An enzyme-loaded reactor using metal-organic framework-templated polydopamine microcapsule

Jing Wang,Yongqin Lv

Beijing Key Laboratory of Bioprocess,College of Life Science and Technology,Beijing University of Chemical Technology,Beijing 100029,China

Keywords:Biocatalysis Enzyme Immobilization Microcapsule Metal–organic framework Template

ABSTRACT Ultrathin polydopamine microcapsules with hierarchical structure and porosity were prepared for the immobilization of multienzymes using metal-organic framework(MOF)as the template.The multienzyme/MOF composite was first prepared using a“one-pot”co-precipitation approach via the coordination and self-assembly of zinc ions and 2-methylimidazole in the presence of enzymes.The obtained nanoparticles were then coated with polydopamine thin layer through the self-polymerization of dopamine under alkaline condition.The polydopamine microcapsules with an ultrathin shell thickness of～48 nm were finally generated by removing the MOF template at acidic condition.Three enzymes were encapsulated in PDA microcapsules including carbonic anhydrase(CA),formate dehydrogenase(FateDH),and glutamate dehydrogenase(GDH).FateDH that catalyzed the main reaction of CO2 reduction to formic acid retained 94.7%activity of equivalent free FateDH.Compared with free multienzymes,the immobilized ones embedded in PDA microcapsules exhibited 4.5-times higher of formate production and high catalytic efficiency with a co-factor-based formate yield of 342%.

1.Introduction

As biocatalysts,enzymes have been widely used for the biotransformation of renewable materials to produce chemicals,fuels,biomaterials,and pharmaceutical products.While enzymatic catalysis has many important advantages compared with chemical catalysis,such as high specificity,high selectivity,and mild operational conditions,the use of free enzymes for industrial applications still features several inherent limitations including low operational stability and the difficulties of recovery and recycling.Immobilization of enzymes on solid carriers can conquer these limitations by providing improved enzyme stability and high reusability[1–17].As a result,the exploration and development of new and efficient solid supports are highly desirable to create immobilized enzymes with enhanced stability and high reusability while retaining its original activity and selectivity.

Microcapsule is one type of promising solid carriers used for the immobilization of enzymes due to its hierarchical properties and structures that allow the modulation of microenvironment by providing compartmentalization[18,19].Enzyme encapsulation in microcapsules can be achieved via different approaches such as self-assembly[20–26],emulsification[27–31],interfacial polycondensation[32–34],and aggregation [35].The templating method is another efficient strategy for the formation of microcapsules due to its universality and versatility.By regulating the size and shape of the templates,the porosity and structures of microcapsules can be easily adjusted.The current reported templates include CaCO3[36–45],SiO2[46],and polystyrene spheres [47].Metal–organic frameworks (MOFs) have emerged as a new type of microporous crystalline material and been extensively applied for the fabrication of enzyme-MOF composites[48–62].Due to its high shape and structure diversity,MOF can also function as a promising template in the preparation of microcapsules[40].In particular,zeolitic imidazolate framework-8(ZIF-8)can be readily synthesized via assembling 2-methylimidazole with Zn2+ions under mild operational conditions [63,64].By immersing in neutral EDTA or acidic aqueous solutions,the ZIF-8 template can be easily decomposed and removed[40].

Inspired by these findings,in this work,we propose the preparation of polydopamine(PDA)microcapsules with hierarchical porosity and structure using ZIF-8 as the template.The PDA microcapsules were utilized as solid carriers for the immobilization of multienzymes containing carbonic anhydrase (CA),formate dehydrogenase (FateDH),and glutamate dehydrogenase(GDH).The multienzyme/ZIF-8 bioconjugate was first synthesized using a“one-pot”co-precipitation approach.The PDA@multienzymes/ZIF-8 composite was then prepared by the self-polymerization of dopamine on the surface of ZIF-8.The removal of ZIF-8 template at acidic condition produced PDA microcapsule that contained three enzymes.The catalytic performances of the immobilized enzymes were evaluated by the transformation of CO2to formic acid.

2.Materials and Methods

2.1.Materials and reagents

Zinc nitrate hexahydrate [Zn(NO3)2.6H2O],2-methylimidazole,dopamine hydrochloride,and 2,3,4,5,6-pentafluorobenzyl bromide were purchased from J&K Scientific Ltd.(Beijing,China).L-glutamic acid was bought from Sinopharm Chemical Reagent Co.,Ltd.(Beijing,China).Carbonic anhydrase(CA,bovine red blood cell),formate dehydrogenase(FateDH,lyophilized),and glutamate dehydrogenase (GDH,bovine liver)were supplied by Sigma-Aldrich(St.Louis,MO,USA).Nicotinamide adenine quinone dinucleotide(NADH,98%) was provided by Aladdin Biotechnology Co.,Ltd.(Shanghai,China).CO2(>99%) was obtained from Beijing Ruyuan Ruquan Technology Co.,Ltd.(Beijing,China).All other chemicals were purchased from Beijing Chemical Factory(Beijing,China).Double-distilled water was applied in all experiments.

2.2.Instrumentation

The transmission electron microscopy(TEM)images of ZIF-8,PDA@ZIF-8,and PDA microcapsule were obtained by using a JEOL 2100F transmission electron microscope (Hitachi,Ltd.,Japan).Scanning electron microscopy(SEM)images of ZIF-8,PDA@ZIF-8,and PDA microcapsule were taken with a JEOL JSM-6700F field emission scanning electron microscope(Hitachi High-Technologies,Tokyo,Japan).Powder X-ray diffractions of ZIF-8,PDA@ZIF-8,and PDA microcapsule were obtained from a D/max-UltimaIII(Rigaku Corporation,Japan).Particle size distributions of ZIF-8 and PDA@ZIF-8 were measured using dynamic light scattering (Zetasizer Nano ZS980,Malvern,UK).A LC 2030 system(Shimadzu,Kyoto,Japan)was employed to evaluate the concentrations of formic acid derivative using a 5020-39001 WondaSil C18column(15 cm×4.6 cm i.d.,5 μm,GL Sciences).The concentrations of NADH and p-nitrophenol were measured in a clear 96-well plate using a Multiskan Spectrum plate reader (Thermo Fisher Scientific,USA).The concentrations of zinc ions were determined using an inductively coupled plasma mass spectrometer (ICP-MS) (Thermo Fisher iCAP Q,USA).

2.3.Synthesis of ZIF-8 and multienzyme/ZIF-8

The synthesis of the ZIF-8 was performed by adding 4 mL of 0.31 mol·L?1zinc nitrate solution to 40 mL of 1.25 mol·L?12-methylimidazole solution.The mixture was stirred at room temperature for 0.5 h,followed by 3 cycles of centrifugation at 6500 r·min?1for 5 min and washing.

The synthesis of the multi-enzyme/ZIF-8 composite was carried out following the approach reported by Ge et al[48].Briefly,4 mL of 0.31 mol·L?1zinc nitrate aqueous solution and 4 mL of enzyme aqueous solution(3 mg·mL?1for CA,3 mg·mL?1for FateDH,and 3 mg·mL?1for GDH)were added to 40 mL of 1.25 mol·L?12-methylimidazole aqueous solution.The mixture was stirred at room temperature for 0.5 h,followed by 3 cycles of centrifugation at 6500 r·min?1for 5 min and washing.

2.4.Synthesis of PDA@ZIF-8 and PAD@multienzymes/ZIF-8

For the synthesis of PDA@ZIF-8,6 mg of dopamine hydrochloride was dissolved in 12 mL of 50 mmol·L?1Tris–HCl buffer(pH 8.5),to which 60 mg freeze-dried ZIF-8 was added quickly.The mixture was then stirred at room temperature for 5.5 h,8 h,and 10 h,respectively,during which dopamine self-polymerized under the alkaline condition.After the reaction,the resultant PDA@ZIF-8 was washed with deionized water at 12000 r·min?1for 3 min to remove the unreacted dopamine.

For the synthesis of PDA@multienzymes/ZIF-8,6 mg of dopamine hydrochloride was dissolved in 12 mL of 50 mmol·L?1Tris–HCl buffer(pH 8.5),to which 60 mg freeze-dried ZIF-8 containing 9 mg enzymes(3 mg CA,3 mg FateDH,and 3 mg GDH)was added quickly.The mixture was then stirred at room temperature for 8 h.After the reaction,the resultant PDA@multienzymes/ZIF-8 was washed with deionized water at 12000 r·min?1for 3 min to remove the unreacted dopamine.

2.5.Synthesis of PDA microcapsule

The PDA microcapsule was prepared by decomposing and removing the ZIF-8 template under acidic conditions.In brief,10 mg of PDA@ZIF-8 composite was incubated in 2 mL PBS buffer solution(50 mmol·L?1,pH 6)at room temperature for 6 h,12 h,18 h,and 24 h,respectively.The concentration of zinc ions in the supernatant was determined by inductively coupled plasma-mass spectrometry(ICP-MS).The PDA microcapsule was washed with deionized water to remove the impurities and separated by centrifugation at 12000 r·min?1for 5 min.

2.6.Preparation of immobilized enzymes encapsulated in PDA microcapsule

The immobilized enzymes encapsulated in PDA microcapsule were obtained by incubating 10 mg PDA@multienzyme-ZIF-8 nanocomposites in 2 mL PBS buffer solution(50 mmol·L?1,pH 6)at room temperature for 18 h.The as-prepared PDA microcapsule encapsulated with enzymes was washed with deionized water to remove the impurities and separated by centrifugation at 12000 r·min?1for 5 min.

2.7.Enzyme activity

To measure the activity of free and immobilized FateDH,2 mL of 2 mg·ml?1NADH solution in PBS buffer(50 mmol·L?1,pH 7)was first purged with N2for 0.5 h,and then bubbled with CO2for 0.5 h.Then 1 mg of free enzyme or 1 mg enzyme immobilized in ZIF-8,PDA@ZIF-8,or PDA microcapsule was separately added to the above solution.The mixture was incubated at 25°C for 30 min.The concentration of residual NADH was monitored in a clear 96-well plate using a Multiskan Spectrum plate reader recorded at 340 nm.The FateDH activity unit was defined as the amount of FateDH consumed to convert 1 μmol NADH to NAD+in 1 min.

To measure the activity of free and immobilized CA,2 mL of 0.2 mmol·L?1p-nitrophenyl acetate solution in PBS buffer(50 mmol·L?1,pH 7) was prepared.Then 0.1 mg of free enzyme or 0.1 mg enzyme immobilized in ZIF-8,PDA@ZIF-8,or PDA microcapsule was separately added to the above solution.The mixture was incubated at 25°C for 5 min.The concentration of p-nitrophenol was monitored in a clear 96-well plate using a Multiskan Spectrum plate reader recorded at 348 nm.The CA activity unit was defined as the amount of CA consumed to convert 1 μmol p-nitrophenyl acetate to p-nitrophenol in 1 min.

To measure the activity of free and immobilized GDH,1 mmol·L?1NAD+and 2 mmol·L-1L-glutamic acid was first admixed in 1 mL PBS buffer(50 mmol·L?1,pH 7).Then 0.05 mg of free enzyme or 0.05 mg enzyme immobilized in ZIF-8,PDA@ZIF-8,or PDA microcapsule was separately added to the above solution.The mixture was incubated at 25°C for 10 min.The concentration of produced NADH was monitored in a clear 96-well plate using a Multiskan Spectrum plate reader recorded at 340 nm.The GDH activity unit was defined as the amount of GDH consumed to convert 1 μmol NAD+to NADH in 1 min.

2.8.Enzymatic catalysis of CO2 to formic acid

Fig.1.Schematic illustration of the preparation of polydopamine(PDA)microcapsules(a)and immobilized enzymes encapsulated in PDA microcapsules(b)using ZIF-8 as the template.

For the conversion of CO2to formic acid using immobilized enzymes,an aqueous mixture containing 10 mmol·L?1L-glutamate and 2 mg·ml?1NADH in 2 ml of PBS buffer (50 mmol·L?1,pH 7)was first purged with N2for 0.5 h and then bubbled with CO2for 0.5 h.Then,3 mg of multienzymes(1 mg CA,1 mg FateDH,1 mg GDH)immobilized in 20 mg of ZIF-8,PDA@ZIF-8,or PDA microcapsule were added quickly to the above solution.The reaction was performed in a sealed flask at 25 °C for 0.5 h.The amount of formic acid product was determined following our previously published method[53].

For the biotransformation of CO2to formic acid using free enzymes,a mixture solution containing 10 mmol·L?1L-glutamate and 2 mg·ml?1NADH in 2 ml of PBS buffer(50 mmol·L?1,pH 7)was first purged with N2for 0.5 h and then bubbled with CO2for 0.5 h.Then,1 mg CA,1 mg FateDH,and 1 mg GDH were added quickly to the above solution.The reaction was performed in a sealed flask at 25°C for 0.5 h.

3.Results and Discussion

3.1.Preparation of PDA microcapsules

The preparation of polydopamine(PDA)microcapsules was illustrated in Fig.1a.ZIF-8 nanoparticle was first prepared via the coordination of 2-methylimidazole and Zn2+.The PDA thin layer was then coated on the surface of ZIF-8 through the self-polymerization of dopamine at alkaline conditions.By incubating the PDA@ZIF-8 nanocomposites in PBS buffer at pH 6 at room temperature for a certain period of time,the ZIF-8 template decomposed due to the cleavage of metal chelation at acidic conditions[40].The PDA microcapsules were generated after the complete removal of ZIF-8 template.

Fig.2.TEM and SEM images of ZIF-8 nanoparticles prepared at a reaction time of 0 min(a,d),5 min(b,e),and 30 min(c,f).

Fig.3.(a)Dynamic light scattering of ZIF-8 and PDA@ZIF-8 nanoparticles dispersed in 50 mmol·L?1 PBS buffer(pH 7).(b)X-ray diffraction patterns of simulated ZIF-8,ZIF-8,PDA@ZIF-8,and PDA microcapsule.(c)The concentration of Zn2+in the supernatant determined by inductively coupled plasma-mass spectrometry after incubating the PDA@ZIF-8 composites in PBS buffer(50 mmol·L?1,pH 6)at room temperature for different periods of time.

The transmission electron microscopy(TEM)and scanning electron microscopy(SEM)images of ZIF-8 in Fig.2(a,d)showed that the direct blending of 2-methylimidazole with Zn2+produced ZIF-8 with spherical nanosheet morphology.Cubic ZIF-8 nanocrystals were formed when the reaction time was 5 min(Fig.2(b,e)).Further elongation of the reaction time to 30 min generated ZIF-8 with the standard rhombic dodecahedral morphology(Fig.2(c,f))and a crystal size of～333 nm as indicated from the dynamic light scattering(DLS)result in Fig.3a.The X-ray diffraction(XRD)patterns of ZIF-8 prepared at a reaction time of 30 min matched well with the simulated one indicating the pure phase of ZIF-8 crystals(Fig.3b).

Fig.4.SEM images of PDA@ZIF-8 nanocomposites(a-c)and PDA microcapsules(d-f)prepared at a self-polymerization time of 5.5 h(a,d),8 h(b,e),and 10 h(c,f).

Fig.5.TEM images of PDA@ZIF-8(a)and PDA microcapsule(b).SEM images of PDA microcapsule prepared by incubating PDA@ZIF-8 in PBS buffer(50 mmol·L?1,pH 6)at room temperature for 18 h(c)and 24 h(d).

The reaction time for the self-polymerization of dopamine was further optimized.The ZIF-8 nanoparticles prepared at a reaction time of 30 min was incubated in the freshly prepared dopamine solution in Tris–HCl buffer (pH 8.5) at room temperature for different times(5.5,8,and 10 h).As shown from the SEM image in Fig.4a,the PDA layer was not fully coated on the surface of ZIF-8 at a reaction time of 5.5 h indicating the insufficient self-polymerization.Further removal of the ZIF-8 template produced broken PDA microcapsules with cracks(Fig.4d).When the reaction time was increased to 8 or 10 h,the ZIF-8 nanocrystals were uniformly covered with PDA layer(Fig.4(b,c)).The PDA microcapsules with defect-free structure and morphology were generated after the subsequent removal of ZIF-8 template(Fig.4(e,f)).In our further study,a reaction time of 8 h was chosen for the selfpolymerization of dopamine.The TEM image of PDA@ZIF-8 was shown in Fig.5a.The DLS result in Fig.3a revealed a particle size of～381 nm for PDA@ZIF-8 nanocomposite implying that the thickness of PDA layer is around 48 nm.The XRD pattern of PDA@ZIF-8 in Fig.3b was consistent with ZIF-8 indicating that coating of PDA thin layer did not change the crystallinity of ZIF-8.

We also optimized the reaction time taken for the removal of ZIF-8 template under acidic condition.PDA@ZIF-8 nanocomposite was immersed in PBS buffer(50 mmol·L?1,pH 6)at room temperature for 6,12,18,and 24 h,respectively.The concentration of Zn2+in the supernatant was determined by inductively coupled plasma-mass spectrometry(ICP-MS)(Fig.3c),which confirmed the complete removal of ZIF-8 template at an incubation time of 18 h.This step produced PDA microcapsules with flawless structure as indicated from the TEM and SEM images in Fig.5(b,c).The XRD pattern of PDA microcapsule was also in accordance with ZIF-8(Fig.2b).However,the increase of incubation time to 24 h also partially dissolved the PDA microcapsules(Fig.5d).As a result,an incubation time of 18 h was selected for the removal of ZIF-8 template.

3.2.Immobilized enzymes encapsulated in PDA microcapsules

Using the same synthetic approach and the optimized conditions,three enzymes including CA,FateDH,and GDH were encapsulated and immobilized in PDA microcapsules(Fig.1b).No residual enzyme was observed in the supernatant as determined from the Multiskan Spectrum plate reader indicating that the enzymes were completely encapsulated within ZIF-8 during the immobilization process.The immobilized quantity of each enzyme was 49.61,46.32,and 107.14 mg·g?1in ZIF-8,PDA@ZIF-8,and PDA microcapsule,respectively(Table 1).

CA is involved to accelerate the hydration of CO2to.FateDH is used to reduceto formic acid.The conversion of CO2to formic acid requires co-factor nicotinamide adenine dinucleotide (NADH) to supply energy,so the use of GDH is to realize the continuous regeneration of NADH co-factor.The multienzymes/ZIF-8 composite was first prepared via a“one-pot”co-precipitation approach by admixing Zn2+,2-methylimidazole,and enzymes (CA,FateDH,and GDH).As revealed from the SEM and TEM images in Fig.6(a,d),the multienzymes/ZIF-8 composite exhibited rhombic dodecahedral morphology similar to ZIF-8.The multienzymes/ZIF-8 was then reacted with dopamine at pH 8.5 and room temperature for 8 h.The SEM and TEM images in Fig.6(b,e)confirmed the successful formation of PDA thin layer on the surface of ZIF-8.The immobilized enzymes encapsulated in PDA microcapsules were finally obtained by incubating the PDA@multienzymes/ZIF-8composites in PBS buffer at pH 6 for 18 h to remove the ZIF-8 template(Fig.6(c,f)).

Table 1 The loading capacity of each enzyme immobilized in ZIF-8,PDA@ZIF-8,and PDA microcapsule,respectively

Fig.6.SEM images of multienzymes/ZIF-8(a),PDA@multienzymes/ZIF-8(b),and multienzymes encapsulated in PDA microcapsule(c).TEM images of multienzymes/ZIF-8(d),PDA@multienzymes/ZIF-8(e),and multienzymes encapsulated in PDA microcapsule(f).

Fig.7.Activities of formate dehydrogenase(FateDH)(a),carbonic anhydrase(CA)(b),and glutamate dehydrogenase(GDH)(c)in solution and immobilized in ZIF-8,PDA@ZIF-8,and PDA microcapsules.

Fig.8.(a)Production amount of HCOOH catalyzed by multienzymes in solution and immobilized in ZIF-8,PDA@ZIF-8,and PDA microcapsules.(b)NADH-based HCOOH yield(YHCOOH )catalyzed by multienzymes in solution and immobilized in ZIF-8,PDA@ZIF-8,and PDA microcapsules.

To confirm the feasibility of our approach,we first immobilized the single enzyme FateDH,CA or GDH in PDA microcapsule and evaluated its activity before and after immobilization.As shown in Fig.7,the immobilization step did not significantly decrease the activity of the three enzymes.For example,the residual activity of FateDH,CA,or GDH encapsulated in PDA microcapsule was 94.7%,91.2%,and 107%of their free counterparts.It is worthy of note that,the coating of PDA layer remarkably enhanced the activities of CA and GDH,that were up to 119% and 116% of their free enzymes.This can be ascribed to the strong affinity of positively charged PDA layer toward the negatively charged substrates like CO2and glutamic acid via electrostatic interactions,which significantly enhanced the local substrate concentration.

3.3.Conversion of CO2 to formic acid

The newly prepared multienzymes containing CA,FateDH,and GDH immobilized in PDA microcapsules were employed to convert CO2to formic acid accompanied by NADH regeneration.The reaction was performed in batch mode containing immobilized multienzymes and NADH,and the reaction time was 0.5 h.The produced formic acid was calculated and compared in Fig.8a.Clearly,the PDA microcapsule based multienzyme system showed the highest HCOOH production amount of(4.4±1.4)mmol·L?1,which is equal to 13.3%conversion yield taking into consideration that the solubility of CO2in water is 33 mmol·L?1[65].For comparison,we also performed the enzymatic reactions catalyzed by free enzymes and immobilized enzymes in ZIF-8 and PDA@ZIF-8.We found that the production amount of HCOOH catalyzed by free enzymes was only(0.96±0.07)mmol·L?1with a conversion of only 2.9%.By using the multienzymes immobilized in ZIF-8 or PDA@ZIF-8 to catalyze CO2,the produced HCOOH was(0.81±0.01)mmol·L?1and (2.1 ± 0.18) mmol·L?1respectively.Obviously,the produced HCOOH catalyzed by the immobilized multienzyme system in PDA microcapsule was more than 4.5-times higher than that of the relevant free enzyme system.The superiority of our new multienzyme system encapsulated in PDA microcapsule can be ascribed to the following two reasons:i)the compartmentalization generated by microcapsule increases the proximity effect of multienzymes,and ii)the PDA shell has high affinity to CO2and provides immobilized enzymes with a high CO2substrate concentration,which allows higher production amount of formic acid.Moreover,no enzyme leakage was observed during the enzymatic reaction process.

During the conversion of CO2to formic acid,the co-factor NADH is used as a terminal electron donor and hydrogen donor.The reduction of CO2to produce 1 mol formic acid consumes 1 mol costly NADH producing NAD+.To achieve the continuous regeneration of NADH,GDH was involved in the multienzyme system.We also calculated the NADH-based HCOOH yield(YHCOOH)according to the following equation[53].

where CHCOOH(mmol·L?1)is the concentration of HCOOH at a reaction time of 0.5 h,and CNADH,initial(mmol·L?1) is the initial NADH concentration.

As shown in Fig.8b,the NADH-based HCOOH yield(YHCOOH)was 158%for multienzymes encapsulated in PDA microcapsule in comparison to 34%of free enzymes.The effects of NADH amount on the production of formic acid were also investigated by varying the concentration of NADH ranging from 0.2 to 2.8 mmol·L?1while maintaining the amount of immobilized enzymes constant.As shown in Table 2,the production of HCOOH increased to 4.43 mmol·L?1when the NADH concentration raised from 0.2 to 2.8 mmol·L?1,while YHCOOHdecreased from 342.0%to 158.5%.This catalytic performance compares well with the values reported for other solid supports elsewhere[66–71].

3.4.Operational stability

The operational stability of immobilized enzymes compared with free ones in solution was also studied by evaluating their residual activity after conditioning at harsh conditions including temperature(90°C)and pH(6 and 10)for 12 h.As illustrated in Fig.9,free FateDH in solution lost more than 95.4%of their initial activities at a temperature of 90°C in contrast to 32.6%loss for immobilized FateDH in PDA microcapsules.With respect to incubating at pH 6 and 10,FateDH in PDA kept up to 83.4%and 40.4%of its initial activity whereas the activity of free one was only 50.6%at pH 6 and 36.1%at pH 10.These results clearly demonstrate that the immobilized FateDH revealed excellent stability under denaturing conditions since the PDA microcapsule supplied a favorablemicroenvironment that assisted protein folding and thus maintaining the enzyme activity.

Table 2 The production of HCOOH at different NADH concentrations

Fig.9.Operational stability of FateDH encapsulated in PDA microcapsule compared with free one in solution(a)at different temperatures(30 and 90°C)and(b)at different pH values(6,7,and 10).

4.Conclusions

We have successfully prepared a new PDA microcapsule that serves as solid carrier for the co-immobilization of multienzymes.This new strategy provides compartmentation that increases the proximity effect of multienzymes.The new PDA microcapsule solid support also exhibits high affinity to CO2and significantly enhances the substrate concentration.The remarkably enhanced catalytic efficiency of immobilized enzymes confirm the great potential of PDA microcapsule functioning as a new type of host matrix material for immobilization of enzymes.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Natural Science Foundation of China(31961133004,21861132017),the National Key Research and Development Program of China (2018YFA0902200),and the Fundamental Research Funds for the Central Universities (PT1917,buctrc201).