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Preparation of Flower-like Nanobiocatalyst System via Biomimetic Mineralization of Cobalt Phosphate with Enzyme Yang Song, Jing Gao, Ying He, Liya Zhou, Li Ma, Zhihong Huang, and Yanjun Jiang Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b03809 • Publication Date (Web): 05 Dec 2017 Downloaded from http://pubs.acs.org on December 6, 2017

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Industrial & Engineering Chemistry Research

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Preparation of Flower-like Nanobiocatalyst System

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via Biomimetic Mineralization of Cobalt Phosphate

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with Enzyme

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Yang Song, Jing Gao, Ying He, Liya Zhou, Li Ma, Zhihong Huang, Yanjun Jiang*

5

School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin,

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300130, China

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*Corresponding Author: E-mail: [email protected]; TEL: +86-22-60204945

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School of Chemical Engineering and Technology, Hebei University of Technology, 8

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Guangrong Road, Hongqiao District, Tianjin, 300130, P. R. China

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ACS Paragon Plus Environment

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Industrial & Engineering Chemistry Research 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

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Abstract

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This study reported a novel and facile method for the spontaneous synthesis of flower-like cobalt

3

phosphate nanocrystals (Co3(PO4)2 nanoflowers) without adding any templates, and the growth

4

mechanism was further studied. Subsequently, an excellent nanobiocatalyst system was

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established via the biomimetic mineralization of cobalt phosphate with Co-type nitrile hydratase

6

(NHase). Owing to the interactions between cobalt ions in the microenvironment of cobalt

7

phosphate and enzyme active site, the encapsulated NHase (NHase@Co3(PO4)2) exhibited high

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catalytic efficiencies and desirable stabilities. The enzymatic activity of encapsulated NHase was

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238.4 U/g and the protein loading amount was 210.73 mg/g. Compared with free NHase, the

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optimum temperature of NHase@Co3(PO4)2 was 40 oC, which was higher than that of the free

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NHase (30 oC). The thermal, pH, anti-proteolytic and storage stability of NHase@Co3(PO4)2

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were all improved. Furthermore, NHase@Co3(PO4)2 could be applied in the production of

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nicotinamide (NAM) with a satisfying yield and it could be reused for 7 times. All these results

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in this work clearly confirmed that the cobalt phosphate nanocrystals, as an original nanocarrier,

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would have promising applications for constructing nanobiocatalyst systems.

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Keywords：：Co3(PO4)2, nitrile hydratase, nanobiocatalyst, enzyme immobilization

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1. Introduction

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Enzymes are recognized as the best present from natural biosome and endowed with ultrahigh

3

catalytic activity, specificity and selectivity. However, enzymes will face harsh conditions

4

including high temperature, strong acid and alkali, etc. if they are applied directly into the

5

industrial process. Hence, immobilized enzyme, which can improve enzyme stability and recycle

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several times for industrial process, has been widely used and studied1-3. Traditional methods are

7

generally immobilizing target enzymes on the pre-existing solid carriers via adsorption, cross-

8

linking or covalent binding approach4, 5. Recently, biomimetic mineralization has been

9

considered as an outstanding process to fabricate advanced materials with diverse morphologies6-

10

9

11

mimetic biomineralizaiton10-15. Thus, the application of biomineralization process in enzyme

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immobilization has gained quite a lot attention due to the particularly mild reaction conditions

13

and ultrahigh enzyme activity recovery16-18. Moreover, thanks to the mild process of

14

biomineralization, enzyme-inorganic hybrid composites could provide reliable stabilization

15

effect for incorporated enzymes and let them exhibit plentiful biological functions. Currently, the

16

biomineralization of metal-phosphates has been proved as an outstanding approach to synthetize

17

nanobiocatalysts. Ge et al. took the lead in synthesizing copper phosphate nanoflowers with the

18

immobilization of enzymes, which exhibited pretty appearance and excellent catalytic

19

properties19. Subsequently, a series of hybrid bio-inorganic nanoflowers were created and

20

various of biocatalyst system were established on the basis of metal-phosphates

21

biomineralization20-26. Nevertheless, the studies about the preparation of cobalt phosphate

22

nanoflowers for immobilizing enzyme have been rarely reported. López-Gallego et al. had

23

reported that cobalt-phosphate sponges were only formed in presence of ploy-histidine tagged

, and it is worth-mentioning that the gel diffusion is an excellently controllable method for


3


1

proteins at their N-terminus, and they believed that the His-tag would selectively drive the

2

mineralization acting as a nucleation site for Co3(PO4)224,

3

synthesized flower-like hierarchical Co3(PO4)2·8H2O via the microwave-assisted method, and

4

the flower-like shape could appear only by using hexamethylenetetramine (HMTA) as

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template28; Kim et al. presented a protein-directed assembly method to prepare BSA-cobalt

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phosphate hybrid flowers and demonstrated that BSA worked as the template to interact with

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cobalt and phosphate29. However, different from the above studies, we found that the cobalt

8

phosphate crystals could autogenously assemble to a flower-like shape in a mild aqueous

9

solution without adding any organic macromolecule as the assistant template. To our knowledge,

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this is the first time that Co3(PO4)2 nanoflowers were synthesized just depending on the cobalt

11

salt and phosphate buffer solution (PBS).

27

; Similarly, Hu et al. had also

12

In addition, to establish an efficient nanobiocatalyst system, the component of excellent

13

enzyme is also an essential part in the whole process. Nitrile hydratase (NHase; EC 4.2.1.84), as

14

a soluble metalloenzyme, has been considered as an excellent bio-catalyst to efficiently catalyze

15

the hydration of a nitrile to an amide30-32. In recent years, NHase has been successfully used in

16

the production of acrylamide (AM), nicotinamide (NAM), and 5-cyanovaleramide, etc.30.

17

However, free NHase is always too fragile to have a wonderful tolerance against the extreme

18

industrial environment, leading to the limitation of the industrial applications. There are two

19

types of NHase, which contains iron ion or cobalt ion at its catalytic center respectively33-35. In

20

this work, the Co-type NHase, which contains a noncorrin cobalt ion, was selected as the model

21

enzyme for immobilization via the co-biomimetic mineralization process with cobalt phosphate.

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In the preliminary jar-test, the enzymatic activity of free NHase was obviously increased after

23

adding a hint of cobalt salts, which could be attributed to the interactions of cobalt ion between


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Co-type NHase and the microenvironment of Co3(PO4)2. Based on this, taking the advantage of

2

the cobalt phosphate nanocrystals to immobilize Co-type NHase will be a better way to obtain a

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quite stable and superb biocatalyst.

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2. Experimental

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2.1 Preparation of Flower-like Co3(PO4)2 nanocrystals and immobilized NHase

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2.1.1 Preparation of pure Co3(PO4)2 nanoflowers: In a typical synthesis, 0.291 mg

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Co(NO3)2·6H2O was added to a flask with well-prepared phosphate buffer solution (10 mM, 100

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ml, pH=7.4), and the molar ratio of Co2+ to PO43- was 1:1. After fully oscillating, cobalt nitrate

9

was dissolved rapidly and the solution turned purple, while purple floc began to precipitate at the

10

same time. Then the precipitates were generated and incubated at 25 oC for 48 h. Finally, the

11

products (pink precipitates) were obtained by centrifugation at 6500 rpm for 5 min and then

12

washed with ultra-pure water for 3 times to remove the impurity. The product was dried at 50 oC

13

for 10 h and then the flower-like Co3(PO4)2 nanocrystals were successfully synthesized.

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2.1.2 Preparation of NHase@Co3(PO4)2: To fabricate immobilized NHase, 10 ml NHase (2

15

mg/ml) was added to 90 ml well-prepared phosphate buffer solution (10 mM, pH=7.4), and then

16

0.291 mg Co(NO3)2·6H2O was added subsequently. In accordance with the experimental

17

operation described above, the mixture was incubated at 25 oC for 48 h. The immobilized NHase

18

(NHase@Co3(PO4)2) was finally obtained by centrifugation at 6500 rpm for 5 min and washed

19

with ultra-pure water for 3 times.

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2.2 Enzyme activity assay

21

2.2.1 Definition of activity: One unit of activity (U) was defined as the amount of NHase which

22

catalyzed acrylonitrile converting to 1µmol acrylamide per minute. Relative activity was a


5


1

percentage, representing the ratio of observed activity to the initial activity (the maximum

2

activity).

3

2.2.2 Activity assay method: In a typical test tube, 1 ml of free NHase or suspension of

4

immobilized NHase was incubated at 30 oC for 5 min, and then 1ml of acrylonitrile solution (250

5

mM) was added and the reaction was initiated under magnetic stirring condition (200 rpm) for 5

6

min. Sequentially, 200 µL of NaOH (2 M) was added to stop the reaction. The resultant of

7

acrylamide was determined by high-performance liquid chromatography (HPLC), which was

8

performed with an Agilent 1200LC system equipped with an Eclipse Plus C18 column (5 µm,

9

4.6×250 mm). The mobile phase was acetonitrile/water (3:7, v/v) and the flow rate was 1

10

ml/min. The UV detector absorbance wavelength was fixed at 230 nm.

11

2.3 Characterization

12

Scanning electron microscopic (SEM) images were recorded on a FEI NanoSEM450

13

microscope. Fourier-transform infrared (FT-IR) spectra were recorded on a Bruker Vector 22

14

FT-IR spectrophotometer using KBr pellets method. Powder X-ray diffraction (XRD) patterns

15

were recorded using a Bruker AXS D8 Discover X-Ray diffractometer with a Cu Kα anode (λ=

16

0.15406 nm) at 40 kV and 40 mA. Confocal laser scanning microscopy (CLSM) images were

17

taken with a Leica TCS SP5 confocal microscope with excitation wavelength of 488 nm and

18

emission wavelength of 525 nm. The thermal gravity analysis (TGA) was measured with a

19

Mettler Toledo TGA/DSC 1 thermogravimetric analyzer under N2 atmosphere and heating rate

20

of 10 oC/min.

21

3 Results and Discussion

22

In a typical synthesis, the rigid granules of Co(NO3)2·6H2O were directly added into the PBS

23

instead of the aqueous solution of Co(NO3)2, which was different from the method that proposed


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previously19-26. The whole process could be considered that the Co(NO3)2 was dissolved in the

2

phosphate buffer solution firstly and then spontaneous crystallized into cobalt-phosphate

3

nanoflowers. Hence, a preliminary formation mechanism for the flower-like Co3(PO4)2

4

nanocrystals was speculated and the schematic diagram was proposed in Fig.1a. Once the

5

granules of Co(NO3)2·6H2O were contacted with PBS, they would be dissolved rapidly and

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release plenty of Co2+ around them. In the meantime, the free PO43- in PBS would combine with

7

cobalt ion as much as possible to self-assemble into Co3(PO4)2 nanosheets. With the time

8

prolonging, there were continuing growth of crystals on the basis of the previous nanosheets and

9

gradually turned more substantial and plentiful. Finally, the Co3(PO4)2 nanoflowers were

10

generated maturely according to the appendix SEM images below. Therefore, the initiation of

11

crystallization was attributed to the enrichment of local concentration of Co2+ released by

12

dissolution and could spontaneously formed Co3(PO4)2 nanoflowers without any assistant

13

templates. On the contrary, all of the previous reported methods were a mixture of two

14

solutions19-26, where the concentration distribution was pretty uniform, so the proteins were

15

needed as template to direct the nucleation and growth of hybrid nanoflowers. Surprisingly, in

16

this work, the Co3(PO4)2 nanoflowers could be fabricated just depending on the cobalt salt and

17

phosphate buffer solution (PBS), and the forming process was started with Co3(PO4)2 nanosheets

18

and gradually self-assembled into flowers, rather than the assistant nucleation of proteins.

19

After that, the nanobiocatalyst system was fabricated via the biomineralization of Co3(PO4)2

20

with NHase. As shown in Fig.1b and Fig.1c, the surface morphologies of both pure Co3(PO4)2

21

nanoflowers and NHase@Co3(PO4)2 were characterized by SEM. Fig.1b showed that the flower-

22

like Co3(PO4)2 crystals had pretty uniform particle size of 20 µm. Additionally, from the enlarged

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image, it was clear to observe that each petal of the cobalt phosphate nanoflowers was utterly


7


1

smooth and dense. Fig.1c was the immobilized NHase (NHase@Co3(PO4)2), which presented

2

daisy-like shapes with 15 µm in diameters. Different from the pure Co3(PO4)2 nanoflowers, the

3

crystal morphology of NHase@Co3(PO4)2 had been changed notably, owing to the addition of

4

enzyme in the process of biomineralization. Moreover, the surfaces of NHase@Co3(PO4)2 were

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completely rougher and cruder than that of the pure Co3(PO4)2, which demonstrated that NHase

6

was successfully immobilized in cobalt phosphate crystals. This phenomenon could be

7

interpreted as the biomineralization in nature, where organic macromolecules such as protein

8

could play a pivotal role in determining the morphology of composites36. Organic

9

macromolecules could stabilize the transient phases, influence the shape, and overcome the

10

intrinsic brittleness of the crystalline phases37. Furthermore, the growth process of pure

11

Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2 in different periods (Fig.S1) was also

12

investigated, which was well consistent with the schematic illustration in Fig.1a. For

13

NHase@Co3(PO4)2, obvious naonosheets could be easily observed in the first 24 hours, and there

14

was a trendency of continued growth. With an increasing time, different from the pure

15

Co3(PO4)2, NHase@Co3(PO4)2 gradually turned more circular and the petals were centralized

16

together from every direction, which could be attributed to the influence of enzymes. The

17

NHase@Co3(PO4)2 did not stabilize until 48 hours later and the morphologies kept constant

18

finally.

19

Besides, several characterization methods were used to confirm that NHase had been indeed

20

incorporated in cobalt phosphate crystals. Firstly, energy dispersive X-ray analysis (EDAX) was

21

used for analyzing the elemental composition (Fig.S2). Obviously, there were two additional

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elements in NHase@Co3(PO4)2 namely carbon and nitrogen elements which were not available

23

in pure cobalt phosphate nanoflowers, verifying the success of enzyme immobilization. N2


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1

adsorption-desorption analysis was also performed and the results were shown in Fig.S3. As can

2

be seen from the N2 adsorption-desorption isotherms, both the pure Co3(PO4)2 nanoflowers and

3

NHase@Co3(PO4)2 were representative type-IV curves, which were the characteristic of

4

mesoporous structure38. According to the inset of corresponding pore size distribution, the

5

average pore size of Co3(PO4)2 nanoflowers was 5.88 nm and the NHase@Co3(PO4)2 was 4.37

6

nm that calculated by using the Barrett-Joyner-Halenda (BJH) model. The cumulative volumes

7

of pores for Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2 were calculated to be 0.0544 cc/g

8

and 0.0355 cc/g. The decreased pore size and pores volume of NHase@Co3(PO4)2 also

9

confirmed that the enzymes were immobilized in the Co3(PO4)2 nanoflowers successfully.

10

Besides, Zeta potentials had been considered as an important role to select the optimum matrix

11

for enzyme immobilization20, 39-41. In this work, the zeta potential of pure Co3(PO4)2 nanoflowers

12

and NHase@Co3(PO4)2 were measured. The results indicated that the zeta potential value of

13

Co3(PO4)2 nanoflowers was about +3.5 mV, and the zeta potential value of NHase@Co3(PO4)2

14

was -1.7 mV under the neutral conditions. It was known that the isoelectric point of NHase was

15

around 5.5 and it could be negative under the neutral conditions42, 43. It could be obviously

16

observed that the zeta potential of NHase@Co3(PO4)2 showed a tendency to be negative owing

17

to the addition of NHase. When the matrix and enzyme were oppositely charged, it would have a

18

maximal binding efficiency between the protein and matrix39. These results further confirmed

19

that the Co3(PO4)2 could be an appropriate matrix for immobilizing NHase. Meanwhile, the

20

samples were characterized by X-ray diffraction (XRD) and the results were corresponded well

21

with the PDF#33-0432 (Co3(PO4)2·8H2O) as shown in Fig.2a. The peaks of Co3(PO4)2

22

nanoflowers and NHase@Co3(PO4)2 were almost the same, which demonstrated that the adding

23

of enzymes could not affect the main crystalline structure of Co3(PO4)2. In addition, the samples


9


1

of Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2 were also characterized by Fourier transform

2

infrared spectrometer (FT-IR) and the spectra were shown in Fig.2b. Compared with the pure

3

cobalt phosphate, there were several extra characteristic absorption peaks in the spectrum of

4

NHase@Co3(PO4)2. The peaks appeared at 1540 cm-1, 2066 cm-1, and 2962 cm-1 were the

5

absorption peaks of -NH2 (bending vibration), -C≡N (stretching vibration), and -CH3 (stretching

6

vibration) respectively, which could verify the enzyme’s incorporation. Likewise, the thermal

7

gravity analysis (TGA) demonstrated the existence of NHase on the basis of the difference

8

between those two relative gravities (%). As shown in Fig.2c, when the temperature rose above

9

100 oC, there would be a sudden slope due to the loss of crystalline water. Then the curve of pure

10

Co3(PO4)2 crystals tended to be steady. However, the curve of NHase@Co3(PO4)2 fell again with

11

the range of 200-350 oC because of the decomposition of proteins, which confirmed the

12

successful incorporation of NHase. Moreover, according to the TGA curves, the content of

13

protein was calculated to be 13.7 wt.%, which was roughly consistent with the value previously

14

measured by Bradford assay (17.67 wt.%). Furthermore, the CLSM image was obtained by

15

making enzymes labelled with fluorescein isothiocyanate (FITC) to prepare immobilized enzyme

16

(Fig.2d). The fluorescence from the images was clearly visible and the surface morphologies

17

could be easily observed, definitely indicating that the enzyme had already been immobilized in

18

the composites.

19

Several catalytic properties of immobilized NHase (NHase@Co3(PO4)2) and free NHase were

20

investigated. Firstly, corresponding to the SEM images in Fig.S1, the effect of immobilized time

21

on the specific activity and the loading amount of NHase was studied (Fig.S4). With the time

22

prolonging, the catalytic activity and the loading amount of NHase were both gradually increased.

23

When the time was extended to 48 h, the enzymatic activity reached the highest value of 238.4


10

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1

U/gimmobilized NHase, and the corresponding protein loading amount was 210.73 mg/gimmobilized NHase.

2

Besides, the effects of different temperature and pH on the activity of free NHase and

3

NHase@Co3(PO4)2 were also investigated (Fig.S5-S6). As shown in Fig.S5, the optimum

4

temperature of free NHase was 30

5

NHase@Co3(PO4)2 was protected by Co3(PO4)2 minerals and the optimum temperature rose to

6

40 oC. Likewise, pH had a great impact on enzymatic activities as well, and the extreme-pH

7

could make the enzyme inactivated. From the Fig.S6, the optimum pH of free NHase was 7.0

8

and the enzymatic activities were mostly lower in acidic and alkaline conditions. However, the

9

optimum pH of NHase@Co3(PO4)2 was shifted to 6.0, which was due to the electrostatic

10

interactions between the matrix and solution, and then leading to the unequal partitioning of H+

11

and OH- concentrations in microenvironment and bulk solution44, 45. In addition, it was worth

12

mentioning that the NHase@Co3(PO4)2 presented the insensitivity to pH according to the trend of

13

curves. Thanks to the tight protection of Co3(PO4)2 minerals, the immobilized NHase would be

14

possibly applied to the wider range of pH environment.

o

C. After the process of immobilization, the

15

Stabilities (thermal, pH, operational stability, etc.) were pretty important parameters for

16

evaluating immobilized enzyme. Higher temperature could effectively improve the reaction rate,

17

but the excessive temperature would destroy the structure and conformation of the enzyme. As

18

shown in Fig.3a, the thermal stability of free NHase was pretty unsatisfactory under the high

19

temperature. When the free NHase was incubated in PBS solution at 45 oC for 30 minutes, the

20

enzymatic activity rapidly decreased to 30.7% of the initial activity. When the temperature rose

21

to 55 oC, the residual activity of free NHase only remain 10.3% after incubated for 1 hour.

22

However, NHase@Co3(PO4)2 could still retain 84.2% and 66.5% of original activity after

23

incubated for 1 hour at 45 oC and 55

o

C, respectively. The improved stability of


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Page 12 of 30

1

NHase@Co3(PO4)2 was attributed to the stable interactions between enzymes and cobalt

2

phosphate minerals, which prevented the NHase from overheat inactivation. Our present finding

3

is similar to the previous results obtained from other protein-inorganic hybrid nanoflowers

4

system. Ge et al had reported that the copper-containing enzyme incorporated in copper

5

phosphate nanoflowers exhibited much higher catalytic activities and stabilities19,

6

Likewise, the pH stability of free NHase was also too terrible to resist the strong acid or alkali

7

environment (Fig.3b). In the first 30 minutes, the activity of free NHase had lost nearly one half

8

of the initial activity but NHase@Co3(PO4)2 maintained 95.3% of the original activity. After

9

incubated for 5 hours, 75.6% of the residual activity for NHase@Co3(PO4)2 in pH 4.0 and 63.9%

10

in pH 10.0 could be maintained, respectively, while free NHase almost lost all the activity.

11

Consistent with the results of Fig.S5 and Fig.S6, the immobilized NHase was able to stabilize the

12

conformation of enzymes and retain the activity when facing the extreme environment48, 49. In

13

addition, the stability of anti-proteolytic digestion was also examined as shown in Fig.3c. The

14

digestion of protease was so intense that the residual activity of free NHase was only 44.2% after

15

1 hour, but NHase@Co3(PO4)2 still remained the equal activity as much as the initial value. It

16

was believed that the Co3(PO4)2 minerals had trapped the enzyme in the interior of particles

17

instead of on the surface bound, and then it could prevent the NHase from proteolytic digestion50.

18

All these stabilities had reflected the superiority of NHase@Co3(PO4)2, which was promising for

19

industrial applications with high enzymatic activity and improved stability.

36, 46, 47

.

20

The kinetic parameters of free NHase and immobilized NHase were also studied and

21

calculated on the basis of Lineweaver-Burk plot method. As shown in Fig.3d, the data were fitted

22

well and the kinetic parameters were easily obtained. As shown in Table 1, the Km value of

23

NHase@Co3(PO4)2 was larger than that of free NHase, which indicated that the affinity between


12

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1

the substrate and enzyme had been weakened after immobilization. The ratio of Kcat/Km was

2

usually considered as an indicator of evaluating the catalytic efficiency. After the process of

3

immobilization, the value of Kcat and Kcat/Km were also decreased in comparison of free

4

NHase, which was due to the mass transfer resistance and lower possibility of forming a

5

substrate-enzyme complex of immobilized enzyme51.

6

As the active form of vitamin B3 and an important component of coenzyme NAD

7

(nicotinamide adenosine dinucleotide), nicotinamide (NAM) has a wide range of applications in

8

the medicinal and food industries such as in preventing diabetes, cancers, leprosy and acen52, 53.

9

Compared with the harsh and extreme conditions in chemical synthetic method, utilizing NHase

10

to catalyze the biotransformation of 3-cyanopyridine to NAM has been commonly recognized as

11

a quite mild and efficient approach for industrial applications. Therefore, taking the advantage of

12

NHase@Co3(PO4)2 to prepare NAM will not only maintain the admirable stability but also

13

achieve the reutilization in comparison of the free NHase. Thus, the effect of free NHase dosage

14

on the yield of NAM was investigated and the results were shown in Fig.4a. When the added

15

amount of free NHase was 3.63U, only 17.5% of NAM would be converted (as shown in Fig.4a,

16

curve a). It was clear that the yield of NAM obviously increased with the increasing activity of

17

free NHase. When the added NHase was around 21.75U, more than 87.5% of NAM yield can be

18

obtained just after reaction for one hour, and the yield would increase to 100% after 3 hours.

19

Likewise, the same research was also studied on NHase@Co3(PO4)2 and the result was similar to

20

free NHase (Fig.4b), indicating the immobilized NHase possess the outstanding catalytic

21

properties as free NHase. When the activity was nearly the same, the performance of

22

immobilized NHase was more favorable than free NHase in the catalytic system, which was

23

consistent with the previous studies54-56. This phenomenon could be explained by that the free


13


1

NHase was more sensitive to the substrate and limited by substrate inhibition56. Hence, it is more

2

appropriate to apply NHase@Co3(PO4)2 to the industrial production of nicotinamide and will

3

gain the satisfactory yield.

4

In general, the reusability of immobilized enzyme is considered as the most commendable

5

superiority over the free enzyme. In order to achieve recycling, the NHase@Co3(PO4)2 was

6

separated by centrifugation. When the NHase@Co3(PO4)2 was reused at the seventh time, the

7

relative activity would still maintain nearly 60% of the initial activity, indicating the feasibility

8

of applying to continuously industrial production and realizing the cost-effective and resource-

9

saving catalytic process (Fig.4c). Moreover, NHase is a kind of enzyme which could be broken

10

down easily by microorganism in the storage process and inactivated in a short time57. Therefore,

11

the storage stability was also an important parameter for immobilized NHase. As shown in

12

Fig.4d, the activity of NHase@Co3(PO4)2 started to decrease gradually after 15th days but could

13

still maintain 64.28% of the initial activity at the last 27th days, while the free NHase was only

14

11.91%. The decline of activity in the long-term storage process might be caused by the change

15

of surrounding environment, the microbial degradation and the destruction of enzyme

16

conformation. Nevertheless, owing to the protection of cobalt phosphate minerals, the

17

NHase@Co3(PO4)2 could retain its catalytic ability and have a promising future for industrial

18

applications.

19

4. Conclusions

20

In conclusion, the pure flower-like Co3(PO4)2 nanocrystals were synthesized for the first time

21

and a preliminary formation mechanism was speculated. After that, an excellent nanobiocatalyst

22

system was established via the biomimetic mineralization of Co3(PO4)2 nanoflowers with Co-

23

type NHase. The NHase was successfully immobilized in the Co3(PO4)2 and exhibited


14

Page 14 of 30

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1

significantly high catalytic efficiencies and desirable stabilities, which could be attributed to the

2

interactions of Co2+ between Co-type NHase and the microenvironment of cobalt phosphate.

3

Therefore, the Co3(PO4)2 nanoflowers, which exhibited the superb protective-effect and

4

stabilities, would have a promising application for industrial enzyme immobilization.

5 6

Supporting Information

7

Experimental details were shown in supporting information which includes experimental

8

materials and the experimental methods of the study on the performance of the enzyme. Figure

9

S1-S6 could be seen in supporting information, this material is available free of charge via the

10

Internet at http://pubs.acs.org.

11

Acknowledgment

12

This work was supported by the National Nature Science Foundation of China (Nos. 21576068,

13

21276060, 21276062, and 21306039), the Natural Science Foundation of Tianjin, China

14

(16JCYBJC19 800), the Natural Science Foundation of Hebei Province, China (B2015202082,

15

B2016202027, and B2017202056), the Program for Top 100 Innovative Talents in Colleges and

16

Universities of Hebei Province, China (SLRC2017029) and Hebei High level personnel of

17

support program, China (A2016002027).

18


15


1 2 3 4 5 6

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11 12


23


1

Figure captions

2

Figure 1 a) Schematic illustration of the crystallization process of cobalt phosphate nanoflowers.

3

b) SEM of Co3(PO4)2 nanoflowers c) SEM of NHase@Co3(PO4)2 (the scale bars of all SEM are

4

10 µm)

5

Figure 2 a) XRD of Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2 b) FT-IR spectra of

6

Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2 c) TGA curve of Co3(PO4)2 nanoflowers and

7

NHase@Co3(PO4)2 d) CLSM image of NHase@Co3(PO4)2

8

Figure 3 a) Thermal stability of free NHase and NHase@Co3(PO4)2 at 45 oC and 55 oC b) pH

9

stability of free NHase and NHase@Co3(PO4)2 at pH 4.0 and 10.0 c) Anti-proteolysis tolerance

10

of free NHase and NHase@Co3(PO4)2 incubated in trypsin d) Lineweaver-Burk plots for

11

determination of apparent kinetic parameters of free NHase and NHase@Co3(PO4)2

12

Figure 4 a) Effect of free NHase activity and reaction time on yield of nicotinamide (a.3.63U

13

b.7.25U c.14.50U d.21.75U e.29.00U) b) Effect of NHase@Co3(PO4)2 activity and reaction time

14

on yield of nicotinamide (a.1.01U b.2.93U c.4.04U d.7.32U e.14.65U) c) Reusability of

15

NHase@Co3(PO4)2 d) Storage stability of free NHase and NHase@Co3(PO4)2

16


24

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1 2

Fig.1 a) Schematic illustration of the crystallization process of cobalt phosphate nanoflowers. b)

3

SEM of Co3(PO4)2 nanoflowers c) SEM of NHase@Co3(PO4)2 (the scale bars of all SEM are 10

4

µm)

5 6


25


1 2

Fig.2 a) XRD of Co3(PO4)2 nanoflowers and NHase@Co3(PO4)2 b) FT-IR spectra of Co3(PO4)2

3

nanoflowers and NHase@Co3(PO4)2 c) TGA curve of Co3(PO4)2 nanoflowers and

4

NHase@Co3(PO4)2 d) CLSM image of NHase@Co3(PO4)2

5


26

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1 2

Fig.3 a) Thermal stability of free NHase and NHase@Co3(PO4)2 at 45 oC and 55 oC b) pH

3

stability of free NHase and NHase@Co3(PO4)2 at pH 4.0 and 10.0 c) Anti-proteolysis tolerance

4

of free NHase and NHase@Co3(PO4)2 incubated in trypsin d) Lineweaver-Burk plots for

5

determination of apparent kinetic parameters of free NHase and NHase@Co3(PO4)2

6


27


1 2

Fig.4 a) Effect of free NHase activity and reaction time on yield of nicotinamide (a.3.63U

3

b.7.25U c.14.50U d.21.75U e.29.00U) b) Effect of NHase@Co3(PO4)2 activity and reaction time

4

on yield of nicotinamide (a.1.01U b.2.93U c.4.04U d.7.32U e.14.65U) c) Reusability of

5

NHase@Co3(PO4)2 d) Storage stability of free NHase and NHase@Co3(PO4)2

6


28

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1


Table 1 The kinetic parameters of free NHase and NHase@Co3(PO4)2

Km (mM)

Kcat (S-1)

Kcat/Km (L·mmol· S-1)

Free NHase

10.23

1.993×103

194.8

NHase@Co3(PO4)2

34.43

1.574×103

45.72

2 3


29


1

Table of Contents graphic:

2

3 4

A promising approach was developed to establish nanobiocatalysts system via biomimetic

5

mineralization of cobalt phosphate nanoflowers and Co-type NHase. The obtained

6

nanobiocatalysts exhibited high catalytic efficiencies and desirable stabilities.

7 8


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Preparation of a Flowerlike Nanobiocatalyst ... - ACS Publications

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