Expression screening, production and functional characterizations of bacterial expansin-related proteins
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Kemian tekniikan korkeakoulu |
Master's thesis
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Authors
Date
2022-08-23
Department
Major/Subject
Biotechnology
Mcode
CHEM3022
Degree programme
Master's Programme in Chemical, Biochemical and Materials Engineering
Language
en
Pages
49+12
Series
Abstract
Expansins 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.Description
Supervisor
Master, EmmaThesis advisor
Haddad Momeni, MajidKeywords
expansins, microbial epansin-like proteins, protein expression, enzyme synergy, protein purification, characterization