CONCLUSION: These results revealed the key role of post-translational modifications in the P. pastors expression system onthe catalytic properties of β-glucuronidase and its potential stability over the prokaryotic expression system which could beapplied as an important tool for the functional enhancement of industrial enzymes.c© 2013 Society of Chemical Industry

Keywords: β-glucuronidase; purification; Escherichia coli ; Pichia pastoris ; glycosylation

INTRODUCTIONβ-glucuronidases (EC 3.2.1.31) are enzymes that catalyse hydrol-ysis of β-linked glucuronides to yield their various derivatives andfree glucuronic acid. There is a great variety of β-glucuronidasesand they have been used extensively for the structural investi-gations of proteoglycans and also as positive selection markers

for transformed plants, bacteria and fungi.1–3 Recently, muchattention has been paid to their exploitation as biocatalystsfor the biotransformation of valuable products. For instance,β-glucuronidase has been used for the selective hydrolysis ofglycyrrhizin (GL) to produce β-D-mono-glucuronide-glycyrrhizin

(GAMG),3–7 which is much safer and with higher biologicalutilization ratio than the substrate GL.8,9 Several β-glucuronidaseswith glycyrrhizin-hydrolyzing activity have been isolated and char-acterized from many organisms including bacteria,5,10,11 fungi,3,7

yeast4 and animals.6 However, it is still a challenging issue to obtainlarge quantities of β-glucuronidases for industrial applications,due to the very low productivity of various wild-type strains.

To enhance the product yield and decrease production costof enzymes, Escherichia coli and Pichia pastoris strains have beenused as the host for the expression and production of industrialenzymes. However, the recombinant proteins from differentexpression systems vary in their post-translational modifications,such as glycosylation which is one of the major naturally occurringmodifications of the covalent structure of proteins in eukaryotic

cells. The recombinant protein expressed in P. pastoris is often

glycosylated with a high proportion of mannosides,12–14 whileprokaryotic cells, such as E.coli, lack organelles for glycoproteinsynthesis, hence express their proteins without post-translationalmodification.15,16

Previously, our research team reported about a newly isolatedfungal strain, Penicillium purpurogenum Li-3,7 that was involvedin producing a specific β-glucuronidase with high chemical bondselectivity and could directly hydrolyze GL to GAMG. In orderto enhance the cell growth and/or enzyme production, theculture conditions (i.e. carbon sources, inducers, initial pH) hadbeen optimized.7 However, despite various efforts at processoptimization, the improvement in enzyme production was stillvery limited and consequently the β-glucuronidase activity

∗ Correspondence to: Chun Li, School of Life Science, Beijing Institute ofTechnology, 5 ZhongGuanCun South Street, 100081 Beijing, PR China.E-mail: lichun@bit.edu.cn

† These authors contribute to this work equally

a School of Life Science, Beijing Institute of Technology, Beijing 100081, People’sRepubic of China

b Institute of Bioengineering, Zhejiang University of Technology, Hangzhou310014, People’s Republic of China

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remained relatively low, thus restricting the application of thisβ-glucuronidase on a practical scale. To resolve this problem,the β-glucuronidase gene (GenBank Accession No. EU095019)from P. purpurogenum Li-3 has been cloned and expressed in E.coli BL21 and P. pastoris G115. Although an improved amountof recombinant enzyme was produced, difference in catalyticproperties was also found between the two recombinant strains.In most cases, the change in catalytic properties between theheterogeneous proteins has been attributed to the differenceof post-translational modification of expression systems, such asglycosylation.17 However, there is little information on the effectsof post-translational modification on the functional and structuralstability of PGUS-P.

In this study, first, two β-glucuronidases from recombinant E. coliand P. pastoris were purified, and their catalytic and glycosylationproperties were thoroughly examined and compared. Thevariations in enzymatic behavior are highlighted on the basisof their expression systems which impart unique properties toproteins. Our aim is to investigate the effects of post-translationalmodifications occurred in different expression systems on thecatalytic properties of recombinant β-glucuronidase. Namely, tofind out whether glycosilation provides, higher stability, andbetter catalytic properties of the enzyme compared with thenon-glycosilated enzyme.

MATERIALS AND METHODSExpression of β-glucuronidases in E. coli and P. pastorisThe recombinant strains E. coli BL21 used in this study wereconstructed in our previous research.18 Briefly, the β-glucuronidasegene (Pgus, GenBank Accession No. EU095019) from Penicilliumpurpurogenum Li-3 was PCR amplified using gene-specific forwardand reverse primers, cloned in the pET28a (+) vector andtransformed to E. coli BL21(DE3) competent cells (Invitrogen,Karlsruhe, Germany). To express the Pgus gene in Pichiapastoris, thegene was inserted into the expression vector pPIC9K (Invitrogen,Carlsbad, CA), and transformed into P. pastoris GS115 (Invitrogen,San Diego, USA) by electroporation (Bio-RAD Genepulser, Gercules,USA). The positive transformants were selected in the presence of1.5 mg mL−1 G418 (Sigma, China) and confirmed by PCR results.

Production and purification of recombinant β-glucuronidasesThe culture of recombinant E. coli BL21 (DE3) (pET28a) was grownin 100 mL LB medium containing kanamycin (50 ug mL−1) in500 mL Hinton flask for 4 h. IPTG was added to a final concentrationof 1 mmol L−1, and incubated at 20 ◦C. Maximum β-glucuronidaseactivity of 90.8 U mL−1 (specifc activity of 10.6 U mg−1) wasobserved after 12 h of growth in LB medium. The cells wereharvested at 6000 g for 10 min, and resuspended in crude extractbuffer (50 mmol L−1 Tris–HCl buffer, pH 7.0). The cells were lysedby sonication and the lysate was centrifuged at 15 000 g at 4 ◦C for20 min. The resulting supernatant was brought to 80% saturationwith (NH4)2SO4, left at 4 ◦C for 2 h, and then centrifuged. Theprecipitate was dissolved in a small volume of 10 mmol L−1

Tris–HCl buffer, pH 7.0, and dialyzed overnight against this buffer.The crude enzyme solution was applied into a Ni-NTA agarose(Invitrogen) and eluted with 10, 50, 100 and 250 mmol L−1

imidazole buffers. The fractions containing the highest activitieswere pooled, concentrated, and applied onto a Sephadex G-100column (1.6 cm × 95 cm; flow rate 10 mL h−1) equilibrated andeluted with 10 mmol L−1 acetate buffer, pH 4.6. The active

fractions were concentrated, and stored at −20 ◦C. The recombi-nant protein extracted from E. coli was designated as PGUS-E.

The culture of recombinant P. pastoris GS115 (pPIC9K) wasgrown in 100 mL BMGY medium in a 500 mL Hinton flask, andthen transferred into 10 mL BMMY medium in a 60 mL baffled flaskwith an initial cell density of 1 OD600, adding methanol to a finalconcentration of 1% (v/v) every 24 h. Maximum β-glucuronidaseactivity of 44.6 U mL−1 (specifc activity of 7.0 U mg−1) was observedafter 72 h of induction. The extracellular β-glucuronidase wasisolated by centrifuging the fermentation broth at 15 000 g at 4 ◦Cfor 20 min, and the supernatant was brought to 80% saturationwith (NH4)2SO4, left for 2 h, and then centrifuged. The precipitatewas dissolved in a small volume of 10 mmol L−1 Tris–HCl buffer,pH 7.0, and dialyzed overnight against the buffer. The crudeenzyme solution was passed through a DEAE-cellulose column(1.6 cm × 20 cm; flow rate 1.0 mL min−1) and bound protein waseluted with a linear NaCl gradient (0–0.5 mol L−1). The fractionscontaining the highest activities were pooled, concentrated, andapplied onto a Sephadex G-100 column (1.6 cm × 95 cm; flowrate 10 mL h−1) equilibrated and eluted with 10 mmol L−1 acetatebuffer, pH 4.6. The active fractions were concentrated, and storedat −20 ◦C. The recombinant proteins extracted from P. pastoriswas designated as PGUS-P.

Determination of enzymatic activity and proteinconcentrationActivities of β-glucuronidase were measured with glycyrrhizinmonoammonium salt as substrate. The reaction medium (1 mLvolume) was buffered with 100 mmol L−1 sodium acetate buffer,pH 5.0. The reaction mixture, containing 200 µL of crudeenzyme solution and 800 µL of 1.25 mmol L−1 glycyrrhizinmonoammonium salt, was incubated at 40 ◦C for 10 min. Thereaction was halted by placing the reaction tubes in boilingwater bath and then centrifuging the reaction mixture at 10000 rpm for 10 min. The supernatant was used to determine theamount of β-D-mono-glucuronide-glycyrrhizin by HPLC analysis.One unit (U) enzyme activity of β-glucuronidase was definedas the amount of enzyme capable of converting glycyrrhizinto 1 µg β-D-mono-glucuronide-glycyrrhizin per hour underspecific conditions. Protein concentrations were determined by aCoomassie Brilliant Blue G-250 dye-binding method using bovineserum albumin as the standard protein.

Molecular weight estimationThe molecular weight of the enzyme was determined by SDS-PAGEand matrix-assisted laser desorption/ionization-time of flight massspectrometry (MALDI-TOF MS).19 SDS–PAGE was performed usinga 12% polyacrylamide gel. The gel was stained with CoomassieBrilliant Blue (CBB) R-250 after electrophoresis. The molecular masswas also determined by MALDI-TOF-MS, using Bruker UltraflexMALDI-TOF/TOF mass spectrometer equipped with a 337 nmnitrogen laser. The matrix was prepared in deionized watercontaining sinapinic acid (10 mg mL−1), 50% acetonitrile and 0.1%TFA. L-asparaginase was mixed with matrix (1:1) and 2 µL of thesample was spotted onto a well of sample plate, dried at roomtemperature and then analyzed.

Glycosylation analysis of the β-glucuronidasesTo determine if the recombinant β-glucuronidases areglycosylated, purifiedβ-glucuronidases were stained with periodicacid-Schiff (PAS) reagent using a GelCode Glycoprotein Staining

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Kit (Thermo Fisher Scientific., Waltham, MA, USA) accordingto the manufacturer’s instructions. Peptide-N-glycohydrolase F(PNGase F, Sigma) was also used to determine if yeast IBP isN-linked glycosylated. Briefly, it was carried out by incubatingreaction mixture of 1 mL β-glucuronidase (1 mg mL−1) and 9 mL50 mmol L−1 Tris–HCl buffer (pH 7.0) with 100 IU PNGase F for24 h at 37 ◦C. The entire procedure and analysis of materials wasrepeated three times which provided comparable results.

Effect of pH and temperatureOptimal pH for the enzyme activity was determined by themeasurement of residual enzyme activity in 50 mmol L−1

sodium acetate buffer (pH 3.0–6.0), Tris–HCl buffer (pH 7.0–9.0),and carbonate buffer (pH 10.0–11.0). The pH stability of theenzyme was estimated by measuring the remaining activity ofβ-glucuronidase after incubation for 1 h in different pH reactionmixtures at 30 ◦C. The optimum enzyme temperature was alsodetermined by examining the enzyme activity in 100 mmol L−1

sodium acetate buffer (pH 5.0) for 10 min over a temperaturerange 25–70 ◦C. And the thermal stability of the enzyme wasdetected by incubating the enzyme for 2 h at a temperature of50–60 ◦C and then samples were withdrawn for enzyme assay atspecific time intervals. The residual activity was determined bytaking the original activity as 100%.

Effect of metal ions and reagents on β-glucuronidase activityThe effect of various metal ions and reagents on enzyme activitywas examined by incubating the reaction mixtures containing 100µL of the enzyme solution and 100 µL of metal ions or reagents(final concentration, 1 mmol L−1) for 20 min at 40 ◦C, and finallythe enzyme activity was assayed under standard conditions.20

Enzymatic activities were expressed as relative values (%) withreference to the activity of the enzyme without any metal ionor reagent.

Kinetic parametersThe Michaelis constant (Km), maximal velocity (Vmax) and turnovernumbers (kcat) of the purified enzyme were determined usingpNPG and glycyrrhizin as substrates in the range 0.1–10 mmol L−1

under the optimal assay conditions. The Michaelis–Mentenequation values for Km and Vmax were determined through non-linear regression analysis using the program Origin 7.5 (OriginLabCorporation, Northampton, MA, USA). The kcat values werecalculated with the formula kcat = Vmax/[E] using an enzymeconcentration ([E]) of 0.1 U mL−1 on the basis of one active

site per 69.717 kDa subunit. Results represent means of threeexperiments ± standard deviation values.

RESULTSPurification of the β-glucuronidasesThe enzyme purification protocol is summarized in Table 1. Theβ-glucuronidase in the crude extract of recombinant E. coli wereconcentrated (NH4)2SO4 and this step achieved 7.0-fold enzymepurification, and 55.8% of the enzyme could be recovered. Then,the concentrated enzyme preparation was fractionated by affinitychromatography using a Ni-NTA sepharose column and thisstep achieved 24.7-fold enzyme purification, and 35.3% of theenzyme was recovered. The remaining protein contaminants wereremoved by gel filtration using Sephadex G-100. The fractions withmaximum enzyme activity were collected. The final step resultedin 35.9-fold purification and the specific activity of purified PGUS-Pwas 380.9 U mg−1, with a net yield of 23%.

Similarly, the culture supernatant of P. pastoris grown for 72 hin BMMY medium was precipitated with (NH4)2SO4 and this stepachieved 6.3-fold enzyme purification, and 61.2% of the enzymewas recovered. Then, the concentrated enzyme preparation wasfractionated by gel filtration chromatography using a SephadexG-100 column, and this step achieved 36.0-fold enzymepurification, and 45.1% of the enzyme was recovered. Theremaining protein contaminants were removed by anionicexchange using DEAE-cellulose DE-52. The fractions with maximalenzyme activity were collected. The final step resulted in 47.4-foldpurification and the specific activity of purified PGUS-P was 335.4U mg−1, with a total yield of 31.6%.

Homogeneity and molecular mass of the β-glucuronidasesThe β-glucuronidases from recombinant E. coli (PGUS-E) andP. pastors (PGUS-P) appeared as single isolated bands havingmolecular masses of 72 kDa and 78 kDa, respectively, as deter-mined by SDS-PAGE analysis (Fig. 1(A)), and these values werequite consistent with the molecular masses (72.43 kDa and78.83 kDa) estimated by MALDI-TOF MS analysis (Fig. 2).

Glycosylation analysis of the β-glucuronidasesTo determine the glycosylation and non-glycosylation profile of therecombinant PGUS-E and PGUS-P, purified β-glucuronidases fromtheir respective sources were stained with periodic acid-Schiff(PAS) reagent using a GelCode Glycoprotein Staining Kit. OnlyPGUS-P depicted a pink colored band when the gel was stained

Table 1. Purification of recombinant β-glucuronidases expressed in E. coli and P. pastoris

Purification steps Total protein (mg) Total activity (U) Specific Activity (U mg−1) Recovery (%) Purification (fold)

From E. coli (PGUS-E)

Crude extract 856.0 9082.5 10.6 100.0 1.0

(NH4)2SO4 precipitation 67.7 5075.0 74.9 55.8 7.0

Ni-NTA Sepharose 12.2 3206.6 262.8 35.3 24.7

Sephadex G-100 5.5 2095.0 380.9 23.0 35.9

From P. pastoris (PGUS-P)

Crude extract 1260.3 8913.2 7.0 100.0 1.0

(NH4)2SO4 precipitation 121.4 5457.6 44.9 61.2 6.3

Sephadex G-100 15.7 4019.6 254.7 45.1 36.0

DEAE-cellulose DE-52 8.3 2813.9 335.4 31.6 47.4

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44.3 kDa

29.0 kDa

A B

97.2 kDa

66.4 kDa

31.2 kDa

M 1 2 3

Figure 1. SDS-PAGE analysis of purified β-glucuronidases expressed in E.coli (PGUS-E) and P. pastoris (PGUS-P) stained by Coomassie blue R-250 (A)and by periodic acid-Schiff (PAS) reagent (B). Lane (M): molecular weightmarker, Lanes (1): PGUS-E, Lane (2) and (3): PGUS-P.

with PAS reagent on SDS-PAGE (Fig. 1(B)), indicating the glycosy-lation of recombinant β-glucuronidase from PGUS-P. On the otherhand, the recombinant PGUS-E and PGUS-P were deglycosylatedwith peptide-N-glycosidase F (PNGase F) under native conditions.The characteristics of deglycosylated GUS are shown in Fig. 3.Initially, PGUS-P band appeared a broad and dark band (∼78 kDa)(lane 1 of Fig. 3). However, after deglycosylation with PNGase-F,the resulting band appeared much sharper and there was a single,unified and darker band shown in lane 2 of Fig. 3, indicating the factthat PGUS-P from the recombinant P. pastoris GS115 was a typical

PGUS-P PGUS-E− + − + PNGase-F

M 197.2 kDa

66.4 kDa

3 42

Figure 3. SDS-PAGE analysis of purified β-glucuronidases treated with N-glycosidase F. Lanes (M) molecular weight marker, (1) PGUS-E, (2) PGUS-Etreated with N-glycosidase F, (3) PGUS-P, (4) PGUS-P treated with N-glycosidase F.

glycoprotein. However, the migration rate of PGUS-E remainedunchanged (lane 3 and 4 of Fig. 3) during the whole incubationtime, consistent with the non-glycosylated nature of PGUS-E.

Effect of pH and temperature on activity of theβ-glucuronidasesThe effects of pH on the activity of purified β-glucuronidases areshown in Fig. 4(A). Both PGUS-P and PGUS-E had similar pH optima(pH 5.0), taken as 100% of relative activities, which had a bell shape,being broader for PGUS-P than for PGUS-E. In example, PGUS-Pwas less sensitive to suboptimal pH values than for PGUS-E.

The optimal temperatures of purified enzymes was determinedby varying the reaction temperature. As can be observed in

A

B

Figure 2. Precise molecular mass of purified β-glucuronidases by MALDI-TOF-MS analysis. (A) PGUS-E; (B) PGUS-P. The matrix was prepared in deionizedwater containing sinapinic acid (10 mg mL−1), 50% acetonitrile and 0.1% TFA. L-Asparaginase was mixed with matrix (1:1) and 2 µL of the sample wasspotted onto a well of sample plate, dried at room temperature and then analyzed.

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3 4 5 6 7 8 9 100

20

40

60

80

100A

B

C

Rel

ativ

e ac

tivity

(%

)

pH

PGUS-P

PGUS-E

20 30 40 50 60 70 80

20

40

60

80

100

Rel

ativ

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tivity

(%

)

Temperature (°C)

PGUS-P

PGUS-E

0 20 40 60 80 100 1200

20

40

60

80

100

PGUS-E (65°C)

PGUS-E (55°C)

PGUS-P (65°C)

PGUS-P (55°C)

Time (min)

Rel

ativ

e ac

tivity

(%

)

Figure 4. Effect of pH and temperature on the activity andthermostability of β-glucuronidases. (A) pH optima; (B) temperatureoptima; (C) thermostability. The activity was assayed under standardconditions (40 ◦C, pH 4.5, 10 min, glycyrrhizin monoammonium salt assubstrate). Results represents means of three experiments, and error barsindicates ± SD.

Fig. 4(B), PGUS-P exhibited a continuous rise in activity from 30to 45 ◦C temperature and then decreased gradually, illustratingits maximum activity at 45 ◦C, while PGUS-E showed increasedactivity at temperatures ranging between 30 and 65 ◦C, displayingmaximum activity at 40 ◦C.

Thermal stability of the β-glucuronidasesThe effect of temperature on the enzyme’s stability with referenceto its activity was examined for PGUS-E and PGUS-P. GUS-E activitywas reduced to a level of 60% whereas PGUS-P showed almostno inactivation even after 2 h of incubation at 55 ◦C, as shown inFig. 4(C). PGUS-E is inactivated after 80 min incubation at 65 ◦C,while PGUS-P retained 80% of its activity after 2 h of incubationat 65 ◦C.

Table 2. Effect of various metal ions and chatropic agents on theactivity of β-glucuronidases

Relative activity (%)

Compound Concentration PGUS-E PGUS-P

Control 100 100

Ca2+ 10 mmol L−1 102.2 ± 0.3 99.8 ± 2.3

Cu2+ 1 mmol L−1 45.4 ± 0.3 32.1 ± 3.7

Fe2+ 10 mmol L−1 95.3 ± 0.8 90.3 ± 2.5

Co2+ 10 mmol L−1 96.3 ± 0.0 93.9 ± 0.8

Mg2+ 10 mmol L−1 99.2 ± 0.6 126.4 ± 0.7

Ni2+ 10 mmol L−1 105.2 ± 0.9 84.3 ± 0.2

Zn2+ 10 mmol L−1 97.7 ± 0.2 98.5 ± 0.7

Mn2+ 10 mmol L−1 97.6 ± 0.9 102.7 ± 0.2

Al3+ 10 mmol L−1 98.3 ± 0.8 94.7 ± 0.9

DTT 10 mmol L−1 80.9 ± 0.9 99.1 ± 0.5

EDTA 10 mmol L−1 51.2 ± 0.4 93.5 ± 0.7

Urea 10 mmol L−1 76.3 ± 0.3 98.5 ± 0.2

SDS 0.5% (w/v) 69.3 ± 0.2 101.5 ± 0.9

Triton X-100 0.5% (v/v) 84.8 ± 0.8 93.6 ± 0.3

Effects of metal ions, inhibitors and surfactantsTable 2 shows the effect of indicated 10 mmol L−1 metal ionsolutions on the activity of both recombinant β-glucuronidases.The activities of PGUS-E and PGUS-P were inhibited strongly byCu2+ (54.6% and 67.9%, respectively). The activity of PGUS-P wasalso slightly inhibited by Ni2+ (15.7%), Fe2+ (9.7%), and Co2+(6.1%), but strongly activated by Mg2+ (26.4%). The activity ofPGUS-E was inhibited by DTT (19.1%), EDTA (48.8%), Urea (23.7%),SDS (30.7%), and Triton X-100 (15.2%), however, PGUS-P showedno effect under the influence of such chatropic agents (Table 2),manifesting the strong inhibition effects of chatropic agents onthe activity of PGUS-E rather than PGUS-P.

Kinetic characterization of the β-glucuronidasesIn order to further understand the difference in their substratepreference, kinetics analysis of PGUS-E and PGUS-P towards4-nitrophenyl-β-D-glucuronide (pNPG) and glycyrrhizin (GL) wasdetermined (Table 3). The Km values of PGUS-E were nearly halfthe values of the Km of PGUS-P towards pNPG and GL, respectively.But the kcat value of PGUS-E toward pNPG was half and towardGL was double, of PGUS-P. Consequently, the kcat/Km ratios, werequite similar for pNPG in PGUS-E and PGUS-P but six times higherfor PGUS-P than PGUS-E, illustrating similar catalytic efficiency ofboth enzyme preparations toward pNPG while PGUS-P showed itssuperior catalytic efficiency toward GL.

DISCUSSIONThe β-glucuronidases expressed in E. coli (PGUS-E) and P. pastors(PGUS-P) had molecular weights of 72 kDa and 78 kDa, respectively(Fig. 1(A)). The variations in molecular weights of β-glucuronidaseshave already been reported in organisms such as Eubacteriumsp. GLH (43 kDa),9 Scutellaria root (55–58 kDa)10,21 and Pomaceasp. (90 kDa).22 These differences could be engendered by geneticvariations present among different species, which play importantroles in determining the size, shape and molecular mass of suchenzymes.

Our results compared the extracellular expression ofβ-glucuronidase in P. pastoris (PGUS-P) and intracellular expression

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Table 3. Kinetic parameters of recombinant β-glucuronidases expressed in E. coli and P. pastoris

Km (µmol L–1) Vmax (µmol/L min−1) kcat (min−1) kcat/Km (L µmol−1 min−1)

E. coli

pNPG 95.2 ± 3.4 (2.43 ± 0.11) × 103 (9.86 ± 0.24) × 103 103.6 ± 4.4

GL 424.3 ± 22.1 85.4 ± 4.2 1.64 ± 0.07 × 103 3.86 ± 1.5

P. pastoris

pNPG 213.1 ± 10.4 (2.18 ± 0.04) × 103 (1.91 ± 0.06) × 104 89.6 ± 4.7

GL 998.7 ± 35.2 53.7 ± 3.4 635.2 ± 27.1 0.64 ± 0.28

Proportions

pNPG 0.45 1.11 0.52 1.16

GL 0.42 1.59 2.58 6.68

in E.coli (PGUS-E). Obviously, β-glucuronidase from P. pastoris(PGUS-P) was glycosylated by cellular organelles and suchmodifications affected its biochemical properties; PGUS-E fromprokaryotic source lacked all such modifications. Although PGUS-E and PGUS-P exhibited similar pH optima (5.0), the activity ofPGUS-E was significantly lower within the pH range 3–10. ThePGUS-P displayed a broad tolerance to pH variations betweenpH 4 and 7 and retained above 80% of its relative activity.However, optimal pH variations have been also displayed byβ-glucuronidases from different sources such as β-glucuronidasefrom A. niger exhibited an optimal pH 3.023 and from callus culturesof Scutellaria baicalensis Georgi showed its maximal activity at dualpHs 7.0 and 8.0.24

The glycosylation difference between these β-glucuronidasesalso influenced their temperature and thermal tolerance profiles.The wild-type β-glucuronidase (PGUS)7 and E.coli expressedprotein (PGUS-E) displayed similar optimum temperature 40 ◦Cbut β-glucuronidase expressed in P. pastoris (PGUS-P) showeda slightly higher optimum temperature (45 ◦C). However, onlythe β-glucuronidase from Scutellaria roots showed optimaltemperature 50 ◦C higher than PGUS-P.10 More stabilized PGUS-Pactivity was observed compared with PGUS-E at temperaturesabove 50 ◦C. Moreover, the glycosylation of PGUS-P improvedits thermo-tolerance capacity between 55 and 65 ◦C comparedwith PGUS-E. The glycosylated PGUS-P retained >50% residualactivity after heating at 65 ◦C for 80 min, whereas no significantactivity was observed in the non-glycosylated PGUS-E after thesame treatment. Previous studies have also demonstrated similareffects of glycosylation on proteins.25–27 The increased levelof glycosylation could cause an improvement in the thermo-

tolerance and structural stability of glycoproteins.28–30 Theglycosylation perhaps confers structural rigidity and decreases thedynamic fluctuations in the glycoproteins, therefore increasingthe thermal tolerance and structural stability.29

PGUS-E and PGUS-P were strongly inhibited by Cu2+ asβ-D-glucuronidases from Scutellaria root21 and StreptococcusLJ-2231 were inhibited by Cu2+. The non-glycosylated PGUS-Eexhibited less sensitivity to metal ions and more sensitivity tochatropic agents compared with PGUS-P (Table 2). Li et al.32

also reported that β-glucuronidase from Aedes aegypti chorionexhibited extreme resistance to peroxidase inactivation andvarious denaturing agents owing to its deglycosylation. Ourprevious study also reported the insensitivity of β-glucuronidaseto metal ions after the removal of N-linked glycans.33 The possiblereason for the sensitivity of glycosylated enzymes toward metalions could be the involvement of glycans to form bonds andconsequently distort the structure of the enzyme. The removal of

glycans could confer inertness to the enzymes toward differentmetal ions by eliminating interacting sites.

The kinetic parameters of PGUS-E and PGUS-P showed promi-nent differences due to their glycosylated and non-glycosylatednature respectively. PGUS-E had Km values nearly half the Km val-ues of PGUS-P toward pNPG and GL. But kcat values of PGUS-Evs. PGUS-P were half toward pNPG and 2.6 times toward GL. Thisshows that glycosilation modifies the catalytic mechanism in astereospecific way. It favors the attack on pNPG and hinders theattack on GL. The values of the kcat/Km ratio were quite similarbetween PGUS-E and PGUS-P for pNGP but six times higher forPGUS-E on GL compared with PGUS-P. Again this shows that gly-cosilation modifies the catalytic mechanism of this glucuronidasein a very specific way. The possible explanations for these resultscould be the presence of oligosaccharide chain, which not onlymake the protein structure more rigid and promote its catalyticefficiency, but also determine the position and role of amino acidson its active site.34,35 Some adjustments might have occurred onthe active site as well which would affect the cleavage and freeenergy of the enzyme, and thus its kinetic parameters.36

CONCLUSIONThis study concludes that glycosylated β-glucuronidase (PGUS-P)from P. purpurogenum, expressed and secreted by P. pastoris, wasmore stable under changes of heat, pH and metal ion denaturationcompared with the active and nonglycosylated β-glucuronidase(PGUS-E) expressed and excreted by genetically transformed E. coli.The study also highlights the importance of glycosylation inenzymatic reactions, which has potential applications for thefunctional enhancement of industrial enzymes.

ACKNOWLEDGEMENTSThis research was partially funded by grants from National ScienceFoundation of China (No. 20776017, No. 20976014), the ScienceFoundation of Beijing (No. 5072028), and the Scientific ResearchFoundation of the Education Department of Zhejiang Province,China (No. 20110410).

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