Expression screening, production and functional characterizations of bacterial expansin-related proteins

dc.contributorAalto-yliopistofi
dc.contributorAalto Universityen
dc.contributor.advisorHaddad Momeni, Majid
dc.contributor.authorJäämuru, Vilma
dc.contributor.schoolKemian tekniikan korkeakoulufi
dc.contributor.supervisorMaster, Emma
dc.date.accessioned2022-08-28T17:14:11Z
dc.date.available2022-08-28T17:14:11Z
dc.date.issued2022-08-23
dc.description.abstractExpansins are small cell wall loosening proteins first discovered in the ‘90s as mediators of “acid growth” in plants. With the discovery of plant expansins, sequence alignment searches revealed expansin-like proteins from non-plant sources like bacteria, fungi and other prokaryotes. Without bona fide catalytic activity, expansins and expansin-like proteins are hypothesized to act on certain “biomechanical hotspots” to induce cell wall creep, slippage and loosening. As a result, expansins have gathered a lot of attention as possible surface-acting proteins that can modify abundant biopolymers like cellulose into different functional materials in various industries. This thesis aims to (1) complete further expression screening and purification of bacterial expansinlike proteins, (2) functionally characterize target bacterial expansin-like proteins, and (3) examine the synergistic activities of target bacterial expansin-like proteins. Small scale production of target proteins was completed with MagicMedia dual temperature protocol or lysogeny broth (LB) media and isopropyl β-d-1-thiogalactopyranoside (IPTG) induction. Large scale production was completed only with LB media and IPTG induction but at various production conditions. Different lysis methods were compared, with sonication being used for large scale production. Characterization of the proteins was done with insoluble polysaccharide pulldown (IPP) assays on commercial substrates (Avicel, chitin, and oat-spelt xylan) as well as hardwood and softwood pulps. Synergistic activities was determined in various reaction conditions with cellulase from Trichoderma reesei and xylanase from a fungal source. From expression screening trials three new bacterial expansin-like proteins were identified. However, large scale production of these proteins were produced with no yield. Production of LT367 and CPO928 was completed successfully with yields of 6 mg/L and 1 mg/L, respectively. All three bacterial expansin-like proteins tested for binding (HE673, LT367 and AJC165) showed significant binding to oat-spelt xylan and both pulps. HE673 also showed significant binding to Avicel and chitin due to its additional carbohydrate binding domain (CBM2). Possible synergistic activity was observed in LT367 and AJC165. The further characterization of LT367 and AJC165 with more binding and synergy studies along with bioreactor production of the protein is recommended. Studies examining the cell wall loosening activity or filter paper weakening are also suggested for further characterization steps.en
dc.format.extent49+12
dc.format.mimetypeapplication/pdfen
dc.identifier.urihttps://aaltodoc.aalto.fi/handle/123456789/116345
dc.identifier.urnURN:NBN:fi:aalto-202208285159
dc.language.isoenen
dc.locationPKfi
dc.programmeMaster's Programme in Chemical, Biochemical and Materials Engineeringfi
dc.programme.majorBiotechnologyfi
dc.programme.mcodeCHEM3022fi
dc.subject.keywordexpansinsen
dc.subject.keywordmicrobial epansin-like proteinsen
dc.subject.keywordprotein expressionen
dc.subject.keywordenzyme synergyen
dc.subject.keywordprotein purificationen
dc.subject.keywordcharacterizationen
dc.titleExpression screening, production and functional characterizations of bacterial expansin-related proteinsen
dc.typeG2 Pro gradu, diplomityöfi
dc.type.ontasotMaster's thesisen
dc.type.ontasotDiplomityöfi
local.aalto.electroniconlyyes
local.aalto.openaccessyes

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