N�

Sa

b

a

ARR1AA

K�PNSU

1

gdi2ootitusa

chtsca

0h

Journal of Biotechnology 164 (2013) 75– 81

Contents lists available at SciVerse ScienceDirect

Journal of Biotechnology

jou rn al hom epage: www.elsev ier .com/ locate / jb io tec

-glycosylation enhances functional and structural stability of recombinant-glucuronidase expressed in Pichia pastoris

huping Zoub,1, Shen Huanga,1, Imdad Kaleema, Chun Lia,∗

School of Life Science, Beijing Institute of Technology, Beijing 100081, People’s Repubic of ChinaInstitute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People’s Republic of China

r t i c l e i n f o

rticle history:eceived 19 September 2012eceived in revised form9 December 2012ccepted 21 December 2012vailable online 11 January 2013

a b s t r a c t

Recombinant �-glucuronidase (GUS) expressed in Pichia pastoris GS115 is an important glycoprotein,encoded by a gene with four potential N-glycosylation sites. To investigate the impact of N-linked car-bohydrate moieties on the stability of recombinant GUS, it was deglycosylated by peptide-N-glycosidaseF (PNGase-F) under native conditions. The enzymatic activities of the glycosylated and deglycosylatedGUS were compared under various conditions such as temperature, pH, organic solvents, detergents andchaotropic agent. The results demonstrated that the glycosylated GUS retained greater fraction of max-

eywords:-Glucuronidaseichia pastoris-glycosylationtability

imum enzymatic activity against various types of denaturants compared with the deglycosylated. Theconformational stabilities of both GUS were analyzed by monitoring the unfolding equilibrium by usingthe denaturant guanidinium chloride (dn-HCl). The glycosylated GUS displayed a significant increase inits conformational stability than the deglycosylated counterpart. These results affirmed the key role ofN-glycosylation on the structural and functional stability of �-glucuronidase and could have potential

onal e

nfolding equilibrium applications in the functi

. Introduction

The unique properties of enzymes to catalyze the reactions withreat specificity, high reaction rates and mild environmental con-itions make them highly suitable for their potential application

n numbers of industrial processes (Nestl et al., 2011; Sheldon,011). Unfortunately, naturally available enzymes are usually notptimally suited for industrial applications. This incompatibilityften related to the intrinsically unstable molecular structure ofhe enzymes under process conditions (Sheldon, 2011). The stabil-ty of an enzyme could be affected easily by many factors, such asemperature, pH, autolysis, surfactants, organic solvents and denat-rants. Hence, there is a great need to develop rational formulationtrategies to improve the stability of the enzymes for industrialpplications (Sheldon, 2011).

Glycosylation is one of the naturally occurring covalent modifi-ations of the eukaryotic proteins. It has been enumerated that overalf of the proteins found in nature are glycosylated, and more thanhree quarters are N-glycosylated (Apweiler et al., 1999). Recent

tudies have shown the key importance of attached carbohydratehains and their vital role in the stability of glycoproteins (Fatimand Husain, 2007; Haga et al., 2011; Hirai-Fujita et al., 2008; Pham

∗ Corresponding author. Tel.: +86 010 68913171; fax: +86 10 68913171.E-mail address: lichun@bit.edu.cn (C. Li).

1 These authors contribute to this work equally.

168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jbiotec.2012.12.015

nhancement of industrial enzymes.© 2013 Elsevier B.V. All rights reserved.

et al., 2008; Price et al., 2010; Sola and Griebenow, 2009; Srimathiand Jayaraman, 2005; Waetzig et al., 2010; Winiarska et al., 2011).These carbohydrate moieties not only increase the thermostabilityof the enzymes but also enhance their resistive capacity againstproteolytic agents. It also contributes as a key factor to improvethe stability of the enzymes against various denaturing conditionsas exhibited by many glycosylated enzymes such as human para-oxonase (Li et al., 2012), dihydrofolate reductase (Tey et al., 2010),bovine deoxyribonuclease (Fujihara et al., 2008) and porcine pan-creatic trypsin (Pham et al., 2008), Momordica charantia peroxidase(Fatima and Husain, 2007), Bacillus licheniformis alpha-amylase(Srimathi and Jayaraman, 2005) and human butyrylcholinesterase(Chilukuri et al., 2005). Obviously, it would be highly advantageousif improved stability could be achieved by N-glycosylation modifi-cation of the industrial enzymes.

�-Glucuronidase (GUS, EC 3.2.1.31) is a glycosidase thathydrolyze �-d-linked glucuronides to yield their various deriva-tives and liberates glucuronic acid. It also has a key role inthe biotransformation of glycyrrhizin (GL) to mono-glucuronide-glycyrrhizin (GAMG) (Akao, 2000; Feng et al., 2006; Kim et al., 1999;Kuramoto et al., 1994; Park et al., 2005). It is much safer and showshigher biological utilization ratio than the substrate GL (Mizutaniet al., 1994). GUS from Penicillium purpurogenum Li-3 exhibited a

highly selective glycyrrhizin-hydrolyzing property and could con-vert GL directly into GAMG, without producing the byproductglycyrrhetic acid (GA) (Feng et al., 2006). In our previous research,we cloned the gene (GenBank accession no. EU095019) from P.

7 otechn

pehtcYcNhvieo

rotcciebi

2

2

Gap

2

mbail1o1cgwtatflaCr7e�rT

2

li

6 S. Zou et al. / Journal of Bi

urpurogenum Li-3 and expressed it in Pichia pastoris GS115 (Qit al., 2011). The recombinant GUS expressed in P. pastoris showedigher thermostability compared to the wild type. In most cases,he recombinant proteins overexpressed in P. pastoris undergo gly-osylatation with high mannose proportion (Stahl et al., 2003;oshimasu et al., 2004). The P. purpurogenum GUS was determinedontaining four potential N-glycosylation sites (N28, N251, N383 and594). The substantial variation in the stability of the wild-type andeterologous GUS has been attributed to the glycosylation. Our pre-ious studies have affirmed that N-glycosylation has an importantnfluence on the catalytic and biochemical properties of GUS (Zout al., 2012). However, there is little information to define the effectsf N-glycosylation on the functional and structural stability of GUS.

In this study, a systematic comparative investigation was car-ied out to determine the effect of glycosylation and deglycosylaionn the enzymatic stability of GUS under wide range of parame-ers such as temperature, pH, detergents and organic solvents. Theonformational stability profiles of two GUS treated with the highoncentrations of guanidinium chloride (Gdn-HCl) was also stud-ed in detail by employing fluorescence spectrometer. This articlencompasses and highlights the changes in the stability of GUSased on the differences of glycosylation and deglycosylation and

ts possible applications in enzymology.

. Materials and methods

.1. Organism and cultivation

The construction and cultivation of recombinant P. pastorisS115 cells expressing the �-glucuronidase (GUS) gene (GenBankccession no. EU095019) from P. purpurogenum Li-3 was reportedreviously (Qi et al., 2011).

.2. Purification of GUS

The extracellular GUS was isolated by centrifuging of the fer-entation broth at 15,000 g at 4 ◦C for 20 min. The supernatant was

rought to 80% saturation with (NH4)2SO4 and stored overnightt 4 ◦C, and then again centrifuged. The precipitate was dissolvedn a small volume of 10 mM Tris–HCl buffer, pH 7.4, and dia-yzed (10,000 molecular weight cut-off) overnight against the0 mM Tris–HCl buffer. The crude enzyme solution was appliednto a DEAE-cellulose (DE 52) column (1.6 cm × 20 cm; flow rate.0 ml/min) equilibrated with the same buffer. After washing theolumn with the buffer, the enzyme was eluted with a linear NaClradient (0.1–0.5 M). The fractions containing the highest activitiesere pooled, brought to 80% saturation with (NH4)2SO4, and cen-

rifuged. The precipitate was dissolved in a small volume of 10 mMcetate buffer, pH 4.6, containing 0.15 M NaCl. The enzyme solu-ion was applied onto a Sephadex G-100 column (1.6 cm × 95 cm;ow rate 10 ml/h) equilibrated and eluted with the buffer. Thective fractions were mixed, concentrated and applied onto a 10 mloncanavalin A Sepharose 4B column (2 cm × 10 cm syringe bar-el). The column was washed with 10 mM Tris–HCl buffer, pH.4, containing 0.5 M NaCl, and the bound glycosylated GUS wasluted with 10 mM Tris–HCl buffer, pH 7.4, containing 0.4 M methyl-d-mannopyranoside. Elution buffer containing the glycosylated

ecombinant GUS was concentrated and dialyzed against 10 mMris–HCl buffer, pH 7.4 and stored at −20 ◦C for further analysis.

.3. Deglycosylation by peptide-N-glycosidase F

Peptide-N-glycohydrolase F (PNGase F, Sigma) was used to deg-ycosylate the recombinant GUS. Briefly, it was carried out byncubating reaction mixture of 1 ml GUS (1 mg/ml) and 9 ml 50 mM

ology 164 (2013) 75– 81

Tris–HCl buffer (pH 7.0) with 100 IU PNGase F for 24 h at 37 ◦C. Deg-lycosylated GUS was separated from non-deglycosylated GUS withCon A Sepharose 4B column. The bound fraction was denoted ascontrol GUS and the unbound fraction as deglycosylated GUS. Thewhole deglycosylation procedure including analysis of materialswas repeated three times which provided comparable results.

2.4. Determination of enzymatic activity and proteinconcentration

�-Glucuronidase activity was measured using a methoddescribed by Zou et al. (2012). The reaction medium (1 ml vol-ume) was buffered with 100 mM sodium acetate buffer, pH 5.0. Thereaction mixture, consisting of 200 �l of crude enzyme solution,800 �l of 0.2% glycyrrhizin mono ammonium salt, was incubatedat 40 ◦C. The reaction was halted by placing the reaction tubes inboiling water bath and then centrifuging the reaction mixture at10,000 rpm for10 min. The supernatant was used to determine theamount of �-mono-glucuronide-glycyrrhizin by HPLC. One unit (U)enzyme activity of �-glucuronidase was defined as the amountof enzyme that is capable of converting glycyrrhizin to 1 �g �-mono-glucuronide-glycyrrhizin per hour under certain conditions.Protein concentrations were determined by a dye-binding method(Bradford, 1976) using bovine serum albumin as the standard pro-tein.

2.5. pH stability and thermostability determination

The stabilities of glycosylated and deglycosylated GUS weredetermined by using different buffers such as glycine–HCl (pH2.0–3.0), sodium acetate (pH 4.0–6.5), sodium phosphate (pH7.0–8.0), glycine–NaOH 0.1 M (pH 9–10.5) at 50 mM concentra-tion. Pure GUS was incubated in different buffers for 2 h at roomtemperature. The original activity was taken as control (100%).

Both GUS dissolved in sodium acetate buffer (50 mM, pH 5.0)were incubated at 55 ◦C and 65 ◦C. And then samples were with-drawn for enzyme assay at specific time intervals. The residualactivity was determined by taking the original activity as 100%.

2.6. Trypsin digestion

Glycosylated and deglycosylated GUS were dissolved at a finalconcentration of 1 mg/ml or less in 20 mM Tris–HCl (pH 8.0)containing 15 mM NaCl. Aliquots of proteins were treated withTPCK-treated trypsin with enzyme/substrate mass ratios of 1:100and 1:50, placed them for overnight as cleavage time period. Thereaction was terminated by heating the mixture in a boiling waterbath and the extent of digestion was monitored on 15% SDS-PAGE.

2.7. Effect of organic solvents

Glycosylated and deglycosylated GUS (0.5 U/ml) were incubatedwith varying concentrations of organic solvents dimethyl sulfox-ide (DMSO) and acetone (10–60%, v/v) in 100 mM sodium acetatebuffer, pH 5.0 at 40 ◦C for 2 h. The activity of enzyme withoutorganic solvent was taken as control (100%) for calculating theremaining percent activity.

2.8. Effect of urea and detergents

Glycosylated and deglycosylated GUS (0.5 U/ml) were incubated

with increasing concentrations of urea (2.0–8.0 M), detergents SDS(0.2–1.2%, w/v) and Triton X-100 (0.2–1.2%, v/v) for 2 h in 100 mMsodium acetate buffer, pH 5.0 at 40 ◦C. The residual activity wasdetermined after each incubation period. The activity of the urea

S. Zou et al. / Journal of Biotechn

Fbp

ut

2

(uFdaatwdbof

F

wvca

�

w(la

�

wco

3

3

scGa

ig. 1. SDS-PAGE analyses of purified GUS treated with PNGase F at different incu-ation time. The glycosylated (78 kDa) and completely deglycosylated (68 kDa) GUSolypeptides are indicated by arrows.

ntreated enzyme was considered as control (100%) for calculatinghe remaining percent activity.

.9. Denaturant induced studies

Samples having varying concentrations of guanidinium chlorideGdn-HCl) were prepared by combining different ratios of denat-rant stock solution and 0.05 M sodium acetate buffer of pH 5.0.or denaturation experiments, different volumes of concentratedenaturant solution were added first to a stock enzyme solutionnd the mixtures were incubated for 12 h and finally buffer wasdded to each reaction mixture to get desired denaturant concen-ration. The final reaction mixtures for denaturation experimentsere further incubated for 12 h before optical measurements. Theenaturation was followed, by monitoring the spectral changesy fluorescence spectroscopy techniques. The denaturation databtained were converted into the fraction denatured FD, using theollowing equation:

D =(

Y − YN

YD − YN

)(1)

here Y is the observed variable parameter and YN and YD are thealues of the variable characteristics of the folded and unfoldedonformations. The difference in free energy between the foldednd unfolded states, �G was calculated by the following equation:

G = −RT ln K = −RT ln(

FD

1 − FD

)(2)

here K is the equilibrium constant, R is the gas constant1.987 cal deg−1 mol−1) and T is the absolute temperature. Theimiting values of transition were determined through regressionnalysis of the line given by

G = �G(H2O) − m [denaturant] (3)

here m is a measure of the dependence of �G on Gdn-HCl con-entration and �G(H2O) is the free energy change in the absencef denaturant.

. Results and discussion

.1. Deglycosylation of GUS with N-glycosidase F

The recombinant GUS expressed in P. pastoris was deglyco-

ylated with peptide-N-glycosidase F (PNGase F) under nativeonditions. The characteristics of glycosylated and deglycosylatedUS are enlisted in Fig. 1. Initially, PGUS-P band appeared broad,nd a dark band (∼78 kDa) and a lighter band were observed in

ology 164 (2013) 75– 81 77

lane 2 of Fig. 1. However, after the deglycosylation with PNGase-F, the band became sharper and a single, unified and darker wasobserved in lane 5 of Fig. 1. It indicates that the oligosaccharidesin glycosylated PGUS-P are heterogeneous. The migration rates ofdeglycosylated GUS exhibited a sequential increasing trend withinthe initial incubation period of 3 h at 37 ◦C with 0.4 U of PNGase-F(lanes 2–5 of Fig. 1). However, further increase in the incubationtime proved ineffective, and the migration rate of GUS remainedunchanged (lane 6 of Fig. 1), indicating that N-linked oligosaccha-rides of glycosylated GUS had been removed virtually after theenzymatic deglycosylation reaction. The molecular weights of gly-cosylated and completely deglycosylated GUS were 78 kDa and68 kDa, respectively, depicting an amount of 14% total carbohydratecontents present in the glycosylated GUS.

These results demonstrated PNGase F is highly effectiveto remove virtually all N-linked oligosaccharides from �-glucuronidase, while some known glycoproteins are very difficultto deglycosylate because steric hindrance of the oligosaccharideslimit the accessibility of PNGase F to the glycosylation sites (Miyataet al., 2012; Skropeta, 2009).

3.2. Effect of N-glycosylation on pH stability and thermostabilityof GUS

The effect of pH on the stability of glycosylated and deglycosy-lated GUS are shown in Fig. 2A. Both enzymes were found to bequite stable within pH range 4.0–8.0 at 40 ◦C for 2 h. However, theglycosylated GUS retained remarkably high activity, 95% after itsexposure to pH 9.0 for 2 h, while the deglycosylated form retainedonly 66% of its original activity under similar incubation conditions.The effects of N-glycosylation on the heat resistance capacity ofboth GUS were evaluated by incubating their reaction mixtures atvarious elevated temperatures. Aliquots were collected from thereaction mixtures after specific time intervals and their residualactivities were determined (Fig. 2B). No distinct activity declinewas observed for both GUS at 55 ◦C. However, the deglycosylatedGUS rapidly lost its activity at 65 ◦C, while its glycosylated counter-part lost its activity much slowly and this pattern shows its internaltolerance for high temperature perhaps conferred by glycosylation.Deglycosylated GUS was inactivated after 2 h of incubation at 65 ◦Cwhereas the glycosylated form retained 80% of its activity at thistemperature. Apparently, N-glycosylation played a vital role in �-glucuronidase’s heat tolerance (Pham et al., 2008; Srimathi andJayaraman, 2005).

Previous studies have referred the improvement in the thermo-tolerance of glycoproteins due to increased levels of glycosylation(Fatima and Husain, 2007; Han and Lei, 1999; Price et al., 2010;Shental-Bechor and Levy, 2008). Jafari-Aghdam et al. (2005)reported that glycosylation provided improvement in functionalcharacteristics of the enzyme, especially in relation to its ther-mostability, and suggested glycosylation is an important factorto protect the enzyme from heat denaturation. Srimathi andJayaraman (2005) reported that glycosylation of Bacillus licheni-formis alpha-amylases increased its thermostability. In otherreport, Somera et al. (2009) reported that N-glycosylation playedcritically important role in the thermal stability of �-xylosidasefrom Aspergillus versicolor. The N-glycosylation enhanced structuralrigidity of the proteins and caused marked decrease in dynamicfluctuations throughout the entire molecule, which led to anincrease its thermal stability (Shental-Bechor and Levy, 2008).

3.3. Effect of N-glycosylation on proteolytic stability of GUS

Both glycosylated and deglycosylated GUS were incubated withvarious concentrations of trypsin and analyzed by electrophoresis(Fig. 3). The glycosylated GUS exhibited high stability to proteolytic

78 S. Zou et al. / Journal of Biotechnology 164 (2013) 75– 81

Table 1Remaining or relative activities of glycosylated and deglycosylated GUS at different concentrations of organic solvents.

Organic solvent (%, v/v) Percent relative activity

DMSO Acetone

Glycosylated GUS Deglycosylated GUS Glycosylated GUS Deglycosylated GUS

0 100 100 100 10010 105.1 ± 2.6 102.3 ± 2.0 105.2 ± 1.7 99.2 ± 1.520 114.3 ± 3.7 104.5 ± 1.8 93.4 ± 1.3 92.5 ± 1.330 126.7 ± 2.0 110.1 ± 2.1 85.3 ± 1.1 74.5 ± 1.6

± 3.5

± 3.3

± 2.3

dphoiotncce

FGlao(

40 107.2 ± 2.5 107.250 96.5 ± 3.2 90.860 56.4 ± 3.6 27.3

egradation and no protein smear was visible even at higherrotease concentrations. In contrast, the deglycosylated GUS wasydrolysed very easily by trypsin (5 g/l) and exhibited an obvi-us smear as could be observed in lane 6 of Fig. 3. These resultsllustrated the profound protective role of carbohydrate moiteisn GUS against proteolytic hydrolysis and in accordance withhe other related studies on the glycoproteins, such as Aspergillusiger glucoamylase (Jafari-Aghdam et al., 2005) and Bovine pan-

reatic ribonuclease B (Rudd et al., 1994). The presence of glycansould provide steric hindrance around the peptide backbone of thenzyme and safeguard the enzyme from the proteolytic hydrolysis.

1 2 3 4 5 6 7 8 9 10 11 120

20

40

60

80

100

Rel

ativ

e ac

tiv

ity

/ %

pH

Deglycosylat ed G US

Glycosylat ed G US

0 20 40 60 80 100 1200

20

40

60

80

100

Re

lative

activity (

%)

Time / min

Glycos ylated GUS (65 C)

Deglycos ylated GUS (65 C)

Glycos ylated GUS (55 C)

Deglycos ylated GUS (55 C)

A

B

ig. 2. (A) Effect of pH on the stability of glycosylated and deglycosylated GUS. BothUS enzymes were incubated in different buffers for 24 h at 25 ◦C. (B) Thermostabi-

ity of glycosylated and deglycosylated GUS. The residual activity was determinedfter incubation in the absence of substrate at 55 ◦C (�), 65 ◦C (�) and in the presencef substrate 55 ◦C (�), 65 ◦C (�) at pH 5. The original activity was taken as control100%). Each value in the panel represents the mean of three ± SD.

64.6 ± 1.1 42.4 ± 1.447.4 ± 0.9 20.4 ± 1.713.5 ± 1.1 0

This might prevent the contact between the surface of the glyco-protein and active site of the protease (Sola and Griebenow, 2009).Rudd et al. (1994) speculated that proteolytic protection of the pep-tide backbone is due to the hydrogen bonding between hydrophilicamino acids and N-glycans.

3.4. Effect of N-glycosylation on organic solvent-tolerance of GUS

The effects of increased concentrations of hydrophilic organicsolvents such as dimethyl sulfoxide (DMSO) and acetone on theactivities of glycosylated and de-glycosylated GUS were examinedand results have been compiled in Table 1. There was a conspicuouselevation in the activation level of glycosyated and deglycosylatedGUS when treated with low concentration of DMSO. The relativeactivities of glycosylated and deglycosylated GUS enhanced 26.7%and 10.1%, respectively, as compared to their initial activity whentreated with 30% (v/v) DMSO. At 60% (v/v) DMSO, the relative activ-ity of glycosylated GUS retained 56.4% of the initial activity whereasdeglycosylated preparation retained only 27.3% of the initial activ-ity. Exposure of glycosylated GUS to 40% (v/v) acetone resulted inthe loss of 56.1% activity and retained 64.6% of the initial activity.The relative activities of glycosylated GUS retained 13.5% of theinitial activity whereas the deglycosylated preparation showed noactivity. These results indicated that N-glycosylation played a vitalrole for organic solvent-tolerance of the GUS.

3.5. Effect of N-glycosylation on chaotropic agent tolerant of GUS

The effect of chaotropic agent such as urea is presented in Fig. 4.There was no significant change in the activity of GUS after their

Fig. 3. Proteolytic cleavage of glycosylated and deglycosylated GUS. The glycosy-lated (lines 1–3) and deglycosylated (lines 4–6) GUS were treated with trypsin andenzyme/substrate ratios of 0, 1:100 and 1:50 for overnight. The reaction was ter-minated by heating the mixture in a boiling water bath and the extent of digestionwas analyzed by 15% SDS-PAGE.

S. Zou et al. / Journal of Biotechnology 164 (2013) 75– 81 79

-2 0 2 4 6 8 100

20

40

60

80

100

Rela

tive a

ctivity (

%)

Urea ( M)

Glycos ylated GUS

Deglycos ylated GUS

Fig. 4. Effect of chaotropic agent on glycosylated and deglycosylated GUS. Glyco-sylated and deglycosylated preparations of GUS (0.4 U/ml) were incubated withincreasing concentration of urea (2.0–8.0 M) in 100 mM sodium acetate buffer, pH5.6 at 37 ◦C for 1 h. The enzyme activity was determined as described in the text. Theactivity of unexposed samples was taken as control (100%) for calculating percentr

iaaaGbfwrttssai

3

ooita1tedu0otro0httc

0.0 0.2 0.4 0.6 0.8 1.0 1.2

40

60

80

100

120

Rel

ati

ve

acti

vit

y (

%)

SDS (w/v)

Glycosyla ted GUS

Deglycosyla ted GUS

0.0 0.2 0.4 0.6 0.8 1.0 1.2

70

80

90

100

110

120

Rel

ativ

e ac

tiv

ity

(%

)

Concentration of T rito n x-100 (v/ v)

Gl ycos ylated GUS

De glycos lated GUS

A

B

Fig. 5. Effect of detergents on glycosylated and deglycosylated GUS. Glycosylatedand deglycosylated preparations of GUS (0.4 U/ml) were incubated with increasingconcentrations of (A) SDS (0.1–1.0%, w/v) and (B) Triton X-100 (0.5–5.0%, v/v) in100 mM sodium acetate buffer, pH 5.6 at 37 ◦C for 1 h. The enzyme activity was

emaining activity.

ncubation in 2.0 M urea for 2 h. However, the change in catalyticctivity became more pronounced in 4.0 M urea concentrationnd onwards. The glycosylated GUS retained remarkably high 90%ctivity after exposure to 8.0 M urea for 2 h whereas deglycosylatedUS retained nearly 70% of the original activity under similar incu-ation conditions. Further incubation of both GUS in 8.0 M ureaor 2 h resulted in a loss of 40% activity of the deglycosylated GUShereas glycosylated GUS retained 85% of the initial activity. These

esults indicated that N-glycosylation played a vital role in the ureaolerance of GUS. Some researchers have already demonstratedhe vital role of carbohydrate residues in glycoproteins for pre-enting a solid resistance to urea mediated inactivation. In a recenttudy, Li et al. (2005) also reported that peroxidase from Aedesegypti chorion was found to be extremely resistant to inactivationsnduced by various denaturing agents.

.6. Effect of N-glycosylation on detergent tolerance of GUS

The effect of increasing concentrations of SDS (0.2–1.2%, w/v)n the activity of both types of GUS is shown in Fig. 5A. The activityf glycosylated GUS was enhanced significantly up to 115% afterts exposure to 0.2% (w/v) SDS whereas deglycosylated prepara-ion was activated to 130% at the same concentration of SDS. Thectivity of glycosylated form was markedly improved up to 72% at.2% (w/v) SDS whereas deglycosylated preparation was activatedo less than half of this value at the same concentration of SDS. Theffect of increasing concentration of Triton-X 100 (0.2–1.2%, v/v) isepicted in Fig. 5B. The activity of glycosylated GUS was enhancedntil a concentration of 0.4% (v/v) reaching a maximum of 120% at.2% (v/v) Triton-X 100 while a closer value of 110% activity wasbserved at 0.4% (v/v) Triton X-100. The deglycosylated prepara-ion was only activated up to a concentration of 0.2% (v/v) where itetained 113% GUS activity. There was a leveling off in the activitiesf both glycosylated and deglycosylated preparation of GUS from.4% (v/v) Triton X-100 to onward. Glycosylated GUS retained aigher enzymatic activity compared to the deglycosylated form, as

he former retained 87% activity at 1.2% (v/v) Triton X-100 whereashe latter reserved only 70% activity under the identical incubationonditions.

determined as described in the text. The activity of unexposed samples was takenas control (100%) for calculating percent remaining activity.

3.7. Effect of N-glycosylation on conformational stability of GUS

The conformational stability GUS was determined by mon-itoring the unfolding equilibrium caused by denaturing agentguanidinium chloride (Gdn-HCl) and analysis is based on the tran-sitional phase in the denaturation curve. The fractional unfoldingof glycosylated and deglycosylated GUS in the presence of Gdn-HCl is shown in Fig. 6A. The influences on the free energy changeof denaturation in the transition state is shown in Fig. 6B. For theglycosylated GUS, the observed sigmoidal change was steep andnarrow and occurred at Gdn-HCl concentrations of 2.1 and 3.1 M,which translated into apparent free energy change �G(H2O) hav-ing a value of 13.06 kJ/mol. The deglycosylated GUS also exhibitedthe typical sigmoidal change, however, the denaturation range forGdn-HCl was from 1.7 to 2.9 M, which led to a lower �G(H2O) valueof 6.30 kJ/mol and was less than half of the glycosylated GUS value(Fig. 5B). The results indicated that N-glycosylation conferred theenhancement of conformational stability to the GUS molecule.

Glycosylation could decrease flexibility or add rigidity to theenzyme structure, which in turn increases the conformational sta-

bility to the enzyme (Rudd et al., 1994; Yoshimasu et al., 2004).Steric interactions between the sugar residues and protein struc-ture have been reported to be involved in stabilizing the effects

80 S. Zou et al. / Journal of Biotechn

0 1 2 3 4 5

0

20

40

60

80

100

Glycosylated GU S

Deglycosylated GU S

Glycosylated GU S

Deglycosylated GU S

Fra

ctio

n N

ativ

e

Gdn -HCl (M)

1 2 3 4

Gdn-HCl (M)

-10

G(

kJ

mol-1

)

40

30

20

10

0

A

B

Fig. 6. Conformational stability of glycosylated and deglycosylated GUS againstGdn-HCl induced unfolding transitions (A) glycosylated GUS (�) and deglycosy-lated GUS (�). Denaturation was followed using enzyme activity and absorbance(300 nm). The graph is modeled based on a two-state derivation of �G assuminga linear dependence of �G on Gdn-HCl concentration. (B) Shows the influence ofds

iMeitY

4

paosfifpgdpTs

Current Opinion in Chemical Biology 15, 187–193.

enaturant concentration on the free energy change of denaturation in the transitiontate.

n many other glycosylated proteins (Fatima and Husain, 2007;uller-Steffner et al., 2010; Srimathi and Jayaraman, 2005). Sev-

ral research groups have referred glycosylation as a major factornvolved in the improvement of the stability of enzyme struc-ure via decreasing the flexibility (Sarkar and Wintrode, 2011;oshimasu et al., 2004).

. Conclusion

One of the challenges for practical applications of therotein biocatalysts is to uplift their stability levels. Therere numerous reports of mutagenesis for the improvementf enzyme function, structure and stability, hence deriving atructure–stability–function relationship, while chemical modi-cation of specific functional group/groups could also induce

avorable changes in function and stability in comparatively inex-ensive ways. In the present study, we have proved the key role oflycosylation in stabilizing the structure of �-glucuronidase and itsegree of strength against various destabilizing agents such as tem-

eratures, pH, organic solvents, detergents and chaotropic agent.his report also supports the stability based modification of enzymetructure to meet the future needs of the biotechnology industry.

ology 164 (2013) 75– 81

Potential applications of these highly stable enzymes in the fieldsof biotechnology and industries still remain to be exploited.

Acknowledgments

This research was partially funded by grants from National Sci-ence Foundation of China (Nos. 20976014 and 21176028), ScienceFoundation of Beijing (No. 2112035), Doctoral Fund of Ministry ofEducation of China (20091101110036 and 2012331712004), StateKey Basic Research and Development Program (973Program, No.2011CBA00800), National High Technology Research and Devel-opment Program of China (863 Program, No.2012AA02A704) andScientific Research Foundation of the Education Department of Zhe-jiang Province, China (No.20110410).

References

Akao, T., 2000. Differences in the metabolism of glycyrrhizin, glycyrrhetic acid andglycyrrhetic acid monoglucuronide by human intestinal flora. Biological andPharmaceutical Bulletin 23, 1418–1423.

Apweiler, R., Hermjakob, H., Sharon, N., 1999. On the frequency of protein glyco-sylation, as deduced from analysis of the SWISS-PROT database. BBA – GeneralSubjects 1473, 4–8.

Bradford, M.M., 1976. A rapid and sensitivemethod for the quantitation ofmicrogramquantities of protein utilizing the principle of protein–dye binding. AnalyticalBiochemistry 72, 248–254.

Chilukuri, N., Parikh, K., Sun, W., Naik, R., Tipparaju, P., Doctor, B.R., Saxena, A., 2005.Polyethylene glycosylation prolongs the circulatory stability of recombinanthuman butyrylcholinesterase. Chemico-Biological Interactions 157, 115–121.

Fatima, A., Husain, Q., 2007. A role of glycosyl moieties in the stabilization of bit-ter gourd (Momordica charantia) peroxidase. International Journal of BiologicalMacromolecules 41, 56–63.

Feng, S.J., Li, C., Xu, X.L., Wang, X.Y., 2006. Screening strains for directed biosynthesisof beta-d-mono-glucuronide-glycyrrhizin and kinetics of enzyme production.Journal of Molecular Catalysis B: Enzymatic 43, 63–67.

Fujihara, J., Yasuda, T., Kunito, T., Fujii, Y., Takatsuka, H., Moritani, T., Takeshita, H.,2008. Two N-Linked glycosylation sites (Asn18 and Asn106) are both requiredfor full enzymatic activity, thermal stability, and resistance to proteolysis inmammalian deoxyribonuclease. I. Bioscience, Biotechnology, and Biochemistry72, 3197–3205.

Haga, Y., Ishii, K., Suzuki, T., 2011. N-glycosylation is critical for the stability and intra-cellular trafficking of glucose transporter GLUT4. Journal of Biological Chemistry286, 31320–31327.

Han, Y.M., Lei, X.G., 1999. Role of glycosylation in the functional expression of anAspergillus niger phytase (phyA) in Pichia pastoris. Archives of Biochemistry andBiophysics 364, 83–90.

Hirai-Fujita, Y., Yamamoto-Hino, M., Kanie, O., Goto, S., 2008. N-glycosylation ofthe Drosophila neural protein Chaoptin is essential for its stability, cell surfacetransport and adhesive activity. FEBS Letters 582, 2572–2576.

Jafari-Aghdam, J., Khajeh, K., Ranjbar, B., Nemat-Gorgani, M., 2005. Deglycosylationof glucoamylase from Aspergillus niger: effects on structure, activity and stability.BBA – Proteins and Proteomics 1750, 61–68.

Kim, D.H., Lee, S.W., Han, M.J., 1999. Biotransformation of glycyrrhizin to 18 beta-glycyrrhetinic acid-3-O-beta-d-glucuronide by Streptococcus LJ-22, a humanintestinal bacterium. Biological and Pharmaceutical Bulletin 22, 320–322.

Kuramoto, T., Ito, Y., Oda, M., Tamura, Y., Kitahata, S., 1994. Microbial-production ofglycyrrhetic acid 3-O-mono-beta-d-glucuronide from glycyrrhizin by Cryptococ-cus magnus Mg-27. Bioscience, Biotechnology, and Biochemistry 58, 455–458.

Li, J.S.S., Cu, L.W., Rock, D.L., Li, J.Y., 2005. Novel glycosidic linkage in Aedes aegyptichorion peroxidase N-mannosyl tryptophan. Journal of Biological Chemistry280, 38513–38521.

Li, T.G., Nana, W., Heng, D., Min, Z., 2012. Polyethylene glycosylation prolongsthe stability of recombinant human paraoxonase-1. Toxicology Letters 210,366–371.

Miyata, T., Harakuni, T., Taira, T., Matsuzaki, G., Arakawa, T., 2012. Merozoite surfaceprotein-1 of Plasmodium yoelii fused via an oligosaccharide moiety of choleratoxin B subunit glycoprotein expressed in yeast induced protective immunityagainst lethal malaria infection in mice. Vaccine 30, 948–958.

Mizutani, K., Kuramoto, T., Tamura, Y., Ohtake, N., Doi, S., Nakaura, M., Tanaka,O., 1994. Sweetness of glycyrrhetic acid 3-O-beta-d-monoglucuronide and therelated glycosides. Bioscience, Biotechnology, and Biochemistry 58, 554–555.

Muller-Steffner, H., Kuhn, I., Argentini, M., Schuber, F., 2010. Identification of theN-glycosylation sites on recombinant bovine CD38 expressed in Pichia pastoris:their impact on enzyme stability and catalytic activity. Protein Expression andPurification 70, 151–157.

Nestl, B.M., Nebel, B.A., Hauer, B., 2011. Recent progress in industrial biocatalysis.

Park, H.Y., Kim, N.Y., Han, M.J., Bae, E.A., Kim, D.H., 2005. Purification and char-acterization of two novel beta-d-glucuronidases converting glycyrrhizin to 18beta-glycyrrhetinic acid-3-O-beta-d-glucuronide from Streptococcus LJ-22. Jour-nal of Microbiology and Biotechnology 15, 792–799.

otechn

P

P

Q

R

S

S

S

S

S

ation on the aspartic proteinase porcine pepsin expressed from Pichia pastoris.

S. Zou et al. / Journal of Bi

ham, V.T., Ewing, E., Kaplan, H., Choma, C., Hefford, M.A., 2008. Glycation improvesthe thermostability of trypsin and chymotrypsin. Biotechnology and Bioengi-neering 101, 452–459.

rice, J.L., Shental-Bechor, D., Dhar, A., Turner, M.J., Powers, E.T., Gruebele, M., Levy,Y., Kelly, J.W., 2010. Context-dependent effects of asparagine glycosylation onpin WW folding kinetics and thermodynamics. Journal of the American ChemicalSociety 132, 15359–15367.

i, F., Dai, D.Z., Liu, Y.L., Kaleem, I., Li, C., 2011. Effects of low-shear modeled micro-gravity on the characterization of recombinant beta-d-glucuronidase expressedin Pichia pastoris. Applied Biochemistry and Biotechnology 163, 162–172.

udd, P.M., Joao, H.C., Coghill, E., Fiten, P., Saunders, M.R., Opdenakker, G., Dwek,R.A., 1994. Glycoforms modify the dynamic stability and functional-activity ofan enzyme. Biochemistry 33, 17–22.

arkar, A., Wintrode, P.L., 2011. Effects of glycosylation on the stability and flexibilityof a metastable protein: the human serpin alpha(1)-antitrypsin. InternationalJournal of Mass Spectrometry 302, 69–75.

heldon, R.A., 2011. Cross-linked enzyme aggregates as industrial biocatalysts.Organic Process Research and Development 15, 213–223.

hental-Bechor, D., Levy, Y., 2008. Effect of glycosylation on protein folding: a dosebook at thermodynamic stabilization. Proceedings of the National Academy of

Sciences of the United States of America 105, 8256–8261.

kropeta, D., 2009. The effect of individual N-glycans on enzyme activity. Bioorganic& Medicinal Chemistry 17, 2645–2653.

ola, R.J., Griebenow, K., 2009. Effects of Glycosylation on the Stability of ProteinPharmaceuticals. Journal of Pharmaceutical Science 98, 1223–1245.

ology 164 (2013) 75– 81 81

Somera, A.F., Pereira, M.G., Souza Guimarães, L.H., Polizeli Mde, L., Terenzi, H.F., MeloFurriel, R.P., Jorge, J.A., 2009. Effect of glycosylation on the biochemical proper-ties of beta-xylosidases from Aspergillus versicolor. Journal of Microbiology 47,270–276.

Srimathi, S., Jayaraman, G., 2005. Effect of glycosylation on the catalytic and con-formational stability of homologous alpha-amylases. The Protein Journal 24,79–88.

Stahl, C.H., Wilson, D.B., Lei, X.G., 2003. Comparison of extracellular Escherichia coliAppA phytases expressed in Streptomyces lividans and Pichia pastoris. Biotech-nology Letters 25, 827–831.

Tey, L.H., Loveridge, E.J., Swanwick, R.S., Flitsch, S.L., Allemann, R.K., 2010. Highlysite-selective stability increases by glycosylation of dihydrofolate reductase. TheFEBS Journal 277, 2171–2179.

Waetzig, G.H., Chalaris, A., Rosenstiel, P., Suthaus, J., Holland, C., Karl, N., Uriarte, L.V.,Till, A., Scheller, J., Grotzinger, J., Schreiber, S., Rose-John, S., Seegert, D., 2010.N-linked glycosylation is essential for the stability but not the signaling functionof the interleukin-6 signal transducer glycoprotein 130. The Journal of BiologicalChemistry 285, 1781–1789.

Yoshimasu, M.A., Tanaka, T., Ahn, J.K., Yada, R.Y., 2004. Effect of N-linked glycosyl-

Glycobiology 14, 417–429.Zou, S.P., Xie, L.P., Liu, Y.L., Kaleem, I., Zhang, G.F., Li, C., 2012. N-linked glycosylation

influences on the catalytic and biochemical properties of Penicillium purpuro-genum beta-d-glucuronidase. Journal of Biotechnology 157, 399–404.