Encapsulation of amine dehydrogenase in hybrid titania nanoparticles by polyethylenimine coating and templated biomineralization

Journal of Biotechnology 241 (2017) 33–41

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Encapsulation of amine dehydrogenase in hybrid titania nanoparticles by polyethylenimine coating and templated biomineralization Hong Ren a , Yonghui Zhang a , Jieru Su a , Peng Lin a , Bo Wang a , Baishan Fang a,b,∗ , Shizhen Wang a,b,∗ a b

Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, Fujian, 361005, PR China

a r t i c l e

i n f o

Article history: Received 18 October 2016 Received in revised form 4 November 2016 Accepted 7 November 2016 Available online 9 November 2016 Keywords: Amine dehydrogenase Polyethylenimine Hybrid immobilization Thermostability Bioinspired mineralization Multimeric enzyme

a b s t r a c t Asymmetric synthesis of chiral amines by amine dehydrogenases (AmDH), which catalyzed reductive amination of ketones with high enantioselectivity, is an ideal route for production of chiral amine. In this study, a facile approach is proposed to immobilize unstable amine dehydrogenase by two steps. Firstly, polyethylenimine (PEI), a cationic polymer, was applied for coating enzyme to inhibit the dissociation of multimeric enzymes. Strong interaction of PEI with AmDH were analyzed and indicated PEI provided the hydrophilic microenvironment for enzyme. The half-life was improved about 18 fold in 50 ◦ C. Secondly, to further stabilize AmDH-PEI, PEI coated on AmDH surface was used as a biological template inducing the hydrolysis and condensation of titanium precursor to form titania. AmDH-PEI-Ti possessed more than 80% of the catalytic activity of free enzyme and the entrapment efficiency can be high up to 90%. A mechanistic illustration of the formation of AmDH-PEI-Ti nanoparticles were proposed. Titania provided a rigid cage pocket for the protection from structure unfolding. This generally applicable strategy offers a potential technique for multimeric enzyme immobilization with the advantages of low cost, easy operation, high reservation of activity and high stability. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Chiral amines are key intermediates for pharmaceuticals and fine chemicals. The asymmetric synthesis of amines from prochiral ketones and free ammonia has been reported as one of the principal aspirational reactions challenging the pharmaceutical industry (Dunn et al., 2007). The application of lipases on the resolution of chiral amines has been restricted by its intrinsic efficiency of kinetic resolution which can only reach 50% (Mahmoudian, 2009). ␻Transaminases exhibit good enantioselectivity, while influenced by reaction kinetics balance and the substrate inhibition (Koszelewski et al., 2008). Amine dehydrogenases (AmDHs) are a new class of enzymes that have recently been obtained by protein engineering of wild-type amino acid dehydrogenases (Abrahamson et al., 2013) and chimeric amine dehydrogenase (Wu et al., 2011). In contrast to the lipases and transaminases which were widely applied in biosynthesis of chiral amine, AmDH dependent on NADH which

∗ Corresponding authors at: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China E-mail addresses: [email protected] (B. Fang), [email protected] (S. Wang). http://dx.doi.org/10.1016/j.jbiotec.2016.11.006 0168-1656/© 2016 Elsevier B.V. All rights reserved.

catalyzed reductive amination of ketones with high enantioselectivity has the advantage such as easy co-enzyme regeneration. When paired a co-enzyme regeneration system with alcohol dehydrogenase (ADH), amine dehydrogenase implemented a redox self-sufficient reaction with high atom efficiency which generated water as the sole by-product (Mutti et al., 2015). Consequently asymmetric synthesis of chiral amines by amine dehydrogenases would be the ideal route to produce chiral amines. However, a pronounced loss of structure occurred in the circular dichroism spectroscopy of AmDH at temperatures above 30 ◦ C, therefore the AmDH is unstable (Abrahamson et al., 2013). Several strategies are currently available for increasing operational stability such as addition of stabilizing additives, chemical modification, immobilization, and medium engineering (Iyer and Ananthanarayan, 2008). Polyethylenimine (PEI) has been reported to prevent dissociation of enzyme multimers, by coating its surface with a polyionic polymer that may simultaneously interact with several enzyme subunits to prevent enzyme dissociation (Poltorak et al., 1998). PEI can interact with enzyme multimers within a certain distance and cover large protein surface, which achieved a complete crosslinking of subunits (Fuentes et al., 2004). Garcia-Galan et al. (Garcia-Galan et al., 2013) have researched that stabilization of glutamate dehy-

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drogenase (GDH) which is a hexameric protein was increased and the activity was maintained in the presence of polyethyleneimine (PEI) because subunit dissociation was restricted. Researchers also have found that PEI-based cross-linking integrated into conventional cross-linked enzyme aggregates (CLEAs) from lipase applied in hydrolysis of fish oil (Yan et al., 2012). PEI coating is also applied for immobilization of glycerol dehydrogenase with the improvement of metal ions coordination and our research has been published (Bastakoti et al., 2014). PEI has been reported as a biological template for silica biomineralization either (Jin and Yuan, 2005). Titania-based materials have received increasing interest for a wide range of applications, due to their excellent pH and thermal stability (Li et al., 2014), low toxicity (Wu et al., 2011), superior mechanical strength (Bastakoti et al., 2014), and biocompatible properties (Chen and Mao, 2007; Thompson and Yates, 2007). Yuan et al. (Jian-Jun and Ren-Hua, 2010) found that PEI is capable of templating and catalyzing the hydrolysis and subsequent polycondensation of titanium precursor to form titania. Moreover, the biomimetic synthesis of titania nanoparticles in vitro induced by protamine was investigated, and a relevant mechanism was tentatively proposed (Jiang et al., 2008). However, to date, there are no reports describing the biomimetic formation of titania induced by oxidoreductase coated with PEI for immobilization. A simple and easy approach was proposed to prepare organic-inorganic hybrid enzyme nanoparticle. Positively charged polyethylenimine (PEI) was coated on the negative surface of amine dehydrogenase, and then the AmDH-PEI was applied as biomimetic template to induce the hydrolysis and polycondensation of titanium precursor to form nanoparticles (Jian-Jun and Ren-Hua, 2010). Morphological and functional studies of the enzyme immobilization by the PEIs induced biomimetic titanification were performed and the mechanism of the improvements in enzyme stability was studied.

2. Materials and methods 2.1. Chemicals NAD+ , NADH, titanium (IV) bis (ammonium lactato) dihydroxide (Ti-BALDH, 50 wt% aqueous solution), polyethyleneimine (branched, MW: 25 KD) and isopropylbeta-d-thiogalactopyranoside (IPTG) were purchased from Sigma Chemical Company (Tianjing, China). Acetophenone, 4-methyl2-pentanone, 1,3-dimethylbutylamine, phenoxy-2-propanone, ␣-methylbenzylamine, and 3-methyl-2-butanone were also obtained from Sigma Chemical Company (Tianjing, China). The LB media were purchased from Sangon Biotech (Shanghai) Co. Ltd. All other chemicals were analytical grade and purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

2.3. Enzyme extraction and purification The cells were centrifuged at 8000 rpm for 10 min, washed twice with Tris-HCl buffer (10 mM, pH 7.4, 4 ◦ C) and resuspended. The cells were then pretreated by ultrasonication for 20 times for working 3 s and cooling for 3 s in an ice bath. To remove cell debris, samples were centrifuged at 10000 rpm for 15 min under 4 ◦ C. The crude extract was filtered through a membrane filter (0.22 ␮m) and loaded onto a 5 mL His-Trap HP affinity column. Ten column volumes of binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) were applied to wash unbound impurities. The column was equilibrated with binding buffer and eluted with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4) at a gradient concentration. The fractions with the desire activity were desalted and concentrated using a Macrosep Advance Centrifugal Device (cut-off 10 kDa, Pall, East Hills, NY, USA). Protein concentrations were determined with a modified Bradford protein assay kit (Sangon Biotech Co. Ltd) using bovine serum albumin as a standard, as described by Bradford (Bradford, 2015). 2.4. Enzyme assay Amine dehydrogenase (AmDH) catalyzed asymmetric reduction of ketone to chiral amine with NADH as cofactor (Fig. 1). Activity of AmDH was measured using Tecan Infinite M200 Pro Spectra Microplate Reader. Measurements were taken at 340 nm and the molar extinction coefficient of NADH (6.22 mM−1 cm−1 ) (Li et al., 2014) was used. One unit of AmDH activity was defined as the amount of enzyme necessary to oxidize 1 ␮mol of NADH per minute under the following conditions. Specific activity for encapsulated enzyme was defined as full enzyme activity in titania particles divide by the concentration of protein amine dehydrogenase in titania particles. For reductive amination, reactions were performed in 200 mM NH4 Cl/NH3 H2 O buffer at pH 9.6, with 0.1 mM NADH and 20 mM of substrate ketone, unless otherwise specified. The catalytic activity of AmDH for reductive amination towards the nonnatural substrates, phenoxy-2-propanone, ␣-Methylbenzylamine, 5-Methyl-2-hexanon, 2-hexanone, 3-methylcyclohexanone, 4methyl-2-pentanone, acetophenone, was evaluated and phenoxy2-propanone was chosen as the representative. For oxidative deamination reactions were performed in 100 mM Gly/NaOH buffer at pH 10.0, with 2 mM NAD+ with 10 mM of the representative amines 1,3-dimethylbutylamine. All reactions were performed at 25 ◦ C unless otherwise specified. For these two reciprocal reaction, reactions were started by the addition of 20 ␮L enzyme solution and the volume of the reaction mixture was 220 ␮L in all cases. Experiments on enzyme activities were determined in 4 replicates. Two kinds of blank controls for reductive amination reaction were set, including reaction mixture (reaction buffer and coenzyme) with ketone or not. Assay mixtures lacking substrates, or the mixtures lacking cell-free extracts, failed to demonstrate any measurable activity.

2.2. Strains and culture conditions

2.5. Preparation of AmDH-PEI-Ti hybrid microcapsules

AmDH gene was obtained from genetic modification of phenylalanine dehydrogenase from Bacillus badius (Abrahamson et al., 2013), which was sequenced and synthesized by Sangon BiotechCo. Ltd. (Shanghai, China). AmDH was expressed in a pET28a, BL21 (DE3) system with a C-terminal His-tag and cultured in LB media supplemented with 50 ␮g/mL kanamycin at 37 ◦ C until OD600 reached 0.6. Isopropyl-beta-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM and incubated 8 h at 25 ◦ C to induce protein expression.

AmDH coated on PEI were studied under different PEI concentration and pH condition. The concentration and pH of PEI were determined by measuring activity of AmDH-PEI. Aqueous solution of AmDH was prepared at 10 mM Tris-HCl buffer (pH 7.4). 2 mL of AmDH solution (protein concentration was about 1 mg/mL) was mixed with 2 mL PEI solution at 4 ◦ C and mixture was shaken constantly for 30 min. The encapsulation was initiated by the addition of 4 mL AmDH-PEIsolution into 4 mL different concentrations Ti-BALDH solution. Resultant precipitate was collected by centrifu-

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Fig. 1. Amine dehydrogenasecatalyzed production of chiral amine.

gation for 10 min at 5000 rpm after agitating reaction mixture for 5 min at room temperature, and then washed three times with Tris-HCl buffer (10 mM, pH 7.4) to remove the unreacted titanium precursor. In control experiment, Tris-HCl buffer (10 mM, pH 7.4) was used in place of PEI and Ti-BALDH. Supernatant was collected after centrifugation and the encapsulation efficiency was determined by Eq. (1): Encapsulation efficiency (%) = (1 −

C[AmDH] supernatant C[AmDH]dissolve

) × 100%

(1)

Where C[AmDH]supernatant and C[AmDH]dissolve were the protein concentration of AmDH in the supernatant and in the original PEI coating AmDH solution, respectively. The encapsulated AmDH was collected after each reaction batch, thoroughly rinsed with Tris-HCl buffer and utilized in the next reaction cycle. The recycling stability of encapsulated AmDH was evaluated by measuring the enzyme activity in each successive reaction cycle and recycling efficiency was determined by Eq. (2): Recycling efficiency(%) =

Enzyme activity of the nth cycle × 100 Enzyme activity of the1st cycle (2)

Fig. 2. Circular dichroism spectrum of AmDHAnalysis condition. AmDH was dissolved in 0.05 M, pH 7.0 Tris-HCl buffer solution and heated one hour at different temperature.

2.6. Characterization

3.2. Structure analysis and prediction of AmDH

Morphology of titania precipitate was observed by scanning electron microscopy (SEM, Zeiss Sigma). For the SEM, 10 ␮L of immobilized enzyme was dropped on a silicon chip and allowed to evaporate overnight, and the sample was then coated with platinum (2-nm thickness using a JEOL JFC 1600, JEOL, Tokyo, Japan) with an electric current of 10 mA for 30 s before imaging with a Zeiss Sigma SEM (Carl-Zeiss AG, Germany). Average particlesize of titania precipitate, PEI and AmDH dissolved in a Tris-HCl buffer (pH 7.4, 10 mM) was measured by ZETA-Potential-Analyzer (Malvern, UK). The interaction between PEI and AmDH was determined by the Isothermal Titration Calorimetry 200 (ITC) (General Electric). For ITC experiment, PEI was added into AmDH which concentration was about 10 ␮M in low ionic strength phosphate buffer (10 mM, pH 7.4) by drops at 25 ◦ C. Circular dichroism spectrum (CD) of AmDH were determined by a Jasco J-810 CD spectropolarimeter (Biologic, Japan) within the range of 190 and 400 nm with an interval of 1 nm. The concentration of AmDH solution was 0.5 mg/mL with the Tris-HCl buffer solution (10 mM, pH 7.4) as the blank control. The scan rate was 1 nm/s. A quartz cell with a 1 cm path length was used, and a constant nitrogen flush was used during wavelength scanning.

Circular dichroism spectrum (CD) was adopted to investigate changes of secondary structures of AmDH with the increasing temperature. Conformational changes in the protein structure of enzymes in particular, may impair their techno-functional and bioactive properties, which are connected with the native state of enzymes (Devi et al., 2011). AmDH solution was splitted in several centrifugal tubes and then treated one hour at different temperature. Fig. 2 depicts the second structural changes of the measured AmDH, indicating that AmDH possess ␣-helix secondary structure at 222 nm notable characteristic peak which is normally used in CD analysis. For the given sequence, the various secondary structure percentages in AmDH was predicted by using PredictProtein (https://www.predictprotein.org/home) (Guy et al., 2014) and the results showed that the secondary structure of AmDH possessed 42% alpha helix proportion, 43% of random coil and beta-sheet only accounted for 15%. It was well known that various secondary structure were maintained through hydrogen bond to constitute tertiary and more advanced structure of protein, and the hydrogen bond content in alpha helix and beta- sheet was more than which in random coil. Therefore, the more content of alpha helix and beta-sheet possessed, the more stabilized structure of protein was. In secondary structure of AmDH, random coil which had good flexibility accounted for the largest proportion, and this characteristics made enzyme protein very sensitive to temperature. Under high temperature circumstances, enzyme protein cannot maintain rigid structure domain which might be the reason of thermal instability. Combination of experimental results and modeling prediction, the thermal unstability of AmDH is due to its random coil rich structure, which indicated the difficulty of the improvement of thermostability by directed molecular evolution and rational design. Therefore, the strategy of organicinorganic hybrid immobilization is simple, convenient and easy way to improve thermostability of AmDH.

3. Results and discussion 3.1. Expression and purification of AmDH AmDH from Bacillus badius was expressed in E. coli BL21 (DE3) at 37 ◦ C, and was purified by a HisTrap HP column and the purification parameters were shown in Table S1. SDS-PAGE analysis at each stage of the purification was shown in Fig. S1. Purified AmDH showed a single band at about 41 kDa, which is consistent with the calculated value. AmDH might be octamer for most of AmDH form a single strip at 330 KDa via the native-PAGE (Fig. S2).

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Fig. 3. The influence of PEI concentration and solution pH on enzyme activity. (A: the influence of PEI concentration; B: the influence of PEI pH) Reaction conditions: NADH, 0.2 mM; NH4 Cl/NH4 OH, 200 mM; phenoxy-2-propanone, 20 mM; pH, 10.0; temperature, 30 ◦ C.

3.3. Coating of amine dehydrogenase with PEI Coating AmDH with PEI was studied. Effect of PEI concentration and solution pH on the activity of AmDH were showed in Fig. 3, which indicated that pH 8.5 and PEI concentration of 0.25 mM was optimal for AmDH coating. Fig. 3A showed that PEI solution with the final concentration of 0.25 mM was the optimal for AmDH coating. Activity of AmDH-PEI composite was decreased with the increase of PEI concentration. PEI solution with high concentration may form large aggregates which can affect the substrate transmission. It was proposed that an excess of PEI likely blocked the active center or tunnel connecting the active sites, or by the close juxtaposition of the sites across the subunits’ interface (Demoss, 1962; Dunn, 2012; Yanofsky and Rachmeler, 1958). Polyethylenimine (PEI) is the cationic polymer contains a high density of ionized amino groups, which is positively charged at pH 7.5 (Huang et al., 2015). PEI has the highest buffer capacity between pH ranging from 8 to 10 (Choosakoonkriang et al., 2003). Positively charged PEI with a high proportion of amine protonation, results in pH value change in the microenvironment of enzymes, and then affecting the activity of enzyme. Therefore, experiment about the range of pH was necessary to find out a peak with minimal impact on the activity of AmDH.

3.4. Interactions of AmDH with PEI Fig. 4 shows that complex formation between PEI and AmDH is a powerful exothermic process. Isothermal titration calorimetry studies showed that the PEI efficiently interacted with AmDH to form polymer-protein assemblies. Affinity constant (K) between PEI and AmDH was about 1.69 ± 0.086 ␮mol−1 . According to reaction enthalpy change (H) and entropy change (S), reaction free energy change (G) can be calculated as Eq. (3), and G was about −27.6 KJ/mol. G = H − TS

(3)

A stoichiometry of PEI per monomer AmDH was 0.7, and was calculated as 5.6 per octamer AmDH. The calculation results were accorded with the experimental results which the reaction energy was powerful exothermic process. PEI interacted with proteins through electrostatic interactions and hydrogen bonds (Garcia-Galan et al., 2013). The result of ITC and the influence of PEI concentration on enzyme activity indicated that polyethylenimine has strong interaction with enzyme.

Fig. 4. Binding process of PEI to AmDH measured using ITC at 25 ◦ C. (a: Titration curve of PEI combined with AmDH by ITC; b:the combination heat to PEI and AmDH mole ratio determined by ITC based on the titration curve fitting).

3.5. Organic-inorganic immobilized amine dehydrogenase Organic-inorganic hybrid immobilization of AmDH were carried out through a biomimetic titanification process induced by PEI coated on enzyme surface. The comparison of titania precipitation induced by AmDH and AmDH-PEI was studied. AmDH and AmDH-PEI were added into Ti-BALDH solution for inducing titania precipitation, respectively (Cole et al., 2006). As described in Fig. S3, when AmDH was added into Ti-BALDH solution, no precipitate was observed at room temperature, while the AmDH-PEI shows a strong ability of titania precipitation. According to the reports, acidic pro-

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teins cannot induce the formation of titania precipitates, while the basic proteins display a stronger ability to precipitate titania particles (Möckel et al., 1999). The polyethylenimine (PEI) which has a high density of ionized amino group is also positively charged under neutral pH. Therefore, anionic group on the protein surface can be neutralized by cationic locked on PEI which can efficiently control the hydrolysis and condensation of inorganic precursors with net negative charges and induced titania precipitate (Belton et al., 2004, 2005; Cole et al., 2006). Furthermore, the amount of titania precipitate increased as the content of PEI coating on the AmDH increased, which indicated that PEI exhibits highly precipitating capability. 3.6. Optimization of immobilization conditions The optimal immobilization condition for the precipitation induced by AmDH-PEI was investigated. AmDH-PEI solution was added into titania precursor solution slowly, and rapid precipitation of titania particles was observed. The effect of Ti-BALDH precursor concentration was investigated considering the encapsulation capacity and enzyme activity which was conducted in 10 mM Tris-HCl buffer (Fig. 5). The concentration of Ti-BALDH solution was selected as 62.5 mM in the subsequent experiments because not only the encapsulation efficiency was upto 90%, but also the activity of immobilized AmDH was highest. Under the low concentration of Ti-BALDH condition, the activity of AmDH was comparatively lower due to the less content of protein encapsulated in titania particles, a part of which was washed off in the process of lavation. However, the amount of titanium precursor concentration increased gradually which might induce excessive titania precipitate and increase the mass transfer resistance. 3.7. Mechanism insight of titania formation induced by amDH-PEI Positively charged proteins have been reported could enrich titanium precursors, promote hydrolysis and polycondensation and induce the formation of titania through electrostatic effect and hydrogen-bond interaction (Manfred, 2002). A similar reaction is induced by cationic peptides (Cha et al., 2000), cationically charged synthetic polymer (Patwardhan and Clarson, 2002) and cationic polyelectrolyte (Patwardhan et al., 2006). Sewell (And and Wright, 2006) and coworkers have extensively studied that the poly (allylamine) which corresponded to a repeat sequence of the silffin precursor polypeptide, for inducing silica and titania mineralization. The residues of template peptides acted as a general acid/basic catalyst for promoting TiO6 octahedral dehydration and condensation. Therefore, PEI molecular as a kind of cationic polyelectrolyte are positively charged in the basic environment, which can concentrate the negatively charged titanium precursor through electrostatic and hydrogen bonding interactions. With the increase of titanium precursor concentration around the AmDH-PEI, the distance of titanium atoms is gradually closer. Hydrogen bonding between Ti OH of the titanium precursor and NH, NH2 of PEI induced nucleophilic substitution of a Ti O oxygen atom on another adjacent titanium atom and polycondensation reaction occurs subsequently (Jiang et al., 2008). As the condensation proceeds, each AmDH-PEI complex can be regarded as a nucleating center to induce titania formation, ultimately resulting in the formation of AmDH-PEI-Ti nanoparticles (Fig. 6). 3.8. Morphology and structure of titania induced by amDH-PEI Morphology of AmDH-PEI-Ti particles and AmDH-PEI were determined by SEM (Fig. 7A and B). Statistical evaluation of the average diameters in Fig. 7A and B were derived from small tools

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of SEM with computer-aided data analysis. AmDH was dissolved in a 0.05 M Tris–HCl buffer, and then added PEI (pH 8.5) solution to make the PEI concentration achieved 0.25 mM. PEIs molecule were branched and aggregations were observed as Fig. 7A. This result might suggest that there are some hydrogen-bond interaction between the surface of AmDH and the amino group of PEI due to the coating of PEI onto AmDH. SEM (Fig. 7A) showed the size of the AmDH-PEI was about 300 nm. Average diameter of the AmDH-PEI was about 300–400 nm measured by Zeta-Potential analyzer (Fig. 7C). The SEM analysis revealed that AmDH-PEI-Ti particles had an average diameter of 100 nm (Fig. 7B), and the average diameter of AmDH-PEI-Ti measured by Zeta- Potentiometer was about 180–200 nm (Fig. 7D). AmDH-PEI and AmDH-PEI-Ti measured by SEM were all smaller than corresponding particles in aqueous solutions through Zeta Potentiometer and it may be due to the hydration shell around the particles (Biedermannová and Schneider, 2015). Whether SEM analysis or Zeta-Potential determine analysis, the size of AmDH-PEI were larger than AmDHPEI-Ti. AmDH-PEI was colloidal structure and could be observed like branched dendritic structure with the size of 300 nm through SEM. This may due to AmDH-PEI complex aggregate with other, while through the condensation reaction, each AmDH-PEI complex act as a nucleating center to induce the formation of AmDH-PEI-Ti nanoparticles. 3.9. Thermostability study Thermostabilities of free AmDH, AmDH-PEI and AmDH-PEI-Ti were compared at 50◦ C and 60◦ C (Fig. 8). Compared with the free enzyme, AmDH-PEI and AmDH-PEI-Ti was more stable. AmDHPEI-Ti exhibited enhanced thermostability at high temperature, as compared with native AmDH, while the AmDH-PEI has certain improvement. Free AmDH can only maintain about 20% activity, but the AmDH-PEI and AmDH-PEI-Ti still possess more than 90% activity about 10 min at 50 ◦ C (Fig. 8A). After incubation for 40 min at 50 ◦ C, AmDH-PEI and AmDH-PEI-Ti still presented nearly 80% of its initial activity while free AmDH almost totally inactivated. The AmDH-PEI-Ti were in the similar tendency to the decay along with time and achieve the relative stability index in a few minutes at 50 ◦ C. The activity of free AmDH were rapidly decreased and even inactibated at 60 ◦ C in about 8 min (Fig. 8B). Significant improvement of thermostability showed at 60 ◦ C, at which incubation for 10 min completely deactivate free AmDH but only caused AmDHPEI to lost about 50% and AmDH-PEI-Ti to miss 40% of its initial activity. Comparatively speaking, the decline velocity of AmDH-PEI exceeded titania encapsulated AmDH. The thermal deactivation kinetics of free AmDH, AmDH-PEI and AmDH-PEI-Ti was investigated (Fig. 8). Therefore, the thermal deactivation kinetics were studied according to Eqs. (4) and (5), where Kdi and E0i are the deactivation rate constant and initial enzyme activity (E0 = 100%), respectively (Maisuria and Nerurkar, 2012; Wang et al., 2013). The deactivation rate constants and halflives t1/2 were calculated based on deactivation process (Eq. (6)). AmDH-PEI-Ti exhibited the enhanced thermostability at high temperature, as compared with native AmDH, while the AmDH-PEI has little improvement. E = E0 · e−Kd t

(4)

E = E01 · e−Kd1 t + E02 · e−Kd2 t

(5)

t 1/2 = ln2/K d

(6)

The thermal inactivation curves indicated that AmDH and AmDH-PEI fitted to a single first-order model more for the graphs best-fitted with a correlation coefficient (R2 ) of 0.9335–0.9996. Similarly it indicated that thermal inactivation of AmDH-PEI-Ti was

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Fig. 5. Encapsulation efficiency and activity of AmDH within titania particles. Reaction conditions: NAD+ , 2 mM; glycine buffer,100 mM; substrate 1,3-DMBA, 20 mM; pH 10.5; temperature 40 ◦ C.

Fig. 6. Biomineralization induced by AmDH-PEI.

not a simple first-order process commonly observed but a multifraction process. The two-fraction first order model (i = 2) fitted the inactivation curves well using a non-liner regression to obtain inactivation rate constants and activity fractions. Thermostability

of AmDH in three type indicated that biomimetic mineralization change the deactivation mechanism AmDH. Two immobilization methods including AmDH-PEI and AmDHPEI-Ti presented lower Kd and longer half-life than in both 50 ◦ C

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Fig. 7. SEM micrographs and average diameter distribution of AmDH-PEI and AmDH-PEI-Ti. (A: SEM micrographs of AmDH-PEI; B: SEM micrographs of AmDH-PEI-Ti; C: average diameter of AmDH-PEI; D: average diameter of AmDH-PEI;).

Fig. 8. The plot for thermal inactivation of AmDH, AmDH-PEI and AmDH-PEI-Ti. (A: incubation at 50◦ C; B: incubation at 60◦ C; 䊏: free AmDH fitted to single first-order model; 䊉: AmDH-PEI fitted to single first-order model; : AmDH-PEI-Ti fitted to two-fraction first order model). Reaction conditions: NADH, 0.2 mM; NH4 Cl/NH4 OH, 200 mM; phenoxy-2-propanone, 20 mM; pH, 10.0; temperature, 30 ◦ C. The relative activity is expressed as a percentage of the original activity as assayed before incubation at the different temperature.

Table 1 Inactivation thermoparameters of AmDH, AmDH-PEI and AmDH-PEI-Ti. Parameter

50 ◦ C

60 ◦ C

Single-fraction

E1 E2 Kd1 Kd2 R2 t1/2 phase I t1/2 phase II

Two-fraction

Single-fraction

Two-fraction

AmDH

AmDH-PEI

AmDH-PEI-Ti

AmDH

AmDH-PEI

AmDH-PEI-Ti

AmDH

AmDH-PEI

AmDH-PEI-Ti

AmDH

AmDH-PEI

AmDH-PEI-Ti

99.65 – 0.109 – 0.9460 6.4 –

99.66 – 0.006 – 0.9335 115.5 –

95.89 – 0.005 – 0.8589 147 –

3.25 96.5 10.25 0.076 0.8659 0.07 9.2

3.87 96.13 0.083 0.0053 0.9003 8.4 131.5

0.59 99.4 0.005 0.0051 0.9577 136.1 135.1

100.0 – 0.562 – 0.9996 1.2 –

99.55 – 0.07 – 0.9980 9.9 –

97.47 – 0.045 – 0.9703 15.4 –

– – – – – – –

0.21 99.5 11.41 0.08681 0.9479 0.06 7.9

0.18 99.8 7.604 0.049 0.9898 0.09 14.1

and 60 ◦ C, which indicated better thermal stability. At the temperature of 60 ◦ C, the Kd of AmDH-PEI was 87% lower than free AmDH and the half-life was increased by 8.3 fold. After the process of titania embedding, AmDH-PEI-Ti extended the half-life about 12 fold and decreased the Kd 91% at 60 ◦ C compared with free enzyme

(Table 1). By contrast, titania immobilization showed more heat resistant and gained significant advantages over AmDH-PEI on the high-temperature stability. In general, conformational transformation was faster with increasing temperature, which can destroy the natural structure of

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Fig. 9. A: The relative activity of free and AmDH-PEI-Ti at different temperatures (䊏: free AmDH; 䊉: AmDH-PEI-Ti) B: Recycling stability of AmDH-PEI-Ti. Reaction conditions: NAD+ concentration 2 mM; glycine buffer 100 mM; substrate 1,3-DMBA 20 mM; pH 10.5.

the enzyme and cause the inactivation (Jiang et al., 2009b). It could be expected that PEI coating preserve the active octamer structure of enzyme with the electrostatic interactions and hydrogen-bond interactions, which improve the stability of AmDH. Compared with free AmDH, the subunits of the octamer which were coating by PEI which maintains the subunits’ interactions under the conditions that would cause dissociation. The thermostability improvement of AmDH-PEI-Ti suggested that AmDH the encapsulated within a certain volume benefited from the so called “cage effect” (Eggers and Valentine, 2001; Gies et al., 2004). AmDH molecules were rigidly held in the titania particles and the rigidity of the cage inhibited AmDH molecules from undergoing thermally denaturation of unfolding-refolding motions. To some content, the cage effect is similar to the excluded volume effects in the natural cells. In addition, the hydrated shell formed by PEI on the surface of AmDH which maintain its activity. 3.10. The optimal temperature and recycling stability of amDH-PEI-Ti Activity assays were carried out to study the temperature influence on AmDH activity. Immobilization has been used for improving enzyme kinetic stability by reducing conformational flexibility (Abian et al., 2004; Srimathi et al., 2006; Villalonga et al., 2006). However, reduced conformational flexibility by modification and immobilization usually results in a significant loss of enzymatic activity (Fernández-Lafuente et al., 2001; Kranz et al., 2007). The activity of AmDH encapsulated in hybrid titania nanoparticles has been decreased compared to free AmDH. Therefore, we chosen the highest activity of free AmDH as 100%, when temperature achieved 40 ◦ C. The highest activity for AmDH-PEI-Ti was achieved in the range of 30 to 50 ◦ C, while the optimal temperature for free AmDH was 40 ◦ C, which demonstrated the great expansion of temperature range of AmDH-PEI-Ti. As temperature increased, the activity of free AmDH decreased significantly, while the activity of AmDH-PEI-Ti maintained a much higher activity until the temperature up to 50 ◦ C. Generally, increased temperature leads to an increase in the thermal motions of enzyme molecule, which would disrupt the amino acid residues interaction that keep the protein structure, and finally leading to enzyme denaturation. The physical cage confinement of AmDH-PEI-Ti attributed to rigidly enzyme holding in the titania matrix and inhibited AmDH molecules from undergoing thermally denaturation of unfolding motions. This suggested that two steps immobilization strategy can stabilize multi-subunits oxidoreductases. Fig. 9 B showed that the activity of titania induced by AmDHPEI was gradually decreased during 6 cycles. AmDH-PEI-Ti retained 60% of its initial activity after 4 repeated cycles, and more than 50.0% of its initial activity was retained after 6 cycles. The high stability

could be attributed to the complete confinement of titania carrier toward AmDH (Jiang et al., 2009a, 2009b). The loss of AmDH activity might be caused by the breakage of the particles after multiple operations under continuous stirring. 4. Conclusion In this study, a facile bio-inspired immobilization method was applied for unstable amine dehydrogenase. The potential of PEIs applied as coating and templating for bioinspired immobilization of multimeric oxidoreductases was studied. The heterofunctional interactions of PEIs with amino acid residues to inhibit multisubunits dissociation were analyzed by ITC. PEI coating on the enzyme surface induced biomineralization of titanium precursor and form AmDH-PEI-Ti hybrid nanoparticles. Morphological analysis and inactivation kinetics of the immobilized enzyme were performed to verify the mechanism insights of the enzyme stability improvements. This novel strategy offers a cheap, simple, high activity reservation, greatly improved thermostability for multimeric enzyme immobilization in biomanufacturing and biosensor as electric enzyme electrodes. Acknowledgment This work was supported by the National Natural Science Foundation of China (No. 41176111, No. 41306124), the State Key Program of National Natural Science Foundation of China (No. 21336009), the Fundamental Research Funds for the Central Universities (No. 2013121029), the Foundation of South Oceanographic Research Center of China in Xiamen (No.: 14GYY011NF11), and the Public science and technology research funds projects of ocean (No.: 201505032-6). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec.2016.11. 006. References Abian, O., Grazú, V., Hermoso, J., González, R., García, J.L., Fernández-Lafuente, R., Guisán, J.M., 2004. Stabilization of penicillin G acylase from escherichia coli: site-directed mutagenesis of the protein surface to increase multipoint covalent attachment. Appl. Environ. Microbiol. 70, 1249–1251. Abrahamson, M.J., Wong, J.W., Bommarius, A.S., 2013. The evolution of an amine dehydrogenase biocatalyst for the asymmetric production of chiral amines. Adv. Synth. Catal. 355, 1780–1786. And, S.L.S., Wright, D.W., 2006. Biomimetic synthesis of titanium dioxide utilizing the R5 peptide derived from cylindrotheca fusiformis. Chem. Mater. 18, 3108–3113.

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