Efficient elimination and detection of phenolic compounds in juice usinglaccase mimicking nanozymes

Hui Huang,Lulu Lei,Juan Bai,Ling Zhang,Donghui Song,Jingqi Zhao,Jiali Li,Yongxin Li

1 College of Food Science and Engineering,Jilin University,Changchun 130025,China

2 College of New Energy and Environment,Jilin University,Changchun 130021,China

Keywords:Nanomaterials Enzyme Mimic laccase Phenols detection Degradation Juice clarification

ABSTRACT Residual phenols in the juice can cause turbidity and affect its sensory quality.Laccase is used to remove phenolic compounds from fruit juices.In order to overcome the shortcomings of natural laccase instability and high cost,in this work,we prepared a laccase mimic enzyme based on copper ion and adenosine monophosphate(AMP-Cu nanozymes).At the same mass concentration(1 mg·ml?1),the catalytic activity of the nanozyme is about 15 times that of laccase.The AMP-Cu nanozymes had a higher Vmax and a lower Km than laccase.The laccase mimic enzyme had a good stability under the condition of 30–90°C and pH ＞6.It also maintained high catalytic activity at high salt concentrations and 9 days storage time.Furthermore,the AMP-Cu nanozymes maintained an initial catalytic activity of about 80%after six consecutive cycles of reaction.The linear range of detection of phenolic compounds by AMP-Cu nanozymes was 0.1–100 μmol·L?1 with a detection limit of 0.033 μmol·L?1.The phenol removal rate of AMP-Cu nanozymes was much higher than that of laccase under different reaction times.When the reaction was performed for 5 h,the phenol removal rate of the fruit juice by AMP-Cu nanozymes was about 65%.The efficient removal of phenolic compounds from different juices by AMP-Cu nanozymes indicates that they have good application prospect in the food juice industry.

1.Introduction

With the improvement of people's consumption level and diet structure,fruit juice beverages are being favored by more and more consumers with their rich vitamin content and balanced nutrition.The juice market is growing rapidly [1].According to the 2018 report of the AIJN(European fruit juice association),the global consumption of fruit juice and nectars was 36.2 billion liters in 2017.The EU remained the largest consumer region,and the growth in juice sales in the Asia-Pacific region was mainly driven by China,spurred by rapidly growing demand for premium juices from Chinese consumers.

The polyphenols in fruit juices are natural antioxidants,but the phenols in fruit juices interact with proteins,forming a fog or sediment that makes the juice cloudy.Enzymatic oxidation and chemical oxidation of phenols cause browning of fruit juice,which affects consumers'acceptance of the product[2,3].Therefore,it is necessary to carry out clarification treatment in the juice production process.Common clarification processes include physical and chemical adsorption[4]or membrane filtration[5].These techniques have disadvantages due to phenolic compounds cannot be removed,resulting in significant turbidity and enzymatic or nonenzymatic browning[6].In recent years,the use of enzymes for the treatment of fruit juices has received increasing attention[7].

Laccase is a polyphenol oxidase containing four copper ions,it belongs to copper blue oxidase and can oxidize various substrates such as polyphenols and aromatic amines[8–10].Laccase has been widely used in biosensors,sewage treatment,dye bleaching,wine clarification and other fields[11–13].Some authors have proposed the use of laccase in juice clarification because it oxidizes most phenolic compounds in juice[14].Purified laccase or immobilized laccase can be used for clarification of fruit juices such as litchi juice and apple juice[15,16].However,the high cost,low stability,short storage life and poor reusability of laccase hinder its wide application in industry[17–19].

In order to solve the shortcomings of the above natural enzymes,people have been committed to the study of enzyme mimics [20].Nanozymes refer to nanomaterials with enzymatic properties,which are widely concerned due to their low-cost,high stability,adjustable catalytic activity and reusability [21,22].Based on these advantages,nanozymes are widely used in biosensors,environmental monitoring,medical diagnosis and treatment [23,24].Recently,many mimetic nanozymes have been synthesized,such as carbon-based nanomaterials[25–27],metal nanomaterials[28,29],metal oxide nanoparticles[30–32]and some other nanocomposites[19].Most of them are simulated natural peroxidase,catalase or superoxide dismutase,and there are not many reports on nanomaterials for simulating polyphenol oxidase[33].

Herein,we successfully synthesized a simulated laccase based on adenosine monophosphate coordinated copper(AMP-Cu nanozymes).The nanozymes were simple to prepare and had better catalytic activity than natural laccase.We found that the AMP-Cu nanozymes had better stability than laccase in temperature,pH,salt concentration and storage.And the AMP-Cu nanozymes could be recycled.Due to the excellent properties of the simulated laccase nanozyme,the AMP-Cu nanozymes were applied for the detection of phenolic substances,as well as removal of phenolic compounds from different juices.

2.Materials and Methods

2.1.Chemicals and instrumentation

Adenosine-5′-monophosphate (AMP),Tris(hydroxymethyl)amino methane (Tris–HCl) and 2-(N-Morpholino)ethanesulfonic acid (MES)monohydrate were purchased from Sangon Biotechnology Co.,Ltd.(Shanghai,China).2,4-Dichlorophenol(2,4-DP)and 4-aminoantipyrine(4-AP)were purchased from Aladdin Co.,Ltd.(Shanghai,China).CuCl2was obtained from Fuchen Chemical(China).Laccase from Aspergillus was purchased from Solarbio Co.,Ltd.Other reagents and chemicals utilized in this study were analytical grade.

Absorbance spectra were measured using a Metash UV-8000S spectrophotometer (Shanghai,China).Fourier transform infrared (FTIR)spectra were recorded with a Shimadzu IRPrestige-21 spectrometer(Japan).Transmission electron microscopy (TEM) images were recorded by the H-600 of Hitachi,Japan.The centrifuge used for centrifugation was HC-2062(Anhui,China).

2.2.Preparation of AMP-Cu nanozymes

AMP-Cu nanozymes were synthesized according to the previous report[34].First,mixing Tris–HCl buffer(100 mmol·L?1,pH 8.5,100 μl),AMP (5 mmol·L?1,200 μl),and CuCl2(50 mmol·L?1,100 μl).Then added 600 μl of pure water to make up 1 ml of reaction system.The solution was centrifuged at 10,000 r·min?1for 5 min,and a blue precipitate was observed at the bottom of the tube.The blue precipitate was washed three times with pure water and dissolved in MES buffer(60 mmol·L?1,pH 6.7).

2.3.Catalytic activity assays of laccase and AMP-Cu nanozymes

The activity of laccase and AMP-Cu nanozymes were measured with 2,4-DP as substrate under the respective optimal reaction conditions.Briefly,2,4-DP(1 mg·ml?1,100 μl)and 4-AP(1 mg·ml?1,100 μl)solutions were added into MES buffer(60 mmol·L?1,pH 6.7,700 μl).AMPCu nanozymes were then added to the system and reacted at room temperature for 1 h.The solution was centrifuged at 10,000 r·min?1for 3 min.The supernatant was taken and its absorbance was measured at 510 nm.The laccase reacted at 50°C,pH 5.5 for 1 h,and other conditions are the same as the AMP-Cu nanozymes.

To estimate the reaction kinetics of laccase and AMP-Cu nanozymes,0.1 mg·ml?1laccase and AMP-Cu nanozymes were respectively added into various concentrations of 2,4-dichlorophenol in the presence of 0.1 mg·ml?14-AP and react under the respective optimal conditions.The initial reaction rates of these solutions were measured.The Michaelis–Menten constant (Km) and the maximal velocity (Vmax)were obtained by the Michaelis–Menten equation:

2.4.Stability and reusability of AMP-Cu nanozymes

To study the effect of temperature on the stability of laccase and AMP-Cu nanozymes,laccase and nanozymes were individually incubated at 30–90°C for 1 h and then catalytic experiments were done at respective optimum temperatures for all the samples.The activity at 30°C was taken as 100%.Then the effect of pH was evaluated by storing laccase and AMP-Cu nanozymes in buffers from pH 4 to 9 for 12 h at room temperature.The relative activity was compared with the activity at pH=7.To study the effect of salt concentration on the stability of laccase and AMP-Cu nanozymes,they were incubated in different concentrations of NaCl(0,100,200,300,400,500 and 600 mmol·L?1)for 10 h.The relative activity was compared to the absence of NaCl.For long-term storage stability,the aqueous solutions of laccase and AMPCu nanozymes were stored at 4°C for 9 days,and the relative activity was compared with the activity at first day.

The reusability of 1 mg·ml?1AMP-Cu nanozymes were studied in the 4 ml reaction system for 6 cycles.2,4-DP(1 mg·ml?1,400 μl)and 4-AP (1 mg·ml?1,400 μl) solutions were added into MES buffer(60 mmol·L?1,pH 6.7,2.8 ml).Then,the AMP-Cu nanozymes(10 mg·ml?1,400 μl)were added and incubated at room temperature for 1 h.The solution was centrifuged at 10,000 r·min?1for 5 min.The liquid supernatant was used for absorbance measurement and the precipitate was used for the next cycle.The residual activity was compared with the first cycle.

2.5.Detection of phenolic compounds

For the detection of phenolic compounds,2,4-dichlorophenol was chosen as an example.Different concentrations of 2,4-DP (100 μl)were added into a series of centrifuge tubes with MES(pH 6.7,700 μl)and 4-AP(1 mg·ml?1,100 μl).AMP-Cu nanozymes(10 mg·ml?1,100 μl) or laccase (10 mg·ml?1,100 μl) solutions were then mixed with the above solution.For AMP-Cu nanozymes,the obtained solution(1 ml)was shaken and reacted at room temperature for 1 h.The solution was centrifuged at 10,000 r·min?1for 3 min before recording the spectral information using a UV–visible spectrophotometer.The laccase reacted at 50°C,pH 5.5 for 1 h,and other conditions are the same as the AMP-Cu nanozymes.Further,a 100-fold dilution of apple juice was used to replace the buffer solution,and the phenolic compound was detected by AMP-Cu nanozymes in the juice system.

2.6.Fruit juice extraction and treatment

Mature and harmless fruits(apples,pears,oranges,kiwis and blueberries)were purchased from a local market in Changchun,China.First,the fruits were carefully washed,then peeled and juiced the fruits with a juicer.After the juice was extracted,it was coarsely filtered with a three-layer filter cloth,and then centrifuged at 10,000 r·min?1for 3 min.

First,catechol(1 mg·ml?1,300 μl)was added to the 50-fold diluted juice system (3.7 ml),then add the AMP-Cu nanozymes or laccase(10 mg·ml?1,1 ml)to react with stirring for different times(1 h,3 h and 5 h)at room temperature.Control groups were reacted without enzyme in the same conditions.The sample was centrifuged at 10,000 r·min?1for 3 min.And the liquid supernatant recovered was assayed.The content of total phenol was measured by Folin–Ciocalteu assay[35].

In order to study the effect of temperature on the removal of phenols by AMP-Cu nanozymes,The experimental group and the control group were stirred at different temperatures(30–90°C)for 1 h.Then the effect of pH was studied.Catechol(1 mg·ml?1,300 μl)was added to 50-fold diluted apple juice(1.7 ml)and then mixed with various pH(4–10)MES buffer solution (2 ml).Add AMP-Cu nanozymes (10 mg·ml?1,1 ml)to the above mixture and stir at room temperature for different times(1 h,3 h,and 5 h).

Fig.1.(a)TEM image of AMP-Cu nanozymes.(b)Synthetic AMP-Cu nanozymes and dispersed into the solution.(c)FTIR spectra of AMP-Cu nanozymes.

3.Results and Discussion

3.1.Characterization of AMP-Cu nanozymes

The morphology of the prepared AMP-Cu nanozymes was studied by transmission electron microscopy(TEM).As shown in Fig.1a,AMP-Cu nanozymes have a network structure and the catalytic part is at the nanoscale[34].Fig.1b shows photographs of AMP-Cu nanozymes and their dried solid under visible light.Compared with the preparation of natural laccase,the laccase mimic enzyme has the advantages of simpler preparation method,lower price and higher yield.About 60 mg of AMPCu nanozymes can be prepared in 30 min.Fig.1c shows FTIR spectra of AMP-Cu nanozymes.The intense and broad characteristic peak at 3410 cm?1occurred because of the vibration of -NH2.The spectrum showed a strong absorption peak at 1645 cm?1because of the stretching vibration of C=C.The stretching vibration of C-H was at 2800 cm?1.The peak at 1455 cm?1was due to the stretching vibration of C=O and the absorption peak at 1111 cm?1represented the C-OCu stretching vibration[36].

3.2.Catalytic activity comparison of AMP-Cu nanozymes and laccase

To test the ability of the prepared AMP-Cu nanozymes to simulate laccase,2,4-DP was used as the substrate in the presence of 4-AP.It has been reported that 2,4-DP can be oxidatively coupled with 4-AP to form an antipyrine dye for the detection of phenolic compounds[37].As shown in Fig.2a,2,4-DP,4-AP,and a mixture of 2,4-DP and 4-AP had no absorption peaks in the visible region.After adding laccase or AMP-Cu nanozymes,a new absorption peak appeared around 510 nm.These results showed that AMP-Cu nanozymes can be used as a laccase mimic.Therefore,we may use AMP-Cu nanozymes instead of laccase for phenol detection and juice clarification.

It is proved that our synthesized AMP-Cu nanozymes have the catalytic ability of laccase.We next compared the catalytic activity of AMPCu nanozymes with laccase.As shown in Fig.2b,with increasing concentrations of laccase and AMP-Cu nanozymes,the catalytic activity gradually increased.The catalytic activity of AMP-Cu nanozymes was significantly higher than that of laccase at each concentration,indicating that the catalytic activity of the simulated enzyme was better than laccase.The high catalytic activity of nanozymes may be due to the higher surface volume ratio of nano-sized materials,which exposes more active sites[38].In the subsequent experiments,the same concentration(1 mg·ml?1)of AMP-Cu nanozymes and laccase were selected for experiments.

Fig.2.(a)UV–vis spectra of 2,4-DP,4-AP,mixture of 2,4-DP and 4-AP,laccase and AMP-Cu nanozymes were added to 2,4-DP and 4-AP,respectively.The concentration of laccase and AMPCu nanozymes are 1 mg·ml?1(b)Comparison of the catalytic properties of AMP-Cu nanozymes and laccase with the enzyme concentration.

3.3.Reaction kinetics

The kinetic parameters of AMP-Cu nanozymes and natural laccase under the optimal conditions were studied.The reaction kinetics were studied at different 2,4-dichlorophenol concentrations.The Michaelis–Menten constant and the maximal velocity were obtained from the Michaelis–Menten equation.As shown in Fig.3a and b,the Kmand Vmaxof native laccase were 0.36 mmol·L?1and 0.28 μmol·L?1·min?1,respectively.Kmvalue and Vmaxvalue of AMP-Cu nanozymes were 0.09 mmol·L?1and 1.30 μmol·L?1·-min?1,respectively (Fig.3c and d).Kmis an important kinetic parameter related to the ability of enzyme to bind to substrate.The Kmof AMP-Cu nanozymes was lower than that of laccase,indicating that the simulated enzyme had stronger affinity with the substrate.The Vmaxof AMP-Cu nanozymes was 4.5 times higher than that of native laccase.The above results indicated that our synthetic AMP-Cu nanozymes had better catalytic activity than laccase at the same mass concentration and can be used in place of laccase in the food industry.

3.4.Stability comparison

We compared the temperature,pH,salts and storage stability of AMP-Cu nanozymes and laccase.The effect of temperature on the stability of the AMP-Cu nanozymes and laccase was examined by incubating at a temperature range of 30–90°C for 1 h.The results are shown in Fig.4a and indicated that the temperature had a significant impact on the stability of laccase and mimic enzyme.At temperatures above 60°C,the activity of laccase was significantly reduced,with almost no catalytic activity at 90°C.However,the activity of mimic enzyme was stable,and its activity remained above 80%at 90°C.This might be due to the high temperature destroying the structure of the laccase,which resulted in irreversible denaturation of native laccase.

The effect of pH was evaluated by storing laccase and AMP-Cu nanozymes in buffers from pH 4 to 9 for 12 h at room temperature,and catalytic experiments were then carried out at respective optimum conditions.As shown in Fig.4b,the mimic enzyme showed higher stability than free laccase in a wide pH range of 4–9,especially in alkaline solution.Probably because the higher pH changed the existence state of metal ions and then the electron transfer rate on nanozymes[39].The results indicated that AMP-Cu nanozymes had good pH-tolerance.

Fig.3.Kinetic parameters for the laccase(0.1 mg·ml?1)(a,b)and AMP-Cu nanozymes(0.1 mg·ml?1)(c,d)catalyze different concentration of 2,4-DP in the presence of 0.1 mg·ml?1 4-AP in their respective optimum conditions.(b)and(d)are double-reciprocal plots of(a)and(c),respectively.

Fig.4.Stability of the AMP-Cu nanozymes compared with laccase at different(a)temperature,(b)pH,(c)NaCl concentration(1 mg·ml?1 of laccase or AMP-Cu nanozymes as the catalysts to catalyze 0.1 mg·ml?1 of 2,4-dichlorophenol at various NaCl concentrations),and(d)storage stability of laccase and AMP-Cu nanozymes.

Next,we also compared the effects of various sodium chloride concentrations on the stability of laccase and AMP-Cu nanozymes by incubating them in different concentrations of NaCl(0–600 mmol·L?1)for 10 h at room temperature.As shown in Fig.4c,the activity of laccase decreased as salt concentration increased and only 40%of the activity was preserved at a NaCl concentration of 600 mmol·L?1.However,the activity of AMP-Cu nanozymes was not reduced,and the catalytic activity was increasing to about 1.5 times.These results may be due to the effect of high concentration of salt solution on protein activity,resulting in reduced laccase activity.For AMP-Cu nanozymes,it is possible that the salting out effect of high concentration of NaCl reduced the solubility of the substrate,making them more susceptible to be adsorbed and conversed by AMP-Cu nanozymes[34].

Storage stability for the aqueous solutions of native laccase and AMP-Cu nanozymes was studied at 4 °C for nine days as shown in Fig.4d.The activity of AMP-Cu nanozymes hardly changed in nine days,whereas the activity of laccase gradually decreased with time.On the ninth day,laccase remained less than half of the initial activity.The good storage stability of mimic enzyme was beneficial to its application in industry.

3.5.Reusability of AMP-Cu nanozymes

The greatest advantage of mimetic enzyme compared to native laccase is that it can be reused.The AMP-Cu nanozymes we prepared can be recovered by centrifugation,which is beneficial to reduce costs.It is of great significance to practical industrial production.The reusability of AMP-Cu nanozymes were studied for six cycles of 2,4-DP oxidation,and the results are shown in Fig.5.The residual activity of mimetic enzyme showed a slight decrease along the cycles,reaching about 80% of the initial activity after six continuous reaction cycles.The decreased in reusability may be due to the loss of AMP-Cu nanozymes by centrifugal operation.

Fig.5.Reusability of AMP-Cu nanozymes(1 mg·ml?1 AMP-Cu nanozymes as the catalyst to convert 0.1 mg·ml?1 of 2,4-DP in the presence of 0.1 mg·ml?1 4-AP at 25°C at pH 6.7).

Fig.6.(a)The UV–vis spectra of 1 mg·ml?1 AMP-Cu nanozymes in different concentrations of 2,4-DP.a–l represent the concentrations of 2,4-DP of 0.0001,0.0005,0.0025,0.005,0.01,0.025,0.05,0.1,0.2,0.3,0.4 and 0.5 mmol·L?1,respectively.(b)Linear relationship between 2,4-DP concentration and absorbance catalyzed by AMP-Cu nanozymes.(c)The UV–vis spectra of 1 mg·ml?1 laccase in different concentrations of 2,4-DP.a–j represent the concentrations of 2,4-DP of 0,0.0025,0.005,0.025,0.05,0.1,0.2,0.3,0.4 and 0.5 mmol·L?1,respectively.(d)Linear relationship between absorbance and concentration of 2,4-DP under the catalysis of laccase.(e)The UV–vis spectra of 1 mg·ml?1 AMP-Cu nanozymes catalyze different concentrations of 2,4-DP in fruit juice system.a–n represent the concentrations of 2,4-DP of 0.0001,0.0005,0.0025,0.005,0.01,0.02,0.025,0.05,0.075,0.1,0.2,0.3,0.4 and 0.5 mmol·L?1,respectively.(f)Linear relationship between 2,4-DP concentration and absorbance catalyzed by AMP-Cu nanozymes.

Table 1 Comparison of different methods for the determination of 2,4-DP

3.6.Detection of phenols

Laccase can catalyze the oxidation of phenolic substrates[40].Since AMP-Cu nanozymes have the catalytic properties of laccase,it can also catalyze phenolic compounds.Here,2,4-DP was used as a substrate and 4-AP as a color developing agent to measure the absorbance of the product at 510 nm for the detection of phenolic compounds.Fig.6a and c respectively showed the UV–vis spectra of different concentrations of 2,4-DP with 1 mg·ml?1AMP-Cu nanozymes and laccase in the presence of 0.1 mg·ml?14-AP.The absorbance of the system increased gradually with increasing 2,4-DP concentration.As shown in Fig.6b,it can be seen that there is a good linear relationship between the absorbance (Abs) at 510 nm and 2,4-DP concentration (c2,4-DP)under the catalysis of AMP-Cu nanozymes in the range of 0.1–100 μmol·L?1.The regression equation was Abs=0.360+6.531c2,4-DP(mmol·L?1).The corresponding regression coefficient of R2=0.996 and the detection limit (LOD) was 0.033 μmol·L?1.As shown in Table 1,our method is more sensitive than the recently reported 2,4-DP detection methods.Under the catalysis of laccase,the absorbance of the system had a linear relationship with the concentration of 2,4-DP in the range of 2.5–400 μmol·L?1.The regression equation was Abs=0.004+0.158c2,4-DP(mmol·L?1).The corresponding regression coefficient of R2=0.995,and the detection limit for 2,4-DP was 0.83 μmol·L?1.These results indicated that the mimetic enzyme we prepared for the 2,4-DP assay was more sensitive than laccase.In addition to 2,4-DP,We have also selected several representative phenolic compounds,including phenol,hydroquino,parachlorophenol,resorcinol,phloroglucinol and catechol.It can be seen from Fig.7 that these phenolic compounds can be oxidized by AMP-Cu nanozymes.Therefore,AMPCu nanozymes have substrate diversity and can be used to detect a variety of phenolic compounds.Our AMP-Cu nanozymes are non-toxic,convenient to prepare and low cost,which have broad application prospects.

Fig.7.Catalytic efficiency of AMP-Cu nanozymes for different substrates (1 mg·ml?1 AMP-Cu nanozymes as the catalyst to catalyze 1 mmol·L?1 of different substrates in the presence of 0.1 mg·ml?1 4-AP at 25 °C at pH 6.7.The absorbance is measured at 510 nm and the background is removEd.)

Furthermore,we studied the effect of AMP-Cu nanozymes on the detection of phenolic compounds in fruit juice systems.A 100-fold dilution of apple juice was used to replace the buffer solution(MES,pH=6.7).Fig.6e showed the UV–vis spectra of 1 mg·ml?1AMP-Cu nanozymes catalyze different concentrations of 2,4-DP in apple juice system.The absorbance of the system increased gradually with increasing 2,4-DP concentration.As shown in Fig.6f,it can be seen that there is a good linear relationship between the absorbance(Abs)at 510 nm and 2,4-DP concentration (c2,4-DP) under the catalysis of AMP-Cu nanozymes in the range of 0.1–100 μmol·L?1.The regression equation was Abs=0.128+7.389c2,4-DP(mmol·L?1).The corresponding regression coefficient of R2=0.992 and the detection limit(LOD)was 0.033 μmol·L?1.The AMP-Cu nanozymes in the juice system did not change the linear range and detection limit of the phenolic compounds compared to the buffer solution.It is indicated that the AMP-Cu nanozymes can still maintain good catalytic activity in the actual juice sample,which has practical significance for the direct detection of phenolic compounds in actual juice in the future.

3.7.Fruit juice clarification

Fig.8.The effect of reducing the phenol content in different juices after adding AMP-Cu nanozymes and laccase at different reaction times.(a),(b)and(c)represent the reaction time of 1 h,3 h and 5 h,respectively.The values are expressed in percentage of remove the phenol content.

Fig.9.(a)Effects of AMP-Cu nanozymes on removal of phenolic compounds from juice at different pH for 1 h,3 h and 5 h.(b)Effects of AMP-Cu nanozymes on removal of phenolic compounds from fruit juice at different temperatures.

To evaluate the phenolic compounds removal effect of the AMP-Cu nanozymes on the actual juice sample.As shown in Fig.8,we selected five different fruits(apples,pears,oranges,kiwis and blueberries)to perform phenol removal experiments on them.The pre-treated juices were incubated at room temperature with laccase and AMP-Cu nanozymes at the same mass concentration for different time(1 h,3 h and 5 h).It can be seen from the figure that as the reaction time increased,the phenol removal content of laccase and AMP-Cu nanozymes also increased.However,the content of phenol removal after adding AMP-Cu nanozymes reaction for one hour is still higher than that after adding laccase reaction for five hours,indicating that the removal rate of AMP-Cu nanozymes in the juice was faster and the effect was better.After 3 h of reaction,the content of phenol removal in juice(apple,orange,kiwifruit and blueberry juice)added with AMP-Cu nanozymes was about 15 times higher than that in juice added with laccase.Among the five fruits,laccase had the best phenolic compounds removal effect on pears,while AMP-Cu nanozymes had better phenolic compounds removal effect on apples than other fruits.These results indicated that AMP-Cu nanozymes were better than laccase for removing phenolic compounds from juice,which was beneficial to maintain the quality of juice.

The stability of natural enzymes is poor,and it is easily denatured and deactivated under the influence of external factors such as temperature and pH.Therefore,we investigated whether the effect of AMP-Cu nanozymes in removing phenolic compounds in juice was affected by temperature and pH.It can be seen from the Fig.9a that as the reaction time increased,the phenolic compounds removal content of AMP-Cu nanozymes also increased.The mimetic enzyme had a good effect of removing phenolic compounds in the pH range of 4 to 10,especially in neutral and alkaline solutions.As shown in Fig.9b,with the increase of temperature,the effect of AMP-Cu nanozymes on the removal of phenol from juice is on the rise.At 70°C,the phenol removal rate is about 80%.As the temperature continues to rise,even at 90°C,the phenol removal rate is still around 75%.This result shows that under high temperature conditions,the mimetic enzyme can effectively remove the phenolic compounds in the juice.This is of great significance for the actual production of fruit juice,because in the processing of fruit juice,high temperature treatment such as sterilization is involved,and there is no need to worry about the high temperature to inactivate the enzyme.Therefore,it is conceivable to carry out the phenolic compounds removal step together with these high temperature treatment steps,which saves time and improves the production efficiency of the juice.

4.Conclusions

In summary,a laccase mimic enzyme of a metal framework structure was prepared based on copper ions and adenosine monophosphate.It had the advantages of stronger catalytic activity and lower price than laccase.The AMP-Cu nanozymes also had a good stability,for example,the catalytic activity remains almost constant over a wide temperature range (30–90 °C).In addition,the laccase mimic enzyme could be recycled,and after 6 consecutive reaction cycles,the residual activity of the AMP-Cu nanozymes still reached about 80%of the initial activity.The concentration range of phenolic compounds detected by AMP-Cu nanozymes was 0.1–100 μmol·L?1,and the detection limit was 0.033 μmol·L?1lower than that of laccase.Therefore,it was conceivable to replace those sensors based on native laccase with our AMP-Cu nanozymes.Furthermore,the proposed mimetic enzyme also be used to remove phenolic compounds from juice and showed a better phenolic compounds removal effect than native laccase.AMP-Cu nanozymes had great potential applications in phenolic compounds detection and juice industry.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China(31772058),the Science and Technology Development Project of Jilin Province,China (20190302088GX and 20190701079GH),the Jilin Provincial Strategic Economic Infrastructure Adjustment fund(2019C043-5 and 2020C023-5)and Fundamental Research Funds for the Central Universities.