Elintarvikebiopolymeerien ristisilloittaminen tyrosinaasilla ja lakkaasilla

Tyrosinases and laccases are copper-containing oxidoreductases, which catalyze oxidation of various mono- and polyphenolic compounds. Tyrosinases oxidize p-monophenols and o-diphenols to quinones, whereas laccases are capable of oxidizing a larger variety of aromatic compounds, such as substituted mono- and polyphenols, aromatic amines and thiol compounds, with subsequent production of radicals. Both tyrosinase and laccase generate reaction products which are prone to react further non-enzymatically, which may lead to polymerization. In addition to the low molecular mass phenolic compounds, the phenolic moieties present in certain biopolymers are susceptible to oxidation by tyrosinase and laccase, which enables crosslinking of the biopolymers. The biochemical properties of a novel fungal tyrosinase from Trichoderma reesei (TrT) were characterized in this work. The substrate specificity and protein crosslinking ability of TrT were compared to other tyrosinases of plant and fungal origin. Furthermore, the suitability of TrT and laccase from Trametes hirsuta (ThL) was examined for hetero-crosslinking of carbohydrates and proteins and for improving wheat breadmaking quality. TrT was over-expressed in its original host under a strong cbh1 promoter and purified with a three step purification procedure, consisting of desalting by gel filtration, and cation exchange and gel filtration chromatography. The purified TrT showed a molecular weight of 43.2 kDa as analyzed by mass spectrometry. TrT was found to be processed from the C-terminus by cleavage of a peptide fragment of about 20 kDa. TrT was active both on L-tyrosine and L-dopa, thus showing typical characteristics of a true tyrosinase. TrT had broad substrate specificity, and the enzyme showed the highest activity and stability in the neutral and alkaline pH range, with an optimum at pH 9. TrT retained its activity relatively well at temperatures of 40 °C and below. When tyrosinases from apple (AT), potato (PT), the white rot fungus Pycnoporus sanguineus (PsT) and the edible mushroom Agaricus bisporus (AbT) were compared to TrT, it was found that the tyrosinases had clearly different features in terms of substrate specificity, inhibition and their ability to crosslink the model protein α-casein. Generally the tyrosinases had lower activity on monophenols than on di- or triphenols. PsT had the highest monophenolase/diphenolase ratio for the oxidation of monophenolic L-tyrosine and diphenolic L-dopa. However, TrT had the highest activity on most of the tested monophenols, and showed clearly shorter lag periods prior to the oxidation of the monophenols than the other enzymes. The activity of AT and PT on tyrosine was undetectable which explains the poor crosslinking ability of α-casein by these enzymes. AbT was also unable to crosslink α-casein, although it could oxidize tyrosine of di- and tri-peptides. Conversely, the activity of PsT on the model peptides turned out to be relatively low, although the enzyme could crosslink α-casein. Of the analyzed tyrosinases, TrT clearly had the best ability to directly crosslink α-casein. However, by after addition of a small molecular weight phenolic compound, L-dopa, to the reaction mixture, the other tyrosinases were also able to crosslink α-casein. It is assumed that L-dopa acted as a bridging compound between the α-casein subunits. The capability of the two different fungal oxidative enzymes, the TrT tyrosinase and the ThL laccase, to catalyze formation of hetero-conjugates between tyrosine side-chains of α-casein and phenolic acids of hydrolyzed oat spelt xylan (hOSX) was studied. TrT was able to crosslink α-casein more efficiently than ThL, whereas only ThL was able to polymerize hOSX. The radical- and quinone-mediated protein crosslinking clearly differed, which was indicated by enhancement of crosslinking by the presence of phenolic acids with ThL, and by inhibition with TrT. Despite the notable differences between the oxidative enzymes in their ability to crosslink the biopolymers, both ThL and TrT were observed to be able to catalyze oxidative hetero-crosslinking of α-casein and xylan. The effects of TrT and ThL were also compared in wheat flour breadmaking. The enzymes were found to act in wheat dough and bread via different crosslinking mechanisms. Both ThL and TrT improved the bread quality, especially when used in combination with xylanase, as indicated by an increase in bread volume and bread crumb softness during storage. The effect of ThL is assumed to be based mainly on the crosslinking of ferulic acid -substituted arabinoxylan with subsequent arabinoxylan network formation, and thus indirectly also strengthening the gluten network of dough. ThL may also have directly oxidized the tyrosyl residues of gluten proteins or enhanced the disulphide bridge formation in gluten polymers via ferulic acid-derived radicals, thus assisting protein aggregation in dough. The effects of TrT in dough and bread are suggested to be due mainly to polymerization of gluten proteins via production of reactive quinones by oxidation of the protein-bound tyrosine residues with consequent formation of crosslinks in the gluten proteins. Tyrosinase may also have influenced the texture properties of dough and bread by oxidizing other phenolic compounds as well as tyrosine of wheat flour, such as p-coumaric and caffeic acids. The oxidative enzymes, tyrosinase and laccase, were shown to have potential in crosslinking of food biopolymers. The T. reesei tyrosinase was found to be an efficient protein crosslinker, especially when compared to the T. hirsuta laccase or to the tyrosinases of plant and fungal origin. On the other hand, ThL was observed to be more efficient in catalyzing the formation of hetero-crosslinks between proteins and carbohydrates, as compared to TrT. It was shown in this work that both types of oxidative enzymes, tyrosinase and laccase, can be applied to generate food biopolymers with added functionalities or novel food structures from diverse raw materials.Tyrosinaasit ja lakkaasit ovat entsyymejä, jotka katalysoivat mono- ja polyfenolisten yhdisteiden hapettumisreaktioita. Reaktio¬tuotteiden jatkoreaktiot voivat johtaa substraattien polymeroitumiseen. Tyrosinaasit ja lakkaasit pystyvät hapettamaan myös biopolymeerirakenteissa esiintyviä fenolisia yhdisteitä, minkä johdosta kyseisten biopolymeerien entsyymiavusteinen ristisilloittaminen on mahdollista. Tässä työssä karakterisoitiin uutta Trichoderma reesei -homeesta peräisin olevaa tyrosinaasia. T. reesei -tyrosinaasin substraattispesifisyyttä sekä kykyä ristisilloittaa maitoproteiini α-kaseiinia verrattiin home- ja kasviperäisiin tyrosinaaseihin. T. reesei -tyrosinaasin kykyä muodostaa heterokonjugaatteja proteiinien ja hiilihydraattien välille sekä entsyymin vaikutuksia vehnäleivonnassa verrattiin Trametes hirsuta -lakkaasiin. T. reesei -tyrosinaasi tuotettiin ylituottomenetelmällä alkuperäisessä tuottoisännässään. Entsyymi puhdistettiin kolmivaiheisella kromatografisella prosessilla. T.reesei -tyrosinaasin molekyylipainoksi määritettiin 43,2 kDa, ja polypeptidin C-terminaalisesta päästä todetiiin katkenneen noin 20 kDa -kokoinen peptidi. T. reesei -tyrosinaasi oli aktiivinen neutraalilla ja emäksisellä pH-alueella. Verrattaessa T. reesei -tyrosinaasia omenasta, perunasta, Pycnoporus sanguineus sekä Agaricus bisporus -homeista peräisin oleviin tyrosinaaseihin havaittiin entsyymeiden substraattispesifisyyden ja kyvyn ristisilloittaa maitoproteiinia eroavan toisistaan huomattavasti. T. reesei -tyrosinaasi hapetti monofenolisia yhdisteitä ja ristisilloitti kaseiinia selvästi tehokkaimmin. Myös P. sanguineus -tyrosinaasi kykeni polymeroimaan kaseiinia. Kun reaktioseokseen lisättiin pienimolekyylipainoista di-fenolia, kaikki tyrosinaasit kykenivät ristisilloittamaan kaseiinia. T. reesei -tyrosinaasia ja T. hirsuta -lakkaasia verrattiin suhteessa niiden kykyyn katalysoida polymerointireaktioita α-kaseiinin ja ksylaanin fenolisten sivuryhmien kautta. Tyrosinaasi kykeni polymeroimaan α-kaseiinia lakkaasia paremmin. Toisaalta lakkaasi kykeni polymeroimaan ksylaania, mutta tyrosinaasi ei. Huolimatta havaituista eroista sekä lakkaasin että tyrosinaasin osoitettiin muodostavan heteroristisidoksia maitoproteiinin ja ksylaanin välillä. Vehnäleivonnassa molemmat entsyymit lisäsivät leivän ominaistilavuutta sekä pehmeyttä. Lakkaasin vaikutus johtui ilmeisesti pääosin vehnän arabinoksylaanin ristisiloittumisesta ja tyrosinaasin gluteeniproteiinien ristisilloitumisesta. Saavutettujen tulosten perusteella voidaan todeta, että T. reesei -tyrosinaasi ja T. hirsuta -lakkaasi soveltuvat elintarvikkeiden rakenne- ja funktionaalisten ominaisuuksien muokkaamiseen.reviewe

Lakkaasin ja ksylanaasin vaikutus vehnäjauho- ja gluteenitaikinan rakenteen muodostumiseen:Diplomityö

In the literature survey, properties of wheat flour and dough as well as the structure formation of wheat bread were discussed, with special emphasis on rheology. Also the theory and a few measurement methods of the rheology were shortly introduced. Considering the influence of enzymes on bread making, a structure formation by an oxidative mechanism was discussed. The enzymes that were viewed more precisely on their effect on the structure formation of wheat bread included lipoxygenase, glucose oxidase, peroxidase, laccase and transglutaminase. In the experimental part of the study, the effect of Aspergillus oryzae and Bacillus subtilis xylanases and Trametes hirsuta and Melanocarpus albomyces laccases, separately and together, on the structure of wheat flour dough and gluten dough were examined. The experiments of added free ferulic acid (FA) were done to clarify the role of FA in dough structure formation. The rheological experiments were performed with Kieffer dough and gluten extensibility rig, when the dough extensibility Ex and the resistance to stretching Rmax, were determined. In a rheology point of view, laccases increased the maximum resistance Rmax of dough and decreased the dough extensibility Ex at Rmax, whereas xylanases decreased the Rmax of dough and increased the Ex at Rmax in flour and gluten doughs. Considering the protein-AX fraction, hardening by laccases and softening by xylanases were weaker in gluten doughs, which was presumably due to the lower AX content in gluten. Like xylanases, the added free FA softened the flour dough structure, as well. As a function of dough resting time, the structure of laccase treated doughs was observed to soften. The softening effect was strengthened as a function of laccase activity. The reason for the softening phenomenon was probably the laccase-mediated depolymerisation of cross-linked arabinoxylan network, resulting from mobile FA radicals. Combined laccase and xylanase experiments led to doughs of higher Rmax, with no remarkable change in Ex. The effect of laccase seemed to be predominant, especially at low xylanase dosages, but when xylanase was added to flour dough at high concentration, the hardening effect of laccase on dough was decreased. Presumably, when the AX fraction was hydrolyzed effectively by xylanase, laccase was not able to create a strong AX network. Similar decrease in laccase mediated hardening in doughs was not seen, when performing the combined laccase and xylanase test with gluten, which was presumably due to low AX content of gluten. The results indicated the critical role of feruloylated arabinoxylan fraction in laccase-catalysed structure formation both in flour and gluten, although in gluten doughs, protein fraction might have also been affected.Työn kirjallisuusosassa tarkasteltiin vehnäjauhon ominaisuuksia sekä vehnätaikinan ja -leivän rakenteenmuodostusta. Kaikkia aihealueita, kuten jauhokomponenttien ominaisuuksia, leivonnan teoriaa ja entsyymejä, käsiteltiin pääasiassa reologiselta näkökannalta. Lisaksi sivuttiin lyhyesti reologian teoriaa ja reologisia mittausmenetelmiä. Leivonnassa käytettävien entsyymien osalta keskityttiin lähinnä oksidatiivisella mekanismilla toimiviin ristisilloittaviin entsyymeihin. Lipoksygenaasi-, glukoosi oksidaasi-, peroksidaasi-, lakkaasi- ja transglutaminaasientsyymejä, sekä erityisesti niiden vaikutusta leivän rakenteenmuodostumiseen tarkasteltiin entsyymeistä yksityiskohtaisimmin. Työn kokeellisessa osassa tutkittiin Aspergillus oryzae ja Bacillus subtius -ksylanaasien sekä Trametes hirsuta ja Melanocarpus albomyces -lakkaasien vaikutusta, yhdessä ja erikseen, vehnäjauho- ja gluteenitaikinan rakenteeseen. Lisäksi vapaan ferulahapon vaikutusta taikinassa pyrittiin selvittämään. Taikinoiden reologiset mittaukset suoritettiin "Kieffer Dough and Gluten Extensibility Rig" -mittauslaitteistolla, jolloin taikinoiden venyvyys Ex ja venytysvastus Rmax, pystyttiin määrittämään. Reologisten mittausten perusteella havaittiin, että lakkaasit lisäsivät ja ksylanaasit vähensivät taikinan venytysvastusta Rmax, kun taas taikinan venyvyys Ex väheni lakkaasien vaikutuksesta ja lisääntyi ksylanaasien vaikutuksesta. Vapaan ferulahapon havaittiin ksylanaasien tavoin pehmentävän jauhotaikinan rakennetta. Tarkastellessa taikinoiden proteiini- ja arabinoksylaani (AX) -osuuksia todettiin, että taikinan kovettuminen lakkaasien vaikutuksesta ja pehmeneminen ksylanaasien vaikutuksesta oli heikompaa gIuteenitaikinoissa, mikä johtui todennäköisesti gluteenin pienemmästä AX-pitoisuudesta. Lakkaasilla käsiteltyjen taikinoiden havaittiin pehmenevän ajan funktiona. Pehmeneminen johtunee lakkaasin vaikutuksesta rakentuneen AX-verkon depolymeroitumisesta, mikä johtui todennäköisesti lakkaasin muodostamien vapaiden F A-radikaalien reaktioista AX-fraktiossa. Lakkaasin ja ksylanaasin yhteisvaikutuksen osalta havaittiin lakkaasin oleva dominoivana pienemmillä aktiivisuuksilla, mutta korkeammilla ksylanaasiaktiivisuuksilla lakkaasin taikinaa kovettava vaikutus heikkeni. Oletettavasti ksylanaasin pilkkoessa tehokkaasti AX-fraktiota lakkaasi ei kyennyt muodostamaan vahvaa AX-verkkorakennetta. Gluteenitaikinoissa vastaavaa heikentymistä ei havaittu, mikä johtui todennäköisesti gluteenitaikinan pienestä AX-pitoisuudesta. Johtopäätöksenä voidaan todeta, että lakkaasin pääasiallisena substraattina toimii AX-fraktio, vaikkakin gluteenitaikinoissa epäiltiin lakkaasin vaikuttaneen myös proteiineihin

Tyrosinase and laccase as novel crosslinking tools for food biopolymer:Dissertation

Tyrosinases and laccases are copper-containing oxidoreductases, which catalyze oxidation of various mono- and polyphenolic compounds. Tyrosinases oxidize p-monophenols and o-diphenols to quinones, whereas laccases are capable of oxidizing a larger variety of aromatic compounds, such as substituted mono- and polyphenols, aromatic amines and thiol compounds, with subsequent production of radicals. Both tyrosinase and laccase generate reaction products which are prone to react further non-enzymatically, which may lead to polymerization. In addition to the low molecular mass phenolic compounds, the phenolic moieties present in certain biopolymers are susceptible to oxidation by tyrosinase and laccase, which enables crosslinking of the biopolymers. The biochemical properties of a novel fungal tyrosinase from Trichoderma reesei (TrT) were characterized in this work. The substrate specificity and protein crosslinking ability of TrT were compared to other tyrosinases of plant and fungal origin. Furthermore, the suitability of TrT and laccase from Trametes hirsuta (ThL) was examined for hetero-crosslinking of carbohydrates and proteins and for improving wheat breadmaking quality. TrT was over-expressed in its original host under a strong cbh1 promoter and purified with a three step purification procedure, consisting of desalting by gel filtration, and cation exchange and gel filtration chromatography. The purified TrT showed a molecular weight of 43.2 kDa as analyzed by mass spectrometry. TrT was found to be processed from the C-terminus by cleavage of a peptide fragment of about 20 kDa. TrT was active both on L-tyrosine and L-dopa, thus showing typical characteristics of a true tyrosinase. TrT had broad substrate specificity, and the enzyme showed the highest activity and stability in the neutral and alkaline pH range, with an optimum at pH 9. TrT retained its activity relatively well at temperatures of 40 ºC and below. When tyrosinases from apple (AT), potato (PT), the white rot fungus Pycnoporus sanguineus (PsT) and the edible mushroom Agaricus bisporus (AbT) were compared to TrT, it was found that the tyrosinases had clearly different features in terms of substrate specificity, inhibition and their ability to crosslink the model protein ?-casein. Generally the tyrosinases had lower activity on monophenols than on di- or triphenols. PsT had the highest monophenolase/diphenolase ratio for the oxidation of monophenolic L-tyrosine and diphenolic L-dopa. However, TrT had the highest activity on most of the tested monophenols, and showed clearly shorter lag periods prior to the oxidation of the monophenols than the other enzymes. The activity of AT and PT on tyrosine was undetectable which explains the poor crosslinking ability of ?-casein by these enzymes. AbT was also unable to crosslink ?-casein, although it could oxidize tyrosine of di- and tri-peptides. Conversely, the activity of PsT on the model peptides turned out to be relatively low, although the enzyme could crosslink ?-casein. Of the analyzed tyrosinases, TrT clearly had the best ability to directly crosslink ?-casein. However, by after addition of a small molecular weight phenolic compound, L-dopa, to the reaction mixture, the other tyrosinases were also able to crosslink ?-casein. It is assumed that L-dopa acted as a bridging compound between the ?-casein subunits. The capability of the two different fungal oxidative enzymes, the TrT tyrosinase and the ThL laccase, to catalyze formation of hetero-conjugates between tyrosine side-chains of ?-casein and phenolic acids of hydrolyzed oat spelt xylan (hOSX) was studied. TrT was able to crosslink ?-casein more efficiently than ThL, whereas only ThL was able to polymerize hOSX. The radical- and quinone-mediated protein crosslinking clearly differed, which was indicated by enhancement of crosslinking by the presence of phenolic acids with ThL, and by inhibition with TrT. Despite the notable differences between the oxidative enzymes in their ability to crosslink the biopolymers, both ThL and TrT were observed to be able to catalyze oxidative hetero-crosslinking of ?-casein and xylan. The effects of TrT and ThL were also compared in wheat flour breadmaking. The enzymes were found to act in wheat dough and bread via different crosslinking mechanisms. Both ThL and TrT improved the bread quality, especially when used in combination with xylanase, as indicated by an increase in bread volume and bread crumb softness during storage. The effect of ThL is assumed to be based mainly on the crosslinking of ferulic acid -substituted arabinoxylan with subsequent arabinoxylan network formation, and thus indirectly also strengthening the gluten network of dough. ThL may also have directly oxidized the tyrosyl residues of gluten proteins or enhanced the disulphide bridge formation in gluten polymers via ferulic acid-derived radicals, thus assisting protein aggregation in dough. The effects of TrT in dough and bread are suggested to be due mainly to polymerization of gluten proteins via production of reactive quinones by oxidation of the protein-bound tyrosine residues with consequent formation of crosslinks in the gluten proteins. Tyrosinase may also have influenced the texture properties of dough and bread by oxidizing other phenolic compounds as well as tyrosine of wheat flour, such as p-coumaric and caffeic acids. The oxidative enzymes, tyrosinase and laccase, were shown to have potential in crosslinking of food biopolymers. The T. reesei tyrosinase was found to be an efficient protein crosslinker, especially when compared to the T. hirsuta laccase or to the tyrosinases of plant and fungal origin. On the other hand, ThL was observed to be more efficient in catalyzing the formation of hetero-crosslinks between proteins and carbohydrates, as compared to TrT. It was shown in this work that both types of oxidative enzymes, tyrosinase and laccase, can be applied to generate food biopolymers with added functionalities or novel food structures from diverse raw materials

Lakkaasin ja ksylanaasin vaikutus vehnäjauho- ja gluteenitaikinan rakenteen muodostumiseen

In the literature survey, properties of wheat flour and dough as well as the structure formation of wheat bread were discussed, with special emphasis on rheology. Also the theory and a few measurement methods of the rheology were shortly introduced. Considering the influence of enzymes on bread making, a structure formation by an oxidative mechanism was discussed. The enzymes that were viewed more precisely on their effect on the structure formation of wheat bread included lipoxygenase, glucose oxidase, peroxidase, laccase and transglutaminase. In the experimental part of the study, the effect of Aspergillus oryzae and Bacillus subtilis xylanases and Trametes hirsuta and Melanocarpus albomyces laccases, separately and together, on the structure of wheat flour dough and gluten dough were examined. The experiments of added free ferulic acid (FA) were done to clarify the role of FA in dough structure formation. The rheological experiments were performed with Kieffer dough and gluten extensibility rig, when the dough extensibility Ex and the resistance to stretching Rmax, were determined. In a rheology point of view, laccases increased the maximum resistance Rmax of dough and decreased the dough extensibility Ex at Rmax, whereas xylanases decreased the Rmax of dough and increased the Ex at Rmax in flour and gluten doughs. Considering the protein-AX fraction, hardening by laccases and softening by xylanases were weaker in gluten doughs, which was presumably due to the lower AX content in gluten. Like xylanases, the added free FA softened the flour dough structure, as well. As a function of dough resting time, the structure of laccase treated doughs was observed to soften. The softening effect was strengthened as a function of laccase activity. The reason for the softening phenomenon was probably the laccase-mediated depolymerisation of cross-linked arabinoxylan network, resulting from mobile FA radicals. Combined laccase and xylanase experiments led to doughs of higher Rmax, with no remarkable change in Ex. The effect of laccase seemed to be predominant, especially at low xylanase dosages, but when xylanase was added to flour dough at high concentration, the hardening effect of laccase on dough was decreased. Presumably, when the AX fraction was hydrolyzed effectively by xylanase, laccase was not able to create a strong AX network. Similar decrease in laccase mediated hardening in doughs was not seen, when performing the combined laccase and xylanase test with gluten, which was presumably due to low AX content of gluten. The results indicated the critical role of feruloylated arabinoxylan fraction in laccase-catalysed structure formation both in flour and gluten, although in gluten doughs, protein fraction might have also been affected.Työn kirjallisuusosassa tarkasteltiin vehnäjauhon ominaisuuksia sekä vehnätaikinan ja -leivän rakenteenmuodostusta. Kaikkia aihealueita, kuten jauhokomponenttien ominaisuuksia, leivonnan teoriaa ja entsyymejä, käsiteltiin pääasiassa reologiselta näkökannalta. Lisaksi sivuttiin lyhyesti reologian teoriaa ja reologisia mittausmenetelmiä. Leivonnassa käytettävien entsyymien osalta keskityttiin lähinnä oksidatiivisella mekanismilla toimiviin ristisilloittaviin entsyymeihin. Lipoksygenaasi-, glukoosi oksidaasi-, peroksidaasi-, lakkaasi- ja transglutaminaasientsyymejä, sekä erityisesti niiden vaikutusta leivän rakenteenmuodostumiseen tarkasteltiin entsyymeistä yksityiskohtaisimmin. Työn kokeellisessa osassa tutkittiin Aspergillus oryzae ja Bacillus subtius -ksylanaasien sekä Trametes hirsuta ja Melanocarpus albomyces -lakkaasien vaikutusta, yhdessä ja erikseen, vehnäjauho- ja gluteenitaikinan rakenteeseen. Lisäksi vapaan ferulahapon vaikutusta taikinassa pyrittiin selvittämään. Taikinoiden reologiset mittaukset suoritettiin "Kieffer Dough and Gluten Extensibility Rig" -mittauslaitteistolla, jolloin taikinoiden venyvyys Ex ja venytysvastus Rmax, pystyttiin määrittämään. Reologisten mittausten perusteella havaittiin, että lakkaasit lisäsivät ja ksylanaasit vähensivät taikinan venytysvastusta Rmax, kun taas taikinan venyvyys Ex väheni lakkaasien vaikutuksesta ja lisääntyi ksylanaasien vaikutuksesta. Vapaan ferulahapon havaittiin ksylanaasien tavoin pehmentävän jauhotaikinan rakennetta. Tarkastellessa taikinoiden proteiini- ja arabinoksylaani (AX) -osuuksia todettiin, että taikinan kovettuminen lakkaasien vaikutuksesta ja pehmeneminen ksylanaasien vaikutuksesta oli heikompaa gIuteenitaikinoissa, mikä johtui todennäköisesti gluteenin pienemmästä AX-pitoisuudesta. Lakkaasilla käsiteltyjen taikinoiden havaittiin pehmenevän ajan funktiona. Pehmeneminen johtunee lakkaasin vaikutuksesta rakentuneen AX-verkon depolymeroitumisesta, mikä johtui todennäköisesti lakkaasin muodostamien vapaiden F A-radikaalien reaktioista AX-fraktiossa. Lakkaasin ja ksylanaasin yhteisvaikutuksen osalta havaittiin lakkaasin oleva dominoivana pienemmillä aktiivisuuksilla, mutta korkeammilla ksylanaasiaktiivisuuksilla lakkaasin taikinaa kovettava vaikutus heikkeni. Oletettavasti ksylanaasin pilkkoessa tehokkaasti AX-fraktiota lakkaasi ei kyennyt muodostamaan vahvaa AX-verkkorakennetta. Gluteenitaikinoissa vastaavaa heikentymistä ei havaittu, mikä johtui todennäköisesti gluteenitaikinan pienestä AX-pitoisuudesta. Johtopäätöksenä voidaan todeta, että lakkaasin pääasiallisena substraattina toimii AX-fraktio, vaikkakin gluteenitaikinoissa epäiltiin lakkaasin vaikuttaneen myös proteiineihin