Conjugation of Candida rugosa lipase with hydrophobic polymer improves esterification activity of vitamin E in nonaqueous solvent

Xiaoyun Hou,Qinghong Shi,2,3,

1 Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China

2 Key Laboratory of Systems Bioengineering and Frontiers Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin 300350, China

3 Haihe Laboratory of Sustainable Chemical Transformations, Tianjin 300192, China

Keywords:Candida rugosa lipase Polymers Biocatalysis Esterification Vitamin E succinate Activity

ABSTRACT We described a novel polymer-lipase conjugate for high-efficient esterification of vitamin E using vitamin E and succinic anhydride as the substrates in nonaqueous media.In this work,the monomer,N-isopropylacrylamide (NIPAM),was grafted onto Candida rugosa lipase (CRL) to synthesize poly(NIPAM) (pNIPAM)-CRL conjugate by atom transfer radical polymerization via the initiator coupled on the surface of CRL.The result showed that the catalytic efficiencies of pNIPAM-CRL conjugates (19.5–30.3 L∙s-1∙mmol-1) were at least 7 times higher than that of free CRL (2.36 L∙s-1∙mmol-1) in DMSO.It was attributed to a significant increase in Kcat of the conjugates in nonaqueous media.The synthesis catalyzed by pNIPAM-CRL conjugates was influenced by the length and density of the grafted polymer,water content,solvent polarity and molar ratio of the substrates.In the optimal synthesis,the reaction time was shortened at least 7 times,and yields of vitamin E succinate by pNIPAM-g-CRL and free CRL were obtained to be 75.4%and 6.6%at 55°C after the reaction for 1.5 h.The result argued that conjugation with pNIPAM induced conformational change of the lid on CRL based on hydrophobic interaction,thus providing a higher possibility of catalysis-favorable conformation on CRL in nonaqueous media.Moreover,pNIPAM conjugation improved the thermal stability of CRL greatly,and the stability improved further with an increase of chain length of pNIPAM.At the optimal reaction conditions (55 °C and 1.5 h),pNIPAM-g-CRL also exhibited good reusability in the enzymatic synthesis of vitamin E succinate and kept～70%of its catalytic activity after ten consecutive cycles.The research demonstrated that pNIPAM-g-CRL was a more competitive biocatalyst in the enzymatic synthesis of vitamin E succinate and exhibited good application potential under harsh industrial conditions.

1.Introduction

Vitamin E,one of the important liposoluble antioxidants,is essential for human health,anti-aging,disease prevention and inflammation elimination [1–6].Native vitamin E is of great economic value due to its antioxidant properties and biological activity.It includes four tocopherols and four tocotrienols,α-tocopherol being the most important biologically active form and either prepared by total chemical synthesis or biosynthetic pathway [7].Since Karrer and Isler [8] firstly disclosed total synthesis of α-tocopherol by condensing trimethylhydroquinone with phytyl bromide,total synthesis and partial synthesis from methyltocols have become major routes to the industrial production of tocopherols [9].However,α-tocopherol and other stereoisomers of vitamin E are unstable and easily oxidized in the presence of light and singlet oxygen.Therefore,vitamin E is usually esterified (e.g.,vitamin E acetate or vitamin E succinate) to protect phenol group against oxidation [10].Vitamin E ester could easily be hydrolyzedviathe pancreatic esterase in the intestine.Industrially,chemical esterification of vitamin E is achieved using a Lewis acid and an organic base as the catalysts [11].It usually suffers from serious problems including harsh conditions,slow reaction rate,acid and caustic corrosion,consumption of a large amount of acid and so on.

Enzymatic synthesis of vitamin E esters provides economic perspective due to mild reaction condition,high selectivity and environmental friendliness.Because vitamin E is a highly lipophilic molecule with a low water solubility,enzymatic esterification is always conducted in nonaqueous media.As the biocatalyst in the esterification,lipase had vast application in enzymatic esterification and other non-aqueous synthesis owing to wide substrate specificity and enantio-selectivity [12].On the surface of lipase,hydrophobic active pocket is covered by a mobile ‘‘lid” domain with one or more helices to protect the active site [13,14].In the presence of hydrophobic interfaces,the lid is predominantly or partially opened in accordance with increased activity [13].However,the lid is essentially in the closed conformation in organic solvents[15].It was an unfavorable conformation for lipase-catalyzed reaction,indicating a longer reaction time and less stability during enzymatic synthesis of vitamin E esters in nonaqueous media.Moreover,the hydration layer on the surface of lipase is unfavorable for mass transfer of hydrophobic substrates to reach active site of the enzyme [16,17].It also reduces the diffusion of product into bulk solvent,leading to product accumulation in the hydration layer.In the esterification catalyzed by lipase,water,ester and acid accumulation always enhance reversible reaction[17,18] and product inhibition[16],and resulted in enzyme inactivation(due to a low pH microenvironment) [17].The catalytic efficiency of lipase in nonaqueous solvents was always 2–6 orders of magnitude lower than that in aqueous solvents[19].Torreset al.[10]reported enzymatic acylation of the phenolic group of tocopherols(vitamin E)by transesterification with vinyl acetate in 2-methyl-2-butanol as the solvent and the reaction was catalyzed byCandida antarcticalipase B (Novozym 435) screened from 15 hydrolases.The result indicated that δ-tocopherol had a higher acylation rate than α-tocopherol,and the yield reached 65%after approximate 16 days.A lower yield(46.95%)was obtained by Jianget al.[20]in the esterification of vitamin E succinate at 55°C for 18 h reaction usingCandida rugosalipase (CRL) as the catalyst.Moreover,the influence of solvents on esterification yield was also investigated by Yinet al.[21],and the highest yield of vitamin E ester was obtained in dimethyl sulfoxide (DMOS) among various nonaqueous media.For enzymatic esterification of vitamin E,therefore,it is great challenge to improve catalytic activity and stability in nonaqueous media [22].

In last decades,several strategies have been developed for improving lipase performance and stability in the enzymatic esterification.These strategies include immobilization [23],sitedirected mutation [24],chemical modification [25] and so on.Among them,lipase immobilization on hydrophobic carriersviainterfacial activation is the paradigm [26],and has extensively been studied for several decades to enhance catalytic performance and increase enzyme stability [27–29].On the other hand,chemical modification has been rekindled as a complementary strategy in the esterification of vitamin E.In the previous research reported by Yinet al.[21],Novozym-435 was modified with acetic,propionic and succinic anhydride in DMSO respectively,and the modified enzyme exhibited a much higher yield of vitamin E succinate than free Novozym-435 after reaction for 48 h.Besides small molecule reagents,polymer with high molecular weight is also studied extensively in chemical modification of lipase.Geet al.[30]conjugated hyper-branched aromatic polyamides with CRL by graftingto method and the conjugate exhibited a significantly enhanced stability and an increased activity at high temperature or in the presence of organic solvent.Currently,grafting-from and grafting-through methods have also been applied in the synthesis of polymer-enzyme conjugates [31–33].Compared with graftingto method,grafting-from method employing atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer polymerization always had great advantage in polymer architecture grafted on enzyme molecules.Since it was firstly reported in the synthesis of polymer-protein conjugate by Leleet al.[34],polymer-lipase conjugatesviagrafting-from approach have been studied by many research groups.Averick and coworkers [35] synthesized ever polymer-lipase conjugateviaphotoinduced electron transfer RAFT using two acrylamide monomers with different hydrophobicity.The result showed that polymer composition had a significant impact on enzyme activity and such influence was not universal between two different lipases.Recently,Chenet al.[36] reported that CRL conjugates with zwitterionic-hydrophobic copolymers increased catalytic activity and stability in aqueous system,demonstrating vital importance of zwitterionic/hydrophobic balance to enzymatic performance.In our experience,however,the application of polymer-lipase conjugates to the esterification of vitamin E in nonaqueous media has not been observed so far.

CRL is well-characterized with lid structure and tunnel for accommodation of substrates [14],and has been investigated by our group as model enzyme in aqueous media[36,37].In this work,N-isopropylacrylamide(NIPAM),a monomer,was conjugated with CRLviaATRP to synthesize polymer-CRL conjugates for the investigation of enzymatic synthesis of vitamin E succinate in nonaqueous media as presented in Fig.1.The influence of polymer chain and density,substrate concentration and temperature on enzymatic performance were discussed in detail.The research not only provided a high-efficient enzymatic synthesis of vitamin E succinate and but helped us deep understanding about the contribution of polymer chain in nonaqueous biocatalysis.

2.Experimental

2.1.Materials

CRL was purchased from Sigma-Aldrich(St.Louis,MO).Succinic anhydride,vitamin E andN-hydroxy succinimide(NHS) were purchased from Shanghai Aladdin Biochemical Technology Co.,Ltd(Shanghai,China).Vitamin E succinate were received from Shanghai Yuanye Bio-Technology Co.,Ltd (Shanghai,China).NIPAM was product obtained from Acros Organics(current Thermo Fisher Scientific,Geel,Belgium).Dimethyl sulfoxide(DMSO),acetic acid and ethanol absolute were the products of Tianjin Jiangtian Chemical Co.(Tianjin,China).Tri(2-dimethylaminoethyl) amine was received from Hynes Bioechnology Co.Ltd (Hefei,China).Acrylamide solution (40%),tetramethylethylenediamine (TEMED,ammonium persulfate (AP),tris(2-dimethylaminoethyl)amine(Me6TREN),triethylamine (TEA)and other reagents were obtained from local distributors.

2.2.Synthesis and characterization of macromolecular initiator

In this work,N-(bromoisobutyryloxy) succinimide (BIBS) was firstly synthesized according to the method as reported previously[37].After 4.45 g NHS and 7.16 ml TEA were dissolved into 100 ml DCM,the solution was incubated in an ice bath,and purged with nitrogen gas.Then,379 mg∙ml-1BIBB in dichloromethane (DCM,20 ml) was added drop by drop in 45 min.The reaction continued for 3 h.After the reaction was terminated by mixing with 200 ml ice-water mixture,the settling organic layer was collected and washed with 0.1 mol∙L-1NaHCO3solution and water.After residual water was dried by MgSO4,DCM was removed by vacuum rotary evaporation,and the product was obtained by recrystallization with hot ether.The product was characterized with electrospray ionization (ESI) mass spectrometry.

Macromolecule initiator was prepared by coupling BIBS onto CRL as presented in Fig.1.In brief,400 mg CRL powder was transferred into 50 mmol∙L-1phosphate buffer(20 ml,pH=7.0),and the solution was centrifuged to remove the insoluble substance.After 60 mg BIBS in 400 μl DMSO was mixed with 0.9 ml deionized water,the mixture was added slowly into CRL solution.The reaction was carried out in in an air bath at 4 °C and 120 r∙min-1for 48 h.The product was dialyzed with a dialysis bag with a MWCO of 14 kDa at 4°C for 48 h to remove small-molecule reactants.Finally,the macromolecular initiator was collected by freeze-drying,and denoted as CRL-Br.The initiator density on CRL was adjusted by the amounts of BIBS (30,50 and 90 mg in 400 μl DMSO) as listed in Table 1,and measured by fluorescamine method [38].

Fig.1. Synthesis process of pNIPAM-g-CRL conjugates via ATRP and enzymatic synthesis of vitamin E succinate.

2.3.Synthesis of pNIPAM-CRL conjugates

pNIPAM-CRL conjugates was synthesizedviaATRP using CRL-Br as the initiator.Prior to the polymerization,40 mg CRL-Br and 199 mg NIPAM were transferred in 25-ml Erlenmeyer flask,and purged with nitrogen gas for at least 20 min.At the same time,1 ml phosphate buffer in 1.5-ml centrifugal tube was purged with nitrogen gas for 5 min,and then 12.6 mg CuBr and 23.5 μl Me6TREN were added.The mixture was purged continuously with nitrogen gas for 5 min,and then transferred into the flask.After mixed well,the mixture was purged with nitrogen gas for 20 min and sealed for reaction in a water bath at 4 °C and 120 r∙min-1for 20 h.The product was exposed to atmosphere for at least 3 h to terminate the reaction.Then,the product was transferred into a dialysis bag with an MWCO of 7 kDa,and the sealed bag was immersed into 1% EDTA solution for 24 h and deionized water for 48 h at 4 °C.The final product was collected by freeze-drying.In this work,fivepNIPAM-CRL conjugates were synthesized,and chain length ofpNIPAM-CRL conjugates was adjusted by the monomer amounts(98,199 and 398 mg in Table 1)added in the polymerization.

2.4.Characterization of macromolecular initiator and pNIPAM- CRL conjugates

The modification degree of amino group in macromolecular initiator was determined by fluorescamine assay as described previously [39,40].Briefly,sample solution was prepared by dissolving CRL-Br and CRL into 50 mmol∙L-1phosphate buffer(pH 8.0)to final protein concentration ranging from 0.05–0.4 mg∙ml-1.Then,200 μl sample solution was transferred in triplicate to 96-well plate,and 2.0 mg∙ml-1fluorescamine in DMSO (20 μl) was added.After the mixture was reacted in the dark for 20 min,fluorescence emissionintensity at 475 nm (excited at 390 nm) was recorded with Tecan Infinite M200 Pro fluorescence plate reader (Salzburg,Austria).

Table1 Composition of five conjugate samples

In this work,pNIPAM-CRL conjugates and CRL were characterized with size exclusion chromatography (SEC) and SDS-PAGE.SEC analysis was conducted in TSK-gel G4000 pwxl column(7.8 mm I.D × 30 cm,10 μm) at a flow rate of 1 ml∙min-1on Agilent 1100 liquid-phase chromatography system (Santa Clara,CA).In the measurement,deionized water was used as the mobile phase,and the eluent was detected at 280 nm.SDS-PAGE analysis was carried out in 10%polyacrylamide gels at a constant voltage of 80 V.Attenuated total reflection Fourier transform infrared (ATRFTIR) spectra of NIPAM,CRL andpNIPAM-g-CRL were recorded in dry state on PerkinElmer Spectrum 100 FTIR spectrometer (Melville,NY) in the range of 4000–400 cm-1.In this work,BCA assay was applied to determine enzyme concentrations of CRL andpNIPAM-CRL conjugates.

2.5.Enzymatic esterification of vitamin E with succinic anhydride

2.5.1.Enzymatic synthesis of vitamin E succinate

In this work,vitamin E succinate was synthesized by enzymatic esterification of succinic anhydride with vitamin E in DMSO.Prior to the experiment,a certain amount ofpNIPAM-CRL conjugates or free CRL was dissolved in 50 mmol∙L-1phosphate buffer (pH 7.0),and then the enzyme solution was freeze-dried for optimization of the enzyme catalysis in non-aqueous media [41].AfterpNIPAMCRL conjugates or CRL was transferred into a 2-ml brown centrifugal tube containing 0.2 mol∙L-1vitamin E and 0.6 mol∙L-1succinic anhydride in DMSO(1.0 ml).The enzyme amounts ofpNIPAM-CRL conjugates and free CRL were 5.43 mg and 4.0 mg,respectively.The reaction was performed at 55 °C and 180 r∙min-1in a water bath.In the experiment,samples were taken at 0.5,1.5,4,8,12 h and diluted 25 times with absolute ethanol for HPLC analysis.The reaction was finally terminated by a 25-times dilution with absolute ethanol.The product was analyzed in Welch Ultimate XB-C18 column (4.6 mm × 250 mm,5 μm) from Welch Materials Inc (Shanghai,China) using methanol-acetic acid mixture with a volume ratio of 50: 0.3 as the mobile phase.In the measurement,10 μl sample was injected at a flow rate of 1 ml∙min-1and signal was monitored at 285 nm.As a control,6 mgpNIPAM and a mixture of 4 mg CRL and 6 mgpNIPAM (denoted as CRL +pNIPAM),rather thanpNIPAM-CRL conjugates or CRL,were added.

2.5.2.Measurement of kinetic parameters

In the esterification of vitamin E with succinic anhydride,succinic anhydride binds first to the enzyme quickly,forming enzyme-acyl acetic acid intermediate,and but no product was released from the active site [42,43].Then,vitamin E binds to the intermediate,and transforms into vitamin E succinate.It is a rate-limiting step and only one product was released.Large molecule substrate diffused at the active site slowly and further worse rate-limiting step.Assuming enzyme-acyl intermediate generated very rapidly,Michaelis-Menten model could be applied in the esterification of vitamin E with succinic anhydride by neglecting substrate inhibition.The enzymatic kinetics of esterification by CRL andpNIPAM grafted CRL were determined at substrate concentrations of 0.1,0.125,0.15,0.2,0.4,0.6,0.8 mmol∙L-1for vitamin E.Kinetic parameters were determined by fitting kinetic data with the following equation,

whereVis the reaction rate (mmol∙L-1∙s-1);Vmaxis the maximum reaction rate (mmol∙L-1∙s-1);Kmis the Michaelis constant(mmol∙L-1);[S] is the substrate concentration (mmol∙L-1).

The conversion number was calculated by Eq.(2),

where [E] is the enzyme concentration in the reaction (mmol∙L-1);Kcatis the reaction rate constant (s-1).

2.5.3.Thermal stability assay

Stability of CRL andpNIPAM-CRL conjugates was measured at 30–60 °C.A certain amount of the enzymes in 50 mmol∙L-1phosphate buffer(pH 7.0)was incubated in a water bath.After 4 h,the residual activity was determined at 55 °C using vitamin E as the substrate as described in Section 2.5.1.Moreover,thermal stability was also measured at different incubation time to investigate the influence of times.A certain amount of the enzymes in 50 mmol∙L-1phosphate buffer (pH 7.0) was incubated in water bath at 55 °C.Aliquots of sample were withdrawn at 0.5,2,3,6,12 and 24 h,and the residual activity was determined at 55 °C using vitamin E as the substrate as described in Section 2.5.1[44].As a control,enzyme activity at 0 h was set as 100%.

2.5.4.Influence of water content and additives

The catalytic activity ofpNIPAM-CRL conjugates was also measured in DMSO with different water contents.In the experiment,pure DMSO or 0.1%,1%and 5%water in DMSO were used as the solvent,and the activity ofpNIPAM-CRL conjugates and free CRL were determined at 55 °C using vitamin E as the substrate as described in 2.5.1.Moreover,different volumes ofn-hexane was mixed with DMSO to final concentration of 0,1%,5%and 10%.The mixture was used as the solvent for enzymatic synthesis of vitamin E succinate to evaluate the influence of solvent polarity the activity ofpNIPAM-CRL conjugates and free CRL inn-hexane/DMSO mixtures were determined at 55 °C using vitamin E as the substrate as described in Section 2.5.1.

2.5.5.Reusability of pNIPAM-CRL conjugates

The activity ofpNIPAM-CRL conjugates was measured in 10 cycles to evaluate its reusability in enzymatic synthesis of vitamin E succinate.In brief,a certain amount ofpNIPAM-CRL conjugates was mixed with substrate solution and the enzymatic esterification bypNIPAM-CRL conjugates was carried out as described in Section 2.5.1.After each cycle,pNIPAM-CRL conjugates was collected by centrifugating at 10000 r∙min-1and then fresh substrate solution with the same volume(1.0 ml)for the next reaction cycle.As a control,the activity ofpNIPAM-CRL conjugates in the first cycle was set as 100%,and the relative activity of the enzyme in following cycles was obtained by comparing to that in the first cycle.

3.Results and Discussion

3.1.Characterization of macromolecular initiator and pNIPAM-CRL conjugates

Fig.2 shows the mass spectrum(MS)of BIBS.The result showed that two major peaks were observed atm/z285.97 andm/z 287.97.Because the theoretical molecular weight of BIBS is 262.98,the spectrum of BIBS atm/z285.97 corresponded to sodium cluster,(BIBS)∙Na+.It was attributed to positive ion electrospray ionization of BIBS.Since bromine has only one stable isotope (81Br isotope)with an isotope ratio of 1:1,another peak atm/z287.97 corresponded to sodium cluster with a bromine isotope.Therefore,it can be confirmed that BIBS is successfully synthesized.

The modification degree of amino group in CRL-Br was determined by a number change of free amino group on CRL during BIBS coupling.In this work,three macromolecular initiators with different modification degrees were synthesized by adding different amounts of BIBS in the coupling as listed in Table 1.The result in Table 2 showed that the number of amino groups on macromolecular initiators decreased with an increase of BIBS amount in the coupling,indicating a successful synthesis of macromolecular initiator.Based on the sequence analysis,CRL contained 20 lysine residues and one α-amino group at N-terminus.BIBS coupling led to a decrease of amino group on CRL to 18 for CRL-Br-L,13 for CRL-Br and 9 for CRL-Br-H,corresponding to the modification degrees of 3 for CRL-Br-L,8 for CRL-Br and 12 for CRL-Br-H.

In this work,pNIPAM-CRL conjugates with different chain lengths and densities were synthesized for the investigation of their performance in enzymatic esterification.Representative FTIR spectra of CRL,pNIPAM andpNIPAM-g-CRL are shown in Fig.3.FTIR spectrum ofpNIPAM in Fig.3 clearly showed characteristic peaks at 1670 cm-1,1530 cm-1and 1260 cm-1corresponding to the amide group C=O (amide I) stretching peak,the N-H (amide II) group bending peak and C—N stretching peak.These characteristic peaks were also evident in FTIR spectrum ofpNIPAM-g-CRL.Compared with FTIR spectrum ofpNIPAM,broad peak around 3280 cm-1was observed in CRL andpNIPAM-g-CRL due to -OH stretching vibration.It corresponded to water in the hydration layer on the surface of CRL.

Fig.2. ESI mass spectrum of BIBS.

Table2 Modification degree of macromolecular initiators

Fig.3. FTIR spectra of CRL, pNIPAM and pNIPAM-g-CRL.

Fig.4 shows SEC chromatograms and SDS-PAGE results of several representative conjugates with different chain lengths.Among CRL and three conjugates,the longest retention time was obtained for CRL,and the retention time of the conjugates decreased with an increase of chain length in Fig.4(a).It indicated a successful grafting of the monomer,NIPAM,on the surface of CRL.Further evidence topNIPAM-CRL conjugates is obtained by SDS-PAGE in Fig.4(b),(c).It revealed more details onpNIPAM-CRL conjugates.In the image of SDS-PAGE,there was only one band for CRL (lanes 4 in Fig.4(b)and lane 2 and 4 in Fig.4(c))with a molecular weight less a little than 60 kDa.It was consistent with the theoretical molecular weight of 57.6 kDa (534 residues in CRL) and previous report [30].In Fig.4(b),the conjugates in lanes 1–3 shifted to higher molecular weights characterized as slower mobilities in electrophoresis.The shorter chain length inpNIPAM-CRL conjugates,the larger the mobility of the conjugates.The result in Fig.4(c) showed that mobility ofpNIPAM-CRL conjugates in lanes 1,3 and 5 likely decreased with an increase of modification degree of the conjugates.Furthermore,all the conjugates in PAGE gel were typical of polydispersity characteristics.Because ATRP was well known as its excellent controllability to chain length and dispersity of polymers [45],the polydispersity ofpNIPAM-CRL conjugates should be attributed to poor mixing in a water bath during the polymerization [46].

Protein contents in crude CRL andpNIPAM-CRL conjugates were determined with BCA method,and esterification activity of CRL andpNIPAM-CRL conjugates were measured at 55°C using vitamin E as the substrate.The results are listed in Table 3.It was seen that the conjugation ofpNIPAM onto CRL led to a significant increase of apparent activity recovery.As listed in Table 3,recovery of esterification activity forpNIPAM-g-CRL was much higher than 100% in DMSO.Clearly,it was attributed more to a significant environmental change on the surface of CRL induced by hydrophobicpNIPAM,rather than simply an increase of purity for target enzyme [47].

3.2.Enzymatic synthesis of vitamin E succinate by pNIPAM-g-CRL

Fig.4. The Characterization of pNIPAM-CRL conjugates with (a) SEC and (b),(c) SDS-PAGE.(a) Size-exclusion chromatographic results,(b) SDS-PAGE images of CRL and pNIPAM-CRL conjugates with different chain lengths.M,Protein marker;Lane 1,pNIPAML-g-CRL;Lane 2,pNIPAM-g-CRL;Lane 3,pNIPAMH-g-CRL;Lane 4,CRL.(c)SDS-PAGE images of CRL and pNIPAM-CRL conjugates different modification degrees.M,Protein marker;lanes 2 and 4,CRL;Lane 1, pNIPAM-g-CRL-L;Lane 3, pNIPAM-g-CRL;Lane 5,pNIPAM-g-CRL-H.

Table3 Protein content,activity recovery and kinetic parameters of CRL and pNIPAM-CRL conjugates

Fig.5. Effect of molar ratio of vitamin E to succinic anhydride on the yield of vitamin E succinate.

In this work,pNIPAM-CRL conjugates were synthesized for the enzymatic synthesis of vitamin E succinate using vitamin E and succinic anhydride as the substrates.The influence of substrates molar ratio on yield is shown in Fig.5.In order to increase efficiency of the key substrate,enzymatic esterification is always carried out using excess succinic anhydride [21].It was seen in Fig.5 that the yield of vitamin E succinate bypNIPAM-g-CRL increased with a decrease in molar ratio of vitamin E to succinic anhydride and the maximal yield was obtained at four-times excess succinic anhydride(corresponding to a molar ratio of vitamin E to succinic anhydride of 1:5).It was consistent with the previous result reported by Yinet al.[21].As economical factor is considered,enzymatic esterification was carried out at a molar ratio of 1:3 in this work.

Fig.6. Effects of reaction time on the yield of vitamin E succinate catalyzed by CRL and pNIPAM-g-CRL.

Fig.6 shows the time trajectory of the yield in enzymatic synthesis of vitamin E succinate by CRL andpNIPAM-g-CRL.It was seen that the yield of vitamin E succinate catalyzed bypNIPAMg-CRL increased rapidly with reaction time to 75% in the initial 1.5 h,and then the yield increased slowly to 84.8%at 12 h.In contrast,the yield of vitamin E succinate catalyzed by CRL was just obtained to be 37% at 12 h.Therefore,the reaction time was set to 1.5 h in the esterification catalyzed bypNIPAM-CRL conjugates.Compared with those previous reports [48,49],the esterification time reduced by at least 7 times in this work.Therefore,p-NIPAM-CRL conjugates offers great advantage in the achievement of acceptable yield in a much shorter reaction time in enzymatic synthesis of vitamin E succinate.

In this work,fivepNIPAM-CRL conjugates (listed in Table 1)with different chain lengths and densities were synthesized for the evaluation of enzymatic synthesis of vitamin E succinate in DMSO.The yields of vitamin E esterification bypNIPAM-g-CRL are shown in Fig.7.The result showed that CRL had a much low yield of 6.6% in the enzymatic synthesis of vitamin E succinate,indicating a much low catalytic efficiency of CRL in non-aqueous synthesis.It was consistent with the statement previously described [19,50].Torreset al.[10] reported ever that the esterification of vinyl acetate withall-rac-α-tocopherol in 2-methyl-2-butanol by Novozym 435 had a yield less than 10%even after reaction for over 150 h.It could be attributed to the fact that hydrophobic substrate was difficult to penetrate through the surface hydration layer of CRL to reach the active site in the esterification[17].On the other hand,a physical mixing ofpNIPAM and CRL(CRL +pNIPAM) led to a little increase of yield in the enzymatic synthesis of vitamin E succinate as shown in Fig.7.In the status,therefore,major challenge for nonaqueous synthesis is how to improve catalytic efficiency.

Fig.7. Yields in enzymatic synthesis of vitamin E succinate by pNIPAM-CRL conjugates with different chain: (a) lengths and (b) densities.

AfterpNIPAM was grafted to CRL,the yield in the enzymatic synthesis of vitamin E succinate increased greatly.Fig.7(a) shows the influence of chain length to the yield in esterification of vitamin E bypNIPAM-CRL conjugates.It was evident thatpNIPAM grafting on CRL increased significantly catalytic efficiency of threepNIPAMCRL conjugates with different chain lengths.Among them,the highest yield was obtained to be 75.4% inpNIPAM-g-CRLcatalyzed synthesis.Although underlying mechanism for polymer conjugation remains poor understood so far,the result in this work demonstrated the great contribution ofpNIPAM to catalytic efficiency.As hydrophobicpNIPAM was grown on the surface of CRL,it destroyed structural order of water in the surface hydration layer of the enzyme,improving the diffusion of hydrophobic substrates to the enzyme.Jianet al.[50] found ever that the conjugation with a soluble polymer improves the flexibility ofCandida antarcticalipase B (CalB) in organic solvents with the help of low-field NMR measurement,leading to an increase in the catalytic efficiency of up to two orders of magnitude.As a hydrophobic polymer,moreover,pNIPAM interaction with internal surfaces of active site on CRL was dominant majorly by hydrophobic interaction in this work,improving conformational change around active site on CRL and thus providing a catalysis-favorable conformation.It was similar interfacial activation of lipase immobilized on hydrophobic supports [26,51].With an increase of chain length as well as chain density,however,more hydrophobicpNIPAM involved in the regulation of structural order of water and substitution of water in vicinity of the enzyme,leading the conformational distortion and even the inactivation of the enzyme [16].As shown in Fig.7(a),a longerpNIPAM chain conjugated onpNIPAMHg-CRL led to a little decrease of yield in enzymatic synthesis.The same tendency was also found in enzymatic synthesis of vitamin E succinate bypNIPAM-CRL conjugates with different densities(shown in Fig.7(b)).It was seen thatpNIPAM-g-CRL had a higher yield thanpNIPAM-g-CRL-L with a lower density.The similar finding was also reported previously in nonaqueous transesterification by CalB conjugated with pluronic F68 at low molar ratios from 0.1 to 1.8 [50].In this work,the result further revealed that too dense or long polymer chain on CRL was likely unfavorable enzymatic synthesis of vitamin E succinate.DenserpNIPAM led to a decrease of the yield ofpNIPAM-g-CRL-H from its maximal value.Therefore,the complex mechanism in term of polymer architecture-function relationship inpNIPAM-CRL conjugates remains obscure and yet,has not to be well characterized.

3.3.Catalytic kinetics of pNIPAM-g-CRL

Lineweaver-Burk curves ofpNIPAM-CRL conjugates with different chain lengths and densities are shown in Fig.8.It could be found thatV-1increased with[S]-1rapidly in enzymatic synthesis by CRL.However,pNIPAM conjugation to CRL led to a great change in the relationship ofV-1and [S]-1.Kinetic parameters of CRL andpNIPAM-CRL conjugates in Table 3 are obtained by fitting experimental data by Eq.(1).The result demonstrated thatpNIPAMCRL conjugates had lowerKmvalues than CRL,indicating thatpNIPAM conjugation improved the substrate affinity to CRL.More importantly,pNIPAM-CRL conjugates had at least six-times higherKcatandVmaxthan CRL.In nonaqueous media,a great reduction of lipase activity is always attributed to conformational rigidity and reduced diffusion of hydrophobic substrate to reach the enzyme.As stated by Jianet al.[50],however,the conjugation with a water-soluble polymer enhanced enzyme dynamics and increased fluctuation of residues located in the substrate channel.In this work,the hydrophobic interaction ofpNIPAM induced conformational change of the lid and increased the possibility of the open conformation of CRL,exhibiting a similar ‘‘interfacial activation”effect in nonaqueous media.On the other hand,hydrophobicpNIPAM chain destroyed the structural order of water in surface hydration layer of the enzyme,improving diffusion of hydrophobic substrate to reach the enzyme.The kinetics result further revealed that an increase of activity ofpNIPAM-CRL conjugates could be achieved by chain length and density.However,too dense or longpNIPAM chain onpNIPAM-g-CRL-H andpNIPAMH-g-CRL led to a little decrease inKcatandVmaxas listed in Table 3.A plausible explanation is that highly hydrophobic polymer led to a serious conformational distortions and even structural destroy.

3.4.Stability of pNIPAM-CRL conjugates

Stability is of importance forpNIPAM-CRL conjugates in the industrial application.Fig.9(a) shows the thermal stability ofp-NIPAM-g-CRL andpNIPAMH-g-CRL as well as CRL after incubation at different temperatures (30–60 °C).After incubation for 4 h,the residual activity of CRL decreased rapidly with an increase of incubation temperature,and only～40%activity of CRL was obtained at a temperature of 50°C and over.However,pNIPAM-CRL conjugates were much more stable than CRL.At 50 °C,pNIPAM-g-CRL had a residual activity of 75% after incubation for 4 h while a higher activity was obtained forpNIPAMH-g-CRL with a longer chain.The result fully reflected the contribution ofpNIPAM conjugation to thermal stability of the enzyme.However,the enzyme underwent considerable deactivation at incubation temperature of 60 °C and residual activity of CRL conjugates decreased greatly.Therefore,enzymatic synthesis of vitamin E succinate was operated at 55 °C in this work.Fig.9(b) further shows the influence of incubation time at 55 °C on the stability ofpNIPAM-CRL conjugates.It was seen that residual activity of the enzymes decreased rapidly with incubation time at initial 0.5 h and residual activities ofpNIPAM-g-CRL and CRL were obtained to be 85% and 71%.After 24 h,the CRL conjugates still retained about residual activities of 47.5% forpNIPAM-g-CRL and 50% forpNIPAMH-g-CRL.In higher incubation temperature(70°C),this residual activity was obtained only after 4 h incubation(data not shown).Our finding was consistent with the result reported previously by Kovaliovet al.[52] inThermomyces lanuginoselipase conjugation with poly-N-[3-(N,Ndimethylamino)propyl] acrylamide.The result revealed thatp-NIPAM-CRL conjugates had higher thermal tolerance than CRL in DMSO.

Fig.8. Lineweaver-Burk diagram of CRL and pNIPAM-CRL conjugates with different (a) chain lengths and (b) densities.

Fig.9. Thermal stability of CRL, pNIPAM-g-CRL and pNIPAMH-g-CRL: (a) effect of incubation temperature,(b) effect of incubation time at 55 °C.

Fig.10. Performance of the esterification of vitamin E in solvents with different (a) water contents and (b) polarities.

In this work,the effect of water content and solvent polarity on stability was also investigated and results are shown in Fig.10.It is well known that essential water was necessary for enzyme flexibility in nonaqueous media.The result in Fig.10(a) showed that the yield of vitamin E succinate by CRL increased with an increase of water content and the yield increased by 100% at 1%water content.However,the influence of water content on the yield ofpNIPAM-g-CRL was very limited,indicating thatpNIPAM conjugation,rather than water content,contributed majorly to the enzyme activityofpNIPAM-g-CRL.The similar mechanism was also found by Jianet al.[50].A further increase in water content improved reverse hydrolysis reaction and the substrate hydrolysis [53].As a consequence,a great decrease in the yield was observed at 5% water content.Fig.10(b) shows the yield of vitamin E succinate at different solvent polarities.DMSO andnhexane had lgPvalues of -1.3 and 3.5,respectively [54].With an increase ofn-hexane content,the yield of vitamin E succinate decreased gradually,indicating that pure DMSO was more favorable for enzymatic synthesis of vitamin E succinate.

3.5.Reuse of pNIPAM-g-CRL

The reusability ofpNIPAM-g-CRL in enzymatic synthesis of vitamin E succinate was studied in ten consecutive cycles.The result in Fig.11 showed that,in initial three consecutive cycles,there was no obvious the loss of enzymatic activity in the synthesis of vitamin E succinate bypNIPAM-g-CRL.At the second cycle,the activity ofpNIPAM-g-CRL was a little higher than 100%.This phenomenon was not persistent in the following cycle,and more likely to be an experimental error.The activity ofpNIPAM-g-CRL decreased to～70%in the fourth cycle.It may be attributed to a long cumulative incubation time(4.5 h corresponding to three cycles)during enzymatic synthesis.In the following cycles,the residual activity fluctuated around 70%up to 10 cycles as shown in Fig.11.Chen and coworkers [55] found that relative activity of PDA@CRL-MSNC,a nanocomposite of enzyme coated metal ion surfactant,decreased by 55% in CRL-catalyzed synthesis of vitamin E succinate after 10 cycles.Caoet al.[56] also achieved yields of 90% in 10 cycles for immobilizedBacillus subtilislipase on Cu-BTC based hierarchically porous metal–organic framework in the esterification of lauric acid and benzyl alcohol.Therefore,the reusability and residual activity was related closely with materials.The result in this work demonstrated sufficient reusability ofpNIPAM-g-CRL in enzymatic synthesis of vitamin E succinate.

Fig.11. Reusability of enzymatic synthesis of vitamin E succinate by pNIPAM-g-CRL in ten cycles.

4.Conclusions

In this work,pNIPAM was conjugated successfully on the surface of CRLviaATRP for the development of high-efficient biocatalyst in enzymatic synthesis of vitamin E succinate.Compared with CRL,pNIPAM-CRL conjugates had much higher catalytic efficiency in DMSO.The result further showed that the synthesis of vitamin E succinate catalyzed bypNIPAM-CRL conjugates was influenced by polymer architecture,water content,solvent polarity and molar ratio of vitamin E and succinic anhydride.In the optimal operation,the reaction time was shortened at least 7 times,and the yield of vitamin E succinate bypNIPAM-g-CRL reached to 75.4% at 55 °C after 1.5 h reaction.It argued that conjugation with a hydrophobic polymer induced hydrophobic interaction with the lid,promoting a similar‘‘interfacial activation”effect,and suggested a higher possibility of catalysis-favorable conformation on CRL in nonaqueous media.On the other hand,hydrophobicpNIPAM chain destroyed the structural order of water in surface hydration layer of the enzyme,improving diffusion of hydrophobic substrate to reach the enzyme.However,too dense or longpNIPAM chain led to conformational distortions and even the inactivation of the enzyme.Moreover,pNIPAM conjugation improved the thermal stability of CRL greatly,and the stability improved further with an increase of chain length ofpNIPAM.pNIPAM-g-CRL exhibited good reusability in the enzymatic synthesis of vitamin E succinate and kept～70% of its catalytic activity after ten consecutive cycles.Therefore,pNIPAM-g-CRL was a more competitive biocatalyst in the enzymatic synthesis of vitamin E succinate and this work exhibited its good application potential under harsh industrial conditions.

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 financially supported by the National Key Research and Development Program of China (2021YFC2102801),National Natural Science Foundation of China (21878221) and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (21621004).We also thank the Haihe Laboratory of Sustainable Chemical Transformations for financial support.