The biotechnological transformation of vegetable oils and animal fats has been successfully developed up to an industrial scale in recent years. However, biotechnology is still a niche technology in ...the oleochemical industry, which classically relies on chemical transformation processes. Nevertheless, the impact of biotechnology is rising, with an increasing number of biotech-based products, especially in the area of lipid-derived specialties, entering the market. In this review we give a summary of the industrially relevant processes in the field of lipid biotechnology.
Linoleic acid hydroperoxides are versatile intermediates for the production of green note aroma compounds and bifunctional ω-oxo-acids. An enzyme cascade consisting of lipoxygenase, lipase and ...catalase was developed for one-pot synthesis of 13-hydroperoxyoctadecadienoic acid starting from safflower oil. Reaction conditions were optimized for hydroperoxidation using lipoxygenase 1 from Glycine max (LOX-1) in a solvent-free system. The addition of green surfactant Triton CG-110 improved the reaction more than two-fold and yields of >50% were obtained at linoleic acid concentrations up to 100 mM. To combine hydroperoxidation and oil hydrolysis, 12 lipases were screened for safflower oil hydrolysis under the reaction conditions optimized for LOX-1. Lipases from Candida rugosa and Pseudomonas fluorescens were able to hydrolyze safflower oil to >75% within 5 h at a pH of 8.0. In contrast to C. rugosa lipase, the enzyme from P. fluorescens did not exhibit a lag phase. Combination of P. fluorescens lipase and LOX-1 worked well upon LOX-1 dosage and a synergistic effect was observed leading to >80% of hydroperoxides. Catalase from Micrococcus lysodeikticus was used for in-situ oxygen production with continuous H2O2 dosage in the LOX-1/lipase reaction system. Foam generation was significantly reduced in the 3-enzyme cascade in comparison to the aerated reaction system. Safflower oil concentration was increased up to 300 mM linoleic acid equivalent and 13-hydroperoxides could be produced in a yield of 70 g/L and a regioselectivity of 90% within 7 h.
Aminoacylases are highly promising enzymes for the green synthesis of acyl-amino acids, potentially replacing the environmentally harmful Schotten-Baumann reaction. Long-chain acyl-amino acids can ...serve as strong surfactants and emulsifiers, with application in cosmetic industries. Heterologous expression of these enzymes, however, is often hampered, limiting their use in industrial processes.
We identified a novel mycobacterial aminoacylase gene from Mycolicibacterium smegmatis MKD 8, cloned and expressed it in Escherichia coli and Vibrio natriegens using the T7 overexpression system. The recombinant enzyme was prone to aggregate as inclusion bodies, and while V. natriegens Vmax™ could produce soluble aminoacylase upon induction with isopropyl β-d-1-thiogalactopyranoside (IPTG), E. coli BL21 (DE3) needed autoinduction with lactose to produce soluble recombinant protein. We successfully conducted a chaperone co-expression study in both organisms to further enhance aminoacylase production and found that overexpression of chaperones GroEL/S enhanced aminoacylase activity in the cell-free extract 1.8-fold in V. natriegens and E. coli. Eventually, E. coli ArcticExpress™ (DE3), which co-expresses cold-adapted chaperonins Cpn60/10 from Oleispira antarctica, cultivated at 12 °C, rendered the most suitable expression system for this aminoacylase and exhibited twice the aminoacylase activity in the cell-free extract compared to E. coli BL21 (DE3) with GroEL/S co-expression at 20 °C. The purified aminoacylase was characterized based on hydrolytic activities, being most stable and active at pH 7.0, with a maximum activity at 70 °C, and stability at 40 °C and pH 7.0 for 5 days. The aminoacylase strongly prefers short-chain acyl-amino acids with smaller, hydrophobic amino acid residues. Several long-chain amino acids were fairly accepted in hydrolysis as well, especially N-lauroyl-L-methionine. To initially evaluate the relevance of this aminoacylase for the synthesis of N-acyl-amino acids, we demonstrated that lauroyl-methionine can be synthesized from lauric acid and methionine in an aqueous system.
Our results suggest that the recombinant enzyme is well suited for synthesis reactions and will thus be further investigated.
Current changes in environmental legislation and customer demands set an urge for the development of more sustainable surfactants. Thus, the objective of this work was the development of novel ...environmentally friendly amino acid surfactants. Combining Diels–Alder cyclization of myrcene with maleic or citraconic anhydride followed by ring opening with amino acids enabled a synthesis route with a principal 100% atom economy. Variation of amino acids resulted in a large structural variety of anionic and amphoteric surfactants. Lysine gave access to either a mono-acylated product bearing a cationic side chain or a bi-acylated gemini surfactant. First, anhydride precursors were synthesized in yields of >90% in a Diels–Alder reaction under microwave radiation and subsequent amino acid coupling in aqueous environment gave fully bio-based surfactants in good yields and purity. Physicochemical characterization showed an enhanced decrease in surface tension upon addition of amino acids to the myrcene–anhydride backbone, resulting in a minimal value of 31 mN·m
for gemini–lysine. Foamabilitiy and foam stability were significantly increased at skin-friendly pH 5.5 by incorporation of amino acids. The carboxylic groups of surfactants with arginine were esterified with ethanol to access cationic compounds. Comparative analysis revealed moderate antimicrobial effects against yeast, Gram-positive bacteria, and Gram-negative bacteria.
Hexanal, hexenal, nonenal and their corresponding alcohols are used as green notes in the fragrance and flavour industry. The production of bio-based hexanal starts from linoleic acid, which can be ...obtained from sunflower or safflower oil. The biocatalytic process utilizes C13-specific lipoxygenase (LOX) for hydroperoxidation and consecutive splitting with hydroperoxide lyase (HPL). In this study, we investigated the chemical splitting of the LOX product 13-HPODE in comparison to HPL catalysis. In addition, 13-HPODE was synthesized using enriched linoleic acid from safflower oil. Varying amounts of soybean flour suspension as a source of LOX yielded up to 60% HPODE with a regioselectivity of 92% towards 13-HPODE. Using low-toxicity Lewis acids like AlCl3 and ZrCl4, cleavage of the produced 13-HPODE was possible. A maximum hexanal yield of 22.9% was reached with AlCl3 under mild reaction conditions, though product degradation was an interfering process. Comparative trials with N-terminal truncated HPL from papaya revealed hexanal recovery within a comparable range. Additionally, we successfully demonstrated the viability of Hock rearrangement of 13-HPODE through heterogeneous catalysts. Notably, Beta zeolite and Montmorillonite K10 exhibited a turnover frequency (TOF) on par with common heterogeneous catalysts employed in industrial processes.
In Escherichia coli, 1-deoxy-D-xylulose (or its 5-phosphate, DXP) is the biosynthetic precursor to isopentenyl diphosphate Broers, S. T. J. (1994) Dissertation (Eidgenössische Technische Hochschule, ...Zürich), thiamin, and pyridoxol Himmeldirk, K., Kennedy, I. A., Hill, R. E., Sayer, B. G. & Spenser, I. D. (1996) Chem. Commun. 1187-1188. Here we show that an open reading frame at 9 min on the chromosomal map of E. coli encodes an enzyme (deoxyxylulose-5-phosphate synthase, DXP synthase) that catalyzes a thiamin diphosphate-dependent acyloin condensation reaction between C atoms 2 and 3 of pyruvate and glyceraldehyde 3-phosphate to yield DXP. We have cloned and overexpressed the gene (dxs), and the enzyme was purified 17-fold to a specific activity of 0.85 unit/mg of protein. The reaction catalyzed by DXP synthase yielded exclusively DXP, which was characterized by 1H and 31P NMR spectroscopy. Although DXP synthase of E. coli shows sequence similarity to both transketolases and the E1 subunit of pyruvate dehydrogenase, it is a member of a distinct protein family, and putative DXP synthase sequences appear to be widespread in bacteria and plant chloroplasts.
Hydroperoxy‐9Z,11E‐octadecadienoic acid (13‐HPODE) can be obtained from safflower oil in an enzyme cascade utilizing lipase, lipoxygenase (LOX), and catalase for in situ oxygen generation. The ...application of immobilized enzymes may open a new path to a cost‐efficient production of 13‐HPODE, which is used for the synthesis of green note aroma hexanal. Ten immobilization supports are compared for immobilization of lipoxygenase‐1 from Glycine max (LOX‐1) and oxirane‐based Immobead 150 P proves to be best with a maximum LOX‐1 activity of 22 470 U g−1. The immobilizate is successfully recycled in eight consecutive batches and maintains full activity over a period of 16 h using a 3D‐printed column reactor. Catalase from Micrococcus lysodeikticus and LOX‐1 are co‐immobilized on Immobead 150 P allowing a constant production of 13‐HPODE for up to six cycles with a maximum product conversion of 45% and a 13‐regioselectivity of 83%
. In this two‐enzyme system with H2O2‐dosage, foam generation is significantly reduced. Co‐immobilization of LOX‐1 and lipaseis possible; however, rapid lipase deactivation occurs. Therefore, a two‐reactor approach with oil hydrolysis in the first reactor is proposed. Immobilized lipases from C. rugosa are suitable for safflower oil hydrolysis and maintain full activity over ten hydrolysis cycles.
Practical applications: Linoleic acid hydroperoxide (13‐HPODE) is the starting material for the synthesis of the green note aroma compound hexanal. The byproduct of the hydroperoxide splitting is ω‐oxododecenoic acid, which is currently not employed industrially. The bifunctional oxodocecenoic acid is interesting as precursor for the synthesis of polymer building blocks. Simple one‐step derivatization of the oxo‐group can yield suitable C12 monomers such as dicarboxylic acids, ω‐amino acids, or ω‐hydroxy acids. Cost‐efficiency is a key parameter to incorporate these new biobased building blocks for polymer applications. In this approach, immobilized enzymes are used for the synthesis of 13‐HPODE starting from safflower oil with in situ oxygen generation to prevent excessive foam formation. A two‐reactor concept is designed to circumvent hydroperoxide‐induced lipase deactivation. Direct comparison of both batch and continuous process is performed and provides information for the implementation of the enzyme cascade and the design of an optimized reactor system.
The synthesis of linoleic acid based hydroperoxide 13‐HPODE is achieved with immobilized lipase and co‐immobilized lipoxygenase (LOX) and catalase starting from safflower oil in an organic solvent‐free system. A reactor cascade is needed to prevent lipase deactivation by the hydroperoxide. In situ oxygen supply for LOX is generated by hydrogen peroxide dosing and catalase activity.
Display omitted
•A two-step-one-pot process with native lipases in deep eutectic solvents was developed.•Ethyl ester yields of >96wt.% with low fatty acid content were achieved without ...purification.•Ester content, free glycerol and acid value comply with biodiesel standard EN 14214 after refinement.•Pure glycerol was obtained in a single distillation step.•Used cooking oils were successfully transesterified with the two-enzyme process.
The synthesis of biodiesel with free lipases starting from refined rapeseed oil and low quality cooking oils was evaluated. A two-enzyme-one-pot system was developed, which gave ethyl ester yields of 96% with less than 1% of residual fatty acids before purification. In the first reaction step Thermomyces lanuginosus lipase was employed, which exhibited high activity of 600nmol/s/mg on triglycerides. Triglyceride turnover was nearly 100% and ethyl ester–glyceride mixtures with 3–4% of fatty acids accumulated. In the second reaction step, Pseudozyma antarctica lipase B dissolved in deep eutectic solvents was applied successfully for residual glyceride transesterification and fatty acid esterification. After phase separation, biodiesel was purified in a one-step refinement to yield a product with 97.6% ethyl ester content. Glycerol of high quality was obtained in a single distillation step from the hydrophilic deep eutectic solvent phase. Comparative trials with several used cooking oils revealed, that the first transesterification step worked equally well, while reaction velocity of the second step was lower than with refined oils. More than 80% ethyl ester yield were obtained with all used cooking oils tested.
Biobased polymers derived from plant oils are sustainable alternatives to petro based polymers. In recent years, multienzyme cascades have been developed for the synthesis of biobased ...ω-aminocarboxylic acids, which serve as building blocks for polyamides. In this work, we have developed a novel enzyme cascade for the synthesis of 12-aminododeceneoic acid, a precursor for nylon-12, starting from linoleic acid. Seven bacterial ω-transaminases (ω-TAs) were cloned, expressed in
Escherichia coli
and successfully purified by affinity chromatography. Activity towards the oxylipin pathway intermediates hexanal and 12-oxododecenoic acid in their 9(
Z
) and 10(
E
) isoforms was demonstrated for all seven transaminases in a coupled photometric enzyme assay. The highest specific activities were obtained with ω-TA from
Aquitalea denitrificans
(TR
AD
), with 0.62 U mg
−1
for 12-oxo-9(
Z
)-dodecenoic acid, 0.52 U mg
−1
for 12-oxo-10(
E
)-dodecenoic acid and 1.17 U mg
−1
for hexanal. A one-pot enzyme cascade was established with TR
AD
and papaya hydroperoxide lyase (HPL
CP-N
), reaching conversions of 59% according to LC-ELSD quantification. Starting from linoleic acid, up to 12% conversion to 12-aminododecenoic acid was achieved with a 3-enzyme cascade comprising soybean lipoxygenase (LOX-1), HPL
CP-N
and TR
AD
. Higher product concentrations were achieved by the consecutive addition of enzymes compared to simultaneous addition at the beginning.
Key points
•
Seven ω-transaminases converted 12-oxododecenoic acid into its corresponding amine.
•
A three-enzyme cascade with lipoxygenase, hydroperoxide lyase, and ω-transaminase was established for the first time.
•
A one-pot transformation of linoleic acid to 12-aminododecenoic acid, a precursor of nylon-12 was achieved.
Graphical Abstract
N-Acyl-amino acids can act as mild biobased surfactants, which are used, e.g., in baby shampoos. However, their chemical synthesis needs acyl chlorides and does not meet sustainability criteria. ...Thus, the identification of biocatalysts to develop greener synthesis routes is desirable. We describe a novel aminoacylase from
Paraburkholderia monticola
DSM 100849 (PmAcy) which was identified, cloned, and evaluated for its N-acyl-amino acid synthesis potential. Soluble protein was obtained by expression in lactose autoinduction medium and co-expression of molecular chaperones GroEL/S. Strep-tag affinity purification enriched the enzyme 16-fold and yielded 15 mg pure enzyme from 100 mL of culture. Biochemical characterization revealed that PmAcy possesses beneficial traits for industrial application like high temperature and pH-stability. A heat activation of PmAcy was observed upon incubation at temperatures up to 80 °C. Hydrolytic activity of PmAcy was detected with several N-acyl-amino acids as substrates and exhibited the highest conversion rate of 773 U/mg with N-lauroyl-L-alanine at 75 °C. The enzyme preferred long-chain acyl-amino-acids and displayed hardly any activity with acetyl-amino acids. PmAcy was also capable of N-acyl-amino acid synthesis with good conversion rates. The best synthesis results were obtained with the cationic L-amino acids L-arginine and L-lysine as well as with L-leucine and L-phenylalanine. Exemplarily, L-phenylalanine was acylated with fatty acids of chain lengths from C8 to C18 with conversion rates of up to 75%. N-lauroyl-L-phenylalanine was purified by precipitation, and the structure of the reaction product was verified by LC–MS and NMR.
Key points
• A novel aminoacylase from Paraburkholderia monticola was cloned, expressed in E. coli and purified.
• The enzyme PmAcy exhibits exceptional temperature and pH stability and a broad substrate spectrum.
• Synthesis of acyl amino acids was achieved in good yields.
PDF
