A cold adapt and ethanol tolerant endoglucanase from a marine Bacillus subtilis☆

Dongsheng Xue ,Xuhao Zeng ,Chunjie Gong ,Dongqiang Lin ,Shanjing Yao ,*

1 Key Laboratory of Fermentation Engineering(Ministry of Education),Hubei Provincial Cooperative Innovation Center of Industrial Fermentation,HubeiKey Laboratory of Industrial Microbiology,Hubei University of Technology,Wuhan 430068,China

2 Department of Chemical and Bioengineering,Zhejiang University,Hangzhou 310027,China

Keywords:Endoglucanase Ethanol tolerant Cold adapt Bioethanol

A B S T R A C T The catalytic properties and thermodynamic kinetics of the endoglucanase from a marine Bacillus subtilis were analyzed.Optimum pH and temperature of the endoglucanase activity were 5.0 and 35°C.The endoglucanase activity,melt point temperature was 1.13 folds(247.02 U·ml-1),2.1 °C higher(39.2 °C)in 6%ethanol solution than that(218.60 U·ml-1),(37.1 °C)in free ethanol.At 40 °C–55 °C,Gibbs free energy,ΔG,and the content of α-helix was higher in 6%ethanol solution than that in ethanol free solution.The increasing of α-helix content led to higher activity and better thermostability in ethanol solution.The cold adapt ethanol tolerant endoglucanase was valuable for bioethanol product by simultaneous saccharification and fermentation process.

1.Introduction

Bioethanol production using carbon source released from cellulose has significance for sustainable development[1,2].Hydrolyzing cellulose with cellulase is a promising method to transform cellulose into glucose,a widely used carbon source.Hydrolyzing cellulose with cellulase has the advantages of high hydrolysis efficiency,low environment pollution and mild conditions[3,4].

Separate hydrolysis and fermentation(SHF)and simultaneous saccharification and fermentation(SSF)are two kinds of process of bioethanol production by hydrolyzing cellulose with cellulase.Separate hydrolysis and fermentation(SHF)process firstly adds cellulase into cellulose material.Cellulase hydrolyzes cellulose material into glucose.After cellulose hydrolysis,yeast is incubated[5,6].Separate hydrolysis and fermentation(SHF)has some inadequacies.During the hydrolysis,high concentration of released glucose inhibits cellulase activity and hydrolysis efficiency.That inadequacies lead to increase the cost of cellulose hydrolysis[7–9].In simultaneous saccharification and fermentation(SSF),cellulase,cellulose material and yeast are simultaneously added into the fermentation mixture.Cellulose material is hydrolyzed into glucose and glucose simultaneously was absorbed by yeast.Glucose maintained a low concentration.A low concentration of glucose cannot inhibit cellulase activity and hydrolysis efficiency.Cellulase can efficiently hydrolyze cellulose[10].High cost is a bottleneck of industrial production of bioethanol.Bioethanol produced by simultaneous saccharification and fermentation(SSF)has a lower cost of bioethanol compared with separate hydrolysis and fermentation(SHF).

Bioethanol fermentation was carried out at the range of 23–37 °C[11–13].The optimum temperature of common cellulase activity was above 50°C[14,15].The hydrolysis efficiency of common cellulase is low at the range of 23–37 °C.The optimum temperature of cold adapt enzyme activity was about 30–40 °C[16,17].Cold adapt cellulalse could efficiently hydrolyze cellulose at the range of 23–37 °C.Cold adapt cellulase was valuable for bioethanol production by simultaneous saccharification and fermentation(SSF).

During the bioethanol fermentation,ethanol concentration continues to increase.High concentration of ethanol decreased the activity and hydrolysis efficiency of common cellulase.Ethanol tolerant cellulase is desired to bioethanol production by simultaneous saccharification and fermentation(SSF).Ethanol tolerant cellulase could remain high activity and thermostability in high concentration of ethanol.Ethanol tolerant cellulase is valuable for bioethanol production.

Cellulase is composed of exoglucanase,endoglucanase and β-glucosidase.Exoglucanase hydrolyzes macromolecular cellulose into small cellulose fragment.Endoglucanase hydrolyzes cellulose fragment into cellubiose.Cellulbiose is transformed into glucose by β-glucosidase[18–20].Endoglucanase is indispensable for hydrolyzing cellulose[21,22].Ethanol tolerantendoglucanase is necessary for bioethanol production by simultaneous saccharification and fermentation(SSF).

Salt tolerant endoglucanase from Stachybotrys microspore[23],alkaline-stable endoglucanase from Paenibacillus sp.[24],and ionic liquid stable endoglucanase from Stachybotrys microsporaInes[23]have been reported.Cold adapt ethanol tolerant endoglucanase has not been reported.

A marine B.subtilis has been isolated from East China Sea.Endoglucanase from a marine B.subtilis was cold adapt and ethanol tolerant.The catalytic properties of the endoglucanase were analyzed under high concentration of ethanol conditions.

2.Material and Methods

2.1.Strain

The bacteria were isolated fromamarine sediments.Based on the ITS DNA sequence and morphologies,the bacteria were identified as B.subtilis.

2.2.Media

Preservation medium was composed of 5.0 g yeast extract,2.0 g glucose,2.0 g peptide,0.2 g(NH4)2SO4,0.01 g MgCl2,0.05 g KCl,20.0 g agar,and 1.0 L tap water.The medium pH was adjusted to 6.5 and sterilized.

Fermentation medium was composed of 2.0 g starch,peptide 0.5 g,CMCNa 10.5 g,(NH4)2SO40.2 g,MgCl20.01 g,KCl 0.05 g,tap water 1.0 L.

2.3.Endoglucanase production and purification

B.subtilis was inoculated on preservation slants and cultivated for 2 d at 35°C.Then B.subtilis was inoculated into fermentation medium.The cell concentration was 1 × 107cells·ml-1.100 ml of fermentation medium was added into a 500 ml flask.Fermentation was carried out at 32°C for 2 d.The fermentation broth was filtrated with filter paper.The filtrate was precipitated with ammonium sulphate(75%,w/v)and centrifugated at 10000 g for 10 min.The result precipitation was dissolved in 20 mmol·L-1Tris-HCl buffer and dialyzed against 20 mmol·L-1Tris-HCl.An ion-exchange column of Q-sepharose was equilibrated with 15 mmol·L-1Tris-HCl buffer.The Q-sepharose column was eluted with 0–1.5 mol·L-1NaCl.The elution fractions were collected and monitored at 280 nm.A fraction with high endoglucanase activity was purified by gel column.The fractions were pooled and concentrated by freeze-drying.

2.4.Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

In order to measure the molecular weight of the endoglucanase,the fraction with high endoglucanase activity was analyzed by 12%gel SDSPAGE.The fraction with high endoglucanase activity was loaded to 12%gel SDS-PAGE(containing 1.2%CMC)for zymogram analysis.The gel was soaking in wash buffer to remove SDS.The wash buffer contained 30 mmol·L-1sodium phosphate buffer and 40%isopropanol(pH 7.2).The gel removed SDS was soaked in equilibrated buffer(containing 30 mmol·L-1sodium phosphate buffer(pH 7.2))for 1.5 h.Then the gel was transferred into renaturation buffer(containing 30 mmol·L-1sodium phosphate buffer,5 mmol·L-1β-mercaptoethanol and 1 mmol·L-1EDTA(pH 7.2))at 4 °C for 16 h.

2.5.Non-denaturating gel electrophoresis and stainness

Native PAGE was performed with 7.5%separating gels and 5.0%stacking gels.Electrode buffer was tris-glycine buffer(pH 8.30).The native-PAGE gel was soaked in 1%carboxymethyl-cellulose in 50 mmol·L-1acetate buffer,at pH 5.0 and 50 °C for 1.2 h.The gel was then transferred to 0.1%solution of Congo red and incubated at 25°C,with constant shaking for 15 min.The gel was destained by washing with 1 mmol·L-1sodiumchloride.The activity band was clearly visible as yellowish clearances against a deep red background.

2.6.Effect of temperature and pH on endoglucanase activity

The optimum temperature of endoglucanase activity was analyzed at the temperature range from 25 °C to 55 °C with increments of 5 °C unit at pH 5.0.

The optimum pH of endoglucanase activity was analyzed at pH 4,5 and 6(50 mmol·L-1Sodium acetate buffer),to 7 and 8(50 mmol·L-1Phosphate buffer)at 30°C.

The endoglucanase activity was performed using 1%(w/v)β-glucan as a substrate.5 ml buffer containing β-glucan and 0.5 ml diluted endoglucanase liquid was mixed.The mixture was incubated for 30 min at designed temperature.Released reduced sugar was measured by DNS method[25].The amount of enzyme activity required to form an amount of reducing sugar corresponding to 1 μmol of glucose per min was regarded as one unit of enzyme activity.

2.7.Effect of ethanol on endoglucanase activity.

Endoglucanase activity was measure at different concentration of ethanol at pH 6.0 and 30°C.The ethanol concentration range was from 2%(w/w)to 10%(w/w).The relative activity was calculated by the endoglucanase activity to the maximum activity.

2.8.Half life time of endoglucanase in different concentration of ethanol solution

Half life time of endoglucanase in different concentration of ethanol solution was determined according to the first order kinetics,which was a single step two stage theory[26].The equation is the following,

where E0is the initial endoglucanase activity.Edis the endoglucanase activity after deactivation for some time in different concentrations of ethanol solution.Kdis first-order deactivation rate constant.t is the deactivation time.From the plot of ln(Ed/E0)versus t,the slope was obtained.The slope was the value of Kd.

Half-life time of an enzyme is the time required by enzyme to lose half of the initial activity,which is calculated by,

where t1/2is half-life time of endoglucanase.

2.9.ΔG of thermodynamic kinetics of ethanol in different concentration of ethanol solution

Activation energy,Ea,is calculated from Arrhenius equation.It was the following[27]:

Eawere the slopes of plot of ln kdversus 1/T.ΔG,Gibbs free energy were calculated by the following equation:

where T,h,kd,kB,R and ΔG were temperature,Plank's constant, first order deactivation rate constant,Boltzman's constant,universal gas constant and Gibbs free energy,respectively.Where h is 6.63 × 10-34J·s-1and kBis 1.38 × 10-23J·K-1and R is 8.314 J·mol-1·K-1.

2.10.Melt temperature measurement

Tmwas melt temperature.Tmof endoglucanase was determined by performing at 220 nm from 25 °C and 55 °C(1 °C·min-1)using 1 mm quartz cells in an Aviv Model 400 spectrophotometer(AVIV Biomedical,USA).Endoglucanase samples were suitably diluted in citric sodium buffer(0.1 mol·L-1,pH 5.0)to a working concentration of 0.31 mg·ml-1.Tmvalues were determined by fitting the curves of circular dichroism signals versus temperature.

3.Results and Discussion

3.1.Endoglucanase purification

There was only one band in SDS PAGE(Fig.1).The fraction with endoglucanase activity contained one kind of protein.Native PAGE gel was stained with congo red.A lighter clearance band in native PAGE gel was visible.The result suggested that the purified protein could hydrolyze CMC.(See Table 1.)

According to SDS PAGE,the molecular weight was about 31000.The endoglucanase from B.subtilis SB13 was 55000[28].Endoglucanase from Thermotoga maritima[29],microorganism from a salt-lake[30],Aspergillus niger[31],Aspergillus flavus[32],and Mucor circinelloides[33]was 70000,61790,33700,35000,27000.Different molecular weights indicate that amino acid sequences were not same.The difference of amino acid sequences leads to different catalysis properties.

3.2.Optimum pH and temperature of endoglucanase activity

Optimum pH and temperature of the endoglucanase from a marine B.subtilis was pH 5.0 and 35°C(Figs.2,3).The endoglucanase from a marine B.subtilis was acidic.The optimum temperature of the enzyme activity was 35°C.The endoglucanase from a marine B.subtilis was called as cold adapt enzymes[34].It was suitable to hydrolyze cellulose under acidic and low temperature condition.

Fig.1.SDS PAGE and native PAGE gel stained with congo red.

Table 1Effect of reagents on endoglucanase activity

The optimum pH and temperature of the endoglucanase from other strains of B.subtilis was pH 6.0 and 15 °C[28],pH 8.0 and 50 °C[35].Optimum pH and temperature of endoglcanase from A.niger,Aspergillus terreus NIH2624 were pH 4.0 and 70 °C[31],pH 5 and 55 °C[36].The optimum activity temperature of the cold adapt endoglucanase from Paenibacillus sp.IHB B 3084 and Eisenia fetid a was 40°C[37,38].

The yeast produces bioethanol at about 32°C[39].The cold adapt endoglucanase from a marine B.subtilis was suitable for bioethanol production by simultaneous saccharification and fermentation process.

Optimum pH and temperature of the endoglucanases from different strains was different.Different amino acid sequences of the endoglucanase lead to different optimum pH and temperature of endoglucanase activity.The cold adapt endoglucanase probably had a flexible structure.The endoglucanase with a flexible structure could efficiently combine and catalyze the substrate at low temperature.Athigh temperature,the endoglucanase with a flexible structure easily changed the 3-dimension structure.The 3-dimension structure change made the endoglucanase not efficiently combine the substrate,so the endoglucanase activity decreased at high temperature.

3.3.Effect of ethanol on endoglucanase activity

In free ethanol solution,the endoglucanase from a marine B.subtilis showed 218.60 U·ml-1activity.In 6% ethanol solution,the endoglucanase showed 247.02 U·ml-1activity.In solution with over 6%ethanol,the endoglucanase remained over 70%activity(Fig.4).The endoglucanase from A.niger was tolerant to 8%ethanol.It could efficiently hydrolyze cellulose in 8%ethanol solution.The concentration of ethanol produced by solid state fermentation was 6.47%[40].The endoglucanase from a marine B.subtilis could efficiently hydrolyze cellulose in the solid state fermentation mixture.

Fig.2.Effect of temperature on endoglucanase activity.

Fig.3.Effect of pH on endoglucanase activity.

β-glucosidase from Sporidiobolus pararoseus was stable up to 15%in 15%ethanol solution[41].Ethanol can act as an acceptor for the intermediate glycosylcation during the substrate hydrolysis[42,43].Increasing of endoglucanase activity probably lied on 3-dimension structure change in ethanol solution.3-dimension structure changes probably lead to easier binding of substrate or easier releasing of product.

3.4.Effect of reagents on endoglucanase activity

Over 90%activity of the endoglucanase was remained in K+,Na+Mg2+,or Fe2+solution.Ca2+and Mg2+slightly enhanced the endoglucanase activity.Ca2+and Mg2+were necessary to increase the substrate binding affinity of the enzyme and stabilize the conformation of the catalytic site[44].The endoglucanase only remained 21%activity in Pb2+solution.Some other works reported that heavy metals decreased endoglucanase activity[45].

The endoglucanase activity was enhanced by ethylenediaminetetraacetate(EDTA,10 mmol·L-1)or dithiothreitol(DTT,10 mmol·L-1).The endoglucanase activity was remained in EDTA solution.Mucor circinelloides endoglucanase activity was enhanced by EDTA or DTT[33].The result indicated that the endoglucanase from a marine B.subtilis was not a metal ion enzyme.

Table 2Substrates specificity of the endoglcanase at 35°C and pH 6.0

3.5.Substrate specificity

The endoglucanase could hydrolyze CMCNa.It could not hydrolyze cellobiose,avicel,and laminarin(Table 2).The endoglucanase had the affinity to the substrate ofCMC.The results concurred with the other reported results.The endoglcuanase from B.subtilis AS3 could only hydrolyze CMC[46].The endoglucanase from Bacillus sp.SV1 could hydrolyze CMC and cellobiose[47].Bacillus amyloliquefaciens endoglucanase could hydrolyze CMC and avicel[48].Hydrolyzing avicel needs exoglucanase activity.Unusual structure of bind domain and catalysis domain endowed some endoglucanase exoglucanase activity.

3.6.Melt points of the endoglucanase in ethanol solution

In ethanol free solution,the melt point of the endoglucanase was 37.1°C.In 6%ethanol solution,the melt point of the endoglucanase was 39.2 °C(Fig.5).The melt point of the endoglucanase was 1.9 °C higher in 6%ethanol solution than in ethanol free solution.The higher the meltpointofthe endoglucanase,the more stable the endoglucanase.The thermostability increasing in ethanol solution indicated that the structure of the endoglucanase changed in ethanol solution.The endoglucanase structure was more stable.It was valuable forbioethanol product by enzymatic hydrolyzing cellulose.

3.7.Thermodynamic kinetics parameters in solution with different concentration ethanol

Thermodynamic kinetics could be used to evaluate enzymes thermostability.Gibbs free energy,ΔG,was an effective parameter to analyze enzyme thermostability.The higher ΔG,is,the more stable enzyme is[49].At 45 °C–60°C,ΔG decreased with temperature increasing(Table 3).The enzyme thermostability decreased with temperature increasing.ΔG was higher in 6%ethanol solution than that in ethanol free solution(Table 2).The endoglucanase was more stable in 6%ethanol solution than in ethanol free solution.The endoglucanase probably changed to a more stable structure in 6%ethanol solution.Ethanol is a polar compound.An oxhydryl of ethanol probably interacted with basic amino acids.The interaction probably made the endoglucanase have a different structures or molecular bridges.Structure or molecular bridges change could increase the stability.

Fig.5.Melt points of the endoglucanase in solution with different concentrations of ethanol.

Table 3Gibbs free energy of endoglucanase in different concentrations of ethanol solution

The reason for the endoglucanase catalysis increasing in ethanol solution may be that ethanol could act as an acceptor for the intermediate glycosyl cation[43,50].

3.8.Secondary structure in different concentrations of ethanol solution

Secondary structure change can lead to 3-dimensional structure change.Secondary structures of enzymes include β-sheet,α-helix,unorder coil.The contents of secondary structures could be analyzed with FT-IR.The absorbance value of the amide peak at approximate 1670 cm-1could be used to measure the content of the enzyme βsheet[51].The absorbance value of the amide peak at 1650–1640 cm-1and could 1660 cm-1be used to measure the content of α-helix content[52],unordered coil[53].

The absorbance at 1660 cm-1and 1670 cm-1was same in ethanol free solution with that in 6%ethanol solution(Fig.6).The results indicated that the content of β-sheet and unordered coil was same in ethanol free solution with that in 6%ethanol solution.The absorbance at 1650–1640 cm-1was higher in 6%ethanol solution than that in ethanol free solution.The results indicated that the content ofα-helix increased.Many research results confirmed that the increasing of α-helix content could enhance the thermostability[18,54].The α-helix content increasing made the endoglucanase thermostability increased.The α-helix content increasing probably led to the 3-dimension structure change.The 3-dimension structure change made the substrate more easily hydrolyzed.To systematically reveal the mechanism for ethanol tolerance of the endoglucanase,3-dimension structure of the endoglucanase should be analysis by NMR in ethanol solution.

Fig.6.FI-IR scan curves of the endoglucanase in different concentrations of ethanol solution.

4.Conclusions

Bottleneck of ethanol industrial scale product was high cost.Ethanol tolerant endoglucanase is valuable for decreasing bioethanol cost.The endoglucanase from a marine B.subtilis cellar showed higher activity and better thermostability in ethanol solution.The increasing of αhelix content probably led to better catalysis properties in ethanol solution.The acidic,thermostable and ethanol tolerant endoglucanase was valuable for bioethanol product by simultaneous saccharification and fermentation process.

Acknowledgments

The work was supported by Science and Technology Research Project of Hubei Provincial Department of Education(B2017041).