Advanced

ω-Transaminase Catalyzed Synthesis of Chiral Amines - Process Improvements Through Whole-cell Immobilization and in situ Product Removal

Rehn, Gustav LU (2013)
Abstract (Swedish)
Popular Abstract in Swedish

Populärvetenskaplig sammanfattning

Den här avhandlingen handlar om förbättringar aven process för att producera sk. kirala aminer -en typ

av molekyler som ingår bl.a. i många läkemedel. Kirala molekyler är de som finns i två former, sk.

enantiomerer, som är varandras spegelbild. Trots sina kemiska och fYsikaliska likheter kan de ha mycket

olika biologisk effekt. Ett välkänt exempel på detta är det tragiska fallet med läkemedlet neurosedyn, där

den ena formen av läkemedlets aktiva substans orsakade allvarliga fosterskador. Genom att producera

endast den ena formen aven substans kan man minska behovet av tester som annars måste göras för... (More)
Popular Abstract in Swedish

Populärvetenskaplig sammanfattning

Den här avhandlingen handlar om förbättringar aven process för att producera sk. kirala aminer -en typ

av molekyler som ingår bl.a. i många läkemedel. Kirala molekyler är de som finns i två former, sk.

enantiomerer, som är varandras spegelbild. Trots sina kemiska och fYsikaliska likheter kan de ha mycket

olika biologisk effekt. Ett välkänt exempel på detta är det tragiska fallet med läkemedlet neurosedyn, där

den ena formen av läkemedlets aktiva substans orsakade allvarliga fosterskador. Genom att producera

endast den ena formen aven substans kan man minska behovet av tester som annars måste göras för att

kontrollera effekten av de båda varianterna.

Enzymer är proteiner som kan katalysera kemiska reaktioner och möjliggör ämnesomsättningen i alla

levande celler. Genom evolutionen har olika enzymer utvecklats för att katalysera specifika reaktioner, och

de kan ofta med stor precision reagera inte bara med rätt molekyler utan också med rätt del av molekylen

och inte minst producera den rätta enantiomeren aven substans. Enzymer kan därför vara mycket

användbara t.ex i framställningen av läkemedelsingredienser, men används också i inom annan kemi- och

livsmedelsindustri.

Enzymer är ofta mycket dyrbara, och man vill därför använda dem så effektivt som möjligt. I vissa fall kan

man använda hela celler, ofta bakterier eller jästceller, som innehåller enzymerna. Genom att fästa celler

(eller rena enzymer) på ett material (eller på varandra) kan man lättare skilja av dem och återanvända dem

i upprepade produktionscykler. I kapitel 3 presenteras en enkel och effektiv metod för att underlätta

återanvändningen av celler genom att binda samman dem m.h.a. chi tosan, som är ett billigt,

cellulosaliknande material som framställs ur bl.a. skalen hos skaldjur.

En kemisk reaktion kan liknas vid en gungbräda. Från början finns bara utgångsmateria1, och

tyngdpunkten är helt förskjuten åt ena sidan. I takt med att utgångsmaterial omvandlas till produkt

föskjuts balansen mot jämvikt, varvid sammansättningen inte längre förändras. Genom att ta bort den

produkt som bildats kan man få mer produkt att bildas för att åter nå jämviktsläget. I kapitel 4

pressenteras en metod för att selektivt separera den önskade produkten (en kiral amin), från reaktorn och

på så sätt öka den mängd produkt som kan bildas. Tekniken som använts kan liknas vid ett perforerat rör,

där porerna är fYllda med ett oljigt lösningsmedel som fungerar som ett membran mellan två vattenfaser.

På rörets utsida finns den lösning där den kemiska reaktionen katalyeras av cellernas enzym, medan en

lösning som kan ta upp den önskade produkten stömmar inuti röret. Jämfört med andra molekyler i

produktlösningen (utgångsmaterial och biprodukt) kan den önskade produkten lättare tränga igenom

membranet och fångas bättre upp i lösningen inuti röret.

Den här tekniken, har tidigare använts för andra tillämpningar men har inte använts i detta sammanhang

visade sig vara effektiv för att förbättra processen för enzymatisk framställning av kirala aminer och kan

förhoppningsvis utvecklas vidare i framtiden. (Less)
Abstract
The industrial use of biocatalysts is increasingly important in the production of e.g. pharmaceuticals, chemicals, fuels and foodstuffs. A successful biocatalytic process is dependent on many factors, such as the biocatalyst properties, the nature of the reaction, reactor type and downstream processing. This work is focused on two important issues: Immobilization of the biocatalyst and in situ product removal.



Different methods were evaluated for immobilization of the biocatalyst, E. coli cells containing a recombinantly expressed ω-TA from A. citreus. It was found that flocculation with chitosan, enhanced by a pH adjustment, was a very simple and effective method, allowing high biocatalyst loading while maintaining good... (More)
The industrial use of biocatalysts is increasingly important in the production of e.g. pharmaceuticals, chemicals, fuels and foodstuffs. A successful biocatalytic process is dependent on many factors, such as the biocatalyst properties, the nature of the reaction, reactor type and downstream processing. This work is focused on two important issues: Immobilization of the biocatalyst and in situ product removal.



Different methods were evaluated for immobilization of the biocatalyst, E. coli cells containing a recombinantly expressed ω-TA from A. citreus. It was found that flocculation with chitosan, enhanced by a pH adjustment, was a very simple and effective method, allowing high biocatalyst loading while maintaining good diffusion properties. The flocculated cells proved useful in both stirred tank and packed bed reactors.



The process considered is the production of chiral amines, which are highly important building blocks in e.g. pharmaceuticals and agrochemicals. The use of ω-transaminases (ω-TAs) for the production of chiral amines has been a rapidly growing field of research during recent years. Asymmetric synthesis is the principally advantageous route because of the high theoretical yield without need for additional reaction steps. However, the equilibrium position is often unfavourable for the amine synthesis. Shifting the equilibrium is therefore a frequently addressed issue. A commonly studied model reaction, conversion of acetophenone to optically pure methylbenzylamine, was employed in this work, using isopropylamine (IPA) as the amine donor.



In this work, in situ product removal was realized by using a supported liquid membrane (SLM). The SLM consisted of n-undecane present in the pores of a hollow fibre allowing three-phase extraction (aqueous : organic : aqueous). The bioreactor effluent was circulated on the one side of the membrane and an acidic stripping phase, capturing the amine product, on the other side. Selective extraction of the amine product, (S)-α-methylbenzylamine, was achieved due to its considerably higher hydrophobicity and its lower pKa value compared to IPA. Thus, similar concentrations of the two amines were extracted despite using a large excess of IPA to drive the reaction. A highly concentrated product, 98 g/l (810 mM), was obtained in 36 hours when using the SLM extraction system coupled to a stirred tank reactor. Ketones are not trapped in the stripping phase. Finally, parameters influencing the product extraction were investigated and discussed.

The use of an SLM system was considered a valuable addition to other existing methods for improving ω-TA catalyzed asymmetric synthesis. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Liese, Andreas, Technical University Hamburg-Harburg, Germany
organization
publishing date
type
Thesis
publication status
published
subject
pages
130 pages
defense location
Lecture hall B, Centre of Chemistry and Chemical Engingeering, Getingevägen 60, Lund University, Faculty of Engineering
defense date
2013-11-19 10:30
ISBN
978-91-89627-97-0
language
English
LU publication?
yes
id
775c8fea-bc2e-4577-adf8-3bc0e177e709 (old id 4121661)
date added to LUP
2013-10-24 08:58:31
date last changed
2016-09-19 08:45:18
@phdthesis{775c8fea-bc2e-4577-adf8-3bc0e177e709,
  abstract     = {The industrial use of biocatalysts is increasingly important in the production of e.g. pharmaceuticals, chemicals, fuels and foodstuffs. A successful biocatalytic process is dependent on many factors, such as the biocatalyst properties, the nature of the reaction, reactor type and downstream processing. This work is focused on two important issues: Immobilization of the biocatalyst and in situ product removal.<br/><br>
<br/><br>
Different methods were evaluated for immobilization of the biocatalyst, E. coli cells containing a recombinantly expressed ω-TA from A. citreus. It was found that flocculation with chitosan, enhanced by a pH adjustment, was a very simple and effective method, allowing high biocatalyst loading while maintaining good diffusion properties. The flocculated cells proved useful in both stirred tank and packed bed reactors.<br/><br>
<br/><br>
The process considered is the production of chiral amines, which are highly important building blocks in e.g. pharmaceuticals and agrochemicals. The use of ω-transaminases (ω-TAs) for the production of chiral amines has been a rapidly growing field of research during recent years. Asymmetric synthesis is the principally advantageous route because of the high theoretical yield without need for additional reaction steps. However, the equilibrium position is often unfavourable for the amine synthesis. Shifting the equilibrium is therefore a frequently addressed issue. A commonly studied model reaction, conversion of acetophenone to optically pure methylbenzylamine, was employed in this work, using isopropylamine (IPA) as the amine donor. <br/><br>
<br/><br>
In this work, in situ product removal was realized by using a supported liquid membrane (SLM). The SLM consisted of n-undecane present in the pores of a hollow fibre allowing three-phase extraction (aqueous : organic : aqueous). The bioreactor effluent was circulated on the one side of the membrane and an acidic stripping phase, capturing the amine product, on the other side. Selective extraction of the amine product, (S)-α-methylbenzylamine, was achieved due to its considerably higher hydrophobicity and its lower pKa value compared to IPA. Thus, similar concentrations of the two amines were extracted despite using a large excess of IPA to drive the reaction. A highly concentrated product, 98 g/l (810 mM), was obtained in 36 hours when using the SLM extraction system coupled to a stirred tank reactor. Ketones are not trapped in the stripping phase. Finally, parameters influencing the product extraction were investigated and discussed. <br/><br>
The use of an SLM system was considered a valuable addition to other existing methods for improving ω-TA catalyzed asymmetric synthesis.},
  author       = {Rehn, Gustav},
  isbn         = {978-91-89627-97-0},
  language     = {eng},
  pages        = {130},
  school       = {Lund University},
  title        = {ω-Transaminase Catalyzed Synthesis of Chiral Amines - Process Improvements Through Whole-cell Immobilization and in situ Product Removal},
  year         = {2013},
}