| 1. |
- Eriksson, Ulrika, 1974-
(författare)
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Impact of autocrine factors on physiology and productivity in Trichoplusia ni serum-free cultures
- 2005
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aim of this study was to increase the understanding of the mechanisms regulating cell proliferation and recombinant protein production in serum-free cultures of Trichoplusia ni (T. ni) insect cells.Conditioned medium (CM) was shown to contain both stimulatory and inhibitory factors (CM factors) influencing cell growth. Metalloproteinase (MP) activity was the major factor responsible for the growth stimulating effect of CM as shown by using the specific MP inhibitor DL-thiorphan. MPs may exist in several different molecular mass forms due to autoproteolysis. Although the main band of the MP was determined to be around 48 kDa, precursor forms above 48 kDa as well as autocatalytic degradation products below the main band could be observed. It is not clear whether all forms of the MP or just the main band is involved in the growth regulation. Further, a proteinase inhibitor could be identified in the inhibitory fraction. Thus, we speculate that the proteinase inhibitor may be part of an autocrine system regulating cell proliferation.Analysis of the cell cycle phase distribution revealed a high proportion of cells in the G1 (80-90 %) and a low proportion of cells in the S and G2/M phases (10-20 %) during the whole culture, indicating that S and G2/M are short relative to G1. After inoculation, a drastic decrease in the S phase population together with a simultaneous increase of cells in G1 and G2/M could be observed as a lagphase on the growth curve and this may be interpreted as a temporary replication stop. When the cells were released from the initial arrest, the S phase population gradually increased again. This was initiated earlier in CM-supplemented cultures, and agrees with the earlier increase in cell concentration. Thus, these data suggests a correlation between CM factors and the cell cycle dynamics.In cultures supplied with CM, a clear positive effect on specific productivity was observed, with a 30 % increase in per cell productivity. The specific productivity was also maintained at a high level much longer time than in fresh-medium cultures. The positive effect observed after 20 h coincided with the time a stimulatory effect on cell growth first was seen. Thus, the productivity may be determined by the proliferation potential of the culture. A consequence of this would be that the secreted MP indirectly affects productivity.Finally, the yeast extract from Express Five SFM contains factors up to 35 kDa which are essential for T. ni cell growth. The optimal concentration was determined to be 2.5-fold that in normal medium, while higher concentrations were inhibitory. However although vital, they were not solely responsible for the growth-enhancing effect, as some other, more general, component present in yeast extract was needed for proliferation as well.
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| 2. |
- Höst, Gunnar, 1976-
(författare)
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Engineering carbonic anhydrase for highly selective ester hydrolysis
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- I denna avhandling presenteras arbete utfört med enzymet humant karboanhydras II (HCAII). Enzymer är en typ av proteiner som accelererar (katalyserar) kemiska reaktioner, vilket är nödvändigt för allt levande. Den naturliga funktionen för HCAII är att katalysera omvandlingen av gasen koldioxid till vätekarbonat, som är löslig i vätska. Detta är viktigt bl.a. för att koldioxid som bildas i kroppen, och fraktas i blodet i form av vätekarbonat, skall hinna över till utandningsluften under den korta tid blodet är i lungorna.Proteiner består av aminosyror som länkats samman i en lång kedja, där varje aminosyra är en av de 20 naturliga aminosyratyperna. Ett proteins struktur och egenskaper bestäms av aminosyrasekvensen, som i sin tur bestäms av genen för just det proteinet. Med genteknik kan ett proteins gen ändras (muteras), så att aminosyrasekvensen ändras, och det har här utnyttjats för att förändra HCAIIs katalytiska egenskaper. Förutom dess naturliga funktion kan HCAII även klyva (hydrolysera) vissa estrar. Mutationer gjordes så att en ’ficka’ i HCAIIs struktur, där molekylerna (substraten) som skall klyvas binder, fick en större volym. På så sätt skapades varianter med en kraftigt ökad kapacitet för att hydrolysera långa estersubstrat jämfört med icke-muterat HCAII. Som en utveckling av detta projekt skapades en mutant av HCAII, som kan hydrolysera ett än mer skrymmande substrat.I ett annat projekt har en ny katalytisk aktivitet skapats i HCAII, som inte utnyttjar enzymets naturliga katalytiska förmåga. Ett nytt estersubstrat konstruerades, med en del som binder kraftigt till HCAII, så att en stark substratbindning erhölls. Sedan muterades vissa aminosyror till en reaktiv aminosyra som heter histidin. Valet av positioner för mutation baserades på en datormodell av enzymet med bundet substrat. Eftersom histidin kan delta i hydrolysreaktioner, får det muterade enzymet möjlighet att klyva substratet. Flera olika mutanter testades, och den effektivaste innehöll ett nära kopplat par av histidiner. Denna mutant undersöktes mer noggrannt, vilket gav viss information om den katalytiska mekanismen.Det långsiktiga målet med detta arbete är att konstruera muterade enzymer som kan klyva giftiga ämnen, eller användas vid framställning av kemikalier. Det finns behov av nya enzymer för olika typer av substrat, och att med rationella metoder skapa nya katalytiska aktiviteter i proteiner är ett svårt vetenskapligt problem som ännu är i ett tidigt utvecklingsskede.
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| 3. |
- Liu, Rong, et al.
(författare)
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Preparation of the four stereoisomers of 3-bromo-2-butanol or their acetates via lipase-catalysed resolutions of the racemates derived from dl- or meso-2,3-butanediol
- 2005
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Ingår i: Tetrahedron. - : Elsevier BV. - 0957-4166 .- 1362-511X. ; 16:15, s. 2607-2611
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Tidskriftsartikel (refereegranskat)abstract
- The four stereoisomeric 3-bromo-2-butanols and/or their acetates were prepared via lipase-catalysed kinetic resolution by hydrolyses of the acetates of the (+/-)-syn- and (+/-)-anti-3-bromo-2-butanols, or via esterifications of the alc hols. The diastereomeric bromoacetates were obtained by syntheses from the dl- and meso-2,3-butanediols, respectively. On a preparative scale, the four stereoisomers, either as the free alcohols or as their acetates, were obtained in > 95% ee, and in 35-40% yield (based on the starting racemates).
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| 4. |
- Marx, Lisa, 1990-
(författare)
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Chemo-enzymatic cascades for the synthesis of chiral high-value chemicals
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chiral amines are frequent in today´s top selling pharmaceuticals. Classical organic synthesis of pharmaceuticals is often work intensive involving many synthesis steps, the use of protection group chemistry, heavy metal catalysts and chiral crystallization techniques. In recent years biocatalysts have proven their outstanding ability to synthesize chiral compounds. In this work the possibility of employing biocatalysts as alternative catalysts for API (active pharmaceutical ingredient) synthesis was explored. Three compounds currently on the market were selected as viable case studies: Cinacalcet (a hyperparathyroidism drug), Vyvanse (an ADHD-drug) and Sertraline (an antidepressant). Two enzyme classes were investigated to directly provide the chiral amines - transaminases and imine reductases. Ketoreductases were also investigated to provide the chiral amine via the chiral alcohol. Laccases and hydrolases were employed to complete the synthesis pathways to the final API. In the case of Vyvanse a true one-pot, two-step enzymatic cascade was achieved by a transaminase and hydrolase. For Cinacalcet a chemo-enzymatic cascade could be demonstrated. Both transaminase and ketoreductase gave excellent enantioselectivities and high yield for the key intermediates, which could then be chemically converted into the final API with good yield. For Sertraline the best yield of one diastereomer precursor could be achieved by a ketoreductase, followed by further enzymatic and chemical steps to the final API. Transaminases and imine reductases both have potential in synthesizing the key amine precursors or the APIs themselves. But to date selectivity and yield are insufficient for industrial application in a lot of cases. This work demonstrates the potential of enzymes to serve as viable alternatives to organo-metallic synthesis. Furthermore enzymes have the potential to simplify work-up because of their excellent enantioselectivity. Finally, a scale-up of a one-step transamination to the key chiral precursor of Cinacalcet demonstrated the enzyme´s applicability in larger volume and at higher substrate concentration.
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| 8. |
- Anderson, Mattias, et al.
(författare)
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Total Synthesis of Capsaicin Analogues from Lignin-Derived Compounds by Combined Heterogeneous Metal, Organocatalytic and Enzymatic Cascades in One Pot
- 2014
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Ingår i: Advanced Synthesis and Catalysis. - : Wiley. - 1615-4150 .- 1615-4169. ; 356:9, s. 2113-2118
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Tidskriftsartikel (refereegranskat)abstract
- The total synthesis of capsaicin analogues was performed in one pot, starting from compounds that can be derived from lignin. Heterogeneous palladium nanoparticles were used to oxidise alcohols to aldehydes, which were further converted to amines by an enzyme cascade system, including an amine transaminase. It was shown that the palladium catalyst and the enzyme cascade system could be successfully combined in the same pot for conversion of alcohols to amines without any purification of intermediates. The intermediate vanillyl-amine, prepared with the enzyme cascade system, could be further converted to capsaicin analogues without any purification using either fatty acids and a lipase, or Schotten-Baumann conditions, in the same pot. An aldol compound (a simple lignin model) could also be used as starting material for the synthesis of capsaicin analogues. Using l-alanine as organocatalyst, vanillin could be obtained by a retro-aldol reaction. This could be combined with the enzyme cascade system to convert the aldol compound to vanillylamine in a one-step one-pot reaction.
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| 9. |
- Gustavsson, Martin, 1984-
(författare)
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Surface expression using the AIDA autotransporter : Towards live vaccines and whole-cell biocatalysis
- 2011
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The area of surface expression has gathered a lot of interest from research groups all over the world and much work is performed in the area. Autotransporters have been used for surface expression in Gram-negative bacteria. One of the more commonly used autotransporters is the Adhesin Involved in Diffuse Adherence (AIDA) of pathogenic Escherichia coli. The surface expression of enzymes and vaccine epitopes offer several advantages. Surface expressed enzymes gain similar properties to immobilised enzymes, mainly simplified handling and separation using centrifugation. Surface expressed vaccine epitopes can have longer half-lives inside the animal that is to be immunized and surface groups on the host cell can act as adjuvants, increasing the immune response and leading to a better immunisation. However, while much basic research is directed towards mechanisms of surface expression using autotransporters there are few reports regarding production of surface expressed protein. Thus the aim of this work was the optimisation of the yield and productivity of surface expressed protein. Protein Z, an IgG-binding domain of Staphylococcal protein A, was used as a model protein for the investigation of which cultivation parameters influenced surface expression. The choice of cultivation medium gave the largest impact on expression, which was attributed to effects based on the induction of the native promoter of AIDA. The AIDA system was then used for the expression of two Salmonella surface proteins, SefA and H:gm, with potential for use as vaccine epitopes. SefA was verified located on the cell surface, and H:gm was found in the outer membrane of the host cell, though only in proteolytically truncated forms lacking the His6-tag used for detection. This proteolysis persisted in E. coli strains deficient for the outer membrane protease OmpT and was concluded to be dependent on other proteases. The removal of proteolysis and further optimisation of the yield of surface-expressed protein are important goals of further work.
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| 10. |
- Abahazi, Emese, et al.
(författare)
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Covalently immobilized Trp60Cys mutant of omega‰-transaminase from Chromobacterium violaceum for kinetic resolution of racemic amines in batch and continuous-flow modes
- 2018
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Ingår i: Biochemical engineering journal. - : Elsevier. - 1369-703X .- 1873-295X. ; 132, s. 270-278
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Tidskriftsartikel (refereegranskat)abstract
- Covalent immobilization of an engineered omega-transaminase mutant Trp60Cys from Chromobacterium violaceum (CvTAW60C) was performed on bisepoxide-activated aminoalkyl resins. Activity of the various CvTAW60C preparations was evaluated in kinetic resolution of four racemic amines (rac-1a–d). The most active EA-G-CvTAW60C preparation (CvTAW60C attached to polymeric resin with ethylamine function activated with glycerol diglycidyl ether—EA-G) could perform the kinetic resolution of racemic 4-phenylbutan-2-amine (rac-1a) over 49% conversion up to 19 consecutive reaction cycles or in media containing up to 50% v/v DMSO as cosolvent in batch mode reactions. The immobilization process of CvTAW60C onto the EA-G resin filled in stainless steel bioreactors was also tested in flow-through mode. Kinetic resolution of three racemic amines containing aromatic moieties (rac-1a-c) was performed in continuous-flow mode resulting in easy-to-separate mixture of the corresponding ketone (2a–c) and the non-converted (R)-amine in high enantiopurity (ee(R)-1a-c ≥ 96%).
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