formation of nitrile hydratase cross-linked enzyme aggregates in mesoporous onion-like silica:...

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Article

Formation of nitrile hydratase CLEAs in mesoporousonion-like silica: preparation and catalytic properties

Jing Gao, Qi Wang, Yanjun Jiang, Junkai Gao, Zhihua Liu, liya Zhou, and Yufei ZhangInd. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/ie503018m • Publication Date (Web): 15 Dec 2014

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1

Formation of nitrile hydratase CLEAs in 1

mesoporous onion-like silica: preparation 2

and catalytic properties 3

Jing Gaoa, Qi Wang

a, Yanjun Jiang

a*, Junkai Gao

b, Zhihua Liu

a, Liya Zhou

a, Yufei 4

Zhangc

5

a. School of Chemical Engineering and Technology, Hebei University of Technology, 6

Tianjin, 300130, China. 7

b. School of Energy and Environmental Engineering, Hebei University of Technology, 8

Tianjin, 300130, China. 9

c. National Key Laboratory of Biochemical Engineering, Institute of Process 10

Engineering, Chinese Academy of Sciences, Beijing 100190, China 11

* Corresponding author: Fax: +86-22-60204294; Tel: +86-22-60204945; 12

E-mail address: [email protected] (Yanjun Jiang) 13

14

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Abstract: 1

Nitrile hydratase CLEAs was formed in mesoporous onion-like silica 2

(NHase-CLEAs@MOS) by using macromolecular dextran polyaldehyde as a 3

cross-linker through the carrier-bound CLEAs method. The effect of preparation 4

parameters on the recovery of enzyme activity was investigated. The properties such 5

as pH, thermal, and storage stability and kinetic parameters of NHase-CLEAs@MOS 6

were also studied. The maximum amount of NHase absorbed in MOS was 535 mg/g. 7

Under optimized conditions, the maximum activity recovery of 8

NHase-CLEAs@MOS was 48.2%. The stabilities of NHase-CLEAs@MOS were 9

improved significantly compared to the NHase@MOS prepared by physical 10

adsorption and free NHase. This work demonstrated that the mesoporous onion-like 11

silica can be efficiently employed as host materials for NHase immobilization and the 12

carrier-bound CLEAs method can lead to enhanced activity and stability of the 13

immobilized enzymes. 14

Keywords: cross-linked enzyme aggregates; nitrile hydratase; enzyme 15

immobilization; mesoporous silica; dextran polyaldehyde 16

17

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1

1. Introduction 2

Nitrile hydratase (NHase; EC 4.2.1.84) is a class of mononuclear iron or cobalt 3

enzymes that catalyze the hydration of nitriles to their corresponding amides.1 NHases 4

are widely used in the industrial production of acrylamide and nicotinamide, and they 5

can also be used in environmental remediation for the conversion of nitrile wastes to 6

less toxic amides.2-6

However, the industrial use of NHase is often limited by the low 7

operational stability, highly sensitive to the change of environment and poor recovery 8

of the free NHase. To overcome these drawbacks, immobilization of NHase on a 9

suitable support is a promising strategy, which not only improves the operational 10

stability of the enzyme, but also facilitates the efficient recovery and reuse.7,8

Until 11

now, different methods including adsorption or covalent binding to a carrier, 12

encapsulation and entrapment or cross-linking have been developed to immobilize 13

enzymes.7,8

However, each immobilization method has its own advantages and 14

disadvantages and a “perfect” universally applicable method for immobilizing 15

enzymes is not available. NHase ES-NHT-118, obtained from E. coli strain that 16

carries the cloned nitrile hydratase gene, is an important enzyme in converting nitriles 17

to amides under physiological conditions. Developing an efficient immobilized 18

method for this kind of NHase is the prerequisite for its industrial applications (i.e. 19

acrylamide production). 20

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In recent years, a new approach which combines the advantages of physical 1

adsorption with cross-linked enzyme aggregates (CLEAs) was developed. The 2

so-called carrier-bound CLEAs can be prepared easily by the following process: 3

enzymes are adsorbed on porous supports firstly, aggregated and then cross-linked, 4

resulting in the formation of CLEAs in the pores.9-11

For example, Kim and 5

co-workers prepared α-chymotrypsin and lipase CLEAs in hierarchically-ordered 6

mesocellular mesoporous silica (HMMS) through “ship-in-a-bottle” approach, and the 7

enzymes showed high loading capacity and increased stability.12

As an extension of 8

this approach, magnetically-separable and highly-stable carrier-bound CLEAs 9

systems were also prepared. Besides the advantages of high enzyme loadings and 10

stability, facile magnetic separation of these immobilized enzyme systems facilitated 11

their repeated usages.13

Hartmann's group reported the successful preparation of 12

cross-linked chloroperoxidase and glucose oxidase aggregates in mesocellular foams. 13

Compared to the immobilized enzymes on the same support through physical 14

adsorption, the carrier-bound CLEAs systems showed increased operational 15

stability.14,15

This approach was also used to prepare an immobilized bi-enzymatic 16

system and the resulting bi-enzymatic system can be successfully used in a 17

continuously-operating fixed bed reactor and the leaching of the enzymes can be 18

suppressed.16

Therefore, the preparation of CLEAs in the pores of a suitable support is 19

a most promising method for enzyme immobilization.9, 17

20

Recently, a novel mesoporous onion-like silica (MOS) was synthesized and used as 21

support for the preparation of nanoscale enzyme reactors by cross-linking adsorbed 22

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lipase. The highly ordered onion-like multilayer and highly curved pore structure of 1

the MOS was effective in improving the lipase stability.17

Further studies of enzyme 2

immobilization in this novel porous materials via carrier-bound CLEAs method and 3

systematic investigation of the resulting biocatalyst are currently still required. 4

Thus, in this study, MOS was synthesized according to the previous report17

and the 5

NHase ES-NHT-118 was immobilized in MOS by adsorption (named NHase@MOS). 6

To avoid the leaching problem and improve the properties of NHase, dextran 7

polyaldehyde (DP) was used to cross-link the adsorbed NHase and then cross-linked 8

enzyme aggregates in the MOS was obtained (named NHase-CLEAs@MOS). This is 9

the first time that successful immobilization of NHase by using the carrier-bound 10

CLEAs method. The preparation conditions were optimized and the effects of pH and 11

temperature on the activity of NHase-CLEAs@MOS were investigated. The pH, 12

thermal and storage stabilities of NHase-CLEAs@MOS were also investigated. 13

2. Experimental 14

2.1 Materials 15

Nitrile hydratase ES-NHT-118 (NHase, EC 4.2.1.84) from E. coli strain that carried 16

the cloned nitrile hydratase gene was purchased from Hangzhou Biosci Biotech Co. 17

(China). Poly(ethlyene glycol)-block-poly-(propylene glycol)-block-poly(ethylene 18

glycol) triblock copolymer (EO)20(PO)70(EO)20 (denoted as P123, Mn=5800), 19

tetraethyl orthosilicate (TEOS) and 1,3,5-Trimethylbenzene (TMB, 98%) were 20

purchased from Sigma-Aldrich (America). SBA-15 was purchased from Nanjing 21

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XFNANO, inc. (China). Dextran (100 kDa) and sodium metaperiodate were 1

purchased from Dingguo Biotechnology Co. (China). Acrylonitrile was purchased 2

from Fuchen Chemical Reagent Factory (China). All reagents were used as received 3

without further purification. 4

2.2 Synthesis and characterization of mesoporous onion-like silica 5

Mesoporous onion-like silica (MOS) was prepared according to the reported 6

procedure.17

Briefly, an amount of P123 (4 g) was dissolved in 150 mL of HCl 7

solution (1.6 M) under stirring condition at room temperature. Then, 2 g of TMB was 8

added and stirred for 5 h. The mixture was heated to 40 oC and then of an amount of 9

TEOS (8.5 g) was added under stirring. After that, the mixture was aged at 40 oC for 10

20 h under stirring condition and further aged at 100 oC under static condition for 24 h. 11

The solid was filtered, washed, and calcined at 550 oC for 4 h, and then the MOS was 12

obtained. 13

Transmission electron microscopic (TEM) images of the MOS were obtained on a 14

Tecnai G2 F20 Transmission Electron Microscope. Scanning electron microscopic 15

(SEM) images were obtained on a FEI NanoSEM450 microscope. N2 16

adsorption/desorption isotherms at 77 K were obtained using a Micromeritic 17

ASAP2020M+C Physisorption Analyzer. Pore size distribution was calculated using 18

the BJH method. Fourier-transform infrared (FT-IR) spectra were recorded on a 19

Bruker Vector 22 FT-IR spectrophotometer using KBr pellets method. The 20

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thermogravimetric analysis (TGA) was performed on a SDT Q600 1

Thermogravimetric Analyzer under air atmosphere and heating rate of 10 oC/min. 2

2.3 Preparation of immobilized NHase 3

MOS (50 mg) was mixed with 10 mL of NHase solution (50 mM sodium phosphate 4

buffer, PBS, pH 7.0) in a centrifuge tube and then incubated in a water bath (25 oC) 5

for several hours under shaking condition (200 rpm). After that, the solid was 6

separated from the supernatant liquid by centrifugation. The obtained solid (referred 7

to as NHase@MOS) was washed with PBS (50 mM, pH 7.0) for three times. The 8

concentration of NHase in the solutions was measured by Bradford assay. Typically, 9

1mL NHase solution was mixed with 5 mL Bradford reagent and allowed to stand for 10

5 min before measuring its absorbance at 595 nm. The final enzyme loading on the 11

MOS was calculated from the difference between the initial NHase amount and the 12

residual amount of NHase in the washing solutions and supernatant. For preparation 13

of NHase-CLEAs@MOS, NHase@MOS was incubated in DP solution for a certain 14

time at a low temperature (<10 oC) with shaking speed of 200 rpm. Then the samples 15

were washed with PBS (50 mM, pH 7.0) for three times, and NHase-CLEAs@MOS 16

was obtained. The preparation conditions of the immobilized NHase were optimized 17

and the used conditions in the different immobilization runs were stated in the text. 18

The immobilized NHase was stored in PBS at 4 oC until use. 19

2.4 Activity assay 20

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The standard reaction mixture (2 mL) contained 125 mM of acrylonitrile, 50 mM of 1

PBS (pH 7.0) and an appropriate amount of enzyme. The reaction was initiated by 2

addition of the free or immobilized NHase and carried out under magnetic stirring at 3

30 oC for 5 min. Then 200 µL of 2 M HCl was added to stop the reaction. The formed 4

acrylamide was determined by high-performance liquid chromatography (HPLC). 5

HPLC was performed with an Agilent 1200LC system equipped with an Eclipse Plus 6

C18 Column (3.5 µm, 4.6×100 mm) operating at 25 oC and at a flow rate of 1 mL/min. 7

The mobile phase was acetonitrile/water (3:7, v/v), and the UV detector absorbance 8

wavelength was fixed at 230 nm. One unit of NHase was defined as the amount of 9

enzyme that catalyzed the formation of 1 µmol of acrylamide per minute. The activity 10

recovery was calculated as follows: 11

Activity recovery (%) = (observed activity of immobilized NHase/starting activity of 12

NHase) 13

2.5 The stabilities and kinetic parameters of the free and immobilized NHase 14

2.5.1 Determination of temperature and pH optima 15

To determine the optimum pH of the free NHase, NHase@MOS and 16

NHase-CLEAs@MOS, their activities were measured in the pH range 3-10 at 30 oC 17

(pH 3–5: 10 mM sodium acetate buffer, pH 6–8: 50 mM PBS, pH 9.0-10.0: 10 mM 18

Tris-HCl buffer). The optimum temperature was determined by measuring their 19

activities in the temperature range from 10-70 oC at pH 7.0 (50 mM PBS) according 20

to the activity assay described above. The results for optimum pH and temperature 21

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were given in relative form, where the relative activity was calculated from the ratio 1

of the activity at each pH or temperature to the maximum activity. 2

2.5.2 Thermal and pH stability measurement 3

To determine the pH stability, the free NHase, NHase@MOS and 4

NHase-CLEAs@MOS were incubated in pH 3.0 and pH 10.0 PBS solutions for 5

different time at 30 oC and the activities were measured following the method 6

described in Section 2.4 (excessive acidity and alkalinity were regulated by HCl and 7

NaOH). To determine the thermal stability, the samples of free NHase, NHase@MOS 8

and NHase-CLEAs@MOS were incubated in PBS (50 mM, pH 7.0) in absence of 9

substrate in a thermostatic bath at 40 and 50 oC. Their activities were measured 10

periodically following the method described in Section 2.4. The results of pH and 11

thermal stabilities were presented as percentage of residual activities, taking their 12

initial activities of the biocatalysts as 100%. 13

2.5.3 Measurement of the stability in shaking condition 14

To determine the stability in shaking condition, the free NHase, NHase@MOS and 15

NHase-CLEAs@MOS were suspended in pH 7.0 PBS under shaking condition (200 16

rpm) at 30 oC. At certain time intervals, the residual activities of each sample were 17

determined following the method described above, taking their initial activities as 18

100%. 19

2.5.4 Measurement of the storage stability 20

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To determine the storage stability, the free NHase, NHase@MOS and 1

NHase-CLEAs@MOS were suspended in pH 7.0 PBS in absence of substrate at 4 oC. 2

At certain time intervals, samples were taken out to determine the residual activities 3

and compared the activities to the initial activities. 4

2.5.5 Determination of kinetic parameters 5

The kinetic parameters of the free NHase and immobilized NHase were calculated 6

according to the Michaelis-Menten equation (1/V vs 1/[S]). To do this, the reactions 7

were carried out in the acrylonitrile concentration range from 2 to 40 mM at pH 7.0 8

and temperature at 30 oC. The concentration of the resulting acrylamide was 9

determined by HPLC. 10

3. Results and discussion 11

3.1 Characterization of the mesoporous onion-like silica supports 12

The SEM image showed that hundreds of nanosize onions particles aggregated into 13

secondary microsize particles (Fig.1a), which was agreed with the previous report.17

14

The TEM image showed that MOS had clearly multilayer structure, and the spacing 15

value between the layers was about 15 nm (Fig.1b). The BET surface area of the MOS 16

was 480 m2/g and the BJH adsorption cumulative volume of pores was 1.35 cm

3/g. 17

The calculated pore size was about 15.8 nm on average, which was consistent with the 18

result of TEM. The pore volume and pore size were large enough to accommodate 19

enzymes. After immobilizing NHase, the BET surface area of the 20

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NHase-CLEAs@MOS was 424 m2/g, the pore volume was 1.27 cm

3/g, and the 1

average pore size was about 12.6 nm. The reduction in BET surface area, pore volume 2

and pore size could be regarded as evidence for the immobilization of NHase into the 3

pores of MOS.18

4

3.2 NHase loading on MOS supports 5

The FT-IR spectra of MOS and NHase@MOS were shown in Fig.2. For MOS, the 6

typical absorption peaks of silica appeared at 1099 cm-1

(Si-O bond stretching), 972 7

cm-1

(Si-OH band) and 462 cm-1

(Si-O bending).19,20

For NHase@MOS, aside from 8

the typical absorption peaks of silica, the curve of NHase@MOS in Fig.2 showed the 9

peaks at 1464 cm-1

and 1545 cm-1

, which can be ascribed to the bending vibration of 10

-C-H and the deformation vibration of –NH2 groups.21

These results indicated that 11

NHase was successfully absorbed into the MOS. When the starting concentration of 12

NHase was 3.00 mg/mL, the volume of NHase solution was 25 mL, and the amount 13

of MOS was 50 mg, under shaking condition (200 rpm) at 25 oC, the maximum 14

amount of NHase absorbed in MOS was 535 mg/g (34.9 wt%). The quantity of 15

enzyme utilised for immobilization was 75.0 mg, and the quantity of enzyme loaded 16

in MOS was ca.26.8 mg, then the efficiency of the enzyme loading (quantity of 17

enzyme utilised for immobilisation vs enzyme loaded in the material) was 35.7%. 18

TGA curves of MOS and NHase@MOS were shown in Fig.S1. According to the 19

TGA curves, NHase@MOS exhibited a 47.4% weight loss, while the weight loss of 20

MOS was only about 10.2% in the same range of temperature. So the enzyme loading 21

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in MOS was about 37.2% as calculated from the TGA results, which was consistent 1

with the calculated value that was measured spectroscopically. 2

Table S1 showed that different starting concentration of free NHase solution can 3

lead to different absorbed performance. When the starting concentration of free 4

NHase was 0.0262 mg/mL, the activity recovery of NHase@MOS can reach 44.6%. 5

However, when the NHase loading in MOS increased, the activity recovery was 6

decreased. This can be explained by the unfavorable aggregation of NHase in MOS 7

that could limit the access of substrates to the active site and thus lower the NHase 8

specific activity.22

Since the concentration of cross-linker and cross-linking time are 9

key parameters in the preparation of NHase-CLEAs@MOS, the optimum cross-linker 10

concentration and cross-linking time were determined. As can be seen in Fig.S2 (the 11

cross-linking time was set as 0.5 h), the activity recovery increased with the increase 12

of cross-linker concentration, and then decreased. Thus, the concentration of 1.43 13

mg/mL was adopted in the subsequent experiments. As shown in Fig.S3, the activity 14

recovery increased with the cross-linking time increased, then decreased with further 15

prolongation of cross-linking time. The highest activity recovery was obtained at the 16

cross-linking time of 2.0 h. These can be explained as follows: at low cross-linker 17

concentration and short cross-linking time, inadequate cross-linking occurred and 18

resulted in operationally unstable CLEAs with low activity23

; While at high 19

cross-linker concentration and prolonged cross-linking time, excessive cross-linking 20

occurred that could rigidify the enzyme molecule, reduce enzyme’s flexibility, and 21

then resulted in the lower activity recovery.24-26

Thus, when the cross-linker 22

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concentration was 1.43 mg/mL and the cross-linking time was 2.0 h, the highest 1

specific activity 5.73 U/mg was obtained and the activity recovery of 2

NHase-CLEAs@MOS was 48.2%. 3

3.3 Optimum pH of free NHase, NHase@MOS and NHase-CLEAs@MOS 4

The activities of free NHase, NHase@MOS and NHase-CLEAs@MOS were 5

measured at different pH values. As shown in Fig.3, free NHase reached its maximum 6

activity at pH 7.0, whereas the NHase@MOS and NHase-CLEAs@MOS reached 7

their maximum activities at pH 6.0. Such shifts in the optimum pH of the enzymes 8

after immobilization have been widely reported in the previous studies and could be 9

attributed to the change of the enzyme’s conformation and the microenvironment 10

upon immobilization.27,28

NHase@MOS and NHase-CLEAs@MOS exhibited a 11

similar curve shape over the pH ranging from 3 to 10. Their relative activities 12

increased rapidly as the pH was increased from 3 to 6, and then decreased at the pH 13

range of 6-10. In addition, for pH below 6, NHase@MOS and NHase-CLEAs@MOS 14

maintained more activity than their free counterpart, which allowed the use of these 15

biocatalysts more efficiently in acidic pH region. 16

3.4 Optimum temperature of free NHase, NHase@MOS and NHase-CLEAs@MOS 17

The activities of free NHase, NHase@MOS and NHase-CLEAs@MOS were 18

measured at different temperatures. As shown in Fig.4, the activities of NHase@MOS 19

and NHase-CLEAs@MOS increased gradually with temperature up to 40 oC, and then 20

decreased. In addition, the temperature profiles of the NHase@MOS and 21

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NHase-CLEAs@MOS were broader than that of the free NHase, which indicated that 1

the activity can be preserved over a wider temperature range through enzyme 2

immobilization. Furthermore, the activity of NHase-CLEAs@MOS was better than 3

that of NHase@MOS when the temperature was above 40 oC. This can be attributed 4

to the presence of multipoint covalent bounds between NHase molecules after the DP 5

treatment, which can maintain the active three-dimensional conformation of the 6

enzyme.29,30

7

3.5 pH stability of free NHase, NHase@MOS and NHase-CLEAs@MOS 8

The pH stabilities of free NHase, NHase@MOS and NHase-CLEAs@MOS were 9

determined by incubating them in pH 3.0 and pH 10.0 buffers at 30 oC for different 10

time intervals. The initial activities of the samples were taken as 100% and the 11

residual activities were shown in Fig.5. It was observed that the decrease of the 12

NHase-CLEAs@MOS activity was slower than that of the free NHase and 13

NHase@MOS. For example, after 6 h of incubation at pH 3.0, free NHase and 14

NHase@MOS only retained 1.1% and 45.4% of the initial activity. In contrast, 15

NHase-CLEAs@MOS maintained more than 66.5% of its initial activity. The same 16

behavior was observed at pH 10.0. The NHase-CLEAs@MOS retained 78.8% of its 17

initial activity after 6 h of incubation, while free NHase and NHase@MOS only 18

retained 10.7% and 56.0% of their initial activities under the same condition. These 19

results demonstrated that NHase-CLEAs@MOS exhibited improved pH stability 20

compared to free NHase and NHase@MOS. This can be attributed to the 21

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immobilization of NHase through the carrier-bound CLEAs method, where the MOS 1

and DP treatment can restrict the conformational change of the NHase induced by H+ 2

or OH- and then stabilize the enzyme.

31 3

3.6 Thermal stability of free NHase, NHase@MOS and NHase-CLEAs@MOS 4

Thermal stabilities of free NHase, NHase@MOS and NHase-CLEAs@MOS were 5

determined by measuring the residual activities of the samples incubated in PBS (50 6

mM, pH 7.0) over different time at 40 oC and 50

oC. The initial activity of NHase was 7

taken as 100% and the results were shown in Fig.6. It can be observed that the 8

decrease rate of NHase-CLEAs@MOS activity was slower than that of the free 9

NHase and NHase@MOS. After 7.5 h of incubation at 40 oC, free NHase and 10

NHase@MOS retained 9.2% and 57.3% of their initial activities, respectively, 11

whereas NHase-CLEAs@MOS maintained 68.6% of its initial activity. The same 12

behavior was observed at 50 oC. NHase-CLEAs@MOS retained 62.4% of its initial 13

activity after 7.5 h of incubation, while free NHase and NHase@MOS only retained 14

4.9% and 33.6%, respectively. These results demonstrated that the thermal stability of 15

NHase-CLEAs@MOS was much better than that of free NHase and NHase@MOS, 16

which can be explained by the protection of the MOS support and the intermolecular 17

and intramolecular chemical cross-linking.32

The MOS can provide a suitable 18

microenvironment and the DP cross-linking can provide a more effective 19

conformational stabilization of NHase in NHase-CLEAs@MOS, which required 20

much more energy to break down this active conformation than free NHase and 21

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[email protected]

As shown in Table 1, the thermal stability of NHase-CLEAs@MOS 1

was compared with some thermophilic and immobilized NHases in previous reports, 2

which indicated that the immobilized NHase ES-NHT-118 in this paper exhibited 3

satisfactory thermal stability. 4

3.7 The stability of free NHase, NHase@MOS and NHase-CLEAs@MOS in shaking 5

and storage conditions 6

Fig.7 showed the stability of free NHase, NHase@MOS and NHase-CLEAs@MOS 7

in an aqueous solution under rigorous shaking conditions (200 rpm). The results 8

indicated that the activity of free NHase decreased fast and almost completely 9

deactivated the next day. In the case of NHase@MOS, about 40.1% of its initial 10

activity was retained after shaking for 7 days. In contrast, the NHase-CLEAs@MOS 11

clearly stabilized the NHase and 81.2% of initial activity was remained under the 12

same conditions. The decrease of activity was due to the denaturation of NHase under 13

rigorous shaking.12

Compared to NHase@MOS, NHase-CLEAs@MOS improved the 14

NHase’s stability, which can be explained by the multipoint covalent linkages 15

between NHase molecules after DP cross-linking. These multipoint covalent linkages 16

were effective enough to prevent the enzyme leaching, conserve the active form of 17

enzyme molecules and then protect the activity of NHase under shaking 18

condition.12,13,17

19

Fig.8 presented the storage stabilities of free NHase, NHase@MOS and 20

NHase-CLEAs@MOS in PBS at 4 oC. Free NHase and NHase@MOS remained 21

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11.0% and 33.2% of initial activity after 30 days, respectively. However, 1

NHase-CLEAs@MOS retained 74.4% of its initial activity at the same conditions. 2

Apparently, NHase-CLEAs@MOS improved the storage stability of NHase under 3

these storage conditions. Overall, this improved stability of NHase-CLEAs@MOS 4

under rigorous shaking and storage conditions demonstrated that the carrier-bound 5

CLEAs approach was most promising in industrial applications. 6

3.8 Kinetic parameters of free NHase and immobilized NHase 7

The kinetic parameters of the free and immobilized NHase were also investigated. 8

As can be seen in Table 2, compared to free NHase, the Vmax values of the 9

immobilized NHase reduced and the Km values increased. These results indicated that 10

the reduction in the affinity of NHase for binding substrate or a lower possibility of 11

forming a substrate-enzyme complex after immobilization.34

The lowered ratios of 12

Vmax/Km of immobilized NHase also confirmed these results.35

Furthermore, the Km 13

value of NHase-CLEAs@MOS was higher than that of NHase@MOS, which may be 14

caused by the steric hindrance upon DP cross-linking which induced the lower 15

accessibility of the substrate to the active sites of the NHase-CLEAs@MOS .34,36

16

For comparison, NHase@SBA-15 and NHase-CLEAs@SBA-15 were prepared by 17

using SBA-15 as support, and their Km values were also measured. The Km values of 18

NHase@SBA-15 and NHase-CLEAs@SBA-15 were higher than those of 19

NHase@MOS and NHase-CLEAs@MOS. This can be explained by the lower mass 20

transfer limitation in the MOS with larger mesocellular pores (ca.15 nm) than that of 21

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SBA-15 (6.65 nm, Fig.S4).17,37

With this favorable characteristic, this method of 1

formation CLEAs in MOS can be easily extended to immobilize other enzymes, and 2

can be applied in various enzyme-based industrial processes. 3

4. Conclusions 4

The active heterogeneous biocatalyst that based on the formation of NHase CLEAs 5

in the pores of mesoporous onion-like silica was successfully prepared in this work. 6

The NHase-CLEAs@MOS was more stable than the biocatalyst prepared by physical 7

adsorption (NHase@MOS) and its free counterpart. NHase-CLEAs@MOS was 8

effective in preventing the leaching of NHase and can retain high activity under 9

rigorous shaking and storage conditions. This method of enzyme immobilization can 10

be easily extended to the stabilization of various other enzymes, and the stabilized 11

NHase potentially provide robust biocatalyst capable of converting a wide variety of 12

nitrile substrates to commercially important chemicals. 13

Supporting Information 14

Table S1 and Fig.S1 to Fig.S4 can be seen in supporting information, this information 15

is available free of charge via the Internet at http://pubs.acs.org/. 16

Acknowledgment 17

This work was supported by the National Nature Science Foundation of China (Nos. 18

21276060, 21276062, and 21106164), the Natural Science Foundation of Tianjin 19

(13JCYBJC18500 and 12JCQNJC06000), and the Science and Technology Research 20

Key Project of Higher School in Hebei Province (YQ2013025). 21

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Figure Captions: 1

Figure 1 SEM image (a) and TEM image (b) of MOS 2

Figure 2 FT-IR spectra of MOS and NHase@MOS 3

Figure 3 Activities of (●) free NHase, (▲) NHase@MOS and (■) 4

NHase-CLEAs@MOS under different pH values. (The 100% relative activity was 5

11.9 U/mg, 8.00 U/mg, and 7.65 U/mg for free NHase, NHase@MOS and 6

NHase-CLEAs@MOS, respectively) 7

Figure 4 Activities of (●) free NHase, (▲) NHase@MOS and (■) 8

NHase-CLEAs@MOS under different temperatures. (The 100% relative activity was 9

11.9 U/mg, 7.03 U/mg, and 7.30 U/mg for free NHase, NHase@MOS and 10


Figure 5 pH stability of (●) free NHase, (▲) NHase@MOS and (■) 12

NHase-CLEAs@MOS at pH 3 and pH stability of (○) free NHase, (△) NHase@MOS 13

and (□) NHase-CLEAs@MOS at pH 10 (The initial activity was 3.57 U, 3.19 U, and 14

3.44 U for free NHase, NHase@MOS and NHase-CLEAs@MOS, respectively) 15

Figure 6 Thermal stability of (●) free NHase, (▲) NHase@MOS and (■) 16

NHase-CLEAs@MOS at 40 oC and thermal stability of (○) free NHase, (△) 17

NHase@MOS and (□) NHase-CLEAs@MOS at 50 oC (The initial activity was 3.57 18

U, 3.19 U, and 3.44 U for free NHase, NHase@MOS and NHase-CLEAs@MOS, 19

respectively) 20

Figure 7 Stability of (●) free NHase, (▲) NHase@MOS and (■) 21

NHase-CLEAs@MOS in a shaking condition (200 rpm) at 30 oC (The initial activity 22

was 3.57 U, 3.19 U, and 3.44 U for free NHase, NHase@MOS and 23


Figure 8 Storage stability of (●) free NHase, (▲) NHase@MOS and (■) 25

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NHase-CLEAs@MOS (The initial activity was 3.57 U, 3.19 U, and 3.44 U for free 1

NHase, NHase@MOS and NHase-CLEAs@MOS, respectively) 2

3

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Tables 1

Table 1 Comparison of thermal stability of NHases (expressed as % residual activity 2

following incubation at given temperatures) 3

Microorganism Temperature

(oC)

Incubation

time (min)

Residual

activity

(%)

Reference

G.pallidus RAPc8 50 150 50 (38)

Bacillus BR449 60 120 100 (39)

B.pallidus DAC521 50 - 50 (40)

P.thermophila 50 120 100 (41)

Eupergit®

C

(EDAC)-immobilized G.pallidus

RAPc8

60 90 80 (42)

PVA/chitosan–GA cross-linked

R. rhodochrous ATCC BAA-870

45 120 70 (43)

NHase-CLEAs@MOS 50 450 62.4 (This

paper)

4

5

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1

Table 2 Kinetic parameters of free NHase and immobilized NHase[a]

2

free

NHase

NHase@MO

S

NHase-CLEAs@

MOS

NHase@SBA-

15

NHase-CLEAs

@SBA-15

Km (mM) 1.35 1.40 1.68 2.19 2.22

Vmax

(mM/s)

5.71 4.61 4.46 4.09 3.96

Vmax/Km

(s-1

)

4.23 3.29 2.65 1.87 1.78

[a]Reactions were carried out at 30

oC in 50 mM PBS (pH 7.0) using 0.202 U NHase. 3

4

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1

Figures 2

3

4

Figure 1 5

6

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4000 3500 3000 2500 2000 1500 1000 500

Tra

nsm

itta

nce

(a.

u.)

Wavenumber (cm-1)

1545cm-1

1464cm-1

1099cm-1

MOS

NHase@MOS

972cm-1

462cm-1

1

Figure 22

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3 4 5 6 7 8 9 10

0

20

40

60

80

100

Rel

ativ

e ac

tiv

ity (

%)

pH

1

Figure 3 2

3

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10 20 30 40 50 60 70

0

20

40

60

80

100

Rel

ativ

e ac

tivit

y (

%)

Temperature (oC)

1

Figure 4 2

3

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0 1 2 3 4 5 6

0

20

40

60

80

100

Res

idual

act

ivit

y (

%)

Time (h)

1

Figure 5 2

3

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0 1 2 3 4 5 6 7 8

0

20

40

60

80

100

Res

idual

act

ivit

y (

%)

Time (h)

1

Figure 6 2

3

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0 1 2 3 4 5 6 7

0

20

40

60

80

100

Res

idual

act

ivit

y (

%)

Time (day)

1

Figure 7 2

3

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0 5 10 15 20 25 30

0

20

40

60

80

100

Res

idual

act

ivit

y (

%)

Time (day)

1

Figure 8 2

3

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formation of nitrile hydratase cross-linked enzyme aggregates in mesoporous onion-like silica:...

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