Design of enzymatic systems for the modification of cellulose fiber surfaces
2003 (English)Doctoral thesis, comprehensive summary (Other scientific)
This thesis describes the design of enzymatic systems forthe modification of cellulose fiber surfaces. A single-enzymeapproach employed a conjugate between theNeocallimastix patriciarumCel6A cellulose-bindingmodule and lipase B fromCandida antarctica(CBM-CALB) to enhance theaccessibility of the enzyme to the target hydroxyl groups. Thecharacteristics of the N.patriciarumfamily 1 CBM along with other wild type andmutated CBMs were investigated. The presence of one tryptophanamong the three aromatic residues on the binding face of afamily 1 CBM enhanced its binding to cellulose while a secondtryptophan did not lead to any further improvement.Transmission electron microscopy analysis indicated thatvarious family 1 and 3 CBMs bound to the exposed hydrophobicsurfaces of cellulose crystals.
Proteolytically stable expression of CBM-CALB in Pichiapastoris was achieved in fed-batch bioreactor cultivationsusing two strategies. Firstly, the linker joining the fusionpartners was engineered to enhance the resistance of CBM-CALBtowards proteolytic cleavage. A 13-residue proline/threoninerich linker was found to be stable during cultivation.Secondly, the cultivation conditions were optimized. Decreasingthe pH from 5.0 to 4.0 and the temperature from 30 ºC to22 ºC resulted in the production of a stable conjugate atan expression level of 1.5 g/L. CBM-CALB could successfullycatalyze the ring-opening polymerization of ε-caprolactoneon paper surfaces. The greatly increased hydrophobicity of thepaper did not arise from covalent attachment of the polymers tothe surface hydroxyl groups, but fromextractable polymerlayers. The polymerization process was highly dependent on thereaction conditions. The water content in a system regulatedthe product molecular weight while the temperature influencedthe reaction rate.
A dual-enzyme system, comprised of CALB andPopulus tremula x tremuloidesxyloglucanendotransglycosylase 16A (PttXET16A), was developed to morestrongly attach functional groups to cellulose. Xyloglucanoligosaccharides (XGOs) were acylated by CALB using vinylstearate or γ-thiobutyrolactone as acyl donors, followedby a PttXET16A-catalyzed incorporation of modified XGOs intohigh molecular weight xyloglucan, which bound tightly tocellulose. The approach enabled the effectively irreversibleintroduction of hydrophobic alkyl chains or reactive sulfhydrylgroups to the cellulose fiber surface.
Key words:Candida antarctica lipase B, Pichia pastoris,cellulose-binding module, proteolysis, carbohydrate ester,xyloglucan endotransglycosylase, fiber modification
Place, publisher, year, edition, pages
Stockholm: Bioteknologi , 2003. , 78 p.
Candida antarctica lipase B, Pichia pastoris, cellulose-binding module, proteolysis, carbohydrate ester, xyloglucan endotransglycosylase, fiber modification
IdentifiersURN: urn:nbn:se:kth:diva-3608ISBN: 91-7283-565-6 (print)OAI: oai:DiVA.org:kth-3608DiVA: diva2:9437
NR 201408052003-09-292003-09-29Bibliographically approved