Bienzyme system immobilized in biomimetic silica for application in antifouling coatings☆

Hongwu Wang ,Yanjun Jiang ,2,*,Liya Zhou ,Jing Gao ,*

1 School of Chemical Engineering,Hebei University of Technology,Tianjin 300130,China

2 National Key Laboratory of Biochemical Engineering,Institute of Process Engineering,Chinese Academy of Sciences,Beijing 100190,China

Keywords:Antifouling Bienzyme H2O2 Coating

ABSTRACT Antifouling coatings are used extensively on vessels and underwater structures.Conventional antifouling coatings contain toxic biocides and heavy metals,which may induce unwanted adverse effects such as toxicity to non-target organisms,imposex in gastropods and increased multiresistance among bacteria.Therefore,enzyme-based coatings could be a new alternative solution.A H2O2-producing bienzyme system was developed in this study.H2O2 can be produced from starch by the cooperation of α-amylase and glucose oxidase,which promotes the hydrolysis of polymeric chain and oxidizes the glucose to produce H2O2,respectively.The encapsulated bienzyme(A-G@BS)exhibits enhanced stabilities of thermal,pH,recycling and tolerance of xylene.The A-G@BS-containing coating releases H2O2 at rates exceeding a target of 36 nmol·cm?2·d?1 for 90 days in a laboratory assay.The results demonstrate that the method is a promising coating technology for entrapping active enzymes,presenting an interesting avenue for enzyme-based antifouling solutions.

1.Introduction

Biofouling is the attachment and growth of various microscopic and macroscopic organisms[1]on the surfaces of equipment immersed in the water.Biofouling has negative effects on submerged structures,such as ships,pipelines,cables, fishing nets,pillars of bridges and oil platforms.Higher fuel consumption,declined heat exchanger performance and material corrosion are caused by increased biofouling.It is essential to use antifouling coatings[2–4].

Traditional antifouling technologies by application of antifouling paints rely on the release of toxins to kill attaching organisms[5].In many cases,metals or organometallic compounds are essential parts of biofouling inhibiting agents.However,the toxins are prone to bioaccumulation and will ultimately be harmful to the environment[6,7].Therefore,it needs to develop environmental-friendly alternatives to metal-based antifouling agents[8].Enzyme-based antifoulings are expected to be environmentally friendly.Enzymatic antifoulings are those antifouling coatings with enzymes playing an essential role in biofouling inhibiting properties[9,10].Based on the functions of enzymes in the coatings,the antifouling approaches are divided into direct and indirect ones[5].

Direct antifouling is usually based on the release of enzymes that are active antifoulants and affect the viability of fouling organisms.Based on the properties of biofouling,polysaccharide-degrading and protein-degrading enzymes are usually utilized[11–13].However,an efficient broad-spectrum direct antifouling coating based on a single or a few enzymes has not been achieved.If a particular adhesive is not degraded by the enzymes in the coating,a heavy fouling produced by a few organisms may occur and the direct adhesive degrading antifouling may be compromised.

A variety of enzymes and reactants are applied to indirect antifouling,where enzyme(s)can convert compounds in water or coating into potent antifoulants.For instance,H2O2and halide ions in the seawater can be converted to hypohalogenic acid by haloperoxidase in an antifouling coating[14].Kiil et al.[15]improved this approach by adding H2O2into the coating.Poulsen and Kragh[16]used a coupled enzyme system to produce H2O2,the toxicity of which can deter fouling organisms.It is essential for an enzyme-activate coating that enzymes with high catalytic capability can be retained in the coating for a period of time[17].Thus,a biomimetic encapsulation procedure for hexose oxidase through polyethylenimine-templated silica co-precipitation was employed to improve the stability and performance of antifouling system[18],but only hexose oxidase was encapsulated and the stability of α-amylase was not improved.

Among different features proposed for enzymatic antifouling,the stability of enzymes in the enzymatic antifoulings is the key problem in their practical applications.In order to improve the stability of enzymes in the coating,in this study,α-amylase and glucose oxidase(bienzyme system)are encapsulated in silica by a biomimetic silicification process.This system can produce H2O2from starch,with α-amylase promoting the hydrolysis of polymeric chain and glucose oxidase(GOD)oxidizing the glucose to produce H2O2,as shown in Fig.1.

In contrast to the previous immobilized enzyme,advantages of this encapsulated bienzyme system are as follows.

(1)Co-encapsulation of the bienzyme can significantly improve the stability of enzymes,so the encapsulated enzymes can maintain an adequate catalytic activity over an extended time-period in the antifouling coating.

(2)In the sequential catalytic reactions of α-amylase and GOD,the product(glucose)of α-amylase is the substrate of GOD.With the co-immobilization of the two enzymes in the system,starch converts to glucose catalyzed byα-amylase and glucose converts to H2O2,enhancing the yield of H2O2.

2.Experimental

2.1.Materials

Poly dimethyl diallyl ammonium chloride[PDADMA,MW100000–200000,20%(by mass)in H2O],α-amylase(23 U·mg?1),glucose oxidase(GOD,5000 U·mg?1),4-aminoantipyrine,phenol,starch,hydrogen peroxide and tetramethoxysilane(TMOS)were obtained from Sigma Chemical Company.Horse radish peroxidase(HRP)(＞150 U·mg?1)was obtained from Shanghai Source Leaf Biological Technology Co.,LTD.

2.2.Encapsulation of α-amylase and GOD in biomimetic silica

TMOS was hydrolyzed in 1 mmol·L?1hydrochloric acid and mixed with an equal amount of PBS(pH=7).The resulting solution was added to the solution(bienzyme and PDADMA mixed in a volume ratio of 1:1)with the volume ratio of 1:10.The mixed solution turned turbid within minutes due to the formation of precipitate and the precipitate was collected via centrifugation at 5000 r·min?1for 5 min.The precipitate was washed with the PBS(pH=7)followed by centrifugation at 5000 r·min?1,repeated for three times.The encapsulated α-amylase and GOD in the biomimetic silica was named A-G@BS.α-Amylase of 4 mg·ml?1and glucose oxidase of 0.3 mg·ml?1were the optimal concentration of bienzyme,A-G@BS was obtain from 0.5 ml bienzyme and the recovery of bienzyme activity was 85.12%.The morphological characterization of composites was observed by scanning electron microscopy(SEM).

2.3.Determination of bienzyme activity

A bienzyme solution of α-amylase(4 mg·ml?1)and glucose oxidase(0.3 mg ·ml?1)was prepared.The concentration of TMOS and PDADMA was 0.4 mol·L?1and 6 mg·ml?1,respectively.The activities of bienzyme and A-G@BS were obtained by measuring the initial release rate of H2O2with a visible infrared spectrometer at 500 nm.0.25 mg of 4-amino diantipyrine,0.25 mg of HRP,0.1 ml of 3%phenol,and 2 ml of 4%starch solution were mixed with 2 ml of PBS(0.1 mol·L?1,pH 7).Then the bienzyme(0.5 ml)or A-G@BS(obtained from 0.5 ml bienzyme)was added to the above mixture solution.The increase of absorbance at 500 nm was measured to determine enzymatic activity[19].

2.4.Bienzyme stability experiments

The thermal stability of free enzyme and A-G@BS was determined by incubating at30°C and 50°C for0,60,180,300,420 and 540 min.For the pH stability,free enzyme and A-G@BS were incubated in buffer solutions at pH 4.5,7.0 and 9 at 50°C for 0,30,60,90,120,150,180 and 210 min.The relative activity was calculated.

A-G@BS was collected by centrifugation after each reaction(30°C,pH=7.0),thoroughly rinsed with buffer solution and reused in the next reaction cycle.The recycling stability of A-G@BS was evaluated by measuring the enzyme activity in each successive reaction cycle expressed by recycling efficiency defined as follows.

The influence of xylene on the activities of free enzyme and A-G@BS was also examined.Both enzymatic preparations were carried out in 1 ml of xylene at 30°C for 0,30,60,90,120,150 and 180 min.

2.5.Construction and application of antifouling coating

In this study,the enzymatic antifouling will be discussed in the light of intended action of enzymes.In order to incorporate A-G@BS and starch into a coating,A-G@BS was formulated as a dry powder by vacuum freeze-drying method.A-G@BS and starch were mixed adequately and then uniformly distributed in the coating.A coating mixed with starch(without enzymes)was used as an enzyme-free reference coating.Enzyme-containing coating was prepared by mixing with 70% antirust paint,6%A-G@BS,and 24%starch.The coatings were applied to sheet iron and submerged in water after dry in the air.

The plates were immersed in beaker with 200 ml distilled water for 0 to 90 days,comparing the release rate of hydrogen peroxide from enzymatic coating.The plates were immersed in the river for 10,20,40 and 80 days,comparing the growing states of biological fouling with time.

3.Results and Discussion

3.1.Characterization of A-G@BS

The particle size of fillers affects the structure and hydrodynamic properties of coated hull,so from a coating technology point-of-view,A-G@BS particles in the paint should not be too large.The particle size of A-G@BS was analyzed by SEM.As shown in Fig.2,the particle is irregular and presents a rough surface and its diameter is about10μm,smaller than the starch granules(about 15 μm)[20,21].Therefore,the A-G@BS will not alter the surface structure of coating.

3.2.Thermal stability of enzymes

Fig.1.Reaction scheme for bienzyme system to produce hydrogen peroxide.

Fig.2.SEM image of A-G@BS particle.

Fig.3.Thermostability of free enzyme and A-G@BS.

For antifouling,A-G@BS must be able to maintain an adequate catalytic activity over an extended time-period at different temperatures.As shown in Fig.3,86.59% of the initial activity of A-G@BS and 85.03% of the free enzyme activity were retained with incubation at30°C for 540 min,which were 66%and 46%,respectively,with incubation at 50°C.Generally,higher temperature can increase thermal motions of enzyme molecules,which would disrupt the covalent bonds that hold the protein structure together and lead to enzyme denaturation[22].Thus the enhanced thermal stability of A-G@BS may be partly explained by the prevention of subunit dissociation via the encapsulation process.

3.3.pH stability of enzymes

Fig.4.The pH stability of free enzyme and A-G@BS at 50°C.

Fig.4 shows the activities of free enzyme and A-G@BS at pH 4.5,7.0 and 9.0.The pH stability is enhanced after encapsulation,maintaining high activity.Under the acidic condition(pH 4.5),the relative activity of A-G@BS reached 66%,while its free counterpart remained 60%;under the alkali condition(pH 9.0),they are 53%and 46%,respectively,suggesting that the A-G@BS is less affected by pH change.The strong resistance of A-G@BS against acidic and alkaline conditions in medium is tentatively explained by the confinement effect of silica cages,in which the pH change is envisaged to be much smaller than that in the bulk solution,providing a milder microenvironment for the enzyme[23].Additionally,the silica cages inhibit to some extent the unfolding–refolding motions of the enzyme inside and preserve its activity.

3.4.Recycling stability of A-G@BS

As shown in Fig.5,77.03%activity of A-G@BS was maintained after recycling 7 times.It may be conjectured that the encapsulation effectively prevents enzyme from leaking.The relatively high recycling efficiency of the bienzyme system is benefited from the protection of silica matrix[24].The slight loss of A-G@BS activity may be attributed to the breakage of particles after multiple operations with continuous stirring[25].

Fig.5.Recycling stability of A-G@BS.

3.5.Stability in xylene

Solvents play an important role in the paint,and xylene is a common organic solvent of painting[26],so the influence of xylene on the activity of A-G@BS was investigated.The enzyme molecule is surrounded by a water-shell with H-bonds,which is the important prerequisite of enzyme inactivation in organic solvent.This hydration shell is an integral part of the enzyme structure and essential for enzyme function.Consequently,in the organic solvent,the bound water is displaced resulting in a denaturation of enzyme structure[27].As shown in Fig.6,in 1 ml of xylene,the activity of free enzyme decreases sharply,while the stability of A-G@BS is improved after encapsulation.It is reasonably believed that the A-G@BS would exhibit a distinct advantage over free enzymes in protecting the spatial structure of enzyme molecules by silica cages.

Fig.6.Stability of free enzyme and A-G@BS in xylene at 30°C.

Fig.7.The release rate of H2O2 from enzymatic coating,mixed dried A-G@BS and starch into paint with 25%(by mass)of wet formulation.

3.6.Application of antifouling coating

The application of antifouling coating relies on the toxicity of H2O2,which can cause oxidative damage to cells.The coating is commonly divided into soluble and insoluble matrices.Soluble matrix antifoulings are based on physical dissolution of rosin into water.The sooner it can trigger the release of H2O2,the shorter the antifouling time of coating.In this study,insoluble matrices are used,which refers to contact leaching and continuous contact coatings.The dissolution of the bienzyme system creates a porous structure in coating because the matrix remains undissolved,increasing the mechanical stability.By the close reaction of A-G@BS,starch and water,coating can give a sequential effect of kill microorganisms by the release of H2O2.

3.6.1.Releasing rate of H2O2 from antifouling coating

Fig.8.Result of antifouling coating.(a)Enzyme-free coating;(b)enzyme-containing coating.

To evaluate the antifouling potential of the coating,the release rate of H2O2is important.A target H2O2release rate of 36 nmol·cm?2·d?1has been proposed in a patent by Cape Cod Research[28].Furthermore,Nippon Paint Co.has detailed a H2O2release rate of 21 nmol·cm?2·d?1[29].The H2O2release rate of our coating with a thickness of 300μm exceeds 36 nmol·cm?2·d?1for approximately 90 days(Fig.7).Without significant self-polishing,the active enzymes in the coating can be expected to have a lifespan of 90 days.

3.6.2.Result of antifouling coating

To illustrate the progress and differences in the trial setup,plates without and with A-G@BS were tested in the north canal of Tianjin(the velocity of water was about 0.5 m·s?1and the temperature was about 25°C)with an immersion depth of 1 m.The A-G@BS-containing coating is compared to the enzyme-free reference coating,with images of plates from10 to 80 as shown in Fig.8.The observations of fouling development showed that the enzyme-containing coating remained relatively clean after immersion for 10 days,while the reference coating was covered by many parts of biofouling.After 40 days,notably denser settlement of biofouling appeared on the enzyme-free reference coating,while the fouling on the A-G@BS-containing coating only increased slightly.After 80 days,the reference coating was covered by all kinds of fouling,while a little fouling appeared on the A-G@BS-containing coating.The results demonstrate that the method developed in this study is a promising coating technology for entrapping active enzymes,presenting an interesting avenue for enzyme-based antifouling solutions.

4.Conclusions

A bienzyme system was immobilized by physical entrapment using the biomimetic method for antifouling applications,which can provide essential aqueous micro-environment to the immobilized enzyme and preserve the native activity.Therefore,an environmentally friendly antifouling based on enzyme can be developed.The enzyme activity in the coating can be retained for a long time(90 days).The A-G@BS-containing coating is able to resist the initial colonization of microscopic and macroscopic organisms.The results suggest that biomimetic silicabased coating is a promising technology for immobilization of enzymes and have a great potential in antifouling applications.