A novel pectin-degrading enzyme complex from Aspergillus sojae ATCC 20235 mutants

A novel pectin-degrading enzyme complex from Aspergillus sojae ATCC 20235 mutants
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  1  5  5 4  ResearchArticle Received: 13 June 2014 Revised: 4 August 2014 Accepted article published: 8 August 2014 Published online in Wiley Online Library: 2 September 2014 ( DOI 10.1002/jsfa.6864 Anovelpectin-degradingenzymecomplexfrom  Aspergillussojae ATCC20235mutants MarcoA.Mata-Gómez, a,b DoreenHeerd, a IñigoOyanguren-García, c FrancisBarbero, c MarcoRito-Palomares b* andMarceloFernández-Lahore a* Abstract BACKGROUND:Inthefoodindustry,theuseofpectinasepreparationswithhighpectinesterase(PE)activityleadstothereleaseof methanol, which is strictly regulated in food products. Herein, a pectin-degrading enzyme (PDE) complex exhibiting lowPE activity of three  Aspergillus sojae  ATCC 20235 mutants (M3, DH56 and Guserbiot 2.230) was investigated. Production of exo-/endo-polygalacturonase(PG),exo-polymethylgalacturonase(PMG)andpectinlyase(PL)bymutantM3and  A.sojae usingtwo different carbon sources was evaluated in solid-state fermentation. Finally, experimental preparations obtained from themutantsandcommercialpectinasesstandardizedtothesamepotencywerescreenedforPDEs.RESULTS: Mutant M3 grown on sugar beet was found to be the best producer of exo-PG, endo-PG, exo-PMG and PL, withmaximumyieldsof1111,449,130and123Ug − 1 ,respectively.AllexperimentalpreparationsexhibitedlowPEactivity,atleast21.5timeslessthancommercialpectinases,andhigherendo-PG(40UmL − 1 ).CONCLUSION:MutantM3wasthebestPDEproducerusingsugarbeet.MutantstrainspresentedaPDEcomplexfeaturinghighendo-PGandverylowPEactivities.Thisnovelcomplexwithlowde-esterifyingactivitycanbeexploitedinthefoodindustrytodegradepectinwithoutreleasingmethanol.©2014SocietyofChemicalIndustryKeywords:  Aspergillussojae ; pectin-degrading enzyme complex; pectin esterase; pectinase; polygalacturonase INTRODUCTION Pectin-degrading enzymes (PDEs) – or pectinolyticenzymes – have been widely exploited in the food industryto degrade pectin, mainly in the beverage industry of wineand fruit juices. 1 Pectin is the most abundant and complex het-eropolysaccharide in nature, after cellulose, constituting the cellwall of plants. It is mainly constituted by  D -galacturonic acidunits (70%, approximately) linked by  D -1,4 bonds. Pectin canpresent a specific degree of esterification (low or high) alongthe backbone that varies depending on pectin source. 2 Due tothe complex structure of pectin, a synergistic action of differentPDEs is required for its complete biodegradation. The enzymesresponsible for pectin degradation are divided in two maingroups according to their mode of action: methyl esterase suchas pectin esterase (PE, EC that attacks on the methylester group from high-esterified pectin, and depolymerases thatbreak down the    -1,4 linkages between  D -galacturonic acid units.The latter includes hydrolases like endo-/exo-polygalacturonase(endo-/exo-PG,EC3.2.1.15/, and lyases such as pectin lyase (PL, EC 3 AmongPDEsproducingmicroorganismsareyeasts,bacteriaandfungi.However,fungiarethebestproducers. 4 Usually,theyexcretedifferent types of PDEs during infection processes of plants thatdegradecellwallpectin,facilitatingpenetrationandcolonization. 5 A fungal complex of PDEs mainly consists of PE, PG and PL,although it may vary between fungal species according to therequirements of habitat and carbon source. 6 Most commercialpreparations are produced by filamentous fungi,  Aspergillus niger  and  Aspergillus aculeatus  being the most used. 7 In general, com-mercial pectinases are well-balanced preparations composed of PG,PL,PMGandPE. 8 Nonetheless,mostofthesepreparationscon-tain high levels of PE activity, resulting in the release of methanol,which is an undesired byproduct. Presence of methanol in theproduct culminates in additional purification steps and in anincrease in production costs. Thus preparations with low levelsof PE activity are preferred. 9 The finding of new PDE complexeswith novel characteristics like low PE activity is of great interestto improve the process efficiency and obtain safer food products.In such a manner, knowledge of the types of enzyme activitiespresentincrudeextractsisimportantinenhancingtheirindustrialapplication. ∗ Correspondence to: Marco Rito-Palomares, Centro de Biotecnología-FEMSA,Tecnológico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501Sur,Monterrey, NL 64849, Mexico; or Marcelo Fernández-Lahore, DownstreamBioprocessing Laboratory, School of Engineering and Science, Jacobs Univer-sity, Campus Ring 1, D-28759, Bremen, Germany. E-mail:; a  Downstream Bioprocessing Laboratory, School of Engineering and Science, JacobsUniversity,D-28759,Bremen,Germany  b  Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Monterrey, NL64849,Mexico c  GuserbiotSL,01015,Vitoria-Gasteiz,Álava,Spain  JSciFoodAgric   2015; 95 : 1554–1561 © 2014 Society of Chemical Industry  1  5  5  5  A pectin-degrading enzyme complex from  A. sojae  mutants  Aspergillus sojae  ATCC 20235, termed  A. sojae  in this study, hasdemonstrateditspotentialtoproducepectin-degradingactivityinsubmergedfermentation(SmF)andsolid-statefermentation(SSF)processes. 10 , 11 Improvement of the  A. sojae  strain by mutagenesiswas recently carried out; the two resulting mutant strains codedas M3 and DH56 showed enhanced PG activity under SSF andSmF, respectively, using sugar beet-based media. 12 Another  A.sojae  mutant strain, Guserbiot 2.230, also showed high PG activityin SmF. Although these mutants have shown great potential todegrade pectin, the complex of PDEs secreted by these newmutant strains is unknown. The purpose of this research was tocharacterize the PDE complex of the  A. sojae  mutants mentionedabove,withspecialattentiontothePDEcomplexfromthemutantstrain M3. EXPERIMENTAL Materials Unmolassed sugar beet pellets (Nordzucker AG, Uelzen, Ger-many), and apple pomace (Döhler Dienter GmbH, Neuenkirchen,Germany), previously freeze dried, were ground using a smallhigh-speed mixing system (0.5L capacity); the apple pomaceconsisted of a mixture of different kinds of apples such as Elstar,Jonagold, Jonagored and Braeburn. An approximate chemicalcomposition of the carbon sources is presented in Table 1. 13 – 15 Powdered carbon sources with heterogeneous particle size wereused in combination with fine wheat bran (90% < 630 μ m) sup-plied by Bremer Rolandmühle Erling GmbH & Co. KG (Bremen,Germany). Sugar beet molasses Goldsaft  ®   (Grafschafter Kraut-fabrik, Mackenheim, Germany), used for preparation of inocula,was purchased from a local market in Bremen, Germany. Com-mercial pectinase preparations were kindly donated by BegerowGmbH & Co. (Langenlonsheim, Germany) in powder presentationsuch as Clair Rapide and Fino G or in a concentrated solutionsuch as Pro Clear, First Yield (Novozymes  ®  ) and Yield MASH(Novozymes  ®  ). Fructozym  ®  P was provided by ERBSLÖH (Geisen-heim, Germany) as a concentrated solution. Polygalacturonicacid ( M r = 25 000–50 000, No. 81325),  D -( + )-galacturonic acidmonohydrate (No. 48280-F), alcohol oxidase from  Pichia pastoris (EC number, 29.5 U mg − 1 protein, product No. A2404),acetyl acetone ReagentPlus  ®   (2,4-pentaendione, No. P7754-A)and 2-thiobarbituric acid (No. T5500-25G) were purchased fromSigma-AldrichGmbH(Schnelldorf,Germany).Citruspectin(ClassicCF 201, 71% degree of esterification) was provided by Herbstreith&FoxKGGmbH(Neuenbürg,Germany).Pierce  ®  Coomassie(Brad-ford)ProteinAssayand10,000NMWCSnakeSkin  ®  dialysistubing(No. 68100) were acquired from Thermo Scientific (Schwerte,Germany). Ruthenium red (No. A3488) and all other chemicals of analytical grade were supplied by Applichem GmbH (Darmstadt,Germany). Microorganisms,propagationandinoculum The  A. sojae  ATCC 20235 strain was purchased from ProcochemInc. (Teddington, United Kingdom), an authorized distributor of theATCC(AmericanTypeofCultureCollection)inEurope.Ithastobe remarked that  A. sojae  ATCC 20235 does not meet the require-ments to be classified as  A. sojae  on the basis of morphologicalparameters, 16 and thus it has been reclassified as  Aspergillusoryzae . 17 The mutant strains M3 and DH 56 were generated byHeerd  etal. 12 The  A. sojae  mutant Guserbiot 2.230 was developedby Guserbiot SLU (Vitoria-Gasteiz, Spain). The strains were prop-agated on yeast malt extract agar plates (10gL − 1 malt extract, Table1.  Approximate chemical composition of the carbon sourcesCarbon sourceComponent (gkg − 1 ) Sugar beet Apple pomaceMoisture 110 a 730 b Ash 51 16-61Total protein 90 24.3–56.7Reducing sugar NA c 303.4Total sugar NA c 359–640Pectin 287 31–143Cellulose 200 72–430Lignin 44 NA ca Value provided by the supplier of sugar beet. b Value determined by drying at 105 ∘ C until constant weight. c Not available. 4gL − 1 yeast, 4gL − 1 glucose and 20gL − 1 agar) and incubated for7 days.The inocula were prepared with the spores obtained from thepropagation step. Spores were inoculated in 50mL conical tubeslants containing sugar beet molasses agar with the followingcomposition (gL − 1 ): glycerol (45), molasses (45), peptone (18),sodium chloride (5), potassium chloride (0.5), ferrous sulfate hep-tahydrate (0.015), potassium phosphate monobasic (0.060), mag-nesium sulfate (0.05), copper sulfate pentahydrate (0.012), man-ganese sulfate monohydrate (0.015) and agar (20). After 7 daysof incubation at 30 ∘ C, spores were harvested in sterile Tween 80(0.2gL − 1 ) and counted on the microscope using a Thoma cham-ber (0.1mm depth, 0.0025mm 2 ). Spore suspension was used asinoculum for the fermentation process. Media,cultureconditionandrecoveryofenzymes Production of PDEs either by SSF or SmF was carried out asdescribed by Heerd  etal. 12 In the case of SSF, Erlenmeyer flasksof 300mL containing the solid substrate (7g wheat bran and 3gsugar beet or apple pomace, wetted with 16mL of 0.2molL − 1 HCl) were sterilized at 121 ∘ C for 15min. The solid medium (10g)was inoculated with 1mL spore suspension (2 × 10 7 spores mL − 1 )from mutant M3 or  A. sojae  and maintained at 30 ∘ C in an incu-bator (Heraeus UB6760, Hanau, Germany). After an appropriateincubation time, enzymes were recovered by adding 100mL dis-tilled water to the solid medium and agitated at 300rpm (Innova4000, New Brunswick Scientific) for 1h. Crude extracts were cen-trifuged (Eppendorf 5810 R, Hamburg, Germany) at 3200 × g  for30min at 4 ∘ C. Supernatants were filtered through filter paper No.1288 (Sartorius AG, Göttingen, Germany) using a Büchner porce-lain funnel. Samples of 2.5mL of the filtrated supernatant weredesalted with PD-10 columns (Sephadex TM G-25, Amersham Bio-sciences, Freiburg, Germany).SmF was carried out in 250mL Erlenmeyer flasks containing30mL medium with the following composition (gL − 1 ): molasses(95), ammonium sulfate (10) and sugar beet pellets (30). ThepH was adjusted at 4.0 by adding diluted HCl solution andflasks were sterilized for 15min at 121 ∘ C. The flasks were inoc-ulated with spores (3.8 × 10 5 spores mL − 1 medium) from themutant strains DH56 or Guserbiot 2.230 and incubated at 30 ∘ Cwith agitation at 250rpm (Innova 4000, New Brunswick Scien-tific) for 4 days. Fermentation broth was clarified as describedabove.  JSciFoodAgric   2015; 95 : 1554–1561 © 2014 Society of Chemical Industry  1  5  5  6 MA Mata-Gómez  etal. Screeningofenzymeactivitiesinexperimentalandcommercialpreparations Cultures from the three mutant strains and its parental strain  A. sojae  (all referred to as experimental preparations), obtainedas indicated further, were filtrated through a 0.45 μ m celluloseacetate membrane (Sartorius, Stedim Biotech GmbH, Göttingen,Germany), dialyzed against water at pH5 for 48h using dialysistubingandsubsequentlylyophilizedinafreezedryer(modelalpha1-4 LSC; Martin Christ  ®   GmbH, Osterode am Harz, Germany). Tocompare experimental preparations and commercial pectinases,both were standardized to the same potency on the basis of exo-PGactivity.Briefly,lyophilizedexperimentalsamplesandcom-mercial preparations were resuspended or diluted in 0.1molL − 1 acetate buffer at pH4.8 in order to prepare enzyme solutionsof approximately 100 U mL − 1 . Enzyme solutions were desaltedusing PD-10 columns (Sephadex TM G-25; Amersham Biosciences,Freiburg, Germany) before enzyme assays. Determinations wereperformed in triplicate. Enzymeassaysanddeterminationoftotalprotein Exo-pectin-degradingactivity,PGorPMG,wasevaluatedbydeter-mining the reducing sugar released after enzyme reaction using2.4gL − 1 polygalacturonicacidorpectinassubstratein0.1molL − 1 acetate buffer at pH4.8. 18 Galacturonic acid was used as stan-dard. One unit of exo-enzyme activity was defined as the amountof exo-PG or exo-PMG that releases 1 μ mol  D -galacturonic acidmin − 1 by hydrolyzing the reducing end of polygalacturonic acidor high-esterified pectin under standard assay conditions.Endo-pectin-degrading activity was estimated by determiningthe amount of hydrolyzed substrate using ruthenium red.  19 Oneunit of endo-enzyme activity was defined as the amount of endo-PG required to hydrolyze, in a random way, the glycosidicbonds of 1mg polygalacturonic acid min − 1 under standard assayconditions.Lyase activity was evaluated in the presence of pectin (10gL − 1 )prepared in 0.1molL − 1 acetate buffer at pH4.8. The reaction mix-ture contained 250 μ L of the sample and 250 μ L of the substrate.After the reaction, the unsaturated galacturonides released weredetected with 2-thiobarbituric acid as described previously. 20 Thefinal reaction mixture (1.65mL) was filtered through a 0.45 μ msyringe filter (Sartorius, Germany) and the absorbance was mea-sured at 550nm using a 1cm quartz cell. Blanks were prepared byadditionofacetatebufferinsteadofenzyme.OneunitofPL,whichacts on high-esterified pectin by trans-elimination, was definedas the amount of enzyme that releases the necessary amount of unsaturateduronicesterunitstoincreasetheabsorbanceby0.01.Esterase activity was evaluated by measuring the methanolreleased in the reaction mixture, which consisted of 10 μ L sam-ple and 90 μ L pectin solution (5gL − 1 ) prepared in 0.05molL − 1 acetate buffer at pH4.8. The reaction was conducted at 25 ∘ C per10min in Eppendorf tubes. The methanol was quantified withalcohol oxidase and acetyl acetone as described by Klavons andBennett. 21 Oneunitofesteraseactivitywasdefinedastheamountof PE that releases 1 μ mol methanol under standard assay condi-tions. Absorbance measurements were performed in a UV-1700Shimadzu spectrophotometer (Duisburg, Germany). Protein con-tent was estimated using a micro-assay based on the method of Bradford 22 and bovine serum albumin was used as standard. Statisticalanalysis Data were validated by statistical analysis. One-way analysis of variance( P  < 0.05)andTukey’spairwisewerecarriedoutusingthestatistical software MINITAB  ®   16.1.1 (Minitab Inc., State College,PA, USA). 23 RESULTSANDDISCUSSION The PDE complex of three  A. sojae  ATCC 20235 mutants – M3,DH56andGuserbiot2.230 – wasinvestigated.Theresultsarepre-sented as follows. First, discussion is focused on PDE productionby the mutant M3 and its parental strain under SSF using two car-bon sources (sugar beet or apple pomace) and how the substrateinfluencedthePDEcomplexregardingitspectinolyticactivitypro-file. Then, a comparison between the pectinolytic activity profilesfound in the standardized experimental preparations obtainedfrom the three  A. sojae  mutants (M3, DH56 and Guserbiot 2.230)and commercial pectinases is presented. Productionofpectinolyticenzymesusingsugarbeet The production of PDEs under SSF by the mutant M3 and itsparental strain  A. sojae  using sugar beet as carbon source ispresented in Fig. 1. Both strains were able to produce a PDEcomplex with potential to depolymerize pectin by degrading its   -1,4 linkages either by hydrolytic (exo-/endo-PG and exo-PMG)ortrans-eliminative(PL)mechanismbutthemutantM3presentedthe highest levels of PDEs. Maximum levels of exo-PG activity,718 and 1111 U g − 1 , were obtained for  A. sojae  and mutant M3,respectively, after 8 days of cultivation (Fig. 1A). The mutant M3produced twofold endo-PG activity (449.20 U g − 1 ) as comparedto  A. sojae  (228.6 U g − 1 ) at day 8 of fermentation (Fig. 1B), whilethe highest exo-PMG activities, 120 and 130 U g − 1 , were observedat days 7 and 6 of cultivation of   A. sojae  and its mutant M3,respectively (Fig. 1C). In the case of PL, the highest activity wasattained at day 8 for both strains  A. sojae  (79 U g − 1 ) and mutantM3 (123 U g − 1 ), as observed in Fig. 1(D). It should be noted thatmutantM3showedagreaterpotentialfortheproductionofPDEs,especially exo-PG, endo-PG and PL activities, in comparison to itsparental strain  A.sojae .Previous studies have demonstrated the capacity of   A. sojae  todegrade pectin by hydrolytic activity of exo-PG 24 – 26 and, morerecently, the  A. sojae  mutant strains M3 and DH56 showed thesame ability. 12 In particular, the mutant M3 has been used forexo-PG production using solid orange peel 25 and wheat bran 27 as carbon sources, with maximum yields of 35 and 535.4 U g − 1 ,respectively, which are lower than the yields of exo-PG activ-ity (1111 U g − 1 ) obtained here by mutant M3 (Fig. 1B). Otherearly studies evidenced the presence of both exo-/endo- PGand exo-/endo- PMG activities in solid orange peel cultures from  A. sojae .  26 In this study, it is demonstrated that both strains, themutant M3 and the parental strain, not only produce a complexof PDEs with hydrolytic (exo-PG, endo-PG and exo-PMG) activ-ity, but also with trans-eliminative (PL) activity (Fig. 1). Similarly,a PDE complex produced by the fungus  Colletotrichum trunca-tum inapectin-basedmediumexhibitedhydrolytic(PMG,PG)andtrans-eliminative(PL)activities. 28 Unlikethisstrain,mutantM3and  A. sojae  produced a higher level of hydrolytic (PG) activity and alower level of PL activity. The high values of activity produced bymutantM3,especiallyhydrolyticactivity(exo-PGandendo-PG),incomparisontootherfungalstrains,suggestthatmutantM3repre-sents an attractive alternative to produce PDEs. Effectofcarbonsourceonpectinolyticenzymeproduction Two carbon sources, i.e. sugar beet and apple pomace, wereinvestigated for their effect on PDE production by mutant M3  © 2014 Society of Chemical Industry  JSciFoodAgric   2015; 95 : 1554–1561  1  5  5 7  A pectin-degrading enzyme complex from  A. sojae  mutants Figure 1.  Production of PDEs (A, exo-PG; B, endo-PG; C, exo-PMG; and D, PL) activities by mutant M3 ( , ) and  Aspergillus sojae  ( , ) under solid-statefermentation using sugar beet as carbon source. Circles and squares represent enzyme activity and specific activity, respectively. Values are the mean of three independent cultures. and its parental strain under SSF. The PDE production profilesobtained at the 8th day of cultivation are presented in Table 2.Sugar beet yielded the highest production levels of all enzymeactivities evaluated for both strains, M3 and  A. sojae , as indicatedby statistical analysis ( P  -value < 0.05), mutant M3 being the bestproducer. Folds of 7.82, 14.92, 1.57 and 3.1 for exo-PG, endo-PG,exo-PMGandPLwereobtained,respectively,whenthemutantM3was grown on sugar beet instead of apple pomace. Meanwhile, asignificantdifference( P  -value < 0.05)wasfoundonlyintheactivityof exo-PG when both strains were grown on apple pomace, whilea non-significant difference was found in the rest of the enzymeactivities.According to the results, sugar beet induced higher levels of enzymeactivity,particularlyendo-PGandexo-PGactivities.Theseenzyme activities have been proved to present more affinityupon low-esterified pectin, especially for sugar beet pectin; 29 hence sugarbeetmay favor theinductionofendo-PGandexo-PGactivities. Conversely, even though it has been reported thathigh-esterifiedpectininducesPLproduction, 30 lowerlevelsofthisenzymeactivitywereobservedwhenapplepomacewasusedasacarbon source. High levels of hydrolytic activity, as a consequenceof both endo-and exo-PG, may suggest that the strains have apreference to utilize low esterified pectin as carbon sources, buttheyarenotlimitedtoactuponhigh-methoxylpectinasPLactivityis also present; besides, both strains grew well on apple pomacecarbon source, which contains highly esterified pectin. It can thenbe deduced that mutant M3 and its parental strain may own acomplex of enzyme activities specialized in the degradation of low-esterified pectin, but not limited to this kind of carbon sourcesince both strains grew well on highly esterified pectin. SomereportsshowthatfungalstrainssecreteaspecificcomplexofPDEsfor a certain carbon source depending on the natural habitat. Forexample, it has been reported that  Rhizopus oryzae  is restrictedto degradation of low-esterified pectin like homogalacturonan, 7 while  Aspergillus  species have enzyme activities that degrade allpectin structural elements. 31 InfluenceofcarbonsourceonthePDEcomplex To investigate whether the type of carbon source influences theproportion in which PDEs are present in the complex of enzymes,crude extracts produced after 8 days of cultivation under SSFby M3 and  A. sojae  strains using apple pomace or sugar beetwere standardized to the same exo-PG activity (100 U mL − 1 )and tested for the different PDEs described above. The pecti-nolyticprofilesfoundinthestandardizedcrudeextractsfrombothstrains are presented in Fig. 2. The carbon source had a significant( P  -value < 0.05) effect on PL and endo-PG activities. In the case of the activity of PL, apple pomace induced a higher activity, whilesugar beet induced the highest endo-PG activity for both strains.A non-significant difference between PE activities of both strainswas found.The results demonstrated that the type of carbon source influ-ences the enzyme activity profile of the PDE complex. PL activ-ity seems to be induced by the high-esterified pectin presentin apple pomace, while the endo-PG seems to be induced bylow-esterified pectin present in sugar beet. Previous studies have  JSciFoodAgric   2015; 95 : 1554–1561 © 2014 Society of Chemical Industry  1  5  5  8 MA Mata-Gómez  etal. Table2.  Effect of carbon source on the production profile of PDE activities in SSFEnzyme activity (U g − 1 )Carbon source Strain Exo-PG Endo-PG Exo-PMG PLSugar beet  A.sojae * 718.1 ± 44.1a 228.5 ± 22.7a 105.4 ± 3.3a 79.3 ± 5.5aM3 1111.7 ± 53.1b 449.2 ± 10.9b 112.8 ± 3.7a 123.1 ± 9.4bApple pomace  A.sojae * 240.0 ± 4.8c 29.1 ± 0.8c 77.4 ± 0.4b 46.7 ± 0.6cM3 142.1 ± 3.5d 30.1 ± 0.4c 71.7 ± 0.7b 39.4 ± 2.3cEnzyme activity assays ware performed in crude extracts obtained after 8 days of cultivation. * Parental strain.Different superscript letters (a–d) indicate significant difference ( P  -value < 0.05) for each activity.The observed values are the average of three determinations with standard error (mean ± SE). Figure 2.  Effect of carbon source (sugar beet, sb; apple pomace, ap) onthe PDE complex from crude extracts produced by mutant M3 (sb orap ) and its parental strain  Aspergillus sojae  (sb or ap ) undersolid-state fermentation. Before enzyme assays, crude extracts obtainedafter 8 days of fermentation were standardized to same exo-PG activity(100UmL − 1 ).Valuesarethemeanofthreedeterminationswithcoefficientsof variation  < 10%. Different letters (a–d) indicate a significant difference( P  -value < 0.05)foreachactivity.Theouterlinerepresentsthelimitofscale. demonstratedthatPLisinducedinthepresenceofhigh-esterifiedpectin, whereas endo-PG is repressed. 30 In addition, a correla-tion between the type of PDEs identified in genomes from 12fungal strains, and the type of pectin from different srcins hasbeen reported. 7 On the other hand, the fact that a highly esteri-fied carbon source like apple pomace did not have any significanteffect on PE activity, as expected, suggesting that these fun-gal strains degrade pectin with a high degree of esterificationby a different mechanism rather than using PE activity, or per-haps they are restricted to degrade low-methoxyl pectin.  Phane-rochaetechrysosporium grewwellonhighcarbonsourcescontain-ing high-methoxyl despite its lack of PE activity, suggesting analternative mechanism to degrade pectin. 7 Screeningofenzymeactivitiesinexperimentalandcommercialpreparations Eight-day SSF crude extracts from mutant M3 and  A. sojae , and4-days SmF culture supernatants from mutants DH56 and Guser-biot 2.230 (referred to as experimental preparations), and com-mercial pectinases were standardized to the same potency asdescribed before and tested for PDEs in order to compare thepectinolytic profiles of experimental preparations to those of commercial pectinases. The enzyme activity profiles found inexperimentalpreparationsofthemutantstrainsandtheirparental  A. sojae  strain are presented in Table 3. According to the statisti-calanalysis,therewasnosignificantdifferencebetweenthepecti-nolytic enzyme activity profiles of the mutant M3 and its parentalstrain, suggesting the mutagenesis process to which  A. sojae  wassubjected did not appear to affect the proportion in which PDEsaresecretedbymutantM3.Asignificantdifference( P  -value < 0.05)was observed between the content of exo-PMG activity of themutants DH56 and Guserbiot 2.230, and that of the mutant M3and  A. sojae , which could be attributed to the type of culture, i.e.SSF or SmF used for PDE production. It has been described thatSSF yields better levels of enzyme activity than SmF; 32 this can beattributed to the fact that SSF has the particularity of minimizingcatabolicrepression.Besides,thegrowthconditions(i.e.humidity)used in SSF are more similar to those found in natural habitats of fungi.Inaddition,thecellmembranediffersaccordingtothetypeof culture, affecting the diffusion of substrate. But the main factorappears to be the difference in diffusion of the substrates; in thecaseofSSF,formationofmicro-gradientsoccurswhenthemicroor-ganismisgrowingandconsumingthecarbonsource,whileinSmFnogradientsareobservedbecauseoftheagitationinvolvedintheculture process. In this manner, glucose acts as a catabolic repres-sor, because it remains near to the mycelial pellets at higher con-centrations for a longer period, repressing the production of theenzymeofinterest. 32 ThemutantGuserbiot2.230wassignificantlydifferent ( P  -value < 0.05) from the rest in the content of PE. Con-sidering that the strain Guserbiot 2.230 was grown in the sameculture medium as the DH56 strain, the difference in the contentof PE activity may be due to the strain itself.The experimental preparations were then compared to com-mercial pectinases (Fig. 3). As expected, the enzyme activity pro-files of experimental samples were different from those of com-mercial pectinases. In relation to the content of enzyme activity,the pectinolytic profiles of experimental and commercial prepa-rations varied considerably with regard to PL, PE and exo-PMGactivities. In spite of the adjustment of all preparations to thesame exo-PG activity, prominent PL activity was found in com-mercial pectinases, particularly in those used for juice clarification(Pro Clear and Fructozym  ®   P) and fruit mashing (First Yield andYield  ®   MASH), while a lower PL activity was observed in experi-mental preparations (Fig. 3A, C). In the case of winemaking com-mercial pectinases (Fino G and Clair Rapide G), PL was slightlyhigher than that of experimental preparations (Fig. 3B, D), butmuch lower than that of commercial pectinases used in fruit and juiceprocessing.Withrespecttoexo-PMG,thehighestactivitywasobservedinthepreparationsFirstYield(Fig.3A,C)andClairRapide  © 2014 Society of Chemical Industry  JSciFoodAgric   2015; 95 : 1554–1561
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