Enhancing the Thermostability of Î²-Glucuronidase by Rationally

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Enhancing the thermostability of #-glucuronidase by rationally redesigning the catalytic domain based on sequence alignment strategy Xu-Dong Feng, Heng Tang, Beijia Han, Bo Lv, and Chun Li Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.6b00535 • Publication Date (Web): 29 Apr 2016 Downloaded from http://pubs.acs.org on May 9, 2016

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Industrial & Engineering Chemistry Research

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Enhancing the thermostability of β-glucuronidase by rationally

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redesigning the catalytic domain based on sequence alignment

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strategy

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Xudong Feng, Heng Tang, Beijia Han, Bo Lv, Chun Li*

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School of Life Science, Beijing Institute of Technology, Beijing 100081, PR China

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*Corresponding author at:

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Tel.: +86 10 68913171; fax: +86 10 68913171. Email: [email protected]

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ACS Paragon Plus Environment


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Abstract

1 2 3

β-Glucuronidase has been widely used in improving the efficacy of the natural

4

glucuronides, but the poor thermostability largely impedes its industrial application. In

5

this study, the thermostability of β-glucuronidase from Penicillium purpurogenum Li-3

6

(PGUS-E) was enhanced by rationally mutating key residues within the catalytic

7

domain based on in-depth structure analysis and sequence alignment. Three mutants

8

F292L/T293K, S35P, R304L were obtained which showed significantly improved

9

thermostability. PGUS-E showed a two-phase thermal deactivation process, and the

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thermal deactivation constants k1 and k2 were solved separately in each phase. The

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mutation of F292L/T293K and S35P contributed more to the maintenance of the

12

enzyme stability in the first deactivation phase, with k1 decreased by one magnitude

13

compared to that of wild-type. Meanwhile, the mutation R304L mainly took effect in

14

the second deactivation phase with the lowest k2 of 0.0021 min-1. In addition, mutant

15

F292L/T293K showed 6.4 times higher kcat/Km than wild-type. The MD simulation

16

indicated that the improved thermostability of the three mutants was due to a unique

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C-terminal fixing effect (F292L/T293K), proline effect (S35P) and hydrophobic

18

interaction (R304L). This study not only promotes the industrial application of

19

β-glucuronidase but also provides new insight into the interplay between structure and

20

stability of β-glucuronidase.

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Keywords:

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mutagenesis; MD simulation

β-glucuronidase;

thermostability; enzyme

2


engineering;

site-directed

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1. Introduction

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β-Glucuronidases (GUS, EC 3.2.1.31), belonging to glycoside hydrolase families GH1,

3

GH2 and GH79, cleave glucuronic acid sugar moieties from the non-reducing termini of

4

glycosides.1 It has been conventionally used in disease diagnosis, gene manipulation,

5

and food industry.2-3 Recently, it has drawn much attention in modifying the natural

6

glucuronides to enhance their efficacy.4-5 Glucuronides are glycoconjugates formed by

7

the attachment of various glycans to the bioactive aglycones displaying in carbon

8

frameworks, which have been widely used in pharmaceuticals, food, cosmetic and feed

9

industry.6 However, the solubility and the associated side effect of glucuronides has

10

largely limited their intake efficiency, and such problems can be solved through partially

11

removing sugar moiety by β-glucuronidases.7

12

Recently, we have identified a β-glucuronidase from Penicillium purpurogenum Li-3

13

and heterologously expressed it in E. coli (PGUS-E). PGUS-E showed high hydrolytic

14

activity to glycyrrhizin (GL), which has been widely used as a herbal medicine and

15

sweetener.8 As shown in Scheme 1, when the glucuronic acid moiety was removed by

16

PGUS-E, GL can be transformed into more valuable glycyrrhetic acid (GA) via a

17

two-step hydrolysis reaction where monoglucuronide glycyrrhetic acid (GAMG) was

18

considered as an intermediate.

19

than GAMG, so the product is mainly in the form of GAMG with little GA formed in

20

the initial period of the reaction. However, the low thermostability of PGUS-E has

21

impeded its application in GL biotransformation at a large scale. In addition, elevating

22

reaction temperature has advantages of high substrate solubility and accelerated reaction

9-12

In addition, PGUS-E has high affinity towards GL

3



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rate.13 Therefore, the thermostability of PGUS-E needs to be enhanced to meet

2

industrial applications. Until now, the improvement of β-glucuronidase thermostability

3

has been achieved by directed evolution based on random-library screening.14-15

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However, such work usually requires expensive high-throughput screening methods,

5

and the possibility to get positive mutant is low.16-17

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Alternatively, semi-rational or rational design with targeted residues based on sequence

7

alignment or structure analysis have been developed.18-21 A general method is to

8

substitute unstable residues or segments with their stable counterparts, thus rigidifying

9

the overall protein structure and enhancing the thermostability. In such protocols,

10

choosing suitable potential hot-spots for mutation is the most critical step, which is

11

usually on the basis of clear understanding of the relationship between the structure and

12

function of enzymes. Currently, more and more protein structures are resolved by

13

experimental technique such as X-ray or predicted with the help of molecular

14

modelling.22 In literature, the selection of potential hot-spots was mainly based on

15

sequence alignment,20 B-factor principle23 and dynamic surface loop,24 etc. Recently,

16

Xie at al. proved that increasing the rigidity of the flexible segment within the active

17

pocket could also improve the enzyme stability.25 Despite the various methods reported,

18

how to precisely and efficiently choose potential residues for substitution is still quite

19

challenging.

20

In this study, we aimed to increase the thermostability of PGUS-E by rational

21

site-directed mutagenesis. Based on the in-depth structure analysis, we mainly focused 4


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on the residues within 30 Å from the catalytic residues of PGUS-E and residues in

2

highly flexible surface loops. Then, through sequence alignment with thermophilic

3

β-glycosidases of GH2 in CAZY database, eight residues were selected as hot-spots for

4

substitution, and three mutants among them showed significantly improved

5

thermostability. Then, the possible mechanism responsible for the thermostability

6

enhancement was extensively investigated by MD simulation. The improvement of

7

PGUS-E thermostability in this study has certain practical significance: it can not only

8

promote the industrial application of glucuronidase but also provide insight into the

9

structure and stability of glucuronidase.

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2. Materials and methods

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2.1. Materials

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E. coli DH5α and E. coli BL21 (DE3) competent cells were purchased from Biomed

13

(Beijing, China). The wild-type pgus gene (GenBank, EU095019.1) was from P.

14

purpurogenum Li-3 (CGMCC, No. 5446).26-27 TransStart FastPfu DNA Polymerase,

15

MgSO4 and dNTP mix were purchased from TransGene Biotech (Beijing, China).

16

Fastdigest DpnI was purchased from Thermo Fisher Scientific (Waltham, USA). The

17

plasmid extraction kit was obtained from Tiangen (Valencia, USA). Glycyrrhizin,

18

glycyrrhetic acid and 4-Nitrophenyl-β-D-glucuronide (pNPG) was purchased from

19

Sigma-Aldrich (St. Louis, USA). All other chemicals were of the highest grade

20

available and were obtained from standard commercial sources.

21

2.2. Site-directed mutagenesis

22

The gene encoding PGUS wild-type was previously cloned into pET-28a(+) and used as

23

the template for site-directed mutagenesis. A schematic diagram of the mutagenesis 5



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protocol used in this study is shown in Fig. S1 (Supporting Information). Briefly, the

2

plasmids were amplified using both forward and reverse primers bearing the mutated

3

nucleotides. All primers used in this study were listed in Table S1 (Supporting

4

Information). The detailed PCR reaction conditions are given in Table S2 and Fig. S2

5

(Supporting Information). The template was digested by DpnI at 37 °C for 1 h (refer to

6

Table S3 for details, Supporting Information) and transformed into E. coli DH5α

7

competent cells for the desired plasmid enrichment. The resulting plasmid was

8

transformed into E. coli BL21 (DE3) competent cells for enzyme expression. The

9

complete gene sequencing was performed to confirm the mutation by Genewiz (Beijing,

10

China).

11

2.3. Enzyme expression and purification

12

The wild-type and mutants enzymes were produced in 300 mL Luria-Bertani medium

13

containing 50 µg mL-1 kanamycin sulfate at 37 °C. When OD600 of the culture reached

14

0.6–0.8, protein expression was induced with 1 mM 1-thio-β-d-galacto-pyranoside

15

(IPTG) for 10 h at 16 °C. The cells were collected by centrifugation (8000 rpm) at 4 °C

16

for 10 min, suspended in binding buffer (50 mM Tris-HCl, pH 7.3, 150 mM NaCl) and

17

lysed by sonication. After centrifugation at 12000 rpm for 20 min to remove

18

precipitated protein and cell debris, the crude enzyme was loaded onto a Ni-NTA

19

affinity column (GE Healthcare, Beijing, China). The target bound protein was eluted

20

by an increasing gradient of elution buffer (50 mM Tris-HCl, pH 7.3, 150 mM NaCl, 1

21

M imidazole) from 0 to 100% and desalted using a HiTrapTM Desalting column with 50

22

mM Tris-HCl (pH 7.3) as elution buffer. All the protein purification was performed by

23

AKTA purifier system (GE Healthcare, Sweden). Enzyme purity was assessed by 6


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SDS-PAGE. Enzyme concentration was determined using the Bradford method at 595

2

nm using bovine serum albumin as the standard.28 The purified enzyme was stored at

3

4 °C until further use.

4

2.4. Enzyme activity assay

5

The activity of PGUS-E wild-type and mutants was determined by hydrolyzing the

6

artificial substrate pNPG into pNP and glucuronide. Typically, 40 µL 1.25 mM pNPG in

7

50 mM acetate buffer (pH 5) was added to 10 µL enzyme solution, and incubated at

8

40 °C for 10 min. Then the reaction was stopped by adding 0.4 M Na2CO3 and the

9

produced pNP was measured at 405 nm with a microplate spectrophotometer (BioTek,

10

USA). One enzyme unit was defined as the amount of enzyme which liberated 1 µmol

11

of p-nitrophenol per minute.

12

2.5. Thermostability assay

13

The purified PGUS-E wild-type and mutants were incubated at 60 °C and 65 °C, and

14

sampled periodically. The enzyme sample was firstly incubated in an ice bath for 20

15

min, and then the residual activity was evaluated by pNPG assay as described in Section

16

2.4. The activity of the non-incubated enzyme was taken as 100%. In this study, the

17

thermal deactivation of PGUS-E showed a two-phase tendency, so the following

18

deactivation scheme was used: 29

19

k1 k2 E  → E1  → E2

20

where E stands for the active form of PGUS-E; E1 stands for the deactivated

21

intermediate of PGUS-E; E2 stands for the final deactivated PGUS-E; k1 and k2 stand for

22

the deactivation kinetic constants in phase 1 and phase 2, respectively. Following this

23

process, the thermal deactivation can be obtained: 30-31 7



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1

a ln   = −k1t  a0 

2

a ln   = −k2t  a0 

3

where a0 is the initial activity; a is the activity at time t during the thermal deactivation.

4

In Eq. (1), t is from 0 to 20 min; in Eq. (2), t is from 20 to 100 min.

(1)

(2)

5

Differential scanning calorimeter (DSC) was employed to measure the melting

6

temperature Tm of PGUS-E wild-type and mutants with a MicroCal VP- DSC (Microcal

7

Inc., USA). The enzyme sample (0.5 g L-1) was prepared in 50 mM Tris-HCl buffer (pH

8

7.4), and scanned from 25 to 80 °C with heating rate of 1 °C min-1. Data was analyzed

9

with Origin DSCITC software (MicroCal Inc., USA).

10

2.6. Kinetics characterization

11

The Michaelis–Menten kinetics of PGUS-E wild-type and mutants were evaluated

12

through the hydrolysis of its natural substrate glycyrrhizin (GL). GL with concentration

13

ranging from 0.1 to 1 g L-1 was prepared in 50 mM acetate buffer (pH 5). The reaction

14

mixture consisting of 100 µL enzyme solution and 400 µl GL was incubated at 40 °C for

15

10 min and then stopped by adding NaOH. Then, the reaction mixture (10 µL) was

16

subjected to reverse-phase HPLC on a C18 column (4.6 × 250 mm, 5 µm particle size,

17

Shimadzu) for the reaction rate quantification. Separation was achieved with mobile

18

phase consisting of a mixture of methanol – 0.6% acetic acid (81:19 v/v) at 40 °C.

19

Elution was monitored with UV detection at 254 nm. The kinetic constants were

20

evaluated by fitting the experimental data to Michaelis–Menten equation through

21

nonlinear regression.

22 23 8


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2.7. The Fluorescence spectroscopy analysis

2

The intrinsic fluorescence was measured using a FluoroMax-4 fluorescence

3

spectrophotometer (HORIBA, USA). The enzyme (0.2 g L-1) was prepared in pH 7.0

4

phosphate buffer (10 mM) and 100 µL sample was loaded onto a quartz cuvette with

5

path length 0.3 cm. An excitation wavelength of 280 nm was applied and the emission

6

spectra were recorded from 300 to 400 nm.

7

2.8. Circular dichroism (CD) analysis

8

The CD measurement was performed with a J-810 CD spectropolarimeter (JASCO,

9

Tokyo, Japan) with Peltier system. The enzyme sample (0.3 g L-1) was prepared in 50

10

mM Tris-HCl buffer (pH 7.4, 25 mM NaCl) and loaded onto a quartz cuvette with path

11

length 0.1 cm. The far-UV CD spectra was recorded with wavelength ranging from 190

12

to 250 nm, and the spectra of buffer blank was subtracted. The secondary structure

13

composition was analyzed with the online server DichroWeb.32

14

2.9. Molecular Dynamics (MD) simulation

15

The PGUS-E structure (PDB ID: 5C70) was used as the starting structure of PGUS-E

16

wild-type for MD simulation. The structures of PGUS-E mutants F292L/T293K, S35P,

17

R304L were constructed individually by VMD software with its inherent script. Then,

18

the PGUS-E wild-type and mutants were subjected to MD simulation at 310 K for 13 ns.

19

In the MD simulation, the NAMD 2.7 and CHARMM36 field force were applied and

20

the structures were put in a cubic box with 30 Å in each XYZ direction. The TIP3P

21

model was used for water molecules and 150 mM NaCl was added. All bonds were

22

constrained using the LINCS algorithm, and periodic boundary conditions were applied.

23

The model systems were relaxed by a series of minimizations and short dynamic

24

simulations. The time step was 2 fs and trajectories were saved every 100 ps. Three 9



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1

independent MD simulations were performed.

2

3. Results and Discussion

3

3.1. Design of PGUS-E mutants

4

Recently, we have resolved the crystal structure of PGUS-E which belongs to GH2

5

family. As shown in Fig. 1, PGUS-E structure is a homotetramer and each monomer is

6

composed of three functional domains: sugar binding domain, immunoglobulin-like

7

β-sandwich domain and TIM-barrel domain, which is similar to the two reported

8

structures of β-glucuronidases from human and E. coli.1-3 According to homology

9

sequence alignment, E414 and E505 located in TIM-barrel domain are identified to be

10

the catalytic residues. In this study, sequence alignment combined with structure

11

analysis was employed to identify the potential hot spots which can improve the

12

thermostability of PGUS-E. We mainly focused on the amino acids on the surface loops

13

within 30 Å from the catalytic residues, since residues within catalytic domain play

14

important roles in the activity and stability of enzyme.25 In addition, a highly conserved

15

loop (Ala24-Ser40) near the sugar binding domain of PGUS-E drew special attention

16

due to its high flexibility with average B-factor of 64.19 (calculated by B-FITTER33), so

17

the residues in this loop were also considered (Fig. 1). Based on the above structure

18

analysis, multiple sequence alignment was performed to find the potential mutation sites.

19

Twenty thermophilic glycosidases in GH 2 family derived from CAZY database were

20

selected as candidates for consensus due to their high homology with PGUS-E (see Fig.

21

S3 and S4 for details, Supporting Information), and eight potential hot spots were

22

selected and subjected to site-directed mutation: A167G, A170W, V239P, A247P,

23

F292L/T293K, N302P, R304L, S35P.

24

For the easy purification, PGUS-E was fused with 6×His-tag at N-terminal. The 10


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PGUS-E wild-type and mutants were purified according to the standard protocol by

2

nickel affinity chromatography followed by desalination34-35. It was found that the

3

mutation did not have effect on the absorbance of PGUS-E onto nickel affinity column,

4

so the data of PGUS-E purification was presented as an example. As shown in Fig. S5a

5

(Supporting Information), the fraction eluted with 0.25 M imidazole showed high

6

activity, so it belonged to PGUS-E. In addition, the purification factors of nickel affinity

7

chromatography were also listed in Table S4. The specific activity of PGUS-E was 250

8

U mg-1, with recovery yield of 80% and purification fold of 39. The purity was greater

9

than 90% as analyzed by SDS-PAGE (Fig. S5b, Supporting Information).

10

3.2. Determination of thermostability

11

Eight PGUS-E mutants were designed totally for the improvement of thermostability.

12

Among them, V239P, A247P, N302P were not successfully expressed which may be due

13

to that the introduction of proline caused significant change to the structure so that the

14

protein could not fold correctly. This point will be further discussed. So only five

15

mutants S35P, A167G, A170W, F292L/T293K, R304L were subjected to further

16

thermostability assay. The residual activity after treatment at 60 and 65 °C was tested

17

with pNPP assay. As shown in Fig. 2a, after treatment at 60 °C for 60 min, mutants

18

F292L/T293K, S35P, R304L showed the best improved thermostability with around 50%

19

higher residual activity than wild-type, and A170W showed 24% higher residual activity

20

than wild-type. Peculiarly, A167G showed 14% decreased thermostability compared to

21

wild-type. As shown in Fig. 2b, F292L/T293K, S35P, R304L still showed good

22

thermostability even at higher temperature of 65 °C. We tried to combine these four

23

mutated sites together (F292L/T293K/S35P/R304L) to further enhance the PGUS-E

24

thermostability, unfortunately, the expressed enzyme was mainly in the form of

25

inclusion body (The SDS-PAGE result is shown in Fig. S6, Supporting Information). 11



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1

Further experiment should be performed to refold the inclusion bodies to recover the

2

enzyme activity, and this is beyond the scope of this paper.

3

In addition, it seemed that PGUS-E showed a two-phase thermal deactivation process,

4

with the initial 20 min considered as the first phase. The two-phase thermal deactivation

5

tendency has also been observed for other enzymes.36 According to the classical enzyme

6

deactivation theory, the thermal deactivation of enzyme usually experiences a multistep

7

series process, so several intermediate forms of enzyme may exist which may be

8

deactivated to the final inactive form. This may lead to the multi-phase deactivation

9

process. In addition, the partial renaturation of the enzyme during storage on ice may

10

also contribute to the two-phase deactivation process. Furthermore, we analyzed the

11

deactivation kinetics separately in two-phase, and in each phase, the deactivation was

12

assumed to follow the first order kinetics (refer to Section 2.5).29, 37 As shown in Fig. 3,

13

the thermal deactivation could be described reasonably well with a two-stage kinetics.

14

PGUS-E was a tetramer, so the deactivation in the first phase may be correlated with the

15

disassembly of the quaternary structure, since the interaction between the monomer was

16

less strong than that within a monomer for a given enzyme. A recent study manifested

17

that increasing the interaction between monomers could confer significantly improved

18

stability to enzyme.38 This may explain the rapid decrease in the activity of PGUS-E

19

wild-type in the first phase, that is, PGUS-E wild-type lost 80% of the original activity

20

during the first phase, with deactivation kinetic constant k1 of 0.0756 min-1 (Table 1).

21

However, for all the three mutants, deactivation was alleviated at different levels in the

22

first phase. The k1 of F292L/T293K and S35P were significantly decreased by one

23

magnitude, while R304L showed a moderate decrease with k1 of 0.0342 min-1. In the

24

second phase, R304L showed a much lower kinetic constant k2 than F292L/T293K and

25

S35P, resulting in that these three mutants maintained a similar activity after 100 min 12


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1

(approximately 40% of the original activity). Therefore, the mutation of F292L/T293K

2

and S35P contributed more to the maintenance of the enzyme stability in the first

3

deactivation phase, while the mutation R304L mainly took effect in the second phase.

4

The thermodynamic stability was evaluated with respect to Tm, which refers to the

5

temperature at which half of the enzyme structure was unfolded. As shown in Table 1,

6

the Tm of the three mutants F292L/T293K, R304L and S35P showed a modest increase

7

of 2-3 °C compared to the wild-type, combining with the deactivation kinetics data, it

8

can be concluded that the mutation contributed more to the improvement of the kinetic

9

stability than the thermodynamic stability of PGUS-E. Tm stands for the resistance of

10

protein to unfolding, so a Tm is usually correlated with a significant structure

11

transformation.39 However, a modest change in tertiary structure during thermal

12

deactivation may result in the significant loss of enzyme activity. In our case, PGUS-E

13

may suffer from severe activity loss before reaching Tm, and this may explain the

14

difference between the thermodynamic and kinetic stability of PGUS-E.

15

3.3. Characterization of the mutants with improved thermostability

16

The effect of reaction temperature on the activity of PGUS-E wild-type and mutants

17

was determined to investigate if the mutation would change the optimal temperature

18

range of PGUS-E. As shown in Fig. 4, the optimal temperature of both wild-type

19

PGUS-E and mutants was approximately 45 °C. Interestingly, PGUS-E wild-type

20

showed the highest activity among all the investigated enzymes when temperature was

21

below 45 °C. As the temperature increased above 45 °C, the mutants showed better

22

performance than wild-type, indicating that the mutation shifted the optimal temperature

23

range to higher temperature, for example, the mutant F292L/T293K showed high

13



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1

activity between 45 and 50 °C. This also corresponds well with the higher

2

thermostability of the mutants.

3

The kinetics of the three thermostability-improved PGUS-E mutants was

4

characterized by GL (the natural substrate of PGUS-E) hydrolysis. Since the kinetic

5

parameters were determined by the initial reaction rate in the first 10 min, the product

6

was mainly in the form of GAMG with little GA formed (refer to Scheme 1). Therefore,

7

Km described the affinity between PGUS-E and GL, and kcat referred to the

8

transformation efficiency of GL into GAMG. As shown in Table 2, at 40 °C (optimal

9

temperature range), F292L/T293K showed similar Km with wild-type (2.47 g L-1)

10

indicating the double mutation at 292 and 293 did not change the binding affinity to GL,

11

while the other two mutants R304L and S35P showed 4 times higher of Km than the

12

wild-type indicating a dramatically decreased binding affinity to GL. Interestingly, all

13

the three mutants had higher kcat than wild-type (0.38 min-1), and F292L/T293K showed

14

the highest kcat of 2.55 min-1. This resulted in the highest catalytic efficiency kcat/Km of

15

F292L/T293K (1.03 min-1 g-1 L), which was 6.4 times higher than that of wild-type

16

(0.16 min-1 g-1 L), while R304L and S35P maintained similar kcat/Km with wild-type.

17

These results indicated that the double mutation at site 292 and 293 can not only

18

enhance the thermostability but also increase the catalytic efficiency. The kinetics

19

beyond the optimal temperature range was also measured at 30 °C and 60 °C. For all the

20

enzymes, the kcat/Km at both 30 °C and 60 °C was decreased compared to that at 40 °C

21

which was in good accordance with the temperature profile in Fig. 4. When the

22

temperature was increased from 30 to 40 °C, kcat showed a dramatic increasing tendency 14


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1

for all the four enzymes, while Km changed little. This may be due to that high

2

temperature could accelerate the molecular motion thus resulting in a high reaction rate,

3

meanwhile, the enzyme was not denatured at this temperature range, so kcat increased as

4

the increase of temperature. When the temperature was further increased to 60 °C, kcat

5

showed little increase associated with the significant increase of Km, which was due to

6

the distortion of enzyme structure at high temperature thus decreasing the affinity

7

between enzyme and substrate. In summary, the increased enzyme activity in the

8

temperature range between 30 to 45 °C (Fig. 4) was mainly correlated to the increased

9

kcat; whereas the decreased activity in the temperature range between 45 and 60 °C (Fig.

10

4) was mainly correlated to the increased Km. This result was also in good agreement

11

with references.36, 40

12

CD analysis was performed to check if the mutation caused significant change to the

13

secondary structure of PGUS-E. As shown in Fig. 5a, the far-UV CD spectrum of

14

PGUS-E wild-type exhibited remarkable absorbance at 194 nm and 220 nm, indicating

15

the secondary structure of PGUS-E is mainly composed of α-helix and β-sheet.41-42 The

16

three mutants showed a very similar CD spectra with wild-type, indicating that the

17

mutation did not cause significant change to the enzyme secondary structure. The

18

potential changes of tertiary structure caused by the mutation were evaluated by the

19

fluorescence spectra (FluoroMax-4, HORIBA, USA). As shown in Fig. 5b, the

20

maximum absorbance wavelength of wild-type and mutants remained the same (334

21

nm), indicating that the tertiary structure was not significantly changed after mutation.

22

This is reasonable since the change in one or couple of amino acids may change the 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

local microenvironment but would not cause dramatic change to the overall structure.

2

Our result is also similar with previous reports, for example, Xie et al. mutated two sites

3

around the active residue of Candida antarctica lipase B to enhance its thermostability,

4

and found that the wild-type and mutant enzyme had very similar overall structure

5

based on the crystal structure analysis.25

6

3.4. Structure analysis by MD simulation

7

MD simulation of the mutants with improved thermostability F292L/T293K, S35P,

8

R304L was performed to obtain the profound insight into the molecular stabilization

9

mechanism. After 13 ns, the structure of mutants became stable with RMSD around 3 Å.

10

The obtained mutant structure was superimposed with PGUS-E wild-type, and no

11

significant structure difference was observed with RMSD of only 1.3 -1.5 Å since

12

RMSD < 3 Å from structure alignment usually indicates that the enzymes have a similar

13

structure.43-44 This is consistent with the result of fluorescence spectra that the three

14

mutations did not cause dramatic change to the overall tertiary structure of PGUS-E.

15

Root mean square fluctuation (RMSF) could reflect the stability of individual residue

16

of protein, and a high RMSF denoted the high flexibility of a given residue. The RMSF

17

values of PGUS-E wild-type and mutants are shown in Fig. 6. For all the three mutants,

18

three regions showed decreased RMSF values compared to wild-type: loop 353-383,

19

loop 467-479 and the C-terminal, so the decreased flexibility of these regions may be

20

responsible for the increased stability of PGUS-E mutants. Apart from the above

21

common feature, each mutation also had its own specific effect on the PGUS-E stability

22

regarding to RMSF which would be further discussed below.

23

Site 35 was located in the loop connecting β3-sheet and β5-sheet in sugar binding 16


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1

domain of PGSU-E, and this loop was found highly dynamic based on B-factor analysis.

2

Such dynamic loops are often hot-spots for enhancing enzyme thermostability. In this

3

study, S35 was mutated to proline according to sequence alignment with thermophilic

4

counterparts, which resulted in the improved thermostability of PGUS-E. As shown in

5

Fig. 7, for both S35 and P35, no obvious interaction was observed with neighboring

6

residues. Therefore, the enhancement of thermostability by substitution of serine with

7

proline at site 35 is not due to the straightforward increased interactions. Nevertheless,

8

the RMSF values around site 35 were decreased for mutant S35P which corresponded

9

well with the increased thermostability (Fig. 6a). It has been well documented that

10

proline has very limited structural geometry within the main chain thus decreasing the

11

conformational freedom of Cα-N rotation, so the introduction of proline at appropriate

12

position may expect to increase the enzyme thermostability.45-46 This may explain the

13

enhanced thermostability of mutant S35P. In addition, the introduction site of proline

14

should be carefully selected with consideration of secondary structure which has been

15

proven to be preferably β-turn.42 In this study, four potential residues were selected for

16

proline substitution: V239, A247, N302, S35, and only mutant S35P was heterologously

17

expressed in E. coli. A247 was not in the β-turn, which may be the reason that mutant

18

A247P was not successfully expressed. The rest three sites were all located in β-turn

19

structure. V239 and N302 both established hydrogen bond with neighboring residues:

20

V239 formed hydrogen bond with Thr195, and Asn302 formed hydrogen bond with

21

Thr301, Arg 304 and Lys575, which may cause the mutants (V239P and N302P) could

22

not fold into the correct structure according to the “hydrogen bond criteria” proposed by 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Yu et al., that is, if the wild-type residue is involved in H-bonding formation, the

2

substitution with proline will lead to destabilization of the protein.46

3

As shown in Fig. 8 (a and b), for PGUS-E wild-type, Phe292 and Thr293 were

4

located at the intersecting point of loop 285-292 and β3-sheet of TIM-barrel structure

5

domain, and these two residues were less than 30 Å away from the active site. Phe292

6

established two hydrogen bonds with Ala545 (located at the loop connecting α8-helix

7

and β10-sheet) and Glu547 (located at β10-sheet), while Thr293 formed three hydrogen

8

bonds with Asn326 (located at loop connecting α1-helix and β5-sheet) and Ser327

9

(located at β5-sheet). This subdomain formed by the hydrogen bonds network may play

10

an important role for the overall stability of the enzyme. When Phe292 was replaced

11

with leucine, no new interaction was formed. When Thr293 was replaced with lysine,

12

the interaction with Asn326 and Ser327 remained the same, but the long side chain of

13

lysine allowed it to establish a new hydrogen bond with Leu585 at C-terminal α9-helix

14

which was not observed in wild-type. This newly formed hydrogen bond may work like

15

a string to fix C-terminal to the aforementioned subdomain, thus increasing the structure

16

stability. This correlated well with the RMSF result. The C-terminal of PGUS-E

17

wild-type was highly flexible according to RMSF analysis, and its RMSF values were

18

significantly decreased for mutant F292L/T293K (Fig. 6b). In addition, the RMSF

19

values of loop 554-566 around C-terminal were significantly decreased for mutant

20

F292L/T293K (Fig. 6b). As reported in literature, C or N-terminal is usually highly

21

flexible region which is responsible for the instability of enzyme, so some researches try

22

to truncate or delete the terminal part to increase the enzyme stability. For example,

23

Damnjanovic et al. deleted the half part of N-terminal of phospholipase D and the

24

thermostability was significantly improved: the half-life at 70 °C was 11.7 times higher

25

than that of wild-type.13 In this study, our result showed that enhancing the C-terminal 18


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1

interaction with other domains can also enhance the thermostability of enzyme. In

2

addition, several researches have shown that lysine on the surface is likely to induce the

3

instability of protein, thus decreasing the thermostability.47-48 Niu et al. substituted all

4

the 12 lysines on the surface of 1,3-1,4-β-glucanase with serines, and obtained a triple

5

mutant (K20S/K117S/K165S) with significantly increased thermostability.49 However,

6

the introduction of lysine in our case showed dramatically improved thermostability due

7

to the C-terminal fixing effect. Therefore, the contribution of lysine to the protein

8

thermostability is not always negative, and it also depends on the local

9

microenvironment.

10

In addition, the mutant F292L/T293K showed 6.9 times higher kcat than wild-type,

11

and the structures of F292L/T293K and wild-type were superimposed to investigate the

12

mechanism. As aforementioned, E414 and E505 are two catalytic residues of PGUS-E

13

and they act as acid/base and nucleophile, respectively. As shown in Fig. 8c, the

14

geometry of the two catalytic residues of mutant F292L/T293K was changed in

15

comparison to wild-type. E505 showed a 1.5 Å motion, and the side chain of E414

16

showed a 1.2-1.6 Å deviation after the mutation. It can be concluded that the new

17

geometry of the two catalytic residues of mutant F292L/T293K may be more favorable

18

for the hydrolysis of the glycosidic bond of the substrate, thus resulting in the high kcat.

19

Interestingly, no direct interaction was observed between site 292/293 and the catalytic

20

residues since their steric distance was around 10-15 Å, so the motion of the catalytic

21

residues may be caused by remote effect via intervening residues. The remote effect of

22

residues on catalytic behavior of enzyme have also been reported for other enzymes,

23

such as metalloenzyme.50-51 It should be noted that a more detailed MD simulation of

24

PGUS-E wild-type and mutants in complex with substrate are required to further

25

elucidate the substrate binding mechanism of PGUS-E, which may give more insights 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

into the increased kcat of mutant F292L/T293K.

2

As shown in Fig. 9, for PGUS-E wild-type, Arg304 established hydrogen bond with

3

Ser48, Gly307 and Asp309 to stabilize the local structure. When arginine at 304 was

4

substituted with leucine, the hydrogen bond was not increased. However, site 304 was

5

surrounded by a number of hydrophobic residues (Ile52-Phe53, Pro44-Ala47), therefore

6

the mutation of hydrophilic arginine to hydrophobic leucine decreased the distance

7

between the neighboring residues Ile52, Ala47 and Leu to 4.0 and 2.7 Å, respectively,

8

which may enhance their hydrophobic interaction in the local region. This may further

9

decrease the flexibility of the adjacent loop 73-81 as indicated in the RMSF analysis

10

(Fig. 6c). This effect may also compensate the increased RMSF in region around site

11

304 caused by the mutation R304L, thus rendering an stabilizing effect on the overall

12

PGUS-E structure. The hydrophobic interaction has long been recognized as an

13

important factor for protein stability.52 The improvement of thermostability by

14

increasing hydrophobic interaction has also been reported in references. Song et al.

15

increased the half-life of GH10 xylanase at 60 °C by 30-fold by enforcing the

16

hydrophobic interactions within N-terminal elements and between N- and C-terminal

17

ends.53

18

4. Conclusion

19

The thermostability of PGUS-E was significantly improved by rational site-directed

20

mutagenesis based on structure analysis and sequence alignment. Three mutants

21

F292L/T293K, S35P and R304L were obtained with both increased kinetic and

22

thermodynamic stability, while mutant F292L/T293K showed 6.4 times higher catalytic 20


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1

efficiency. This indicates that the constructed β-glucuronidase mutants are more suitable

2

for industrial application in terms of both thermostability and activity. The MD

3

simulation indicated that the improvement of thermostability was due to a unique

4

N-terminal fixing effect, proline effect and hydrophobic interaction. The study

5

demonstrated that the precise selection of even a few residues for mutagenesis could

6

effectively enhance the enzyme thermostability.

7

Acknowledgements

8

This research was funded by grants from National Natural Science Foundation of China

9

(No. 21506011, No. 21425624), China Postdoctoral Science Foundation (No.

10

2015M570038) and the Foundation of Key Laboratory for Industrial Biocatalysis

11

(Tsinghua University), Ministry of Education (No. 2015202).

12 13

Content of Supporting Information

14

Fig . S1 The schematic diagram of the mutagenesis protocol. Fig. S2 The temperature

15

profile of the PCR reaction. Fig. S3 & Fig. S4 Sequence alignment of PGUS-E with

16

thermophilic glycosidases in GH 2. Fig. S5 The purification of PGUS-E wild-type and

17

mutants by nickel affinity chromatography, and the corresponding SDS-PAGE analysis.

18

Fig. S6 The SDS-PAGE analysis of combined mutant (F292L/T293K/S35P/R304L).

19

Table S1 The primers used in this study. Table S2 The PCR reaction conditions. Table

20

S3 The reaction conditions for template digestion. Table S4 Enzyme yields and

21

purification factors of nickel affinity chromatography

22 23

21



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proline substitution and surface loop deletion in the extended active site of human carbonic anhydrase ii. Febs J. 2015, 282, 1445-1457. (46) Yu, H. R.; Zhao, Y.; Guo, C.; Gan, Y. R.; Huang, H. The role of proline substitutions within flexible regions on thermostability of luciferase. BBA-Proteins Proteomics 2015, 1854, 65-72. (47) Niu, C.; Zhu, L.; Wang, J.; Li, Q. Simultaneous enhanced catalytic activity and thermostability of a 1,3-1,4-beta-glucanase from Bacillus amyloliqueformis by chemical modification of lysine residues. Biotechnol. Lett. 2014, 36, 2453-2460. (48) Khajeh, K.; Naderi-Manesh, H.; Ranjbar, B.; Moosavi-Movahedi, A. A.; Nemat-Gorgani, M. Chemical modification of lysine residues in Bacillus alpha-amylases: Effect on activity and stability. Enzyme Microb. Technol. 2001, 28, 543-549. (49) Niu, C.; Zhu, L.; Zhu, P.; Li, Q. Lysine-based site-directed mutagenesis increased rigid β-sheet structure and thermostability of mesophilic 1,3–1,4-β-glucanase. J. Agric. Food Chem. 2015, 63, 5249-5256. (50) Tiwari, M. K.; Kalia, V. C.; Kang, Y. C.; Lee, J.-K. Role of a remote leucine residue in the catalytic function of polyol dehydrogenase. Mol. Biosyst. 2014, 10, 3255-3263. (51) Tiwari, M. K.; Singh, R. K.; Singh, R.; Jeya, M.; Zhao, H. M.; Lee, J. K. Role of conserved glycine in zinc-dependent medium chain dehydrogenase/reductase superfamily. J. Biol. Chem. 2012, 287, 19429-19439. (52) Pace, C. N.; Fu, H. L.; Fryar, K. L.; Landua, J.; Trevino, S. R.; Shirley, B. A.; Hendricks, M. M.; Iimura, S.; Gajiwala, K.; Scholtz, J. M.; Grimsley, G. R. Contribution of hydrophobic interactions to protein stability. J. Mol. Biol. 2011, 408, 514-528. (53) Song, L. T.; Tsang, A.; Sylvestre, M. Engineering a thermostable fungal GH10 xylanase, importance of N-terminal amino acids. Biotechnol. Bioeng. 2015, 112, 1081-1091.

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25



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1

Figure Legends

2

Scheme 1 The schematic diagram of the two-step enzymatic hydrolysis of glycyrrhizin

3

(GL) into glycyrrhetic acid (GA) with monoglucuronide glycyrrhetic acid (GAMG) as

4

the intermediate.

5 6

Figure 1 The structure of PGUS-E monomer containing three domains: the sugar

7

binding domain (green), immunoglobulin-like β-sandwich domain (magentas), and TIM

8

barrel domain (cyan). The catalytic residues Glu414 and Glu505 are shown in yellow

9

spheres. The eight mutation residues within 30 Å from catalytic residues Ala167,

10

Asn302, Ala247, Val239, Ser35, Arg304 and Phe292/Thr293 are displayed in red

11

spheres. Ser35 located in the highly flexible loop 24-40 is shown in blue spheres.

12

13

Figure 2 The thermostability of PGUS-E wild-type and mutants at 60 °C (a) and 65 °C

14

(b). Values are the average of three independent replicates; error bars represent average

15

± one standard deviation.

16 17

Figure 3 The two-phase thermal deactivation of PGUS-E wild-type and mutants at

18

65 °C: (a) phase 1; (b) phase 2. Values are the average of three independent replicates;

19

error bars represent average ± one standard deviation.

20 21

Figure 4 The effect of reaction temperature on the activity of PGUS-E wild-type and

22

mutants. The enzyme activity was determined by glycyrrhizin (GL) hydrolysis.

23

Substrate concentration: 0.8 g L-1, buffer: 50 mM acetate buffer (pH 5), reaction volume: 26


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1

500 µL, reaction time: 10 min. Values are the average of three independent replicates;

2

error bars represent average ± one standard deviation.

3 4

Figure 5 The circular dichroism spectra (a) and fluorescence spectra (b) of PGUS-E

5

wild-type and mutants.

6 7

Figure 6 Root mean squared fluctuations (RMSF) measured during a 13 ns MD

8

simulation: (a) S35P mutant; (b) F292L/T293K mutant; (c) R304L mutant. The regions

9

with change in RMSF are highlighted with green box.

10 11

Figure 7 (a) The superimposition of PGUS-E wild-type and S35P mutant structure, S35

12

is shown in magentas and P35 is shown in cyans. (b) The interaction between Val239

13

and Thr195 of PGUS-E. (c) The hydrogen bond network around Asn302 of PGUS-E.

14 15

Figure 8 The local hydrogen bonds network within subdomain of α1-helix, α8-helix,

16

C-terminal α9-helix, β3-sheet, β5-sheet and β10-sheet of PGUS-E wild-type (a) and

17

F292L/T293K mutant (b). Residues Phe/Leu 292 and Thr/Lys 293 are shown in

18

magentas and green, respectively. The structure of F292L/T293K mutant was obtained

19

by 13 ns MD simulation. The yellow dashed line stands for the hydrogen bond. (c) The

20

superimposition of the catalytic residues E414 and E505 between PGUS-E wild-type

21

(red) and F292L/T293K mutant (magentas). The moved distance of E414 and E505 is

22

shown in yellow dashed line. 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Figure 9 The local interaction network around site 304 of PGUS-E wild-type (a) and

2

R304L (b). The hydrophobic residues are shown in yellow. Residue at 304 is shown in

3

magentas. The distance between Ile52, Ala47 and Arg/Leu 304 is shown in red dashed

4

line.

5

28


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1

Table 1 The thermostability measurement of PGUS-E wild-type and mutants. PGUS-E S35P F292L/T293K R304L

2 3

k1 (min-1)

k2 (min-1)

Tm (°C)

0.0756±0.0075 0.0052±0.0009 0.0073±0.0011 0.0342±0.0022

0.0031±0.0005 0.0112±0.0006 0.0063±0.0003 0.0021±0.0001

71.3±0.03 74.4±0.02 74.5±0.01 73.4±0.02

The k1 and k2 stand for the thermal deactivation kinetic constants at phase 1 and phase 2, respectively. Uncertainties are denoted as the average±one standard deviation.

4 5 6 7 8

9

Table 2 The kinetic parameters of PGUS-E and mutants. Enzyme

Temperature (°C)

Km (g L-1)

kcat (min-1)

kcat/Km (min-1 g-1 L)

Wild-type

30

2.83±0.12

0.32±0.02

0.11

F292L/T293K

1.86±0.07

0.43±0.01

0.23

R304L

7.71±0.69

0.92±0.08

0.12

S35P

8.84±0.58

0.77±0.04

0.087

Wild-type F292L/T293K R304L S35P

40

2.47±0.16 2.46±0.39 9.95±0.85 8.74±0.61

0.38±0.05 2.55±0.43 1.81±0.12 0.95±0.11

0.16 1.03 0.18 0.11

Wild-type F292L/T293K R304L S35P

60

5.98±0.48 3.26±0.13 13.4±0.57 11.2±1.25

0.57±0.07 2.23±0.31 1.92±0.07 1.14±0.08

0.095 0.68 0.14 0.10

Uncertainties are denoted as the average±one standard deviation.

10

29



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1 2

TOC graphic

3

30


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Scheme 1 The schematic diagram of the two-step enzymatic hydrolysis of glycyrrhizin (GL) into glycyrrhetic acid (GA) with monoglucuronide glycyrrhetic acid (GAMG) as the intermediate. 20x5mm (600 x 600 DPI)



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Figure 1 The structure of PGUS-E monomer containing three domains: the sugar binding domain (green), immunoglobulin-like β-sandwich domain (magentas), and TIM barrel domain (cyan). The catalytic residues Glu414 and Glu505 are shown in yellow spheres. The eight mutation residues within 30 Å from catalytic residues Ala167, Asn302, Ala247, Val239, Ser35, Arg304 and Phe292/Thr293 are displayed in red spheres. Ser35 located in the highly flexible loop 24-40 is shown in blue spheres. 36x30mm (300 x 300 DPI)


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Figure 2 The thermostability of PGUS-E wild-type and mutants at 60 °C (a) and 65 °C (b). Values are the average of three independent replicates; error bars represent average ± one standard deviation. 77x32mm (600 x 600 DPI)



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Figure 3 The two-phase thermal deactivation of PGUS-E wild-type and mutants at 65 °C: (a) phase 1; (b) phase 2. Values are the average of three independent replicates; error bars represent average ± one standard deviation. 69x30mm (600 x 600 DPI)


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Figure 4 The effect of reaction temperature on the activity of PGUS-E wild-type and mutants. The enzyme activity was determined by glycyrrhizin (GL) hydrolysis. Substrate concentration: 0.8 gL-1, buffer: 50 mM acetate buffer (pH 5), reaction volume: 500 µL, reaction time: 10 min. Values are the average of three independent replicates; error bars represent average ± one standard deviation. 68x58mm (600 x 600 DPI)



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Figure 5 The circular dichroism spectra (a) and fluorescence spectra (b) of PGUS-E wild-type and mutants. 65x25mm (600 x 600 DPI)


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Figure 6 Root mean squared fluctuations (RMSF) measured during a 13 ns MD simulation: (a) S35P mutant; (b) F292L/T293K mutant; (c) R304L mutant. The regions with change in RMSF are highlighted with green box. 199x499mm (600 x 600 DPI)



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Figure 7 (a) The superimposition of PGUS-E wild-type and S35P mutant structure, S35 is shown in magentas and P35 is shown in cyans. (b) The interaction between Val239 and Thr195 of PGUS-E. (c) The hydrogen bond network around Asn302 of PGUS-E. 49x14mm (300 x 300 DPI)


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Figure 8 The local hydrogen bonds network within subdomain of α1-helix, α8-helix, C-terminal α9-helix, β3sheet, β5-sheet and β10-sheet of PGUS-E wild-type (a) and F292L/T293K mutant (b). Residues Phe/Leu 292 and Thr/Lys 293 are shown in magentas and green, respectively. The structure of F292L/T293K mutant was obtained by 13 ns MD simulation. The yellow dashed line stands for the hydrogen bond. (c) The superimposition of the catalytic residues E414 and E505 between PGUS-E wild-type (red) and F292L/T293K mutant (magentas). The moved distance of E414 and E505 is shown in yellow dashed line. 50x13mm (300 x 300 DPI)



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Figure 9 The local interaction network around site 304 of PGUS-E wild-type (a) and R304L (b). The hydrophobic residues are shown in yellow. Residue at 304 is shown in magentas. The distance between Ile52, Ala47 and Arg/Leu 304 is shown in red dashed line. 49x21mm (300 x 300 DPI)


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Enhancing the Thermostability of Î²-Glucuronidase by Rationally

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