Improving the glycosylation potential of sucrose phosphorylase through enzyme engineering

Glycosidic compounds are chemical structures that consist of sugars molecules or that have a sugar attached to another molecule. They are now already used in various industries and have the potential to serve an even much wider range of applications. They can be synthesised chemically, but with the right enzymes, production processes can often be much cleaner and more efficient. Glycoside phosphorylases are such enzymes that can be used to synthesise glycosides and in that respect sucrose phosphorylase is highly interesting. Sucrose phosphorylase is namely not restricted to its wild-type substrates. It can also transfer a sugar moiety to a variety of different acceptor molecules. In addition, it uses sucrose as a cheap, renewable and reactive donor substrate, which makes it an attractive biocatalyst for the production of special sugars and glucoconjugates. Activities on relevant components are unfortunately poor and in many cases not even exceeding the unwanted hydrolytic side activity. Moreover, for industrial processes increased stability is also desired. It is thus clear that sucrose phosphorylase offers many possibilities, but that still some improvements are required to fully exploit its potential. Therefore, in this thesis enzyme engineering was applied to alter the specificity and enhance the stability of sucrose phosphorylase. Knowing how specificity is controlled, would allow to optimise alternative activities in a more efficient way. Therefore, a map of the acceptor site of the SP from Bifidobacterium adolescentis was first created by substituting each residue by alanine and analysing the influence on the affinity for both the natural (inorganic phosphate and fructose) and alternative acceptors (D-arabitol and pyridoxine). All residues examined were found to contribute to the specificity for phosphate (Arg135, Leu343, Tyr344), fructose (Tyr132, Asp342) or both (Pro134, Tyr196, His234, Gln345). Alternative acceptors that are glycosylated rather efficiently e.g. d-arabitol were found to interact with the same residues as fructose, whereas poor acceptors like pyridoxine did not seem to make any specific interactions with the enzyme. Furthermore, it was shown that SP is already optimised to outcompete water as an acceptor substrate, meaning that it will be very difficult to lower its hydrolytic activity any further. Consequently, increasing the transglycosylation activity towards alternative acceptors seems to be the best strategy, although that would probably require a drastic remodelling of the acceptor site in most cases. Regioselectivity can most likely be engineered more easily than completely shifting specificity, but it can nevertheless be equally important. Kojibiose (Glc-α1,2-Glc) for instance is a very expensive and hardly available compound with prebiotic properties, while its regioisomer maltose (Glc-α1,4-Glc) is cheap and has little added value. Natural sucrose phosphorylases produce a mixture of both and a variant that only makes kojibiose would of course be of great interest. To that end, ten positions in the acceptor site were randomised individually and screened with a high performance anion exchange chromatography (HPAEC) based screening procedure. Several improved mutants were obtained from this first round of mutagenesis and the best mutant L341I displayed a selectivity of 79%. Rational combination as well as combinations predicted by a statistical ProSAR model could further improve the selectivity, although the former approach yielded the best mutants. Two double (L341I_Q345S and L341I_Q345N) and one triple mutant (L341I_Y344A_Q345N) were obtained with selectivities of 93 to 95%. Activities were only slightly lower than the wild-type enzyme and therefore the variants created in this work will allow the development of a cost-effective and scalable process for the enzymatic synthesis of kojibiose from the readily available and low-cost substrates sucrose and glucose. As discussed above, stable biocatalysts are a real benefit for industrial processes. Thermophilic organisms are a rich source of stable enzymes, but for sucrose phosphorylase this area has not yet been explored. Hence, in this study, the putative sucrose phosphorylase from the thermophile Thermoanaerobacterium thermosaccharolyticum was recombinantly expressed and fully characterised. The enzyme showed significant activity on sucrose (optimum at 55°C), and with a melting temperature of 79°C and a half-life of 60 hours at the industrially relevant temperature of 60°C, it is far more stable than known sucrose phosphorylases. Substrate screening and detailed kinetic characterisation revealed however a preference for sucrose 6’-phosphate over sucrose. The enzyme can thus be considered as a sucrose 6’-phosphate phosphorylase, a specificity not yet reported to date. Homology modelling and mutagenesis pointed out particular residues (Arg134 and His344) accounting for the difference in specificity. Moreover, phylogenetic and sequence analysis suggest that glycoside hydrolase 13 subfamily 18 might harbour even more specificities. In addition, the second gene residing in the same operon as sucrose 6’-phosphate phosphorylase was identified as well, and found to be a phosphofructokinase. The concerted action of these both enzymes implies a new pathway for the breakdown of sucrose, in which the reaction products end up at different stages of the glycolysis. In addition to the development of applications, techniques like ancestral reconstruction, correlated mutation analysis and in silico prediction of stability were evaluated for their general utility in protein engineering. Present-day enzymes have evolved from a common ancestor that is believed to be promiscuous and to have an appropriate mutational background to evolve. Ancestral enzymes would accordingly be ideally suited to create new (related) specificities by directed evolution. To investigate whether an ancestral enzyme could be a good starting point for mutagenesis and what the role of promiscuity is, a full-length ancestor of sucrose phosphorylase (SP) and sucrose 6’-phosphate phosphorylase (SPP) enzymes was reconstructed. The ancestor behaved like a sucrose phosphorylase, but its activity was one to three orders of magnitude lower than present-day SP’s. This was mainly due to a low kcat (200 mM). In addition, promiscuity was very similar in qualitative terms, but activities were in general lower. Swapping SP specific residues with their SPP counterpart could furthermore not introduce SPP activity in the ancestor. However, the same mutations could enable a specificity switch in a thermostable present-day SP, showing that for directed evolution a stable template might be more appropriate than a reconstructed ancestor. Another aspect of evolution that could be useful for enzyme engineering, is coevolution of specific positions in related enzymes. Statistical analysis of large multiple sequence alignments can reveal such correlated positions, i.e. positions that house specific combinations of residues more prevalent than expected from random distribution. They often form complex networks and are likely to be involved in activity, specificity and stability. Their exact role is however currently still obscure. Therefore, in this work we compared correlated mutations in two related specificities (sucrose and sucrose 6’-phosphate phosphorylase) and experimentally evaluated hypotheses on different aspects of the correlation network. Distribution across the structure suggests that correlated positions might be involved in supporting the different active site topologies, rather than directly being involved in substrate contact. Retracing the evolution showed that correlated positions that are close to each other in the structure or that are strongly correlated often not evolve simultaneously. Introduction of the active site of sucrose 6’-phosphate phosphorylase in sucrose phosphorylase enable this latter enzyme to catalyse the former reactions. The activity was however very low en therefore correlated positions were mutated as well. This caused a decrease in activity (up to fifty- fold) in most cases, but in one double mutant the activity could selectively be increased by a factor of two. Mutation of correlated positions can thus alter the specificity, but general guidelines on which positions to mutate and which residues to introduce, could unfortunately not be derived and will need further research. Stable enzymes are important as already discussed previously. They can be obtained from natural diversity, but not every specificity is available and therefore stable variants need to be created by engineering existing enzymes. In that respect, in silico prediction of stabilising mutations could greatly reduce time, cost and effort compared to current in vitro screening procedures. Here, we evaluated the potential of the FoldX algorithm. All possible point mutations were introduced in a sucrose phosphorylase and those predicted to improve the free energy of folding or the dimer interaction energy were visually inspected for unreasonable mutations. Two thirds were rejected during this manual selection, mostly because hydrophobic residues became too solvent exposed. From the remaining mutants, the nine most promising were experimentally tested. Unfortunately none proved to be more stable: four appeared to be neutral or nearly neutral, while five were deleterious. Remarkably, the mutant predicted to be the most stabilising drastically impaired stability. To explain these findings, molecular dynamics (md) simulations were performed. Root mean square fluctuations (rmsf), representing local flexibility, were found to be bad predictors of stability. Examining if the interactions responsible for the predicted stability are maintained over a period of time in contrast could indeed explain the observed effects in many cases

The Depression Conundrum and the advantages of uncertainty

According to the WHO (2012), the prevalence of unipolar depressive disorders is rising, even in those places where mental health treatments are widely available. The WHO predicts that these disorders will be the leading contributor to the global burden of disease by 2030. This sobering projection fits poorly with how psychological treatments for depression are presented in the mainstream scientific literature: as highly effective therapies, based upon a sound understanding of the causes of distress. There is a clear discrepancy between the rising prevalence figures on the one hand, and the confident claims of this effectiveness research on the other. This discrepancy prompts a set of complex interlinked questions, which we have called 'The Depression Conundrum.' In search of a partial answer, the aim of our study was to critically analyze five meta-analytic studies investigating the effectiveness of psychological EBTs for depression, all of which had been published in high impact factor journals. Our examination established a number of methodological and statistical shortcomings in every study. Furthermore, we argue that the meta-analytic technique is founded upon problematic assumptions. The implications of our analysis are clear: decades of quantitative research might not allow us to conclude that psychological EBTs for depression are effective. The uncertainty and questions raised by our findings might act as a catalyst to broaden the way in which depression and associated therapies are researched. In addition, it might contribute toward a more vigorous and interdisciplinary debate about how to tackle this soon-to-be global public health priority number one

Health economic evaluations of interventions to increase physical activity and decrease sedentary behavior at the workplace : a systematic review

Objective The workplace is an ideal setting to implement public health strategies, but economic justification for such interventions is needed. Therefore, we performed a critical appraisal and synthesis of health economic evaluations (HEE) of workplace interventions aiming to increase physical activity (PA) and/or decrease sedentary behavior (SB). Methods A comprehensive search filter was developed using appropriate guidelines, such as the Peer Review of Electronic Search Strategies (PRESS) checklist, and published search algorithms. Six databases and hand searches were used to identify eligible studies. Full HEE of workplace interventions targeting PA/SB were included. Methodological quality was assessed using the Consensus Health Economic Criteria (CHEC) list. Two researchers independently performed all procedures. Hedges' g was calculated to compare intervention effects. Outcomes from HEE were recalculated in 2017 euros and benefit-standardized. Results Eighteen HEE were identified that fulfilled on average 68% of the CHEC list criteria. Most studies showed improvements in PA/SB, but effects were small and thus, their relevance is questionable. Interventions were heterogeneous, no particular intervention type was found to be more effective. HEE were heterogeneous regarding methodological approaches and the selection of cost categories was inconsistent. Indirect costs were the main cost driver. In all studies, effects on costs were subject to substantial uncertainty. Conclusions Due to small effects and uncertain impact on costs, the economic evidence of worksite PA/SB-interventions remains unclear. Future studies are needed to determine effective strategies. The HEE of such interventions should be developed using guidelines and validated measures for productivity costs. Additionally, studies should model the long-term costs and effects because of the long pay-back time of PA/SB interventions

What are the economic dimensions of occupational health and how should they be measured? A qualitative study.

BACKGROUND Decision makers want to know if there is a financial benefit in investing scarce resources in occupational health management (OHM). Economic evaluations (EEs) of OHM-strategies try to answer this question. However, EEs of OHM-strategies which are strongly marked by quantitative methods may be limited by contextual, qualitative residuals. Therefore, the objectives of this study were to (1) explore important economic dimensions of OHM and (2) to discuss the methods used in current EEs for measuring these dimensions. METHODS In this explorative qualitative study, OHM-specialists were recruited via the Swiss organisation for health promotion. Thirteen semi-structured interviews were performed from November 2020 until May 2021. Videotapes were transcribed verbatim and organised by using an open coding strategy. Codes were clustered and synthesised as themes (i.e. the dimensions of EEs of OHM) through a mix of inductive and deductive content analysis. Member check with eight participants was accomplished to validate the results. RESULTS The interviews had an average duration of 70.5 min and yielded 609 individual codes. These codes were merged into 28 subcategories which were finally categorised into five main themes: Understanding of OHM, costs, benefits, environmental aspects, and evaluation of OHM. Participants stated that the greater part of costs and benefits cannot be quantified or monetised and thus, considered in quantitative EEs. For example, they see a culture of health as key component for a successful OHM-strategy. However, the costs to establish such a culture as well as its benefits are hard to quantify. Participants were highly critical of the use of absenteeism as a linear measure of productivity. Furthermore, they explained that single, rare events, such as a change in leadership, can have significant impact on employee health. However, such external influence factors are difficult to control. CONCLUSIONS Participants perceived costs and benefits of OHM significantly different than how they are represented in current EEs. According to the OHM-specialists, most benefits cannot be quantified and thus, monetised. These intangible benefits as well as critical influencing factors during the process should be assessed qualitatively and considered in EEs when using them as a legitimation basis vis-à-vis decision makers

Communication satisfaction and job satisfaction among critical care nurses and their impact on burnout and intention to leave : a questionnaire study

To investigate the relationship between communication and job satisfaction and their association with intention to leave and burnout among intensive care unit nurses.A multicentre questionnaire study.Intensive care nurses (n = 303) from three Flemish hospitals.Communication satisfaction assessed by the Communication Satisfaction Questionnaire, intention to leave through the Turnover Intention Scale (from the Questionnaire for the Perception and Assessment of Labour) and burnout by the Maslach Burnout Inventory. Job satisfaction was measured by a visual analogue scale.Average job satisfaction was 7.66 ± 1.34/10. Nurses were most satisfied about 'Communication with supervisor' (68.46%), and most dissatisfied about 'Organisational perspectives' (34.12%). Turnover intention was low among 49.5% (150/290) and high among 6.6% (20/290). Three percent (9/299) of intensive care nurses were at risk for burnout. All dimensions of communication satisfaction were moderately associated with job satisfaction, intention to leave and burnout.This study demonstrated high levels of communication and job satisfaction in a sample of nurses in Flanders. Intention to leave and burnout prevalence were low. To a certain extent, communication satisfaction might be associated with job satisfaction, intention to leave and burnout

Correlated positions in protein evolution and engineering

Statistical analysis of a protein multiple sequence alignment can reveal groups of positions that undergo interdependent mutations throughout evolution. At these so-called correlated positions, only certain combinations of amino acids appear to be viable for maintaining proper folding, stability, catalytic activity or specificity. Therefore, it is often speculated that they could be interesting guides for semi-rational protein engineering purposes. Because they are a fingerprint from protein evolution, their analysis may provide valuable insight into a protein's structure or function and furthermore, they may also be suitable target positions for mutagenesis. Unfortunately, little is currently known about the properties of these correlation networks and how they should be used in practice. This review summarises the recent findings, opportunities and pitfalls of the concept