采用相应曲面法分析反胶束萃取淀粉酶的应用.pdf

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1、ORIGINAL PAPERApplication of reverse micelle extraction processfor amylase recovery using response surface methodologyManav B.Bera Parmjit S.Panesar Reeba Panesar Bahadur SinghReceived:16 July 2007/Accepted:22 October 2007/Published online:7 November 2007?Springer-Verlag 2007AbstractThe effect of di

2、fferent process variables ofreverse micelle extraction process like pH,addition ofsurfactant(AOT)concentration and potassium chloride(KCl)concentration on amylase recovery has been studiedand analysed.Solid-state fermentation was used for theproduction of amylase enzyme.Response surface meth-odology

3、(RSM)using central composite rotatable design(CCRD)was employed to analyse and optimize theenzyme extraction process.The regression analysis indi-cates that the effect of AOT concentration,and KClconcentration were significant,whereas the effect of pHwas non-significant on enzyme recovery.For the ma

4、ximumrecovery of enzyme,the optimum operating condition forpH,AOT concentration(M)and KCl concentration were10.43,0.05 and 1.00,respectively.Under these optimalconditions,the enzyme recovery was 83.16%.KeywordsAmylase?Reverse micelle extraction?Optimization?Response surface methodologyIntroductionEn

5、zymes make ideal catalysts in food industries owing totheir specificity,mild reaction condition and non-toxicity.Micro-organisms are the most convenient source forenzyme production.There are several micro-organisms,which serve as sources for enzyme production andapproved by the FDA for food use.Gene

6、rally,in the industry,crude enzymes are producedafter the centrifugation of the fermentation broth.Theenzymatic activity of these crude enzymes is very low andso it is required in large quantities for food processing.Thecrude enzymes usually have off-flavour,require morespace during storage and tran

7、sportation.Therefore,down-stream processing of these crude enzymes is required toextract the enzyme from the broth,purify and dry them,sothat it can be conveniently stored and used in smallquantities 1.Commercial interest in cost-effective meth-ods that can separate,concentrate and purify proteinsco

8、ntinuously and be easily scaled-up,has increasedmarkedly in recent years.In this context,liquidliquidextraction using reverse micellar organic solvents has beenfound to have this potential 2.It has been extensivelystudied with model proteins such as cytochrome C,ribo-nuclease,myoglobin,bovine serum

9、albumin,etc.However,not much work has been done on the actual system offermentation broth 36.Optimization of parameters by the conventional methoddoes not depict the interactive effect of all the variables andinvolvechangingoneindependentvariablewhileunchanging all others at a fixed level,which is e

10、xtremelytime-consuming and expensive for a large number ofvariables 7.Statistical methodologies can be applied inbiotechnological processes to observe the effect andinteractions of the major factors.Response surface meth-odology(RSM)is a collection of statistical techniques fordesigning experiments,

11、building models,evaluating theeffect of factors and searching for optimum conditions fordesirable responses.RSM can identify and quantify thevarious interactions among different parameters and it hasbeen applied for optimization of medium and culturalM.B.Bera?P.S.Panesar(&)?R.Panesar?B.SinghBiotechn

12、ology Research Laboratory,Departmentof Food Engineering and Technology,Sant Longowal Instituteof Engineering and Technology,Longowal 148106,Punjab,Indiae-mail:123Bioprocess Biosyst Eng(2008)31:379384DOI 10.1007/s00449-007-0172-6conditions in bioprocesses 8,9.Keeping the above inview,the RSM was empl

13、oyed in the present work tooptimize various process variables,i.e.pH,AOT(Aerosol-OT bis(2-ethyl hexyl)Sulphosuccinate sodium saltconcentration and potassium chloride(KCl)concentrationof reverse micellar phase composition for extraction andpurification of crude amylolytic enzyme.Materials and methods

14、Procurement of micro-organismAn amylolytic enzyme producing strain of micro-organismAspergillus niger(NCIM 641)obtained from the NationalChemical Laboratory,Pune(India),was used for the pro-duction of a-amylase enzyme.Cell cultivation and production of enzymeNutrient solution used for solid-state fe

15、rmentation wasprepared by mixing 4.45 g potassium di-hydrogen phos-phate,9.3 g ammonium sulphate and 2.3 g urea in 96 ml ofwater and pH was adjusted to 5.0 using 5 N ammoniasolution.The solution was sterilized and used for furtherexperimentation.The method applied for the production ofamylase using

16、wheat bran under solid-state fermentationtechnique 10 was adopted.Briefly,in this method,500 gof sterilized wheat bran was taken into the sterilized con-ical flask containing 500 ml of nutrient solution and mixedthoroughly.The media was inoculated with 4 9 106g-1concentration of A.niger NCIM 641 spo

17、res.The conicalflask was incubated in the BOD incubator at of 36?Ctemperature.After fermentation,entire fermenting mediumwas mixed with equal volume of distilled water andimmediately centrifuged at 6,000 rpm for 10 min at roomtemperature.The supernatant was collected and used ascrude enzyme for furt

18、her experiments.The amylaseactivity of crude enzyme preparation was found to be201.80 IU/ml.Extraction of enzyme from crude enzymeThe reverse micelle system was used for the extraction ofcrude enzyme.Reverse micelle system was developed bymixing predetermined levels of Aerosol-OT bis(2-ethylhexyl)Su

19、lphosuccinate sodium salt in isooctane,KCl andcrude enzyme(aqueous phase)(Table 2).The pH of thesystem was adjusted by addition of acid(1 N HCl)or alkali(5N NaOH).The entire mixture was taken in a 250-mlconical flask.The flask containing mixture was stirred in amagnetic stirrer for 30 min.The revers

20、e micellar systemwas allowed to settle for 30 min,which led to separation intwo phases.The volume of each phase was recorded.Measurement of a-amylase was done by estimating theincrease in the reducing power of a solution of solublestarch by DNSA method 11.All the experiments wereperformed in triplic

21、ate and the mean values have beentaken into consideration.Experimental designFor the optimization of enzyme recovery process,theexperiments were conducted according to Central Com-posite Rotatable Design(CCRD)12 with three variablesat five levels each.The design was generated by DesignExpert,Trial V

22、ersion 6.0,Stat-Ease INC.,Minneapolis,MN,USA statistical software.The variables were pH,AOT concentration and KCl concentration for enzymerecovery process.The low level and high level in the actual(un-coded)form were taken as 4.510.5(pH),0.050.1 M(AOT concentration)and 0.51.0 M(KCl concentration),re

23、spectively.The relationships between coded and un-coded independent process variables are given in Table 1.The experiment plan in coded and un-coded form ofprocess variables is as given in Table 2.The experimentswere conducted randomly.Statistical analysis and optimizationThe second order polynomial

24、 equation was fitted to theexperimental data of each dependent variable as givenbelowYk Bk0Xni1BkixiXni1Bkiix2iXn?1i1Xnji1Bkijxixj ek;1Table 1 Levels of different process variables in coded and un-codedform for reverse micelle processCoded levelsUn-coded values of process variablespHAOT Conc(M)KCl C

25、onc(M)(x1)(x2)(x3)-1.6822.4540.03290.329-1.0004.5000.0500.5000.0007.5000.0750.7501.00010.5000.1001.0001.68212.5460.1171.170380Bioprocess Biosyst Eng(2008)31:379384123whereYk=responsevariableY1=Enzymerecovery(%),xis represent the coded independent variablesx1=pH,x2=AOT conc(M),x3=KCl conc(M).WhereBko

26、is the value of fitted response at the centre point ofdesign,i.e.point(0,0,0),Bki,Bkiiand Bkijwere the linear,quadraticandcross-productregressioncoefficients,respectively.The regression analysis of the experimental data wascarried out to observe the significance of the effect ofvarious process param

27、eters on the various responses by thestatistical software 13.The relative effect of each processparameter was compared from the b-values correspondingto that parameter,the b coefficients being the regressioncoefficients obtained by first standardizing the processvariables to a mean of zero and stand

28、ard deviation to one.The advantage of using b coefficient(as compared to Bcoefficients which are not standardized)is that the mag-nitudes of these values help to compare the relativecontribution of each independent variable in the predictionof the dependent variable.The higher the positive value ofb

29、 of a parameter;the higher would be the effect of thatparameter and vice versa.For checking the validity ofempirical models for all the enzyme recovery processes,regression analysis was performed.The response surface and contour plots were generatedfor different interactions of any two independent v

30、ariables,while holding the value of the third variable as constant.Such three-dimensional surfaces could give accurate geo-metrical representation and provide useful informationabout the behaviour of the system within the experimentaldesign.Results and discussionDiagnostic checking of fitted model a

31、nd surface plotsThe results of second order response model in the form ofanalysis of variance(ANOVA)are given in Table 3.TheANOVA results indicated that the quadratic regressionto produce the second order model was significant(P0.0001).Thelackoffitwasnon-significant(P=0.0048),and only 0.48%of the to

32、tal variations werenot explained by model(R2=98.52%).The Model F-valueof 74.3595 also implies that the model is significant.Thevalue of the adjusted determination coefficient(adjustedR2=97.20%)was high to advocate a high significance ofthe model 14.This suggested that the model accuratelyrepresents

33、the data in the experimental region.This alsoindicated that second-order terms were sufficient andhigher-order terms were not necessary.The magnitude of P-values in Table 3 indicates that thelinear and quadratic and interaction terms of all the processvariables have significant effect on enzyme reco

34、very at 5%level of significance(P0.05).The relative magnitude ofb-values indicates the maximum positive contribution ofKClconcentration(b=0.3164)followedbypH(b=0.1570)on enzyme recovery.The effect of AOTconcentrationonenzymerecoverywasnegative(b=-0.2323).It implies increased enzyme recovery withincr

35、ease of KCl concentration and pH,and decreasedenzyme recovery with increase of AOT concentration.Theincrease of enzyme recovery with increase of pH is due tothe fact that the pH of the solution affects the solubilizationcharacteristics of the protein primarily in the way in whichit modifies the char

36、ge distribution over the protein surface.With increasing pH the protein becomes less positivelycharged until it reaches its iso-electric point.At pH abovethe iso-electric point the protein would take on a net neg-ative charge 15.At higher pH maximum protein/enzymemight have solubilized which increas

37、ed the recovery ofenzyme.The decrease of enzyme recovery with increase ofAOT concentration in the phase may be due to theincreased partitioning of biomaterial into other phase.The addition of surfactant(AOT)helps in changing theTable 2 Experimental designs in coded form of process variables forand v

38、alues of experimental data for optimization for Reverse micelleprocessCoded variablesUn-coded variablesResponsepH(x1)AOT(M)(x2)KCl(M)(x3)pH(X1)AOT(M)(X2)KCl(M)(X3)Enzymerecovery(%)(Y1)-1.000-1.000-1.0004.5000.0500.50055.2601.000-1.000-1.00010.5000.0500.50038.420-1.0001.000-1.0004.5000.1000.50063.530

39、1.0001.000-1.00010.5000.1000.50059.340-1.000-1.0001.0004.5000.0501.00068.4201.000-1.0001.00010.5000.0501.00083.150-1.0001.0001.0004.5000.1001.00038.1101.0001.0001.00010.5000.1001.00074.720-1.6820.0000.0002.4540.0750.75022.7101.6820.0000.00012.5460.0750.75030.9900.000-1.6820.0007.5000.03290.75077.980

40、0.0001.6820.0007.5000.11700.75068.7400.0000.000-1.6827.5000.0750.32952.9600.0000.0001.6827.5000.0751.17077.5200.0000.0000.0007.5000.0750.75040.5300.0000.0000.0007.5000.0750.75042.4700.0000.0000.0007.5000.0750.75039.4700.0000.0000.0007.5000.0750.75040.1200.0000.0000.0007.5000.0750.75040.1200.0000.000

41、0.0007.5000.0750.75040.000Bioprocess Biosyst Eng(2008)31:379384381123structure of the reversed micelles,changes in their adapt-ability in size and surface charge density 1.The increasein enzyme recovery with increase of KCl concentrationmay be due to the Salting in effect,brought in by therequired i

42、onic strength of KCl and further increase in saltconcentration of aqueous solution did not improve therecovery of enzyme.It is a well-known fact that increase inelectrolyte concentration in the aqueous phase causes adecrease in the water solubilized in reverse micelles 16,and this factor plays an im

43、portant role in protein/enzymeextraction.The relative magnitude of b-value indicates that theeffect of pH on enzyme recovery was less when comparedto AOT and KCl concentrations.The concentration of KClwas the most effective factor on enzyme recovery.Thequadratic terms of AOT and KCl have positive ef

44、fect andpH has negative effect on enzyme recovery.The maximumpositive contribution of quadratic term was of KCl con-centration(b=0.5286)followed by AOT concentration(b=0.4597)and negative effect of quadratic term of pH(b=-0.1534).The interaction terms of pH and KClconcentration,has more positive eff

45、ect(b=0.335642)followed by negative effect of the interaction term ofAOT and KCl concentration effect(b=-0.3150)onenzyme recovery.The effect of interaction term of pHand AOT was positive and lowest(b=0.16014)whencompared to the effects of other interaction terms.The b-values as well as Figs.13 indic

46、ate the highinteractive effect of pH,AOT and KCl concentration onenzyme recovery.Figures 1 and 2 indicate that with anincrease in pH,there is an increase in enzyme recovery athigh concentration of AOT and KCl.Figure 1 indicatesthat,with the increase in AOT concentration,the enzymerecovery decreased

47、up 0.08 M concentration and thereafterit increased.Similarly,Fig.3 indicates that with theincrease in AOT and KCl concentrations,there is adecrease and increase in the enzyme recovery,respectively.The equation of the model fitted for enzyme recovery inthe coded form of process variables is:Enzyme re

48、covery%40:32 3:24?x1?1:84?x2 6:53?x3?3:95?x21 12:50?x22 9:62?x23 4:32?x1?x2 9:05?x1?x3?8:49?x2?x3:The equation of the model fitted for enzyme recovery in theactual form of process variables is:Enzyme recovery%216:34479?5:70192?X1?2485:01615?X2?193:43966?X3?0:43873?X12 19;992:36617?X22 153:99001?X32

49、57:55?X1?X2 12:06167?X1?X3?1358:60?X2?X3Optimization of reverse micelle processIn order to optimize the process conditions for enzymerecovery process by numerical optimization technique,themain aim was to get maximum recovery of enzyme.Theoptimum operating conditions for pH,AOT and KClTable 3 Regres

50、sion summery and ANOVA table for Enzyme recovery for coded values of process variablesSourcedfbBSum of squaresF-valueP-levelModel95,943.8774.35950.0001Constant140.18402x110.1570183.238846143.280216.13230.0025x21-0.23235-4.7926846.09385.1898230.0459x310.3164726.527918582.021665.531350.0001(x1)21-0.15