Encapsulation of lipases on coordination polymers and their catalytic performance in glycerolysis and esterification

Can Zeng,Nanjing Zhong

School of Food Science,Guangdong Pharmaceutical University,Zhongshan 528458,China

Keywords: Lipase Coordination polymers Encapsulation Glycerolysis Esterification

ABSTRACT In this study,lipases of CALB(Candida antarctica lipase B),TLL(Thermomyces lanuginosa lipase),RML(Rhizomucor miehei lipase),CALA(Candida antarctica lipase A)and LU(Lecitase®Ultra)were encapsulated into the nucleotidehybrid metal coordination polymers (CPs) for diacylglyerols (DAG) preparation.Guanosine 5′-monophosphate(GMP) and adenosine 5′-monophosphate (AMP) were used as coordinating molecules,and metal ions of Fe3+,Ba2+,Mn2+,Ni2+and Cr3+were applied to prepare matrix.Results indicated that,besides Ba2+with AMP,all other metal ions can coordinate with AMP and GMP to generate CPs.In addition,the AMP/Ni was amorphous when standing temperature was 4°C,while it was crystalline when standing temperature was from 30 to 180°C.DAG content from 47.55%to 64.99%was obtained from glycerolysis by CALB@GMP/Ba,RML@GMP/Ba,TLL@GMP/Ba,RML@GMP/Mn and TLL@GMP/Mn.Additionally,CALB@GMP/Fe showed selectivity towards DAG formation in the esterification and DAG content up to 61.88%was obtained.

1.Introduction

Enzymes are natural biocatalysts,they are widely used in food processing,biopharmaceuticals,green energy and other fields,due to their high catalytic efficiency,gentle reaction conditions,substrate specificity and environmental friendliness[1].However,free enzymes are sensitive to external environments,leading to poor stability and low reusability and in turn high cost,which limits their applications in large-scale industries.Immobilization facilitates the recovery of enzymes and permits the continuous use in industry.As for the lipases,immobilization also allows their applications in hydrophobic media.In addition to the simple recovery,immobilization is also an efficient strategy for enzyme feature improvement.Enzymatic features of lipases,including stability,activity,selectivity,specificity,and resistance to inhibitors and chemical reagents,were reported to be improved efficiently after immobilization with proper design[2–4].Lipases(triacylglycerol hydrolases;EC 3.1.1.3)are the most widely used enzymes in biocatalysis.They are special enzymes based on interfacial activation[5,6].In aqueous medium,the active centers of lipase molecules tends to be covered by a polypeptide chain(called lid),and the active centers are isolated from reaction medium.Lipases are in closed form and inactive in this case.While in the presence of a hydrophobic surface,lipases becomes adsorbed on the surface and the active centers are fully exposed,therefore they can hydrolyze drops of oils.Lipases in this case are in the so-called open form and they are active.

Among the immobilization methods,encapsulation is a physical process,it is simple,easy and cheap.Additionally,encapsulation could retain the enzyme in its original conformation and bring good stability[7].Carriers are one of the key factors affecting the properties of their supported enzymes.Coordination polymers(CPs)are organic-inorganic hybrid materials built from the self-assembly of metal ions and organic bridge ligands through ligand interactions[8,9].Metal coordination and enzyme encapsulation take place under mild conditions without toxic radicals or organic solvents usage,CPs are therefore competitive candidates for enzyme immobilization[10].In addition,the specific surface area of CPs is large and pore size is tunable.Moreover,the functional groups on the backbone of CPs can activelyparticipate the catalytic process,making the enzyme-CPs composites more selective for substrate molecules,and in turn greatly enhancing the catalytic activity of enzyme-CPs complexes[11,12].Therefore,CPs are potential carriers for enzyme immobilizaition,and it has become a popular research topic in recent years[13,14].Many enzyme-CPs composites exhibit excellent catalytic performance,far outperforming free enzymes in many aspects.

Liang and co-authors had studied the co-immobilization of multiple enzymes by metal coordinated nucleotide hydrogel nanofibers,and they found the stability of Candida rugosa lipase had been improved after immobilization [15].In our previous study,we have developed a series of nucleotide-hybrid metal CPs for lipases encapsulation.The organic ligand used was guanosine 5′-monophosphate(GMP)and metals were zinc,copper,iron,cadmium,zirconium,lanthanides,yttrium and scandium.We found that the CPs encapsulated Candida antarctica lipase B(CALB)(CALB@CPs)samples were highly selective for esterification while poor performance in glycerolysis reaction.Of which,the CALB@GMP/Tb transformed over 98%of oleic acid into glycerides in esterification,while in glycerolysis the triacylglycerols (TAG) conversion was <5% [16].Interestingly,the GMP/Tb encapsulated other lipases,like Aspergillus oryzae lipase(AOL),Thermomyces lanuginosa lipase(TLL),Rhizomucor miehei lipase(RML),Lecitase®Ultra(LU),Candida antarctica lipase A(CALA)and lipase from Burkholderia cepacia(BCL),exhibited opposite catalytic behaviors:they catalyzed the glycerolysis reaction efficiently,but could not initiate the esterification[17].As a supplement and continuation of these studies,in this work,CPs constructed by GMP or adenosine 5′-monophosphate(AMP) with other metal ions,including Fe3+,Ba2+,Mn2+,Ni2+,and Cr3+,were studied for lipases encapsulation.Then the glycerolysis and esterification performances of the lipase@CPs were carefully studied.

According to the Report on the Status of Nutrition and Chronic Diseases of the Chinese Population in 2020,obesity has become a major public health problem in China.Being overweight or obese was associated with an increased risk of many chronic diseases [18].TAG account for over 95%of the cooking oil consumed on a daily basis,they cause fat accumulation in the body when consumed in excess or not digested and absorbed in a timely manner,leading to overweight or obesity[19,20].On the other hand,diacylglycerols(DAG),especailly 1,3-DAG,have been claimed to be capable of reducing postprandial serum TAG levels and preventing fat accumulation and in turn the obesity,without loss of taste and processing functions[21].

Enzymatic glycerolysis is a primary route for DAG production,due to its high-space cost efficiency.Despite extensive studies on the enzymatic DAG production had been carried out in recent years,the enzymatic glycerolysis efficiency as well as the selectivity towards DAG generation were still needed improvement [22].Since the enzymatic properties of the immobilized lipases were influenced by the immobilization conditions and supports,modification or exploring new carriers was a potential way to increase DAG selectivity.

Therefore,in this study,GMP and AMP were used as coordinating molecules,and metal ions of Fe3+,Ba2+,Mn2+,Ni2+and Cr3+,were applied to prepare matrix for lipase encapsulation.The possibility of generating CPs(solid precipitation or gel)through coordination by the self-assembly of the present metal ions (Fe3+,Ba2+,Mn2+,Ni2+,and Cr3+) and GMP (or AMP) was firstly evaluated.The effects of standing temperature on the AMP/Ni formation were then studied.Solid CPs were used for lipases(of CALB,TLL,RML,CALA and LU)encapsulation.The obtained encapsulated lipases(lipase@CPs)were then applied to catalyze glycerolysis and esterification for DAG preparation,and their performances were carefully studied.

2.Materials and methods

2.1.Materials and reagents

Commercial soybean oil was purchased from a local supermarket.Tributyrin with a purity of over 97%,commercial TLL and RML solutions were purchased from Sigma-Aldrich (Shanghai,China).Commercial CALA,CALB and LU solutions were obtained from Novozymes(Beijing,China).AMP disodium salt(>99%),GMP disodium salt hydate(>98%),TbCl3·6H2O (>99.9%),FeCl3(>99.9%),MnCl2(>99%),NiCl2(>99%),BaCl2·2H2O (>99.9%),and N-2-hydroxyethylpiperazine-N′-2-ethyl sulfonic acid(HEPES)(>99%)were purchased from Aladdin Reagents Co.Ltd.(Shanghai,China).Glycerol with a purity of>99.0%was purchased from Sinopharm Chemical Reagent Co.Ltd.(Shanghai,China).All other solvents and reagents were of analytical or chromatographic grade.

2.2.Preparation of CPs

The GMP or AMP (0.1 mol/L,3 mL) solution was mixed quickly with required amount of metal ion (0.1 mol/L,for the preparation of AMP/Ni and GMP/Ni,the Ni2+and GMP or AMP concentrations were 0.5 mol/L)solution,the mixture was vortexed completely and standing at room temperature for 1 h.In the preparation of AMP/Ni,the mixture after vortex was standing for 12 h,and the standing temperatures at 4,30,40,50,60,70,80,180 °C were studied.After that,the sample was centrifuged at 10,000 rpm for 5 min and washed with Milli-Q water to remove unreacted chemicals.The precipitants were finally freeze-dried for 16 h.

2.3.Preparation of lipase@CPs

Required amount of the commercial lipase solution was dispersed into HEPES buffer(100 mmol/L,pH 7.0)to make a HEPES-lipase solution(the concentration of the commercial lipase solution in the HEPESlipase solution was 600 mg/mL).The HEPES-enzyme solution (3 mL)was mixed with GMP or AMP solution (100 mmol/L,3 mL),then required amount of metal ion solution(0.1 mol/L;for the preparation of AMP/Ni and GMP/Ni,the Ni2+concentration was 0.5 mol/L) was added into this mixture quickly and vortexed completely.After that,the mixture was standing for 1 h(In the preparation of AMP/Ni,the mixture after vortex was standing for 12 h)at room temperature.The mixture was finally centrifuged at 10,000 rpm for 5 min and washed with Milli-Q water to remove unreacted chemicals,and the obtained encapsulated lipases were freeze-dried for 16 h.

2.4.Characterization

Wide-angle powder X-ray diffraction (XRD)was performed on a D8 advance X-ray diffractometer(Bruker,Karlsruhe,Germany)with Cu-Kα radiation(λ=0.15418 nm)at 40 kV and 40 mA.The scanning step was 0.02°and speed was 5°/min.

2.5.Enzymatic glycerolysis of soybean oil

In a typical reaction,3.520 g(0.004 mol,the molecular weight of soybean oil 880)of soybean oil and 0.184 g(0.002 mol)of glycerol were mixed in a 50 mL round-bottom flask(molar ratio of soybean oil to glycerol 2:1),and incubated in an oil bath at constant temperature,with magnetic stirring at 200 rpm.Then the present encapsulated lipase was added(4%,wt,based on substrates) to proceed the reaction.After 12 h reaction,the reaction products were placed at 100 °C for 15 min to deactivate the lipase,then filtrated to remove the lipase and stored at 4°C.Experiments were carried out in duplicate.

2.6.Enzymatic esterification of oleic acid and glycerol

In a typical reaction,2.82 g(0.01 mol,the molecular weight of oleic acid 282)of oleic acid and 0.46 g(0.005 mol)of glycerol were mixed in a flask(molar ratio of oleic acid to glycerol 2:1).The flask was placed in an oil bath at 70°C and the mixture was agitated magnetically at 200 rpm.Then 0.15 g of the encapsulated lipase was added and the reaction was lasted for 12 h under a stream of nitrogen gas.After the reaction,the reaction products were placed at 100°C for 15 min to deactivate the lipase,then filtrated to remove the lipase and stored at 4°C.Experiments were carried out in duplicate.

2.7.Reusability of the encapsulated lipases

Reusability of TLL@GMP/Mn in glycerolysis and CALB@GMP/Fe in esterification was studied.Reaction conditions for TLL@GMP/Mn catalyzed glycerolysis were:soybean oil 3.520 g,glycerol 0.184 g,TLL@GMP/Mn 0.150 g,reaction temperature 60°C and time 12 h with magnetic stirring at 200 rpm.While conditions for CALB@GMP/Fe catalyzed esterification were as follows:oleic acid 2.82 g,glycerol 0.46 g,CALB@GMP/Fe 0.15 g,reaction temperature 70°C and time 12 h under nitrogen gas with magnetic stirring at 200 rpm.After each cycle,the mixture was centrifuged and the separated,and the encapsulated lipases were washed with 10 mL hexane and then used for the next run under the selected conditions.The relative activity of the lipase was defined as the ratio of DAG content obtained from each cycle to the DAG content obtained from the first cycle.

2.8.Determination of lipid profiles by reversed phase high-performance liquid chromatography(RP-HPLC)

The glyceride components were determined according to the RP-HPLC method with minor modifications[23].Before analysis,20 μL of the reaction mixture was withdrawn and added into 4 mL mobile phase(acetonitrile: hexane: isopropanol 27:8:10,V:V:V).Catalysts remained in the mixture were removed by filtrating through a microfilter(0.45 μm).The chromatographic separation was carried out with a Purospher®STAR RP-18e column(250×4.6 mm i.d.,particle size 5 μm),and the column temperature was held constant at 40°C.Isocratic elution was used,the mobile phase consisted of acetonitrile,hexane,and isopropanol (acetonitrile: hexane: isopropanol 27:8:10,V:V:V),the injection volume was 10 μL,and the elution flow rate was 1.0 mL/min.ELSD detection conditions:the drift tube temperature was 35°C,and the nitrogen gas pressure was 350 kPa.Quantitation and identification of the components were described in detail in our previous study[24].Double determinations were performed.

2.9.Statistical analysis

The data was analyzed by SPSS 13.0 and then expressed as average±SD.Tukey's test was used to detect the differences,and P<0.05 was considered significant.

3.Results and discussion

3.1.CPs constructed by metal ions with AMP and GMP

In order to obtain solid CPs for lipase encapsulation,CPs constructed by the present five metal ions with AMP and GMP were firstly studied.Metal ion solution and AMP(or GMP)solution(the molar ratio of AMP/GMP to metal ion was 2:1)was added to the test tube and then quickly mixed to see whether CPs(solid or hydrogel)could be generated.Results were presented in Table 1 and Fig.1.CPs of GMP/Fe,GMP/Ba,GMP/Mn,GMP/Ni and AMP/Fe were quickly generated and they were solid(Fig.1a–1e).On the other hand,no precipitation or hydrogel was observed from AMP and Ba2+solutions(Fig.1f),indicating that AMP cannot coordinate with Ba2+,and no CPs were generated.To look into this phenomenon,we changed the molar ratio of AMP to Ba2+to 1:1,however,the solutions were still clear and no CPs were generated.As for the AMP/Mn,GMP/Cr and AMP/Cr,CPs were generated and all of them were hydrogel (Fig.1g–1i).In addition,solid pellets of AMP/Ni were interestingly observed,they were attached to the wall of the tube (Fig.1j).However,they were less even with morestanding time(12h),suggesting that the AMP can coordinate with Ni2+but quite slowly.Therefore,besides Ba2+with AMP,all other metal ions can coordinate with AMP and GMP to generate solid or hydrogel CPs.In the present study,solid CPs were used for lipases encapsulation.

Fig.1.Graphs of the CPs constructed(if coordinated and CPs generated)by GMP and AMP with five different metals.(f)was the AMP with Ba2+solutions,AMP cannot coordinate with Ba2+and no CPs was generated.AMP,adenosine 5′-monophosphate;GMP,guanosine 5′-monophosphate;AMP/metal or GMP/metal,CPs constructed by AMP or GMP with metal ions;CPs,coordination polymers.

Table 1 CPs constructed by the present metal ions with GMP and AMP.

To look into the AMP/Ni,effects of standing temperature(the temperature at the 12 h standing period,the standing period was that after quickly mixing of AMP with Ni2+solutions,the mixture was standing for a period)on the AMP/Ni morphology was studied.Results indicated that with standing temperature from 30 to 50°C,the generated AMP/Ni was in the form of pellets;while temperature from 50 to 80°C,the pellets would be linked into chains;further increasing to 180°C,the AMP/Ni was powder.XRD patterns suggested that the AMP/Ni was amorphous when standing temperature was 4°C(Fig.2a),while the AMP/Ni was crystalline when standing temperature was from 30 to 180°C(Fig.2b–2e).

Fig.2.XRD patterns of AMP/Ni generated at different standing temperatures.AMP,adenosine 5′-monophosphate;AMP/Ni,coordination polymers constructed by AMP with Ni2+.

3.2.Enzymatic glycerolysis of soybean oil by the present lipase@CPs

3.2.1.Enzymatic glycerolysis by lipase@GMP/Ba

Table 2 presented the glycerolysis performance from CALB@GMP/Ba and RML@GMP/Ba.During the lipase@GMP/Ba preparation,GMP to Ba2+molar ratio from 1:1 to 3:1 was studied.When GMP to Ba2+was 2:1,CALB@GMP/Ba exhibited better performance,with DAG content at 47.55% and TAG conversion 52.67% observed.In addition,DAG/MAG ratio was 9.36,indicating that it was selective for DAG preparation.A decrease in DAG content(36.62%)and TAG conversion(47.57%)was obtained when the molar ratio of GMP to Ba2+was 1:1.While poor performance was observed when GMP to Ba2+was 3:1,with TAG conversion at only 11.86%obtained.

As for the RML@GMP/Ba,better performance was observed when GMP to Ba2+was 1:1,with DAG content at 51.92%and TAG conversion 60.02%observed.And poor performance was obtained when GMP to Ba2+ratios were 2:1 and 3:1.

The glycerolysis performance from CALA@GMP/Ba and LU@GMP/Ba was also studied,with GMP to Ba2+molar ratio from 1:1 to 3:1 during the lipase@GMP/Ba preparation.However,they all exhibited poor performance,with DAG content<2%and TAG conversion<3%obtained(data not presented in detail).

Glycerolysis performance at 60,70 and 80 °C by the TLL@GMP/Ba(GMP to Ba2+molar ratio 1:1)was studied,and better performance was obtained from 70°C,in which DAG content was 54.62%and TAG conversion 66.61%(Table 3).In addition,when GMP to Ba2+molar ratio was 2:1,a higher DAG content and TAG conversion was interestingly observed,which was 62.41%and 76.71%respectively.

3.2.2.Enzymatic glycerolysis by lipase@GMP/Mn

Table 4 listed the enzymatic glycerolysis performance from GMP/Mn encapsulated TLL,RML and LU samples.With GMP to Mn2+molar ratio at 1:1 and 2:1,TLL@GMP/Mn showed good performance,DAG content over 60%and TAG conversion at about 85%was obtained.The GMP to Mn2+molar ratio at 1:1 was more suitable for RML@GMP/Mn to catalyze glycerolysis,DAG content at 54.09%and TAG conversion 60.47%was obtained.A decrease in DAG content and TAG conversion was observed when GMP to Mn2+molar ratios were 2:1 and 3:1.The LU@GMP/Mn exhibited poor glycerolysis performance,DAG content from 11.36% to 23.27% and TAG conversion from 27.45% to 12.45% was observed,when GMP to Mn2+molar ratios were 1:1 and 2:1.

We have also studied the glycerolysis performance from CALB@GMP/Mn and CALA@GMP/Mn,with GMP to Mn2+molar ratio from 1:1 to 3:1 during the lipase@GMP/Mn preparation.However,they all exhibited poor performance,with DAG content<2%and TAG conversion<3%obtained(data not presented in detail).

3.2.3.Enzymatic glycerolysis by lipase@GMP/Fe

Table 2 Enzymatic glycerolysis of soybean oil by CALB@GMP/Ba and RML@GMP/Ba a.

Glycerolysis of soybean oil by GMP/Fe encapsulated TLL,RML,CALA and LU samples was studied,with GMP to Fe3+molar ratio from 1:1 to 3:1 during the lipase@GMP/Fe preparation.Unfortunately,they all exhibited poor performance,DAG content and TAG conversion was respectively<2%and 3%(data not presented in detail).The CALB@GMP/Fe exhibited relatively“better”glycerolysis performance compared to the GMP/Fe encapsulated TLL,RML,CALA and LU samples,nevertheless,DAG content at only 30.74%and TAG conversion 36.42%was obtained(Table 5).Therefore,the present lipase@GMP/Fe samples showed poor glycerolysis performance,and they were not suitable for MAG or DAG preparation through glycerolysis.

3.2.4.Enzymatic glycerolysis by lipase@GMP/Ni

DAG content at 15.43% and 5.79%,TAG conversion 16.47% and 7.63%,was respectively obtained from TLL@GMP/Ni and CALB@GMP/Ni catalyzed glycerolysis (Table 6).In addition,DAG content <2% and TAG conversion <3% was observed from the GMP/Ni supported RML,CALA and LU samples(data not presented in detail).

Some lipases need a certain amount of water to maintain their structure and flexibility,since lipases act at the oil-water interface,rendering the formation of an acyl-enzyme complex[25,26].Therefore,water content at 10%and 30%(based on glycerol)was added into the reaction system to see if the glycerolysis could be enhanced.However,no increase in DAG content and TAG conversion was observed(data not shown).Therefore,like the above lipase@GMP/Fe samples,the lipase@GMP/Ni samples were not suitable for MAG or DAG preparation through glycerolysis either.

Table 5 Enzymatic glycerolysis of soybean oil by the CALB@GMP/Fe a.

3.2.5.Enzymatic glycerolysis by lipase@AMP/Ni

Table 7 listed the glycerolysis performance by lipase@AMP/Ni.RML@AMP/Ni samples showed poor glycerolysis performance,with DAG content<10%and TAG conversion about 10%obtained.Additionally,water addition had no significant effect on the glycerolysis performance.

With AMP:Ni2+molar ratio at 1:1,DAG content<7%and TAG conversion<8%was observed from TLL@AMP/Ni samples,and water addition had no effect on the glycerolysis.When AMP:Ni2+molar ratios were at 2:1 and 3:1,a significant improvement on DAG content and TAG conversion was obtained with water content increasing to 10% and 30%.Nevertheless,the highest DAG content among these was only 32.35%.

DAG content<2%and TAG conversion<3%was obtained from AMP/Ni supported CALA and LU samples.Therefore,the present lipase@AMP/Ni samples exhibited poor glycerolysis performance and they were not suitable for MAG or DAG preparation through glycerolysis.

3.3.Enzymatic esterification by lipase@CPs

Esterification of oleic with glycerol was studied by the present lipase@CPs samples,results were listed in Table 8.To enhance the reaction,water generated from the esterification was evaporated by a stream of N2gas to shift the reaction equilibrium towards the glycerides formation side.

CALB@GMP/Fe exhibited high DAG selectivity,with DAG content up to 61%and oleic acid conversion at about 73%obtained.It was interesting and it could be considered for DAG preparation through esterification.The CALB@GMP/Ba(GMP:Ba2+molar ratio 2:1)also showed DAG selectivity in esterification,yet its esterification activity was lower(oleic acid conversion 40.92%),and DAG content at 34.32%was obtained.

Other the lipase@CPs samples,including GMP/Ba encapsulated TLL,RML,CALA and LU,GMP/Mn encapsulated TLL,RML,CALA,LU and CALB,GMP/Ni encapsulated TLL,RML,CALA,LU and CALB,AMP/Ni encapsulated TLL,RML,CALA,LU and CALB,as well as GMP/Fe encapsulated TLL,RML,CALA and LU samples,all exhibited quite poor performance in esterification,with oleic acid conversion lower than 2%.Interestingly,oleic acid conversion over 98%was obtained from CALB@GMP/Tb catalyzed esterification in our previous study[16].In addition,oleic acid at 90.77%was obtained from Novozym 435 catalyzed esterification[27].

3.4.Resuability of the lipase@CPs

The reusability of the immobilized lipase is quite important in practical applications [28,29].As presented,TLL@GMP/Mn retained 50.48% ±3.21%of its initial glycerolysis activity after five cycles of reuse(Fig.3a);while CALB@GMP/Fe ratained 65.33%±2.63%of its initial esterification activity after five cycles of reuse(Fig.3b).The results indicated TLL@GMP/Mn and CALB@GMP/Fe were potential in practical applications.Interestingly,94.67%and 89.85%of their initial glycerolysis activity was respectively retained from TLL@GMP/Tb and RML@GMP/Tb,after five cycles of reuse in our previous study[17].In addition,the Novozym 435 can be reused 11 times in esterification without significant activity decrease[27].

Fig.3.Reusability of the encapsulated lipases.(a)Reusability of TLL@GMP/Mn in glycerolysis.Reaction conditions:soybean oil 3.520 g,glycerol 0.184 g,TLL@GMP/Mn 0.150 g,reaction temperature 60°C and time 12 h with magnetic stirring at 200 rpm.(b)Reusability of CALB@GMP/Fe in esterification.Reaction conditions:oleic acid 2.82 g,glycerol 0.46 g,CALB@GMP/Fe 0.15 g,reaction temperature 70°C and time 12 h under nitrogen gas with magnetic stirring at 200 rpm.

4.Conclusions

Besides Ba2+can not coordinate with AMP,all other metal ions can coordinate with AMP and GMP to generate solid or hydrogel CPs.In addition,the AMP/Ni was amorphous when standing temperature was 4°C,while it was crystalline when standing temperature was from 30 to 180°C.The present encapsulated lipases were used for DAG preparation through glycerolysis and esterification.With proper molar ratio of GMP to metal ions(Ba2+and Mn2+),DAG content from 47.55% to 64.99% could be obtained from glycerolysis by CALB@GMP/Ba,RML@GMP/Ba,TLL@GMP/Ba,RML@GMP/Mn and TLL@GMP/Mn.As for the esterification,CALB@GMP/Fe showed selectivity towards DAG formation and DAG content up to 61.88%was obtained.

Table 7 Enzymatic glycerolysis of soybean oil by lipase@AMP/Ni a.

CRediT Authorship Contribution Statement

Can Zeng:Methodology,Software,Investigation.Nanjing Zhong:Supervision,Conceptualization,Methodology,Funding acquisition,Writing–original draft.

Declaration of Competing Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China(31772000).