Biocatalysis is a field rapidly expanding to meet a demand for green and sustainable chemical processes. As the use of enzymes for synthetic chemistry becomes more common, the construction of multistep enzyme reactions is likely to become more prominent providing excellent cost and productivity benefits. However, the design and optimisation of multistep reactions can be challenging. An enzyme toolbox of well-characterised enzyme parts is critical for the design of novel multistep reactions. Furthermore, while whole-cell biocatalysis offers an excellent platform for multistep reactions, we are limited to the use of mesophilic host organisms such as Escherichia coli. The development of a thermophilic host organism would offer a powerful tool allowing whole-cell biocatalysis at elevated temperatures. This study aimed to investigate the construction of a multistep enzyme reaction from well-characterised enzyme parts, consisting of an esterase, a carboxylic acid reductase and an alcohol dehydrogenase. A novel thermostable esterase Af-Est2 was characterised both biochemically and structurally. The enzyme shows exceptional stability making it attractive for industrial biocatalysis, and features what is likely a structural or regulatory CoA molecule tightly bound near the active site. Five carboxylic acid reductases (CARs) taken from across the known CAR family were thoroughly characterised. Kinetic analysis of these enzymes with various substrates shows they have a broad but similar substrate specificity and that electron rich acids are favoured. The characterisation of these CARs seeks to provide specifications for their use as a biocatalyst. The use of isolated enzymes was investigated as an alternative to whole-cell biocatalysis for the multistep reaction. Additional enzymes for the regeneration of cofactors and removal of by-products were included, resulting in a seven enzyme reaction. Using characterised enzyme parts, a mechanistic mathematical model was constructed to aid in the understanding and optimisation of the reaction, demonstrating the power of this approach. Thermus thermophilus was identified as a promising candidate for use as a thermophilic host organism for whole-cell biocatalysis. Synthetic biology parts including a BioBricks vector, custom ribosome binding sites and characterised promoters were developed for this purpose. The expression of enzymes to complete the multistep enzyme reaction in T. thermophilus was successful, but native T. thermophilus enzymes prevented the biotransformation from being completed. In summary, this work makes a number of contributions to the enzyme toolbox of well-characterised enzymes, and investigates their combination into a multistep enzyme reaction both in vitro and in vivo using a novel thermophilic host organism.

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Argonautes (AGOs) are present in all domains of life. Like their eukaryotic counterparts, archaeal and eubacterial AGOs adopt a similar global architecture and bind small nucleic acids. In many eukaryotes, AGOs, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. We find that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15–18 nt DNA guides derived from the chromosomal region where replication terminates, and TtAgo complexed to short DNA guides enhances target finding and prefers to bind targets with full complementarity. Additionally, TtAgo associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of both gyrase and TtAgo activity slows growth and produces long, segmented filaments in which the individual bacteria are linked by DNA. Furthermore, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. Finally, at physiologic temperature in vitro, we find TtAgo possesses highest affinity for fully complementary targets. We propose that terminus-derived guides binding in such a fashion localize TtAgo, and that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.

This diploma thesis deals with the biodegradation of whey on selected microbial products (carbohydrates, lactic acid, acetic acid and ethanol) thermophilic bacteria of genus Thermus aquaticus and mesophilic bacteria of genus Lactobacillus casei and Bacillus coagulans. For cultivation was used as medium whey, from which the proteins were removed and which was enriched with nutrients. On the basis of culture in the fermentor were determined growth curve and the HPLC method were determined individual bioremediation products.

We have carried out structural studies on bovine pancreatic phospholipase A2 (BPLA2) and two proteins involved in molybdenum cofactor (Moco) biosynthesis pathway. In addition, molecular-dynamics simulations and other analyses have been performed to corroborate the findings obtained from the crystal structures. Crystal structures of the three active-site mutants (H48N, D49N and D49K) of BPLA2 were determined to understand the mechanism by which the mutant H48N is able to catalyze the reaction of phospholipid hydrolysis and to see the effect of the loss of Ca 2+ ion in the active site of D49N and D49K mutants. We found that Asp49 could possibly play the role of a general base instead of His48 in the case of the H48N mutant. In the case of D49N and D49K mutants, the active site of the enzyme is perturbed, whereas the overall tertiary structure of these mutants is intact. In addition, a total of 24 invariant water molecules were identified in all of the crystal structures of BPLA2 available in its archive, PDB. Out of these, four water molecules are essential for the catalytic activity, whereas, the remaining water molecules play a role in the stability of the enzyme. In addition, structural studies on two proteins MoaC and MogA involved in Moco biosynthesis pathway have been carried out. For the first time, crystal structure of MoaC bound with GTP molecule has been reported. The gene id TTHA0341, which is mentioned as MoaB in the CMR database, was annotated as MogA based the comparative analysis of sequences and structures (with the present work and the structures available in the literature). The role of N-and C-termini of MoaB and MogA proteins were proposed that these residues might stabilize the substrate and/or product molecule in the active site. In addition, the residues involved in the oligomerization are compared with MD simulations. The molecular docking studies show that MoaB proteins show more preference to GTP than ATP. The comparison of the two active (MPT and AMP-binding) sites revealed that MPT-binding site is preferred over AMP-binding site for nucleotide binding.