Enzyme catalyzed synthesis of renewable polyamides was investigated using Candida antarctica lipase B. A fatty acid-derived AB-type functional monomer, having one amine and one methyl ester functionality, was homopolymerized at 80 and 140 °C. Additionally, the organobase 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) was used as a catalyst. The results from the two catalysts were comparable. However, the amount of lipase added was 1.2 × 10³ times lower, showing that the lipase was a more efficient catalyst for this system as compared to TBD. Moreover, the AB-type monomer was copolymerized with 1,12-diaminododecane to synthesize oligoamides of two different lengths.

An efficient one-pot one-step biocatalytic amine transaminase/acyl transferase cascade for the formation of amides from the corresponding aldehydes and ketones in aqueous solution has been developed. N-benzyl-2-methoxyacetamide has been synthesized utlilizing the developed cascade in conversions up to 97%. The cascade was also evaluated for the synthesis of chiral amides.

Biocatalysis is an ever evolving field that uses enzymes or microorganisms for chemical synthesis. By utilizing enzymes that generally have evolved for specific reactions under mild conditions and temperatures, biocatalysis can be a more environmentally friendly option compared to traditional chemistry.

Amide-type chemistries are important and bond formation avoiding poor atom economy is of high priority in organic chemistry. Biocatalysis could potentially be a solution but restricted substrate scope is a limitation. Esterases/lipases usually display broad substrate scope and catalytic promiscuity but are poor at hydrolyzing amides compared to amidases/proteases. The difference between the two enzyme classes is hypothesized to reside in one key hydrogen bond present in amidases, which facilitates the transition state for nitrogen inversion during catalysis.

In this thesis the work has been focused on introducing a stabilizing hydrogen bond acceptor in esterases, mimicking that found in amidases, to develop better enzymatic catalysts for amide-based chemistries.

By two strategies, side-chain or water interaction, variants were created in three esterases that displayed up to 210-times increased relative amidase specificity compared to the wild type. The best variant displayed reduced activation enthalpy corresponding to a weak hydrogen bond. The results show an estimated lower limit on how much the hydrogen bond can be worth to catalysis.

MsAcT catalyze kinetically controlled N-acylations in water. An enzymatic one-pot one-step cascade was developed for the formation of amides from aldehydes in water that gave 97% conversion. In addition, engineered variants of MsAcT with increased substrate scope could synthesize an amide in water with 81% conversion, where the wild type gave no conversion. Moreover, variants of MsAcT displayed up to 32-fold change in specificity towards amide synthesis and a switch in reaction preference favoring amide over ester synthesis.

A novel enzyme engineering strategy for accelerated catalysis based on redesigning a water network through protein backbone deshielding is presented. Fundamental insight into the energetic consequences associated with the design is discussed in the light of experimental results and computer simulations. Using water as biobricks provides unique opportunities when transition state stabilisation is not easily attained by traditional enzyme engineering.

The present study describes the use of an epoxy functional fatty acid, 9,10-epoxy-18-hydroxyoctadecanoic acid (EFA), extracted from birch (Betula pendula) outer bark to produce thermosets. The purified epoxy fatty acid was polymerized by enzyme-catalyzed polycondensation utilizing Candida antarctica lipase B (CalB) to form oligomers with targeted degrees of polymerization (DP) of 3, 6, and 9 and obtained DPs of 2.3, 5.9 and 7.3, respectively. It was determined that it is possible to first enzymatically polymerize and aliphatically endcap the epoxy functional fatty acid resulting in controlled oligomer lengths while also maintaining the epoxy functionality for further reaction by main-chain homo-epoxy cationic photopolymerization. The enzymatic polymerized oligomers were characterized in terms of conversion of the residual epoxy groups (FT-IR), the thermal properties (DSC, TGA) and the purity by MALDI-TOF and ¹H-NMR. The amorphous thermoset films with varying degrees of crosslinking resulting from the cationically photopolymerized oligomers, were characterized in terms of their thermal properties and residual epoxy content (FT-IR ATR). The crosslinked polyesters formed insoluble, amorphous, and transparent films. This work demonstrates that thermoset films with designed properties can be effectively made with the use of forest products to reduce the petroleum-based plastics market.

Some esterases/lipases display high acyl transfer activity, favoring alcoholysis over hydrolysis, which make them valuable catalysts for synthesis reactions in aqueous media. An esterase from Mycobacterium smegmatis, MsAcT, has been characterized as an efficient catalyst for ester synthesis in water. The acyl donor specificity for MsAcT was however found to be very narrow and the enzyme displayed no activity towards esters with larger acyl group than butyrate. With rational engineering, the narrow acyl donor specificity of wild type MsAcT enzyme was altered and variants displaying extended substrate scope were generated. A double mutant, T93A/F154A, could accommodate methyl nonanoate as substrate, i.e. five carbons longer acyl group as compared to wild type, without compromising the acyl transfer capabilities. With similar selectivity towards a broad range of acyl donors (propionate to nonanoate) this is a more applicable catalyst than the wild type. Furthermore, the T93A/F154A variant was an efficient catalyst for synthesis of N-benzylhexanamide in water using methyl hexanoate as acyl donor, which is not a substrate for the wild type enzyme. The conversion reached 81% and the enzyme variant could potentially be used to produce amides in water with a wide variety of acyl donors.

Enzyme catalyzed synthesis towards renewable polyamides was investigated using Candida antarctica lipase B. A fatty acid-derived AB-type functional monomer, having one amine and one methyl ester functionality was homopolymerized at 80 and 140°C. Additionally, the organobase 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) was used as catalyst. The results from the two catalysts were comparable. However, the amount of lipase added was 1200 times lower showing that the lipase was a more efficient catalyst for this system as compared to TBD. Moreover, the AB type monomer was copolymerized with 1,12-diaminododecan to synthesize oligoamides of two different lengths.

The esterase/acyltransferase from Mycobacterium smegmatis, MsAcT, display high acyltransfer capacity in water media with demonstrations found for both ester and amide syntheses. However, it has recently been discovered that esterases in contrast to amidases lack a key hydrogen bond in the transition state, donated by the scissile NH-group of the substrate. Esterases with improved amidase performance have been achieved with the introduction of amino-acid side chains or water network as hydrogen bond acceptors. Using the esterase from Mycobacterium smegmatis, MsAcT, the influence of this hydrogen bond was studied in both amide hydrolysis and synthesis, using a rational engineering approach. Two positions were selected for mutagenesis and enzyme variants with improved performance in amide synthesis and hydrolysis were generated. Compared to the wild-type, variant F154A had the highest absolute increase in amidase specificity (11-fold) and I194Q had the greatest change in relative amidase versus esterase reaction specificity (160-fold). The relative reaction specificities for amide over ester synthesis followed a similar trend as that of hydrolysis and the best variant was I194Q with a 32-fold increase compared to wt. Based on MD-simulations water seems to play an important role in the transition state as a hydrogen bond bridge between the NH-group of the amide substrate and the enzyme.