Enrichment and Coimmobilization of Cofactors and His-Tagged Ï

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Enrichment and Co-immobilization of Cofactors and His-Tagged #-Transaminase into Nanoflowers: A Facile Approach to Constructing Self-sufficient Biocatalysts Guangxiu Cao, Jing Gao, Liya Zhou, Ying He, JiaoJiao Li, and Yanjun Jiang ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00626 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 13, 2018

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ACS Applied Nano Materials

Enrichment and Co-immobilization of Cofactors and His-Tagged ω-Transaminase into Nanoflowers: A Facile Approach to Constructing Self-sufficient Biocatalysts

Guangxiu Caoa,b, Jing Gaoa,b, Liya Zhoua,b, Ying Hea,b, JiaoJiao Lia,b, Yanjun Jianga,b,*

a School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, P.R. China b Hebei Provincial Key Lab of Green Chemical Technology and High Efficient Energy Saving, Hebei University of Technology, Tianjin 300130, PR China

*Corresponding Author: E-mail: [email protected] (Yanjun Jiang). Fax: +86-22-60204294; Tel: +86-22-60204945

1

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ABSTRACT: ω-transaminase (ω-TA), a cofactor-dependent enzyme, has been shown to be an outstanding biocatalyst for the production of chiral amines. The need for improved enzyme stability and cofactor (PLP) recyclability is an important goal in the biocatalysis

process.

In

this

study,

a

novel

self-sufficient

biocatalyst,

ω-TA-PLP@Co3(PO4)2, was constructed by co-immobilizing ω-TA and PLP into Co3(PO4)2

nanoflowers

for

the

first

time.

The

preparation

process

of

ω-TA-PLP@Co3(PO4)2 was investigated in detail, and the formation mechanism was clarified. The resulting ω-TA-PLP@Co3(PO4)2 was characterized by various analytical techniques, such as scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and X-ray photoelectron spectroscopy (XPS). Compared with free ω-TA, the ω-TA-PLP@Co3(PO4)2 showed improved catalytic efficiency, thermal stability, pH stability and storage stability. Furthermore, ω-TA-PLP@Co3(PO4)2 could maintain about 72% of initial overall activity after twelve catalytic cycles. All these results confirmed the feasibility of enrichment and co-immobilization of cofactors and His-tagged ω-TA into Co3(PO4)2 nanoflowers for constructing self-sufficient biocatalysts. KEYWORDS:

Cofactor;

His-tagged

ω-transaminase;

Co-immobilization;

Enrichment; Co3(PO4)2 nanoflowers

2


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INTRODUCTION Chiral amines are important chemicals for synthesizing pharmaceuticals, such as immunological, neurological and anti-hypertensive drugs.1 Production of chiral amines using ω-transaminase (ω-TA) as a catalyst is an attractive topic in the pharmaceutical industry because it possesses environmentally friendly processes and exhibits excellent enantioselectivity.2,3 Nevertheless, there are several limitations associated with free ω-TA in industrial applications, such as low stability, difficult recovery, and high production cost. Immobilization of ω-TA is known as an effective strategy to overcome these problems. Currently, the immobilized enzyme has been widely exploited in the industrial process due to the outstanding advantages, including higher stability and better reusability compared to its free counterparts. The development of suitable immobilization methods is conducive to the production of valuable chemicals. If the enzyme is enriched before immobilization, the overall process cost of immobilized enzyme in biotransformations will fall dramatically. However, this decrease requires separation devices and chromatographic instruments that are frequently used in enriching intracellular enzymes, which not only consumes numerous of chemicals but also affects the activity of labile enzymes.4 Therefore, developing a simple and cost-effective method for one-pot enrichment and immobilization of enzymes will be very attractive to industrially related enzymes. The His-tag integrated into recombinant proteins can allow for the enrichment and immobilization of enzymes. Based on immobilized metal affinity chromatography (IMAC), metal ions (Co2+, Ni2+, Cu2+) have been used to enrich and immobilize His-tagged enzymes.5 For example, Ren et al. synthesized Ni2+-functionalized 3



Fe3O4@polydopamine

magnetic

nanoparticles

for

Page 4 of 35

selectively

immobilizing

His-tagged ω-TA BJ110, and the immobilized ω-TA exhibited enhanced specific activity and improved stability.6 In our previous report, Ni2+-functionalized magnetic silica nanoflowers were prepared for purifying and immobilizing His-tagged ω-TA in one step.7 This Ni2+-functionalized nanomaterials displayed good specificity to His-tagged ω-TA and was considered to be a proper carrier to improve the properties of immobilized ω-TA. In these reports, metal ions were anchored on pre-existing supports with several limitations, including difficult manipulations and long separation time. Hence, development of an easy and efficient approach to enriching ω-TA in the support synthesis process will be beneficial for extended enzyme immobilization. To date, the preparation of enzyme-containing metal-phosphates has gained widespread attention due to the ultrahigh activity and excellent stability of embedded enzymes.8,9 Ge et al. first developed an enzyme-Cu3(PO4)2 hybrid system10 and synthesized several types of hybrid nanoflowers for efficient immobilization of α-lactalbumin, lipase, laccase, and carbonic anhydrase.11 Ke et al. reported a lipase-Ca3(PO4)2 hybrid nanocatalyst with increased activity.12 López-Gallego et al. discovered that His-tagged alcohol dehydrogenase could trigger the mineralization of Co3(PO4)2-sponges.13 Patel et al. first purified His-tagged enzymes from crude extract using the Ni-NTA column and later prepared the enzyme-Cu3(PO4)2 hybrid nanobiocatalyst.14 It should be noted that there are few reports on constructing a

4


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metal-enzyme hybrid biocatalytic system for one-step enrichment and immobilization of the enzyme. ω-TA requires 5′-pyridoxal phosphate (PLP) as a cofactor in the catalytic reaction. The recycling of expensive PLP is important for the implementation of enzymatic catalysis at the industrial level. Preparation of biocatalysts by co-immobilizing ω-TA and PLP has been reported. For example, Andrade and collaborators exploited methacrylate beads to immobilize E. coli cells containing ω-TA and PLP to enable continuous flow reactions in an organic solvent.15 The organic solvent restrained the leakage of PLP. Velasco-Lozano reported the following reaction sequence: aldehyde activated agarose microbeads bound ω-TA and polyethylenimine (PEI); a reduction reaction stabilized aldehyde-PEI/ω-TA imines; then, the PLP was ionically adsorbed to cationic polymer.16 However, the reported methods to co-immobilize PLP and ω-TA on the solid carriers required multistage procedures and organic solvents, which led to the loss of enzyme activity and high production costs. In this work, the recombinant His-tagged ω-TA gained successful expression in E.coli Rosetta (DE3). Next, for the first time, the co-immobilization of His-tagged ω-TA and PLP in one pot was performed through biomineralization of Co3(PO4)2 with ω-TA and PLP. Co-immobilization was based on the affinity interaction between the His-tag and cobalt ions and the electrostatic interaction between the negatively charged phosphate group of PLP and the positively charged cobalt ions. As shown in Scheme 1, the one-pot enrichment and immobilization of His-tagged ω-TA directly 5



from cell lysates was performed. Moreover, the

Page 6 of 35

nanobiocatalyst (named

ω-TA-PLP@Co3(PO4)2) consisted of organic (PLP and ω-TA) and inorganic (cobalt phosphate) components, whereas the immobilized ω-TA was catalytically active without the need for external PLP. The ω-TA-PLP@Co3(PO4)2 was characterized by such techniques as SEM, CLSM, and XPS. The properties of ω-TA-PLP@Co3(PO4)2 were

investigated

and

compared

with

free

ω-TA.

The

fabrication

of

ω-TA-PLP@Co3(PO4)2 via biomimetic mineralization is unprecedented and opens a new research direction for the immobilization of cofactor-dependent enzymes.

Scheme 1 Schematic illustration of the formation of ω-TA-PLP@Co3(PO4)2 nanobiocatalyst

EXPERIMENTAL SECTION

Materials The gene for the N-terminal His-tagged ω-TA from Chromobacterium violaceum was synthesized and cloned into pET-28a by General Biosystems, Inc. (China). E. coli Rosetta (DE3) expressing the gene encoding ω-TA was obtained from Miaoling Biotechnology Co., Ltd. (China). Co(NO3)2·6H2O was obtained

from

Shanghai

Macklin

Biochemical

Co.,

Ltd.

(China).

Na2HPO4·12H2O and NaH2PO4·2H2O were purchased from Tianjin Chemical Corporation

(China).

5′-pyridoxal

phosphate

(PLP),

6


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Isopropyl-β-D-thiogalactopyranoside

(IPTG),

(S)-α-Phenylethylamine,

pyruvate and other reagents were purchased from Beijing Solarbio Science & Technology Co., Ltd. (China).

Expression of His-tagged ω-TA E. coli Rosetta (DE3) cells were transformed with the recombinant plasmid pET-28a harboring the ω-TA gene. The resulting strains were cultivated in Luria-Bertani broth containing kanamycin (50 µg/mL) at 37 °C. When the OD600 reached approximately 0.6, IPTG (1 mM) was added to induce the expression of His-tagged ω-TA. After 12 h induction at 30 °C, the cells were harvested. The cell lysates were obtained by breaking up the cells using glass beads according to a previous reported method.7

Preparation of Co3(PO4)2 nanoflowers Co3(PO4)2 nanoflowers were synthesized by the precipitation method as described in our previous report.8 Typically, 0.1455 g of Co(NO3)2·6H2O was added to a conical flask and dissolved in 50 mL of phosphate buffer solution (PBS, 10 mM NaH2PO4·2H2O and Na2HPO4·12H2O, pH 7.4). The solution was fully oscillated, and purple floccules appeared. After incubating at 25 °C for a certain amount of time, the pink precipitate was generated. Finally, Co3(PO4)2 nanoflowers were separated by centrifugation and rinsed with ultra-pure water.

Preparation of ω-TA-PLP@Co3(PO4)2 First, 5 mL of cell lysates containing His-tagged ω-TA were mixed with 45 7



Page 8 of 35

mL of PBS (10 mM, pH 7.4) to prepare the enzyme solution. Next, 0.1455 g of Co(NO3)2·6H2O and a certain amount of PLP were added to the conical flask directly. Subsequently, the enzyme solution was slowly added to conical flask. Finally, the mixed solution was incubated at 25 °C for some time. The particles that co-immobilized ω-TA and PLP (denoted as ω-TA-PLP@Co3(PO4)2) were obtained through centrifugation and rinsed three times with ultra-pure water.

Characterization SEM images were obtained using an FEI Nano SEM450 microscope. XPS spectra were measured with a Thermo VG Scientific Sigma Probe spectrometer. XRD patterns were recorded on a Bruker AXS D8 Advance X-ray diffractometer using Cu-Ka radiation. Raman spectra were recorded with the Horiba LabRAM HR Evolution Raman spectrometer at 532 nm excitation. Nitrogen

adsorption-desorption

experiments

were

performed

on

a

micromeritics ASAP 2020 gas sorptometer at 77 K. CLSM micrographs were recorded using a Leica TCS SP5 confocal microscope. Thermogravimetric analyses (TGA) were measured with a Netzsch TG 209 F3 instrument under nitrogen atmosphere and a heating rate of 10 °C/min.

Determination of enzyme activity One unit of enzyme activity (U) was defined as the amount of ω-TA that catalyzed the formation of 1 µmol of acetophenone at 30 °C per minute. The enzymatic activities of free ω-TA and ω-TA-PLP@Co3(PO4)2 were 8


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determined by monitoring the concentration of acetophenone. Typically, free ω-TA or ω-TA-PLP@Co3(PO4)2 was immersed in PBS (50 mM, pH 7.5), the reaction was performed by adding cofactor PLP (or needless) and 50 mM of pyruvate and (S)-α-phenylethylamine. After 15 min of the oscillatory reaction (180 rpm), 1% trifluoroacetic acid was added to terminate the reaction. Acetophenone concentration was quantified by HPLC analysis (with an Eclipse Plus C18 column, 4.6 × 250 mm) at 210 nm. The mobile phase was PBS (pH 6.0, 20 mM)/methanol (2:3, v/v) and the flow rate was 0.6 mL/min. The concentration of PLP was measured at 390 nm by the UV detector. The residual immobilized PLP (%) was calculated using the following equation: Residual immobilized PLP (%) =

residual amount of immobilized PLP initial amount of immobilized PLP

×100%

The activity recovery was calculated using the following equation: Activity recovery =

activity of immobilized enzyme initial activity of free enzyme

×100%

Determination of kinetic parameters For comparison, the immobilized ω-TA (ω-TA@Co3(PO4)2) was also prepared (using the preparation method for ω-TA@Co3(PO4)2 provided in the Supporting Information). Km and Vmax values were evaluated by determining the

initial rates of free ω-TA, ω-TA@Co3(PO4)2 and ω-TA-PLP@Co3(PO4)2 with different (S)-α-phenylethylamine concentrations ranging from 25 mM to 125 mM and 50 mM of pyruvate (a fixed value). The kinetic parameters were obtained by using the Michaelis-Menten nonlinear regression model.

9



Page 10 of 35

Optimum temperature and pH To

investigate

the

optimum

temperature

of

free

ω-TA

and

ω-TA-PLP@Co3(PO4)2, the samples were measured in the temperature range of 20-70 °C in PBS (50 mM, pH 7.5). The optimum pH of free ω-TA and ω-TA-PLP@Co3(PO4)2 were measured in the pH range of 6-10 at 30 °C (50 mM PBS). The results were transformed into relative activity (%) in comparison to the maximum activity (100%). Thermal stability and pH stability The thermal stability was assessed by incubating free ω-TA and ω-TA-PLP@Co3(PO4)2 at 50 °C and 60 °C for different amounts of time. Subsequently, the samples were carried for 5 min in an ice bath for cooling down, and the activity was measured when the temperature dropped to 30 °C. The pH stability of free ω-TA and ω-TA-PLP@Co3(PO4)2 was tested by incubating samples in 50 mM acetate buffer (pH 4) and PBS (pH 10.0) at 30 °C for different amounts of time. Subsequently, the residual activity was determined according to the activity assay.

Storage stability To

investigate

the

storage

stability

of

free

ω-TA

and

ω-TA-PLP@Co3(PO4)2, the samples were stored at 4 °C for several days. At predetermined time intervals, the sample was taken out to measure the relative activity (%) in comparison to the initial activity (100%).

10


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Reusability The reusability of ω-TA-PLP@Co3(PO4)2 was investigated by performing the catalytic reaction repeatedly. ω-TA-PLP@Co3(PO4)2 was separated from the reaction mixture by centrifugation and rinsed with PBS three times. The recovered ω-TA-PLP@Co3(PO4)2 was employed in the next reaction measurement immediately. RESULTS AND DISCUSSION

Co-immobilization of PLP and His-tagged ω-TA from cell lysates The engineered plasmid pET-28a harboring the ω-TA gene was transformed into E. coli Rosetta (DE3) to obtain His-tagged ω-TA. Next, the cell lysates containing His-tagged ω-TA were mixed with cobalt nitrate and PBS (10 mM, pH 7.4) to prepare ω-TA@Co3(PO4)2. As shown in Fig. S1, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the protein fractions of ω-TA@Co3(PO4)2. The distinct band of target

protein presented in lane 2 demonstrated that the selective enrichment of His-tagged ω-TA from cell lysates could be realized in the preparation of ω-TA@Co3(PO4)2, which may be due to the specific interaction between cobalt ions and the His-tag. Subsequently, the co-immobilized His-tagged ω-TA and PLP were prepared. For easy observation, the rhodamine-labelled enzyme and PLP were used to prepare ω-TA-PLP@Co3(PO4)2 and CLSM was used to evaluate the successful co-immobilization of ω-TA and PLP. As presented in Fig. 1, the fluorescent signal of rhodamine and blue autofluorescence of PLP 11



Page 12 of 35

were detected in the ω-TA-PLP@Co3(PO4)2. Combining CLSM images with SDS-PAGE analysis (Fig. 2), we found that the ω-TA and PLP co-immobilized in the ω-TA-PLP@Co3(PO4)2 successfully.

Fig. 1 CLSM images of ω-TA-PLP@Co3(PO4)2. The enzyme is labeled with rhodamine and the PLP presents autofluorescence.

Fig. 2 SDS-PAGE analysis. “M” indicates the protein marker (130, 95, 72, 55, 43 and 34 kDa). Lane 1: Cell lysates containing His-tagged ω-TA. Lane 2: Protein fractions from the ω-TA-PLP@Co3(PO4)2 mixed with the loading buffer.

Characterization of ω-TA-PLP@Co3(PO4)2 The morphologies of the synthesized Co3(PO4)2 nanoflowers, ω-TA@Co3(PO4)2 and ω-TA-PLP@Co3(PO4)2 were observed by SEM. As shown in Fig. 3a and 3b,

the Co3(PO4)2 nanoflowers had an average size of approximately 20 µm, flowerlike morphologies and regular structures. As shown in Fig. 3c and 3d, the 12


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ω-TA@Co3(PO4)2 had a daisy-like structure with an average size of approximately 16 µm, which was different from Co3(PO4)2 nanoflowers. As shown in Fig. 3e and 3f, the ω-TA-PLP@Co3(PO4)2 had a dandelion-like structure with a uniform size of ca. 13 µm. It was obvious that the participation of ω-TA and PLP in formation process

had significantly changed the morphology, resulting in different morphologies of the ω-TA-PLP@Co3(PO4)2 in comparison with the ω-TA@Co3(PO4)2 and Co3(PO4)2 nanoflowers. SEM images in Fig. 4 presented the growth process of the ω-TA-PLP@Co3(PO4)2 with different precipitation times. Many nanosheets could be clearly observed at 6 h and the self-assembly of nanosheets appeared at 12 h. With time, the symmetric growth of nanosheets was observed around the center. At 72 h, the nanosheets were closely arranged in every direction, and the shape of the ω-TA-PLP@Co3(PO4)2 became circular and unchanged. Based on the above results, the formation mechanism of the ω-TA-PLP@Co3(PO4)2 was speculated. First, the His-tag of ω-TA bound Co2+ via coordination bonds and the negatively charged phosphate group of PLP attached onto Co2+ through electrostatic interaction, forming specific metal-organic complexes. These complexes served as

specific nucleation points to induce the mineralization of Co3(PO4)2,13 forming the nanoplates. Second, such nanoplates aggregated together and started the self-assembly of hybrid nanoflowers, which might have been dominated by the binding sites of Co2+.17 Finally, the growth of hybrid nanoflowers was continuously

carried

out

until

complete

formation

of

well-ordered

ω-TA-PLP@Co3(PO4)2.18 13



Page 14 of 35

Fig. 3 SEM images of Co3(PO4)2 nanoflowers: (a) low-resolution, (b) high-resolution. SEM images of ω-TA@Co3(PO4)2: (c) low-resolution, (d) high-resolution. SEM images of ω-TA-PLP@Co3(PO4)2: (e) low-resolution, (f) high-resolution.

Fig. 4 SEM images of ω-TA-PLP@Co3(PO4)2 at different precipitation times (a: 6 h; b: 12 h; c: 24 h; d: 36 h; e: 48 h; f: 72 h). 14


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Fig. 5 SEM images of the synthesized ω-TA-PLP@Co3(PO4)2 with different initial concentrations of PLP: (a) PLP 0.02 mM, (b) PLP 0.05 mM, (c) PLP 0.1 mM (d) PLP 0.25 mM.

The effect of initial concentration of PLP on the formation of ω-TA-PLP@Co3(PO4)2 was analyzed (Fig. 5). When the concentration of PLP increased from 0.02 to 0.25 mM, the size of ω-TA-PLP@Co3(PO4)2 decreased from 43 to 11 µm (Fig. 5a-d). It was also found that the central vein of the petals became thin and dense gradually when the initial concentration of PLP increased from 0.02 mM to 0.25 mM. These phenomena were presumably attributed to the fact that the different additions of organic components could influence the number of nucleation sites and the crystalline phases.19,20 XPS analysis was performed to investigate the surface element composition and chemical bonds of ω-TA-PLP@Co3(PO4)2. The XPS spectra in Fig. 6 showed that there were Co, P, O, C and N elements in ω-TA-PLP@Co3(PO4)2. The C and N elements were probably derived from PLP and proteins. The Co 2p peaks at 781.7 eV and 797.8 eV corresponded to 15



Co 2p3/2 and Co 2p1/2 with two satellite peaks located at 786.3 eV and 803.2 eV, which

were

consistent

with

the

characteristic

peaks

of

Co2+

in

Co3(PO4)2·8H2O.21,22 The P 2p peak at 133.2 eV corresponded to metal phosphate bonding.23 The two binding energy peaks of O 1s were detected at 531.4 eV and 532.5 eV, which were obtained from the phosphate species24 and hydrated oxide species.25 The 284.6 eV strong peak of C 1s was assigned to sp2 C=C bond,26 originating from PLP. The other C 1s peaks at 285.9 eV and 287.9 eV were attributed to C-N bond and C=O bond,27 respectively. Moreover, EDAX analysis also demonstrated that the ω-TA-PLP@Co3(PO4)2 had two additional C and N elements which were not detected in Co3(PO4)2 nanoflowers (Fig. S2).

(b) Co 2p Intensity (a.u.)

Intensity (a.u.)

(a) full scan

O 1s Co 2p C 1s N 1s

1200

1000

800

600

400

P 2p

200

810

0

800

2

4

6

790

780

770

Binding Energy (eV) 8

10 10

(c) O 1s

0

8000

14000

8

12000

2

4

6

8

10 10

9000

(d) C 1s 8

Intensity (a.u.)

0 16000

2p3/2 2p1/2

Binding Energy (eV)

Intensity (a.u.)

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

Page 16 of 35

7000 6000

6

10000

6

5000

8000

4000

4

6000 4000 2 2000

2000

2

1000 0

0

540

4

3000

535

530

Binding Energy (eV)

525

0

0

295

290

285

280

Binding Energy (eV)

Fig. 6 XPS spectra of ω-TA-PLP@Co3(PO4)2: (a) full scan, (b) Co 2p, (c) O 1s, (d) C 1s.

Raman spectroscopy was employed to characterize the Co3(PO4)2 16


Page 17 of 35

nanoflowers and ω-TA-PLP@Co3(PO4)2. As shown in Fig. S3, Raman peaks at 965 cm-1 belonged to the P-O stretching of Co3(PO4)2 (·),13 and the peaks at 382 cm-1 and 1335 cm-1 were assigned to CNC in-plane bending (◆) and C-O stretching (△) of PLP,28,29 respectively. Furthermore, the signal peaks at 750 cm-1 and 1675 cm-1 corresponded to Trp residues (⊕) and the amide I band (*),13,30 respectively. The results confirmed that the presence of PLP and enzyme

in

ω-TA-PLP@Co3(PO4)2.

As

presented

in

Fig.

S4,

N2

adsorption-desorption isotherms of Co3(PO4)2 nanoflowers were type-IV, which indicated the existence of the characteristic mesoporous structure.31 From the pore size distributions in the insets, the average pore size for Co3(PO4)2 nanoflowers and ω-TA-PLP@Co3(PO4)2 was estimated to be 5.9 nm and 3.4 nm using the BJH model. There was an apparent change in the pore size since ω-TA and PLP were incorporated in ω-TA-PLP@Co3(PO4)2. In addition, the BET surface area and pore volume of ω-TA-PLP@Co3(PO4)2 were 67 m2/g and 0.073 cm3/g, respectively, indicating that the hybrid nanoflowers possessed a high surface-to-volume ratio. a

b

Co3(PO4)2 nanoflowers

Co3(PO4)2 nanoflowers

100

ω-TA-PLP@Co3(PO4)2

ω-TA-PLP@Co3(PO4)2

Weight (%)

Intensity (a.u.)

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


90

80

70

60 10

20

30

40

50

60

0

200

400

600

o

2θ (degree)

Temperature ( C)

Fig. 7 (a) XRD patterns of Co3(PO4)2 nanoflowers and ω-TA-PLP@Co3(PO4)2. (b) TGA curves of Co3(PO4)2 nanoflowers and ω-TA-PLP@Co3(PO4)2. 17



The

crystalline

nature

of

Co3(PO4)2

Page 18 of 35

nanoflowers

and

ω-TA-PLP@Co3(PO4)2 was investigated by XRD analysis. As shown in Fig. 7a, XRD patterns of Co3(PO4)2 nanoflowers and ω-TA-PLP@Co3(PO4)2 were approximately the same and matched with Co3(PO4)2·8H2O (JCPDS file no. 41-0375),22,32 indicating that the incorporation of the enzyme and PLP did not impact the crystal structure of Co3(PO4)2 nanoflowers. To further verify the existence of the enzyme and PLP in hybrid flowers, TGA was conducted. TGA curves in Fig. 7b showed that the samples had obvious weight loss above 100 °C, which was related to the desorption of water. For the Co3(PO4)2 nanoflowers, there was no further weight loss, indicating that the Co3(PO4)2 did not decompose below 600 °C. For ω-TA-PLP@Co3(PO4)2, subsequent weight loss occurred from 200 °C to 600 °C and was due to the decomposition of PLP and proteins.

Optimal concentration of PLP for ω-TA-PLP@Co3(PO4)2 Effect of initial concentration of PLP on its loading amount and activity of ω-TA-PLP@Co3(PO4)2 was also investigated. As shown in Fig. S5, the PLP loading was gradually increased and the activity of ω-TA-PLP@Co3(PO4)2 was first increased and then decreased with the initial concentration of PLP increasing. The results were ascribed to the adverse impact of excess PLP on the activity of ω-TA.33,34 When the initial concentration of PLP was increased to 0.1 mM, the activity of ω-TA-PLP@Co3(PO4)2 reached the maximum of 1582 U/g and the activity recovery was approximately 89%. The corresponding PLP loading was 18


Page 19 of 35 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


0.094 mmol/gsupport. Thus, an initial PLP concentration of 0.1 mM was employed

for the preparation of ω-TA-PLP@Co3(PO4)2 in the subsequent experiments.

Stability of the immobilized PLP The leakage of immobilized cofactor commonly occurs in enzymatic processes in an aqueous medium, and thus the stability of the immobilized cofactor in the solid-phase is important for continuous operations. Therefore, the residual PLP bound to the ω-TA-PLP@Co3(PO4)2 was determined. As shown in Fig. S6, we observed that residual immobilized PLP was more than 97% after three reaction cycles, indicating less than 3% of PLP leakage from the ω-TA-PLP@Co3(PO4)2 under reaction conditions. After seven reaction cycles, 91% of immobilized PLP was retained in nanoflowers. The leakage of PLP from the ω-TA-PLP@Co3(PO4)2 was less than 10%, which was beneficial for the reutilization of PLP. The low leakage of PLP in nanoflowers was ascribed to the electrostatic interaction between the negatively charged phosphate group of PLP and the positively charged cobalt ions, which favored PLP remaining in the ω-TA-PLP@Co3(PO4)2. Previous reports showed that metal ions could bind with negatively charged phosphate groups.35,36

Kinetic parameters The Km and Vmax values of free ω-TA, ω-TA@Co3(PO4)2 and ω-TA-PLP@Co3(PO4)2

are

shown

in

Table

1.

The

Km

values

of

ω-TA@Co3(PO4)2 and ω-TA-PLP@Co3(PO4)2 were smaller than that of free 19



Page 20 of 35

ω-TA, which showed that the immobilized ω-TA had a better affinity for the substrate. It has been reported that enzyme immobilization by the metal-organic hybrid method contributes to enzyme-substrate affinity.18,37 Compared to free ω-TA, the Vmax values of ω-TA@Co3(PO4)2 and ω-TA-PLP@Co3(PO4)2 increased slightly. This may be due to the accumulation of more substrates inside or around the metal-enzyme hybrid nanoflowers, making it easy to form the substrate-enzyme complex. In addition, we found a lower Km and higher Vmax of ω-TA-PLP@Co3(PO4)2 compared to ω-TA@Co3(PO4)2. The results showed that the co-immobilization of ω-TA and PLP inside the porous hybrid nanoflowers could increase the enzyme-substrate affinity and promote the accessibility of PLP to the active sites of ω-TA. Similar results showing a faster reaction rate of co-immobilized ω-TA and PLP than immobilized ω-TA and additional PLP have been reported.16 The increased catalytic performance of the ω-TA-PLP@Co3(PO4)2 could be ascribed to the vicinity effect between the enzyme and cofactor, which contributed to the direct transport of intermediates.38 Numerous studies indicated that properly co-immobilization of multiple enzymes and/or cofactor could exhibit an enhancement in overall activity compared to free enzymes.39-41

Considering that the kinetic performance of ω-TA-PLP@Co3(PO4)2 was better than that of ω-TA@Co3(PO4)2, the properties of ω-TA-PLP@Co3(PO4)2 were studied in the subsequent experiments.

20


Page 21 of 35

Table 1 The kinetic parameters for free ω-TA, ω-TA@Co3(PO4)2 and ω-TA-PLP@Co3(PO4)2 (S)-α-phenylethylamine affinity Kinetic parameters Free ω-TA

ω-TA@Co3(PO4)2

ω-TA-PLP@Co3(PO4)2

Km (mM)

62.2

57.6

53.8

Vmax (mM/min)

2.45

2.58

2.77

Optimum temperature and pH The

optimum

temperature

and

pH

of

free

ω-TA

and

ω-TA-PLP@Co3(PO4)2 were investigated. As presented in Fig. 8a, the optimum temperature of both free ω-TA and ω-TA-PLP@Co3(PO4)2 was 30 °C. We observed that the ω-TA-PLP@Co3(PO4)2 displayed a wider application of temperature range than that of the free ω-TA, enhancing the practicality of ω-TA in industrial production. Fig. 8b shows the pH profiles of free ω-TA and ω-TA-PLP@Co3(PO4)2. The optimum pH of ω-TA-PLP@Co3(PO4)2 was pH 7.5, which was the same as that of free ω-TA. In contrast, with the free ω-TA, the ω-TA-PLP@Co3(PO4)2 displayed a broad pH range of 6.0-10.0, which benefited from the protection of the protein-Co3(PO4)2 hybrid systems. 120 100

Free ω-TA ω-TA-PLP@Co3(PO4)2

80 60 40 20

b Relative Activity (%)

a Relative Activity (%)

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



100 80 60 40 20 0

0 20

30

40

50

60

70

6

7

8

9

10

pH

o

Temperature ( C)

Fig. 8 (a) Optimum temperature of free ω-TA and ω-TA-PLP@Co3(PO4)2. (b) Optimum pH of free ω-TA and ω-TA-PLP@Co3(PO4)2.

Stability and reusability 21



Page 22 of 35

As presented in Fig. 9a, the thermal stability of free ω-TA and ω-TA-PLP@Co3(PO4)2 at 50 °C and 60 °C for different amounts of time were examined. After 1 h of incubation, the free ω-TA and ω-TA-PLP@Co3(PO4)2 displayed

increased

activity,

which

was

probably

attributed

to

the

temperature-related refolding of the enzyme that recovered its natural conformation.42 Previous reports have also shown increased activity of the enzyme when incubated at high temperatures.7,43 After 3 h incubation at 50 °C, the activity of ω-TA-PLP@Co3(PO4)2 increased to 144% of its initial activity, whereas the free ω-TA maintained 115% of the initial activity. When incubated at 60 °C for 4 h, the free ω-TA and ω-TA-PLP@Co3(PO4)2 retained 34% and 75% of the initial activity, respectively. Compared to the free ω-TA, the thermal stability of ω-TA-PLP@Co3(PO4)2 was remarkably improved. The reason for this could be that the cobalt phosphate crystals confined the protein within the nanospace,44

which led to the

increase

of

enzyme

rigidity against

conformational changes, avoiding the overheating inactivation.8 Fig. 9b shows the pH stability of free ω-TA and ω-TA-PLP@Co3(PO4)2. Compared to the free ω-TA in the strong acid and alkali conditions, the ω-TA-PLP@Co3(PO4)2 exhibited significantly improved stability. This prominent pH resistance was probably attributed to the affinity interaction between His-tagged ω-TA and Co2+ that could reduce the mobility of enzyme molecules, stabilizing the conformation of ω-TA.17 In addition, the immobilization could provide an appropriate microenvironment for the immobilized enzyme and thus prevent 22


Page 23 of 35

the denaturation of the enzyme in extreme environments. a

160

b

120 100 80

o

50 C Free ω-TA o 50 C ω-TA-PLP@Co3(PO4)2

60

o

60 C Free ω-TA o 60 C ω-TA-PLP@Co3(PO4)2

40

100

Relative Activity (%)


140

80 60 40 pH 4.0 Free ω-TA pH 4.0 ω-TA-PLP@Co3(PO4)2

20

20

pH 10.0 Free ω-TA pH 10.0 ω-TA-PLP@Co3(PO4)2

0 0

1

2

3

4

0

1

Time (h)

2

3

4

5

Time (h)

Fig. 9 (a) Thermal stability of free ω-TA and ω-TA-PLP@Co3(PO4)2. (b) pH stability of free ω-TA and ω-TA-PLP@Co3(PO4)2.

a

b Relative Activity (%)

100


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


80

60

40


20

100

80

60

40

20

0

0 0

2

4

6

8

10

12

14

0

16

Time (d)

2

4

6

8

10

12

Recycling number

Fig. 10 (a) Storage stability of free ω-TA and ω-TA-PLP@Co3(PO4)2. (b) Reusability of ω-TA-PLP@Co3(PO4)2.

The long storage stability of the enzyme can lower the costs for its large-scale industrial applications. As presented in Fig. 10a, the storage stability of ω-TA was examined in detail. The free ω-TA and ω-TA-PLP@Co3(PO4)2 were stored at 4 °C for 15 days and the residual activity was investigated. The storage stability of free ω-TA was not satisfactory, losing nearly 30% of the initial activity after storage for 3 days. In contrast, the ω-TA-PLP@Co3(PO4)2 still retained 96% of the initial activity under the same conditions. After a 15-day

storage

period,

81%

of

initial

overall

activity

for

the 23



Page 24 of 35

ω-TA-PLP@Co3(PO4)2 could be retained, while the free ω-TA lost almost half of its activity. The enhanced storage stability of the enzyme-inorganic nanoflowers has also been described in previous literature.45,46 This could be ascribed to the encapsulation of the enzyme in Co3(PO4)2 nanoflowers, which minimized the alteration of the enzyme active sites47 and kept the enzyme stable in the storage process. Similarly, the reusability of the enzyme is an important factor for economic feasibility. As shown in Fig. 10b, successive cycles of experiments were performed to evaluate the reusability of ω-TA-PLP@Co3(PO4)2. With the increase in cycle number, the activity of the recycled ω-TA-PLP@Co3(PO4)2 declined slowly, and we observed that about 72% of the initial activity could be maintained after twelve catalytic cycles. The excellent reusability was probably due to a confined space for the enzyme encapsulated in the hybrid flowers, protecting

the

enzyme

from

deactivation.48

The

reduction

of

the

ω-TA-PLP@Co3(PO4)2 activity might be caused by the leakage of the immobilized PLP from the carrier and the enzyme inactivation during the washing process and recycling assay.49,50

Conclusions In this study, an efficient and self-sufficient biocatalyst integrating ω-TA and PLP into Co3(PO4)2 nanoflowers was developed for the first time using a facile approach. The enrichment and co-immobilization of PLP and His-tagged ω-TA

24


Page 25 of 35 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


were based on the affinity interaction between the His-tag and cobalt ions and the electrostatic interaction between the negatively charged phosphate group of PLP and the positively charged cobalt ions. The integrated nanobiocatalyst exhibited

desirable catalytic performance, high stability and reusability. Therefore, the facile approach not only provides a new route for the enrichment and immobilization of His-tagged enzymes but also develops a promising method to co-immobilize the cofactor and enzyme for self-sufficient biocatalyst construction. ASSOCIATED CONTENT Supporting Information Fig. S1 SDS-PAGE analysis. “M” indicates the protein marker (130, 95, 72, 55, 43 and 34 kDa). Lane 1: Cell lysates containing His-tagged ω-TA. Lane 2: Protein fractions from the ω-TA@Co3(PO4)2 mixed with the loading buffer. Fig. S2 EDAX analysis of (a) Co3(PO4)2 nanoflowers and (b) ω-TA-PLP@Co3(PO4)2. Fig. S3 Raman spectra of Co3(PO4)2 nanoflowers and ω-TA-PLP@Co3(PO4)2. Fig. S4 Nitrogen adsorption-desorption isotherms and corresponding pore size distributions of (a) Co3(PO4)2 nanoflowers, and (b) ω-TA-PLP@Co3(PO4)2. Fig. S5 Effect of initial concentration of PLP on its loading and activity of ω-TA-PLP@Co3(PO4)2. Fig. S6 Residual immobilized PLP after recycling of ω-TA-PLP@Co3(PO4)2.

25



Page 26 of 35

ACKNOWLEDGMENT

This work was supported by the National Nature Science Foundation of China (Nos. 21576068, 21276060, 21276062, and 21306039), the Natural Science Foundation of Tianjin City (16JCYBJC19800), the Natural Science Foundation of Hebei Province (B2015202082, B2016202027, and B2017202056), the Program for Top 100 Innovative Talents in Colleges and Universities of Hebei Province (SLRC2017029), the Hebei High Level Personnel of Support Program (A2016002027), and the Graduate Innovation Support Program in Hebei Province (CXZZSS2017024).

26


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Graphical Abstract Self-sufficient biocatalyst was constructed using co-immobilization of His-tagged ω-TA and cofactors.

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Enrichment and Coimmobilization of Cofactors and His-Tagged Ï

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