SarojiniChiluvuri
Department
ABSTRACT
hasgiventothevariables.Toconfirmtheseresults,experimentswerecarriedoutusingRSMthesenutrientswithsame concentrationsanditwasobservedthatthemeanvalueofalkalineproteasewas10.13U/mLascomparedtothepredictedvalueof10.76U/mLusingMINITAB.The finaloptimizedmediumhasgivenapproximatelythree-foldincreaseinalkalineproteaseproductionincomparisonwithone-factor-at-a-time(5.12U/mL)method.
KEYWORDS:MINITAB,Taguchi,PenciliumoxalicumKRSS-S-FP10,PDAmedia,optimization.
INTRODUCTION:
Currentlyenzymescanbeusedinbasicandappliedarenasofresearchaswellas inawiderangeofproductdesignandmanufacturingprocesses,suchasfood,beverage, pharmaceutical, detergent, leather processing and peptide synthesis industrieswiththeestimatedvalueoftheglobalsalesover3billionUSD(Jordan Chapmanetal.,2018).Ofthewholeindustrialenzymes,75%arehydrolytic.
Threesoilsampleswerecollectedindepthof5-6cmfromsoil.AlkalinepHwas observed across the sampling sites and a total of 3 samples were found with plentyofmicrofloraoccurrence(AshokPandeyetal.,(2013)).Thetotalmicroorganismcountsofthesoilswereestimatedbystandarddilutionplatetechnique. The isolated microbes were identified by their cultural and fungi and actenomycetes were obtained from the positive three soil samples (Dilution WorksheetandProblems(2021)).Theseare10fungalformsfromKakinada,9 fromVishakhapatnamand40fromTenaliblackfieldmorphologicalcharacteristics.Total 74 microorganism forms (includes bacteria soils.Alkaline proteases fungiwereisolatedusingmilkagarplateassayconsistsof0.5%caseinfromdifferent soils collected from Kakinada, Vishakhapatnam beach soil and Tenali blacksoilfieldsofAndhraPradesh.Threesoilfungalisolateswereexaminedfor protease producing microorganism. Isolation and RAPD-identification of the highest alkaline protease producer under submerged fermentation, using PDA media.
MATERIALSANDMETHODS:
Reagents:Unlessotherwisestated,thechemicalsandmediumingredientused inthisstudywerepurchasedfromSigmaChemicalCo.(St.Louis,Mo,USA)and BDBioscience(LePontdeClaix,France).Allotherchemicalswerealsoofanalyticalgrade.
Optimizationofenzymeproduction:Characterizationofthedifferentfactors foralkalineproteaseproductionwasoptimizedbyapplyingRSM.ThenstatisticalmodelwasobtainedusingCentralcompositedesign(CCD)withthreeindependent variables alkaline protease (X), (X) and MnSO concentration (X). 1 2 4 3 CCDmaximizestheamountofinformationthatcanbeobtainedwhilelimiting thenumberofindividualexperiments(KunamneniandSingh,2005).Eachfactorinthedesignwasstudiedatfivedifferentlevels(Table-1).Asetof20experimentswereperformed.Allvariablestakenatacentralcodedvalueswerelistedin Table-2 Uponcompletionofexperiments,theaverageofalkalineproteaseproductionwastakenasthedependentvariableorresponse.
Statisticalanalysis:OnefactorANOVAinTaguchiandMINITABsoftwarewas usedtoanalysetheexperimentalresults.Statisticalanalysisisconsideredasan importanttooltointerpretandsummarisetheexperimentaldata.Alltheexperiments results were carried out in duplicate and data obtained is represented as mean±SDinthispaper P-valuesoftheexperimentaldataarereportedrespective figures.The P-value less than 0.05 is considered to be statistically significant.
RESULTSANDDISCUSSION: 4.5.1OptimizationofthefactorsofTaguchiL :12
UsingL orthogonalarraydesignapproach,therelationshipsbetweenmedium 12 componentvariablesandtheirconcentrationscouldbeworkedout.Theoptimal combinationsandtheconcentrationofthefactorsrequiredtoachievethehighest alkalineproteaseactivitywasrepresentedinTable4.5.1.1.Thealkalineprotease activity was in between 2.0 U/mLto 10.76 U/mLat different levels of various independent factors. The Taguchi approach suggests to analyze variation by usinganappropriatelychosensignal-to-noise(S=N)ratioandalsoservetoanalyzetheaverageresponseforeachrunintheinnerarray TheTable-4.5.1.2and 4.5.1.3 represent the response table for mean and S/N ratio to understand the deltaandtherankvalueofthesystem.TheDeltavalueindicatestheeffectofthat componentwhereasrank(Table-4.5.1.4)basedonthedeltavaluesserializedthe factorsfromthegreatesteffecttotheleasteffectontheresponse.
Inthepresentstudy,itcanbeseenthatforeachofthesevenvariablesatthreelevels, one level increases the mean compared to the other level (Table-4.5.1.5). Thus the factors NaNO at level-1, MgSO at level-1, ZnSO at level-1, TAat 3 4 4 level-1,KHPO atlevel-2,FeSO atlevel-1,KClatlevel-1showsamaineffect. 2 4 4 Theselevelsalsorepresenttheoptimalconcentrationsoftheindividualcomponents in the medium. The Fig.10 and Fig.11 represent the main effect plot of means and S/N ratio in the system. Final optimized levels of each factor in the mediumforalkalineproteaseproductionhadshowninTable-4.5.1.1.Toconfirm theseresults,experimentswerecarriedoutusingthesenutrientswithsameconcentrations and it was observed that the mean value of alkaline protease was 10.13U/mLascomparedtothepredictedvalueof10.76U/mLusingMINITAB. Thefinaloptimizedmedium(Table-4.5.1.6)hasgivenapproximatelythree-fold increaseinalkalineproteaseproductionincomparisonwithone-factor-at-a-time (5.12U/mL)method.Therefore,itshouldbeconsideredthattheselectedconditionswerethemostsuitableinpracticeandNaNO,caseinandKClwereidenti- 3 fiedasthemajorinfluencingfactorsintheenzymeproduction.
10 10 1 1 0.005 0.050 1 1 6 10 2 8.240 8.520
11 10 10 1 0.050 0.005 1 1 6 10 2 10.630 10.270 12 10 1 4 0.005 0.005 4 4 6 10 6 10.760 10.130
Table4.5.1.2.ResponseTableforSignaltoNoiseRatios
1 40.4238 43.5218 40.6685 25.1055 28.9432 22.9226 18.0618 39.9127 37.3846 41.8684
2 36.6501 36.4160 36.0426 36.9830 37.0424 34.8195 35.8304 35.4223 36.5373 35.5176 3 31.9141 30.1763 30.6532 31.2489 33.0206 31.4222 31.6511 30.6031 30.9134 30.1116 4 35.9046 36.1387 36.5120 35.5717 35.5123 37.7351 36.7242 37.1324 36.0173 37.0370 5 9.5424 39.4626 33.2552 37.5012 0.0000 36.3909 36.9020 34.9638 31.5957 42.3454 Delta 30.8814 13.3455 10.0152 12.3958 37.0424 14.8126 18.8402 9.3096 6.4712 12.2338 Rank 2 5 8 6 1 4 3 9 10 7
52*C^2+200 31*ABD158 81*ACD+181 31*BCD130 35*A^2D396 40*B^2 D138.66*C^2D.
FinalEquationinTermsofActualFactors(pH9.0): R1=+1.22166E+0051.83737E+006*MnCl21884.72947*Casien+14828.82972 *NaMoO42420.00000*MnCl2*Casien2.48718E+005*MnCl2*NaMoO4530.7 6923*Casien*NaMoO4+1.01484E+007*MnCl2^2+27.95051*Casien^2+6.73 725E+005*NaMoO4^2and
FinalEquationinTermsofActualFactors:pH10.0: R1=+37550 07733-76878 05410*MnCl2-2039 89282*Casien+2 87974E+ 005*NaMoO4+5592.50000*MnCl2*Casien3.50641E+006*MnCl2*NaMoO4 +1328 84615*Casien*NaMoO42 79959E+005*MnCl2^2+20 02245*Casien^ 25597.90761*NaMoO4^2
Table4.5.2.1:Actualandpredictedvalues
Fig.11:MainEffectsPlotforMeans
4.5.2.
Fig.12:MainEffectsPlotforSNratios
OptimizationoftheselectedmediumcomponentsbyRSM:
TheTaguchiresultsshowedthatamongsevendifferentculturecomponents,four have significant impact on enzyme production (Mishra, Rashmi, (2020)). Responsesurfacemethodologywasusedtoinvestigatetheeffectsoffourcomponentsviz.MnCl,Casein,NaMoO andpHbytakingasindependentvariables 2 4 onalkalineproteaseenzymeproduction(MalhotraandChapadgaonkar,2020).It wasfoundthatalkalineproteaseproductionwasenhancedupto10.76U/mLin themediumoptimizedbyRSM.
TheCCDmatrixintermsofcodeandactualvaluesofindependentvariablesis giveninTable-4.5.2.1.Thesecondordermodelequationwasquantifiedbyusing Analysisofvariance(ANOVA).ANOVAofthequadraticmodelindicatedthat themodelissignificant.InthisstudyitwasfoundthatA,C,D,A2,H2,AB,AC, AH, BH and CH (A=MnCl 0.005 g/L, B=casein 13.1521g/L, C= NaMoO 2 4 0.0205g/L,andD=pH9.0/10.0)arethemodelterms.Thesecondorderresponse modelwasmadeafteranalysisofregression.Themodelcanbeillustratedasfollows.
FinalEquationinTermsofCodedFactors: R1=+523.9752.65*A3642.48*B45.32*C173.27*D+79.31*AB183.06*AC118. 23*AD+77.81*BC+842.31*BD101.88*CD+123.35*A^2+2398.65*B^2+117.
The significance of second order model equation was verified by F (ANOVA) test (Table-4.5.2.3.). For this model, if the F test is significant at 5% level i.e. P<0.0001, then the model 5 is considered as fit.ANOVAanalysis can explain whetherthemorecomplexmodelisrequiredforabetterfitanditgivesthevalues of the model (Rouaa Daou et al., 2021).According to theANOVO of the quadraticregressionmodel,itwasidentifiedthatthepresentmodelishighlysignificant and it is justified by the F test value of 49.79 with low probability value (Pmodel>Fis0.0001)(Table-4.5.2.3).The"PredR-Squared"of0.9793isnotas close to the "Adj R-Squared" of 0.9596as one might normally expect (Abiola EzekielTaiwo et al., 2020). Thismayindicatealargeblockeffectorapossible problem with the model and/or data. Things to consider are model reduction, response tranformation, outliers, etc "Adeq Precision" measures the signal to noiseratio.Aratiogreaterthan4isdesirable(SunilChamoli,2015).Aratioof 31.246indicatesanadequatesignal(Table-4.5.2.4.).Thethreedimensionalplots oftheresponsesweredrawntopredictthealkalineproteasefordifferenttestvariable values to understand the interaction among the independent factors (Ram 2 2 2 Kumaretal.,2018).TheplotsofA,B,C,AC,BC,BDwereconstructedbytakingresponse(alkalineproteaseproduction)atz-axisagainsttwovariables(Fig.
12) It was observed that medium containing MnCl 0 005 g/L, casein 2 13.1521g/L,NaMoO 0.0205g/L,andpH9.0/10.0. Thepredictedresultscom- 4 parativelyshow0.5-foldincreasewithTaguchi(10.76to15.82U/mL)alkaline protease activity (Hammami, Bayoudh, and Abdelhedi, 2018). Similarly, the effectsofparameterssuchas,relativehumidity,pHoftheliquidmedium,andvolumeofinoculumswerepositiveandsmallerthantheerrorlimitsandthechange in mean effect was also small, in which case according to the decision-making procedure of the evolutionary operation technique it was advisable to select a new search region and start a new phase of experiments reported (Dhayalan, Velramar,Govindasamy,2022).RSMhasbeenappliedfordesigningofexperiments to evaluate the interactive effects through a full 31 factorial design and reportedthesimilarresultsbyGilmour,Steven,intheyear(2006).Theoptimum conditionswerecaseinconcentration,3.22%;fermentationperiod,96h;tempero 2 ature,30C;andpH9.0.Thehighvalueoftheregressioncoefficient(R=0.9793) indicateexcellentevaluationofexperimentaldatabysecond-orderpolynomial regressionmodel.TheRSMrevealedthatamaximumalkalineproteaseproductionof15.82U/mLwasobtainedattheoptimumconditionsandfoundthesimilar resultswithGomaa,(2013).
Table4.5.2.2:ANOVAforResponseSurfaceReducedcubicmodel Analysisofvariancetable.
Table4.5.2.3.Responsecurvestandardswithfactor1andR1
Fig.(a)
Fig.(b) Fig.(c) Fig.(d) Fig.(e)
Fig.(i)
Fig.(f)
Fig.(j)
Fig.(g)
Fig.(k)
Table4.5.2.4:StandardDeviation,Mean
Fig.(h)
CONCLUSION:
Presentinvestigationaimstodevelopnewbioprocessandeconomicallyfeasible media by using optimization of production medium composition. It effects the costofproductionmediumandfoundeffectiveproductionofenzyme.Bytaking PDAmediaandseriallyoptimizedthemediabyusingsomestatisticaltoolsare Taguchi,RSMandMinitabreducestheunwantedvariablesinthemediaforgood fermentation,thisstepreducesthecostofproduction.Asaresult,useofdependentandindependentvariablesenhancetheproductionofalkalineproteaseshake flaskfermentation.Thebioprocessoptimizationpredictedresultscomparatively show0.5-foldincreasewithTaguchitoRSM(10.76to15.82U/mL)alkalineproteaseactivityandalsohaveprepareddifferentproductionmediaforenzymefer-
mentation.
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