Xuebin Feng, Siyi Hong, Hongbo Zhao, Thu V. Vuong, Emma R. Master
{"title":"多糖胺化的生物催化级联。","authors":"Xuebin Feng, Siyi Hong, Hongbo Zhao, Thu V. Vuong, Emma R. Master","doi":"10.1186/s13068-024-02477-6","DOIUrl":null,"url":null,"abstract":"<div><h3>Background</h3><p>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 <i>Fusarium graminearum</i> for oxidation, this study compared the performance of CvATA (from <i>Chromobacterium violaceum</i>) and SpATA (from <i>Silicibacter pomeroyi</i>) 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.</p><h3>Results</h3><p>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.</p><h3>Conclusions</h3><p>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.</p><h3>Graphical Abstract</h3><div><figure><div><div><picture><source><img></source></picture></div></div></figure></div></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":null,"pages":null},"PeriodicalIF":6.1000,"publicationDate":"2024-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02477-6","citationCount":"0","resultStr":"{\"title\":\"Biocatalytic cascade to polysaccharide amination\",\"authors\":\"Xuebin Feng, Siyi Hong, Hongbo Zhao, Thu V. Vuong, Emma R. Master\",\"doi\":\"10.1186/s13068-024-02477-6\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Background</h3><p>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 <i>Fusarium graminearum</i> for oxidation, this study compared the performance of CvATA (from <i>Chromobacterium violaceum</i>) and SpATA (from <i>Silicibacter pomeroyi</i>) 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.</p><h3>Results</h3><p>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.</p><h3>Conclusions</h3><p>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. 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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.
期刊介绍:
Biotechnology for Biofuels is an open access peer-reviewed journal featuring high-quality studies describing technological and operational advances in the production of biofuels, chemicals and other bioproducts. The journal emphasizes understanding and advancing the application of biotechnology and synergistic operations to improve plants and biological conversion systems for the biological production of these products from biomass, intermediates derived from biomass, or CO2, as well as upstream or downstream operations that are integral to biological conversion of biomass.
Biotechnology for Biofuels focuses on the following areas:
• Development of terrestrial plant feedstocks
• Development of algal feedstocks
• Biomass pretreatment, fractionation and extraction for biological conversion
• Enzyme engineering, production and analysis
• Bacterial genetics, physiology and metabolic engineering
• Fungal/yeast genetics, physiology and metabolic engineering
• Fermentation, biocatalytic conversion and reaction dynamics
• Biological production of chemicals and bioproducts from biomass
• Anaerobic digestion, biohydrogen and bioelectricity
• Bioprocess integration, techno-economic analysis, modelling and policy
• Life cycle assessment and environmental impact analysis