| 1. |
- Adlercreutz, Patrick
(författare)
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Comparison of lipases and glycoside hydrolases as catalysts in synthesis reactions
- 2017
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 101:2, s. 513-519
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Forskningsöversikt (refereegranskat)abstract
- Lipases and glycoside hydrolases have large similarities concerning reaction mechanisms. Acyl-enzyme intermediates are formed during lipase-catalyzed reactions and in an analogous way, retaining glycoside hydrolases form glycosyl-enzyme intermediates during catalysis. In both cases, the covalent enzyme intermediates can react with water or other nucleophiles containing hydroxyl groups. Simple alcohols are accepted as nucleophiles by both types of enzymes. Lipases are used very successfully in synthesis applications due to their efficiency in catalyzing reversed hydrolysis and transesterification reactions. On the other hand, synthesis applications of glycoside hydrolases are much less developed. Here, important similarities and differences between the enzyme groups are reviewed and approaches to reach high synthesis yields are discussed. Useful strategies include the use of low-water media, high nucleophile concentrations, as well as protein engineering to modify the selectivity of the enzymes. The transglycosylases, hydrolases which naturally catalyze mainly transfer reactions, are of special interest and might be useful guides for engineering of other hydrolases.
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| 2. |
- Börner, Tim, et al.
(författare)
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A Process Concept for High-Purity Production of Amines by Transaminase-Catalyzed Asymmetric Synthesis: Combining Enzyme Cascade and Membrane-Assisted ISPR
- 2015
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Ingår i: Organic Process Research & Development. - : American Chemical Society (ACS). - 1083-6160 .- 1520-586X. ; 19:7, s. 793-799
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Tidskriftsartikel (refereegranskat)abstract
- For the amine transaminase (ATA)-catalyzed synthesis of chiral amines, the choice of donor substrate is of high importance for reaction and process design. Alanine was investigated as an amine donor for the reductive amination of a poorly water-soluble ketone (4-phenyl-2-butanone) in a combined in situ product removal (ISPR) approach using liquid-membrane extraction together with an enzyme cascade. This ISPR strategy facilitates very high (>98%) product purity with an integrated enrichment step and eliminates product as well as coproduct inhibition. In the presented proof-of-concept alanine shows the following advantages over the other frequently employed amine donor isopropyl amine: (i) nonextractability of alanine affords high product purity without any additional downstream step and no losses via coextraction, (ii) higher maximum reaction rates, and (iii) broader acceptance among ATAs.
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| 3. |
- Börner, Tim, et al.
(författare)
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Explaining Operational Instability of Amine Transaminases : Substrate-Induced Inactivation Mechanism and Influence of Quaternary Structure on Enzyme-Cofactor Intermediate Stability
- 2017
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 7:2, s. 1259-1269
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Tidskriftsartikel (refereegranskat)abstract
- The insufficient operational stability of amine transaminases (ATA) constitutes a limiting factor for high productivity in chiral amine synthesis. In this work, we investigated the operational stability of a tetrameric ATA with 92% sequence identity to a Pseudomonas sp. transaminase and compared it to the two commonly used dimeric ATAs from Chromobacterium violaceum and Vibrio fluvialis. In the presence of substrate, all three ATAs featured reduced stability in comparison to their resting stability, but the tetramer showed slower inactivation rates than the dimeric ATAs. Kinetic and thermodynamic analysis revealed an amine donor induced inactivation mechanism involving accumulation of the less stable aminated enzyme-cofactor intermediate. Dissociation of the enzyme-PMP complex forms the unstable apoenzyme, which can rapidly unfold. Crystal structure analysis shed light on the structure-function relationship suggesting that the cofactor-ring binding element is stabilized in the quaternary structure conferring higher operational stability by minimizing PMP leakage and apoenzyme formation. In contrast to the common practice, increasing the amine acceptor content improved the stability and substrate turnover of dimeric ATAs. An extra supply of the pyridoxal cofactor (PLP) enhanced the stability of dimeric and tetrameric ATAs but reduced the transamination activity. The ATA inactivation mechanism described here provides valuable aspects for both process development and protein engineering.
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| 4. |
- Börner, Tim, et al.
(författare)
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Generic HPLC platform for automated enzyme reaction monitoring : Advancing the assay toolbox for transaminases and other PLP-dependent enzymes
- 2016
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768. ; 11:8, s. 1025-1036
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Tidskriftsartikel (refereegranskat)abstract
- Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes.
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| 5. |
- Börner, Tim, et al.
(författare)
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Three in One : Temperature, Solvent and Catalytic Stability by Engineering the Cofactor-Binding Element of Amine Transaminase
- 2017
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227. ; 18:15, s. 1482-1486
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Tidskriftsartikel (refereegranskat)abstract
- Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate-induced inactivation mechanism involving dissociation of the enzyme-cofactor intermediate. Here, we report on engineering the cofactor-ring-binding element, which also shapes the active-site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme-cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5′-phosphate supply, thus making large-scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural "hotspot" is a common feature of other transaminases it could serve as a general engineering target.
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| 6. |
- Canet, Albert, et al.
(författare)
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Exploring substrate specificities of a recombinant Rhizopus oryzae lipase in biodiesel synthesis
- 2017
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784. ; 39, s. 59-67
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Tidskriftsartikel (refereegranskat)abstract
- The alcoholysis of triolein was used to explore the specific features of a recombinant Rhizopus oryzae lipase (rROL) for biodiesel synthesis. For this purpose, different acylglycerols were compared as substrates in lipase-catalysed transesterification. rROL was shown to exhibit a higher specificity towards 1-monoolein than triolein compared to other R. oryzae lipases, being more than 4-fold more specific; in contrast, rROL did not accept 2-monoolein as substrate, concluding that it is highly 1,3-positional specific. Comparing ethanol and methanol as acyl-acceptors, it was observed that the latter caused more lipase inactivation. Regarding alcohols, it was also demonstrated that acyl migration occurred in moderate alcohol concentrations.
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| 7. |
- Cao, Xi, et al.
(författare)
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Acyl migration in enzymatic interesterification of triacylglycerols : Effects of lipases from Thermomyces lanuginosus and Rhizopus oryzae, support material, and water activity
- 2016
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Ingår i: European Journal of Lipid Science and Technology. - : Wiley. - 1438-7697 .- 1438-9312. ; 118:10, s. 1579-1587
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Tidskriftsartikel (refereegranskat)abstract
- The enzymatic interesterification of a solvent-free, equimolar mixture of trilaurin and 1,3-palmitin-2-olein was studied using three immobilized lipase preparations as catalysts. Analysis of triacylglycerol (TAG) content and fatty acid (FA) distribution monitored the lipase-catalyzed interesterification in sn-1,3 positions and FA exchange in the sn-2 position caused by acyl migration. Lipase from Rhizopus oryzae immobilized on polypropylene showed high sn-1,3 regioselectivity, and minimal exchange in the sn-2 position. With lipase from Thermomyces lanuginosus on silica (Lipozyme® TL IM), completely randomized FA distribution was obtained in 24 h. T. lanuginosus lipase on polypropylene caused a moderate rate of FA exchange in the sn-2 position. Thus, the T. lanuginosus lipase and silica promoted randomization of FA distribution, whereas the R. oryzae lipase and polypropylene did not. Higher water activity promoted hydrolysis and thereby increased concentrations of partial acylglycerols, but at the same time a decrease in the acyl migration rate of these intermediates was also observed. The net result was that at a certain degree of interesterification, there was no significant effect of water activity on the degree of exchange in the sn-2 position. On the other hand, a low water activity had the major advantage of giving a high yield of TAG. Practical applications: Both the type and the position of FA affect the properties of TAG. In TAG interesterification, there are thus different requirements for regiospecificity in FA exchange, depending on which product is desired. According to our results, lipase-based catalysts can be used for TAG interesterification either to achieve unchanged FA composition in sn-2 position or fast FA randomization in all positions. This helps to broaden the application of lipases in interesterification. The production of TAG can be tailor-made for lipid mixtures with particular TAG composition and FA distribution using proper lipases, support materials, water activity, and reaction time. The enzymatic LLL–POP model reaction was used to investigate effects of lipase, support material, and water activity on acyl migration. (Fig. A): effect of Lipozyme® TL IM, TLPP and ROPP at aw 0.26, (Fig. B): effect of aw between 0.15 and 0.80 using Lipozyme® TL IM as the example.
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| 8. |
- Chen, Shan, 1986-
(författare)
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Stability and inactivation mechanisms of two transaminases
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the past decades, more and more enzymes are employed as biocatalysts in industrial processes because of their advantages, such as high efficiency, substrate selectivity and stereoselectivity. Among them, amine transaminases (ATAs) are pyridoxal 5’-phosphate (PLP) dependent enzymes. ATAs have gained attention for their excellent performance in chiral amine synthesis, and their broad substrate acceptance. However, the low operational stability of amine transaminases still limits their application in industry.The amine transaminase from Chromobacterium violaceum (Cv-ATA) has been selected for further investigation for its relatively low operational stability. Co-solvents and various additives have been added to the enzyme storage solution to improve its storage stability at various temperatures. Co-lyophilization of Cv-ATA with surfactants has been applied to improve its enzymatic activity in neat organic solvents.As a PLP-dependent dimeric enzyme, the Cv-ATA is not primarily inactivated due to tertiary structural changes. Instead, both dimer dissociation and PLP release may affect the enzyme stability. Therefore, the inactivation pathway of the Cv-ATA during operational conditions was explored. The unfolding of the enzyme was detected by several methods, and the detection of fluorescence intensity spectrum of tryptophan is extensively applied for its high sensitivity. The phosphate group of PLP can be coordinated into the phosphate group binding cup, which may influence the enzyme structural stability. Therefore, the effect of both PLP and inorganic phosphate ions (present in phosphate buffer) on the enzyme stability was explored.The amine transaminase from Vibrio fluvialis (Vf-ATA) is another amine transaminase, which catalyses the same biocatalytic reaction and has a similar substrate scope as Cv-ATA. However, there is still a lack of data on the stability of Vf-ATA. Consequently, the operational stability of Vf-ATA in various environments was studied.
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| 9. |
- Falck, Peter, et al.
(författare)
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Arabinoxylanase from glycoside hydrolase family 5 is a selective enzyme for production of specific arabinoxylooligosaccharides
- 2018
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Ingår i: Food Chemistry. - : Elsevier BV. - 0308-8146. ; 242, s. 579-584
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Tidskriftsartikel (refereegranskat)abstract
- An arabinose specific xylanase from glycoside hydrolase family 5 (GH5) was used to hydrolyse wheat and rye arabinoxylan, and the product profile showed that it produced arabinose substituted oligosaccharides (AXOS) having 2-10 xylose residues in the main chain but no unsubstituted xylooligosaccharides (XOS). Molecular modelling showed that the active site has an open structure and that the hydroxyl groups of all xylose residues in the active site are solvent exposed, indicating that arabinose substituents can be accommodated in the glycone as well as the aglycone subsites. The arabinoxylan hydrolysates obtained with the GH5 enzyme stimulated growth of Bifidobacterium adolescentis but not of Lactobacillus brevis. This arabinoxylanase is thus a good tool for the production of selective prebiotics.
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| 10. |
- Falck, Peter, et al.
(författare)
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Characterization of a family 43 β-xylosidase from the xylooligosaccharide utilizing putative probiotic Weissella sp. strain 92.
- 2015
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 26:2, s. 193-202
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Tidskriftsartikel (refereegranskat)abstract
- In this work we present the first XOS degrading glycoside hydrolase from Weissella, WXyn43, a two-domain enzyme from GH43. The gene was amplified from genomic DNA of the XOS utilizing Weissella strain 92, classified under the species pair Weissella cibaria/W.confusa, and expressed in Escherichia coli. The enzyme is lacking a putative signal peptide and is, from a homology model, shown to be composed of an N-terminal 5-fold ß-propeller catalytic domain and a C-terminal ß-sandwich domain of unknown function. WXyn43 hydrolysed short (1-4)-β-D-xylooligosaccharides, with similar kcat/KM for Xylobiose (X2) and xylotriose (X3) and clearly lower efficiency in xylotetraose (X4) conversion. WXyn43 displays the highest reported kcat for conversion of X3 (900 s(-1) at 37°C) and X4 (770 s(-1)), and kcat for hydrolysis of X2 (907 s(-1)) is comparable to or greater than the highest previously reported. The purified enzyme adopted a homotetrameric state in solution, while a truncated form with isolated N-terminal catalytic domain adopted a mixture of oligomeric states and lacked detectable activity. The homology model shows that residues from both domains are involved in monomer-monomer hydrogen bonds, while the bonds creating dimer-dimer interactions only involved residues from the N-terminal domain. Docking of X2 and X3 in the active site show interactions corresponding to sub-sites -1 and +1, while presence of a third subsite is unclear, but interactions between a loop and the reducing-end xylose of X3 may be present.
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