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Author:Mikander, Saara
Title:The mechanism of cellulase adsorption on isolated lignin fractions
Sellulaasien adsorptiomekanismi eristettyihin ligniinifraktiohin
Publication type:Master's thesis
Publication year:2010
Pages:vii + 95 + [4]      Language:   eng
Department/School:Biotekniikan ja kemian tekniikan laitos
Main subject:Bioprosessitekniikka   (Kem-70)
Supervisor:Leisola, Matti
Instructor:Marjamaa, Kaisa ; Kruus, Kristiina
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Location:P1 Ark Aalto  3513   | Archive
Keywords:cellulase
CBM
lignin
adsorption
sellulaasi
ligniini
adsorptio
Abstract (eng): Unproductive binding of cellulolytic enzymes to lignin is one of the restricting factors in the cost-effective production of lignocelluloses bio ethanol.
The aim of the present work was to study the mechanism of cellulase adsorption on lignin.
Lignin was isolated from steam pre-treated spruce and wheat straw both enzymatically (EnzP-lignin) and by alkali extraction (alkali-lignin).
The adsorption of a thermostable Melanocarpus albomyces endoglucanase Ce145A and its fusion proteins containing a native or mutated carbohydrate binding module (CBM) and an associated linker peptide from Trichoderma reesei Ce17A was studied by quantifying the amount of unadsorbed protein using SDS-PAGE combined with a Criterion Stain Free Gel Imaging system.
The most interesting variants were purified to homogeneity and used for the construction of Langmuir adsorption isotherms.

The adsorption followed the Langmuir isotherm relatively well.
EnzP-lignin from spruce showed higher adsorption capacity (GAMMAmax=1.66 - 3.31 µmol/g lignin) compared to wheat straw EnzP-lignin (GAMMAmax=0.38 - 74 µmol/g lignin).
Both alkali-lignins adsorbed the enzymes very efficiently and the differences between the enzymes were so small that reliable conclusions could not be made.
The CBM with the linker peptide increased significantly the adsorption of Ce145A on EnzP-lignin's.
In contrast, the replacement of tyrosine amino acids (Y31, Y32) in the CBM by neutral alanines decreased the level of adsorption.
Amino acids Y31 and Y32 have been shown to be crucial in the binding of the CBM to crystalline cellulose.
The replacement of the tyrosine residue by a more hydrophobic tryptophan at position 31 increased the adsorption of the enzyme on EnzP-lignins.
Furthermore, a deletion removing practically all of the linker peptide significantly decreased the binding of the enzyme to lignin.
The results strongly indicate that the CBM has a significant effect on the adsorption of the enzymes on lignin and that the CBM binds to lignin mainly with a similar mechanism which binds it to crystalline cellulose.
Abstract (fin): Sellulolyyttisten entsyymien epäspesifinen sitoutuminen ligniiniin on eräs tekijä, joka rajoittaa merkittävästi lignoselluloosaan perustuvan bioetanolin taloudellista tuotantoa.
Työn tarkoituksena oli tutkia sellulaasien sitoutumismekanismia ligniinin.
Ligniini eristettiin höyryräjäytetystä kuusesta ja vehnän oljesta entsymaattisesti (EnzP -ligniini) sekä alkaliuutolla (allcaliligniini).
Adsorptiota tutkittiin termostabiilin Melanocarpus albomyces endoglukanaasin (Ce145A) sekä erilaisten fuusioproteiinien avulla.
Fuusioproteiinit sisälsivät Trichoderma reesei -sienen CeI7A- entsyymin natiivin tai muokatun hiilihydraattiin sitoutuvan moduulin (CBM) ja siihen liittyvän linkkeripeptidin.
Adsorptiota eri ligniinifraktioihin tutkittiin määrittämällä sitoutumattoman proteiinin määrä SDS-PAGE:n ja Criterion Stain Free Gel Imaging -systeemin avulla.
Mielenkiintoisimmat entsyymivariantit puhdistettiin ja puhdisteiden adsorptiota tutkittiin määrittämällä niille Langmuirin adsorptioisotermit.

Adsorptio noudatti Langmuirin mallia suhteellisen hyvin.
Kuusesta eristetyn EnzP -ligniinin adsorptiokapasiteetti (GAMMAmax=l.66 - 3.31 µmol/g ligniini) oli huomattavasti suurempi oljen EnzP -ligniiniin verrattuna (GAMMAmax=0.38 - 0.74 µmol/g ligniini).
Molemmat alkaliligniinifraktiot sitoivat entsyymejä erittäin voimakkaasti, ja erot eri mutanttien välillä olivat hyvin pieniä.
CBM:n ja linkkeripeptidin liittäminen CeI45A:han kasvattivat entsyymin adsorptiota EnzP -ligniineihin huomattavasti.
CBM:ssä sijaitsevien tyrosiiniaminohappojen (Y31, Y32) vaihtaminen neutraaleiksi alaniineiksi heikensi adsorptiota.
Aminohappojen Y31A ja Y32A on osoitettu olevan hyvin keskeisessä asemassa CBM:n sitoutuessa kiteiseen selluloosaan.
Y31:n muuttaminen hydrofobisemmaksi tryptofaaniksi sitä vastoin kasvatti adsorptiota EnzP -ligniineihin.
Linkkeripeptidin poistava mutaatio heikensi merkittävästi entsyymin sitoutumista ligniiniin.
Tulosten perusteella CBM:llä on merkittävä vaikutus entsyymin ja ligniinin välisessä adsorptiossa.
Tulokset myös viittaavat vahvasti, että CBM:n sitoutuminen ligniiniin tapahtuu samanlaisella mekanismilla, joka sitoo CBM:n kiteiseen selluloosaan.
ED:2010-12-20
INSSI record number: 41459
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