Biocatalytic cascade to polysaccharide amination

dc.contributorAalto Universityen
dc.contributor.authorFeng, Xuebin
dc.contributor.authorHong, Siyi
dc.contributor.authorZhao, Hongbo
dc.contributor.authorVuong, Thu V.
dc.contributor.authorMaster, Emma R.
dc.contributor.departmentDepartment of Bioproducts and Biosystemsen
dc.contributor.groupauthorProtein Technologyen
dc.contributor.organizationUniversity of Toronto
dc.contributor.organizationUniversity of Helsinki
dc.descriptionPublisher Copyright: © The Author(s) 2024.
dc.description.abstractBackground: Chitin, the main form of aminated polysaccharide in nature, is a biocompatible, polycationic, and antimicrobial biopolymer used extensively in industrial processes. Despite the abundance of chitin, applications thereof are hampered by difficulties in feedstock harvesting and limited structural versatility. To address these problems, we proposed a two-step cascade employing carbohydrate oxidoreductases and amine transaminases for plant polysaccharide aminations via one-pot reactions. Using a galactose oxidase from Fusarium graminearum for oxidation, this study compared the performance of CvATA (from Chromobacterium violaceum) and SpATA (from Silicibacter pomeroyi) on a range of oxidized carbohydrates with various structures and sizes. Using a rational enzyme engineering approach, four point mutations were introduced on the SpATA surface, and their effects on enzyme activity were evaluated. Results: Herein, a quantitative colorimetric assay was developed to enable simple and accurate time-course measurement of the yield of transamination reactions. With higher operational stability, SpATA produced higher product yields in 36 h reactions despite its lower initial activity. Successful amination of oxidized galactomannan by SpATA was confirmed using a deuterium labeling method; higher aminated carbohydrate yields achieved with SpATA compared to CvATA were verified using HPLC and XPS. By balancing the oxidase and transaminase loadings, improved operating conditions were identified where the side product formation was largely suppressed without negatively impacting the product yield. SpATA mutants with multiple alanine substitutions besides E407A showed improved product yield. The E407A mutation reduced SpATA activity substantially, supporting its predicted role in maintaining the dimeric enzyme structure. Conclusions: Using oxidase–amine transaminase cascades, the study demonstrated a fully enzymatic route to polysaccharide amination. Although the activity of SpATA may be further improved via enzyme engineering, the low operational stability of characterized amine transaminases, as a result of low retention of PMP cofactors, was identified as a key factor limiting the yield of the designed cascade. To increase the process feasibility, future efforts to engineer improved SpATA variants should focus on improving the cofactor affinity, and thus the operational stability of the enzyme. Graphical Abstract: (Figure presented.).en
dc.description.versionPeer revieweden
dc.identifier.citationFeng, X, Hong, S, Zhao, H, Vuong, T V & Master, E R 2024, ' Biocatalytic cascade to polysaccharide amination ', Biotechnology for Biofuels and Bioproducts, vol. 17, no. 1, 34 .
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dc.publisherBioMed Central
dc.relation.ispartofseriesBiotechnology for Biofuels and Bioproducts
dc.relation.ispartofseriesVolume 17, issue 1
dc.subject.keywordAminated polysaccharide
dc.subject.keywordAmine transaminases
dc.subject.keywordEnzymatic cascade
dc.subject.keywordTransaminase activity assay
dc.titleBiocatalytic cascade to polysaccharide aminationen
dc.typeA1 Alkuperäisartikkeli tieteellisessä aikakauslehdessäfi