Microbial and enzymatic syntheses of polymer building blocks through selective transformations of polyols and furans
Författare
Summary, in English
Abstract
Transition from fossil- to bio-based economy is a critical step towards reduction of greenhouse gas emissions and climate change, and hence for achievement of sustainable communities and environment. In order to be fossil-free, the chemical and material industry is in need of carbon-neutral building blocks from renewable resources for the diverse array of products that are currently produced from olefins and aromatics. Hence, new pathways for producing the same or novel chemical structures are needed. Industrial biotechnology offers a key technology area for transformation of biomass components or derivatives to chemical building blocks by the use of microorganisms or their enzymes.
The thesis introduces new routes for microbial and enzymatic biotransformations of trimethylolpropane (TMP) and 5-hydroxymethyl furfural (HMF) to building blocks for polymers. TMP is an important industrial polyol with three hydroxyl groups produced from butyraldehyde and formaldehyde that can be potentially biobased, while HMF, a dehydration product of sugar, is totally biobased. The building block molecules produced from TMP include 2,2-bis(hydroxymethyl)butyric acid (BHMB), six membered cyclic carbonates, and from HMF are 5-hydroxymethyl-2-furan carboxylic acid (HMFCA), 5 formyl-2-furan carboxylic acid (FFCA) and 2,5-furan carboxylic acid (FDCA).
Growing cells of Mycobacterium sp. MS1601 (previously Corynebacterium sp. ATCC 21245) was the only bacteria that showed the ability to selectively oxidize only one hydroxyl group of TMP to form BHMB at high yield. After optimization of the process parameters and employing high cell density cultivations in a sequential batch mode with cell recycling and cell bleeding, the volumetric productivity of BHMB was improved from 0.02 g/L.h to 0.2 g/L.h to yield 21 g/L BHMB, the highest amount reported so far. Moreover, BHMB was recovered from the reaction medium by anion exchange resin at 78% yield. Transesterification of TMP with methacrylic acid and its derivatives including methyl, ethyl vinyl and dimethyl carbonate (DMC) was investigated using immobilized lipase (Novozym435) in solvent free medium in order to produce methacrylate functionalized six-membered cyclic carbonates. The results obtained from the experimental part and in-silico analysis indicate that methyl and ethyl methacrylate were preferable substrates for the enzyme to give the product with 61.3 % yield and 73% selectivity after 9 hours reaction. Also, the functionalized cyclic carbonate was purified from the reaction solution using silica chromatography at 60.5% yield. Even the production of bio-based TMP under mild conditions was demonstrated by condensation of bio-based butyraldehyde with formaldehyde produced by oxidation of the corresponding alcohols using Gluconobacter oxydans cells and alcohol oxidase, respectively.
Oxidation of crude 5-HMF to HMFCA at 100 % selectivity and yield was achieved using resting cells of G. oxydans DSM 50049. The bacteria show the ability to oxidize 31.5 g/L of crude HMF completely to HMFCA after only 6 h of the reaction, indicating that the bacteria is tolerant to the antimicrobial activity and high concentration of HMFCA. The product was recovered from the reaction with 98% purity using a simple liquid-liquid extraction step. On the other hand, Mycobacterium sp. MS1601 cells activated by growth in glycerol, oxidized HMF to FDCA and HMFCA with 60% and 40% yield, respectively. A gene sequence encoding HMF oxidase (HMFO) like enzyme was identified in the genome sequence of Mycobacterium sp. MS1601, cloned and expressed in E.coli BL21 (DE3), and the enzyme was purified and characterized. The enzyme oxidized HMF to diformyl furan (DFF) followed by conversion to FFCA at 100 % yield without further conversion to FDCA. In-silico analysis of the HMFO-Myc1 indicated that catalytic histidine is positioned at 445 and tyrosine 444 and 443 residues, which are directing the substrate into the right position in the active site, hinders FFCA from being accommodated in the right position, which motivates further studies on engineering the enzyme to enable conversion of FFCA to FDCA.
Transition from fossil- to bio-based economy is a critical step towards reduction of greenhouse gas emissions and climate change, and hence for achievement of sustainable communities and environment. In order to be fossil-free, the chemical and material industry is in need of carbon-neutral building blocks from renewable resources for the diverse array of products that are currently produced from olefins and aromatics. Hence, new pathways for producing the same or novel chemical structures are needed. Industrial biotechnology offers a key technology area for transformation of biomass components or derivatives to chemical building blocks by the use of microorganisms or their enzymes.
The thesis introduces new routes for microbial and enzymatic biotransformations of trimethylolpropane (TMP) and 5-hydroxymethyl furfural (HMF) to building blocks for polymers. TMP is an important industrial polyol with three hydroxyl groups produced from butyraldehyde and formaldehyde that can be potentially biobased, while HMF, a dehydration product of sugar, is totally biobased. The building block molecules produced from TMP include 2,2-bis(hydroxymethyl)butyric acid (BHMB), six membered cyclic carbonates, and from HMF are 5-hydroxymethyl-2-furan carboxylic acid (HMFCA), 5 formyl-2-furan carboxylic acid (FFCA) and 2,5-furan carboxylic acid (FDCA).
Growing cells of Mycobacterium sp. MS1601 (previously Corynebacterium sp. ATCC 21245) was the only bacteria that showed the ability to selectively oxidize only one hydroxyl group of TMP to form BHMB at high yield. After optimization of the process parameters and employing high cell density cultivations in a sequential batch mode with cell recycling and cell bleeding, the volumetric productivity of BHMB was improved from 0.02 g/L.h to 0.2 g/L.h to yield 21 g/L BHMB, the highest amount reported so far. Moreover, BHMB was recovered from the reaction medium by anion exchange resin at 78% yield. Transesterification of TMP with methacrylic acid and its derivatives including methyl, ethyl vinyl and dimethyl carbonate (DMC) was investigated using immobilized lipase (Novozym435) in solvent free medium in order to produce methacrylate functionalized six-membered cyclic carbonates. The results obtained from the experimental part and in-silico analysis indicate that methyl and ethyl methacrylate were preferable substrates for the enzyme to give the product with 61.3 % yield and 73% selectivity after 9 hours reaction. Also, the functionalized cyclic carbonate was purified from the reaction solution using silica chromatography at 60.5% yield. Even the production of bio-based TMP under mild conditions was demonstrated by condensation of bio-based butyraldehyde with formaldehyde produced by oxidation of the corresponding alcohols using Gluconobacter oxydans cells and alcohol oxidase, respectively.
Oxidation of crude 5-HMF to HMFCA at 100 % selectivity and yield was achieved using resting cells of G. oxydans DSM 50049. The bacteria show the ability to oxidize 31.5 g/L of crude HMF completely to HMFCA after only 6 h of the reaction, indicating that the bacteria is tolerant to the antimicrobial activity and high concentration of HMFCA. The product was recovered from the reaction with 98% purity using a simple liquid-liquid extraction step. On the other hand, Mycobacterium sp. MS1601 cells activated by growth in glycerol, oxidized HMF to FDCA and HMFCA with 60% and 40% yield, respectively. A gene sequence encoding HMF oxidase (HMFO) like enzyme was identified in the genome sequence of Mycobacterium sp. MS1601, cloned and expressed in E.coli BL21 (DE3), and the enzyme was purified and characterized. The enzyme oxidized HMF to diformyl furan (DFF) followed by conversion to FFCA at 100 % yield without further conversion to FDCA. In-silico analysis of the HMFO-Myc1 indicated that catalytic histidine is positioned at 445 and tyrosine 444 and 443 residues, which are directing the substrate into the right position in the active site, hinders FFCA from being accommodated in the right position, which motivates further studies on engineering the enzyme to enable conversion of FFCA to FDCA.
Avdelning/ar
Publiceringsår
2018
Språk
Engelska
Fulltext
Dokumenttyp
Doktorsavhandling
Förlag
Department of Chemistry, Lund University
Ämne
- Engineering and Technology
Nyckelord
- Mycobacterium sp. MS1601, G. oxydans, oxidase, lipase, trimethylolpropane (TMP), 2,2- bis(hydroxymethyl)butyric acid (BHMB), 5-hydroxymethyl furfural (5-HMF), 5-hydroxymethyl-2-furan carboxylic acid (HMFCA), 2,5-furandicarboxylic acid (FDCA), cyclic carbonate , estrification, Biotransformation, polymer building blocks, product recovery, selective oxidation, bioprocess engneering, cell recycling, cell bleeding, DNA recombinant technology, in-silico analysis
Status
Published
Projekt
- STEPS – Sustainable Plastics and Transition Pathways, Phase 1
Handledare
ISBN/ISSN/Övrigt
- ISBN: 978-91-7422-611-9
- ISBN: 978-91-7422-610-2
Försvarsdatum
17 december 2018
Försvarstid
10:15
Försvarsplats
lecture hall C, Kemicentrum, Naturvetarvägen 14, Lund University, Faculty of Engineering LTH, Lund
Opponent
- Jin-Byung Park (Professor)