Search

Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Special Research Fund (Bijzonder Onderzoeksfonds, BOF) Hasselt University. This study is part of Limburg Clinical Research Center, supported by the foundation Limburg Sterk Merk, province of Limburg, Flemish government, Hasselt University, Ziekenhuis Oost-Limburg and Jessa Hospital. Background Atrial fibrillation (AF) is a major cause of ischaemic stroke. Oral anticoagulation is recommended in stroke survivors with AF to prevent recurrence. Prolonged ECG monitoring using insertable cardiac monitors (ICMs) has been shown to increase the detection rate of AF compared to standard 24-hour ECG monitoring in cryptogenic stroke patients. However, prolonged ambulatory ECG monitoring is underutilized, likely contributing to an underdiagnosis of AF and missed anticoagulation treatment opportunities for secondary stroke prevention. Purpose This study aims to evaluate how our evolving cardiac monitoring strategy affected AF detection rates one year after cryptogenic ischaemic stroke or transient ischaemic attack (TIA) in a tertiary care centre. Methods We retrospectively identified all consecutive cryptogenic stroke or TIA patients admitted to our centre between 1/01/2017 – 1/01/2022. Patients with a pacemaker or implantable cardioverter-defibrillator were excluded from the analysis. Data were collected from the electronic medical record. Available cardiac monitoring modalities included 24-hour Holter monitoring, 7-day Holter monitoring, and insertable cardiac monitors (ICM). After October 2020 (i.e., period 2), the latter became part of our routine diagnostic workup for AF detection in case extended (7-day) Holter monitoring was negative. Results All 691 cryptogenic stroke or TIA patients admitted to our hospital during the inclusion period were considered. These were elderly patients (69.7 ± 13.2 years) with a CHA2DS2-VASc score of 3 [2 - 5]. Figure 1 shows evolving trends in the use of the different cardiac monitoring tools before and after October 2020. In particular, the use of 7-day Holter monitoring and long-term continuous monitoring with ICM increased after implementing our new diagnostic protocol compared to 24-hour Holter monitoring (p Conclusions The increased use of prolonged cardiac monitoring in cryptogenic stroke or TIA patients resulted in a two-fold increase in AF detection one year after stroke. Therefore, our results underscore the need to implement guideline-recommended prolonged rhythm monitoring using ICMs in addition to short-term ECG monitoring to ensure adequate secondary stroke prevention.

Funding Acknowledgements Type of funding sources: None. Background Heart failure with preserved ejection fraction (HFpEF) frequently coexists with atrial fibrillation (AF). The gold-standard test for HFpEF diagnosis remains invasive assessment of left ventricular end-diastolic pressures but noninvasive diagnostic scoring systems have been developed to aid accurate diagnosis. However, the diagnosis of HFpEF in patients with AF is more difficult because some diagnostic criteria for HFpEF may be associated with AF instead of HFpEF per se. Purpose To compare the guidelines and scoring systems for HFpEF diagnosis in patients with AF, and to indicate the most relevant clinical and echocardiographic parameters supporting the diagnosis of HFpEF in an AF cohort. Methods Consecutive patients with paroxysmal or persistent AF scheduled for catheter ablation were included. Clinical characteristics and transthoracic echocardiography examination was collected at baseline visit. The European Society of Cardiology (ESC) guidelines from 2016 and 2021 as well as scoring systems (HFA-PEFF and H2FPEF scores) were used to diagnose HFpEF. In the H2FPEF score, patients with AF alone, score of 3, were assessed as group with the low risk for HFpEF (instead of intermediate risk according to the H2FPEF score), as all enrolled patients had AF. Multiple logistic regression was performed to find optimal parameters predicting HFpEF, that was defined as diagnosis of HFpEF according to all following scores: ESC guidelines from 2016 and 2021 and high risk in HF2FPEF and HF-PEFF scores. Results We analysed 325 patients (median age 65 [59-72], 36% women). Considerable differences were observed in HFpEF diagnoses when stratifying patients according to ESC 2016 and ESC 2021 guideline definitions; the percentage of HFpEF patients reduced from 55% (n=180) to 28% (n=90). The agreement of HF2PEF score relative to HFA-PEFF score was 46% (n=25/55), 54% (n=115/213) and 35% (n=20/57) of patients regarding low, intermediate and high risk for HFpEF, respectively. Overall, 5.8% of patients (n=19) had HFpEF (Figure 1). These 19 patients were older (median age 70 vs 65, p Conclusions The differences in HFpEF definition in the guidelines and various scoring systems lead to considerable difference in stratification of HFpEF in AF patients. In our population, just 5.8% of AF patients were diagnosed with HFpEF regarding ESC guidelines and high risk for HFpEF in scoring systems.

Funding Acknowledgements Type of funding sources: None. Introduction Chronic obstructive pulmonary disease (COPD) is a well-recognized risk factor for atrial fibrillation (AF) and negatively impacts symptom burden and AF rhythm control treatment. Nevertheless, there are currently no guideline recommendations or practical guides about whether or not and how to screen for COPD. Purpose To describe the implementation of an integrated screening and management pathway for COPD in patients with diagnosed AF, into the existing pre-ablation work-up embedded in an AF outpatient clinic infrastructure. Methods Unselected consecutive AF patients scheduled for catheter ablation were prospectively screened for airflow limitation using handheld (micro)spirometry devices. Patients with a forced expiratory volume (FEV) in one second (FEV1)/FEV in six seconds ratio of ≤0.73 or a FEV1/forced vital capacity ratio of Results In total, 232 patients (median age 66 [59-71], 31.9% female, 33.6% self-reported dyspnoea) were included in this screening and management pathway. Screening (micro)spirometry yielded interpretable results in 88.8% of patients. Airflow limitation was observed in 47 patients (20.3%) of whom 29 (12.5%) opted for referral to the pulmonologist and 17 (7.3%) received a final diagnosis of chronic respiratory disease, either COPD or asthma (11 newly diagnosed). The majority of these 17 patients (70.6%) scored high on the CAT (score ≥10), whereas 14 (82.4%) had a moderate-to-highly increased risk of COPD based on the RHSQ (score ≥16.5). Conclusions A COPD care pathway can successfully be embedded in an existing AF patient clinic infrastructure, using (micro)spirometry and remote analysis of results. Although all patients chose to perform handheld (micro)spirometry, only 60% of those in whom referral was advised opted for referral. Preselection of patients based on respiratory symptoms as well as increasing patient involvement might increase patient acceptance for respiratory analysis, and requires further research.

Funding Acknowledgements Type of funding sources: None. Background Catheter ablation (CA) has been established as an effective therapeutic option to achieve rhythm control in patients with atrial fibrillation (AF). However, AF recurrence occurs in approximately 45% of the patients undergoing CA. The association between AF recurrence after CA and left atrial (LA) strain, which is a surrogate marker of LA mechanical dysfunction already appearing before the enlargement of the LA, remains less clear. Aim The purpose of this study was to evaluate the association between LA function assessed by strain echocardiography and AF recurrence after CA. Methods This sub-study of ISOLATION (NCT04342312), involved consecutive patients with paroxysmal or persistent AF scheduled for first cryoballoon or radiofrequency CA. Clinical characteristics and echocardiography examination were collected at baseline visit before CA. Patients were followed for 12 months. Follow-up consisted of outpatient visits and heart rhythm monitoring (48 day Holter, 12-lead electrocardiogram, 7 day photoplethysmography-based rhythm app) at 3, 6, and 12 months. Results We analyzed 312 patients (median age 65, 36% women). More than half (52%; n=162) underwent radiofrequency CA and 84 (27 %) had AF recurrence within one year follow-up. No statistically significant differences in baseline characteristic were observed between patients with and without recurrent AF, expect higher prevalence of persistent AF prior to CA in patients with recurrence of AF (75% vs. 46%, p Conclusions LA function assessed by LA strain is associated with AF recurrence after CA, even in patients with non-dilated LA. Whether integration of results derived from systematic preprocedural LA strain analysis into patients’ selection strategies may help to identify patients profiting from CA required further study.

Funding Acknowledgements Type of funding sources: None. Background The diagnostic work-up for atrial fibrillation (AF) catheter ablation is often complex and expensive. Improving the organization of this work-up may help to streamline patient journeys and to reduce administrative burden and costs. Additionally, reorganizing the existing work-up provides the opportunity to integrate (translational) research into routine clinical care, hereby contributing to improved patient selection and improved care for future patients. Purpose The aim of this project was to optimize a care pathway for patients considered for AF ablation, with the goals to improve the patient journey and simultaneously integrate research into the clinical process. Methods The Lean Six Sigma approach was used to map the pre-existing process, identify constraints in the process and formulate countermeasures. The impact of this pathway optimization was evaluated using four outcome measures: (1) efficient use of medical resources, defined as the percentage of patients receiving the pre-ablation work-up and eventually undergoing AF ablation, (2) number of hospital visits and consultations with a cardiologist, (3) pathway compliance, defined as the percentage of patients that completed the work-up with a maximum of 2 outpatient hospital visits, a maximum of 1 cardiologist consultation, laboratory results available, and a complete echocardiogram available, and (4) completeness of scientific data, defined as the availability of a predefined set of relevant variables (clinical indicators, medication, laboratory results and echocardiography derived data). Results Five root causes for constraints in the pre-existing work-up were identified and corresponding countermeasures were formulated (Figure 1). The impact of the countermeasures was studied for 33 patients before and 26 patients after pathway optimization. After optimization, efficient use of resources increased from 44% to 95% (p Conclusions This optimization project resulted in a more efficient care pathway for patients considered for AF ablation. The structural integration of research into the care pathway may lay the foundations for further improvements of AF care in the future.

Funding Acknowledgements Type of funding sources: None. Background Untreated obstructive sleep apnoea (OSA) contributes to progression of atrial fibrillation (AF) and reduces the success rate of heart rhythm control strategies. OSA remains one of the most frequently underdiagnosed modifiable risk factors in AF patients due to a lack of standardized screening methods and low awareness. Purpose To assess the impact of implementation of a structured remote OSA screening and management pathway on the prevalence of OSA in AF patients scheduled for AF ablation procedures. Methods In October 2020, a novel remote OSA screening and management pathway (VIRTUAL-SAFARI) was introduced in two AF outpatient clinics in the Netherlands. Consecutive patients scheduled for AF ablation were offered OSA screening consisting of sending a portable home sleep test to patients’ homes to perform a remote sleep recording for one night, analysis by a sleep physician, discussion of results with the patient, and initiation of treatment (when applicable). The impact of this structural screening strategy was assessed by comparing the prevalence of concomitant OSA (defined as apnoea-hypopnoea index ≥5) for patients scheduled for AF ablation in the year before and after introduction of the OSA management pathway. Results A total of 733 patients was studied, 308 in the year before (Oct ’19 - Sep ’20) and 425 in the year after (Oct ’20 - Sep ’21) introduction of the VIRTUAL-SAFARI pathway. Median age was 65 [58-71], 64% was male and median body mass index (BMI) was 27 [25-30] kg/m2. Baseline characteristics were comparable for the groups before and after introduction of the pathway (Table 1). In the cohort before pathway introduction, OSA had been diagnosed in 26 patients (8%, Figure 1) and was treated with positive airway pressure in 10 cases (3%). In the cohort after pathway introduction, OSA had previously been diagnosed in 53 patients (12%). Eighty-eight percent of patients without previous OSA screening was referred via the remote pathway. Results of the sleep recordings were available for 213 (59%) at the time of this analysis. Previously unknown OSA was diagnosed in 184 patients (86% of available recordings), increasing the prevalence of confirmed OSA to 237 (55%). For 22% of patients, results of sleep recordings are pending. Absence of OSA was confirmed in 9%, and 13% of patients had not been screened (e.g. because of patient preference or logistical reasons). After pathway introduction, 82 patients (19%) were treated or received advice to start treatment with positive airway pressure. Treatment decisions are pending for 9% of the cohort. Conclusion After the implementation of structured remote OSA screening in a well characterized cohort of consecutive patients scheduled for AF ablation, the prevalence of diagnosed OSA increased from 8% to 55%. Whether appropriate risk factor management, including treatment of OSA identified by structured screening, will improve AF outcomes needs to be tested in future studies.

Funding Acknowledgements Type of funding sources: None. OnBehalf TeleCheck-AF Investigators Aims TeleCheck-AF is a multicentre international project initiated to maintain care delivery for patients with atrial fibrillation (AF) during COVID-19 through teleconsultations supported by an on-demand photoplethysmography-based heart rate and rhythm monitoring app (FibriCheck®). We describe the characteristics and experiences from recruited patients. Methods Self-reported patient characteristics were obtained from the app. A survey exploring patient experiences completed by 826 patients. Results Within 28 weeks, 1930 AF patients were recruited by 38 centers in 14 countries, mainly for remote AF control (31% of patients) and AF ablation follow-up (42%). One-third of patients was in the age range 60-69 years. The most common comorbidity was hypertension (42% of all patients). More than 70% of patients were treated with oral anticoagulation. In total 59.858 heart rate and rhythm measurements were recorded. During the one-week FibriCheck® use, patient adherence was high. Patients performed between 2 and 5 recordings a day and the median number of measurements per patient was 21 [15-29]. The highest average number of measurements per patient was seen in patients older than 80years of age. Patients agreed that the FibriCheck® app was easy to use (94%) and easy to install (89%). The app gave patients a safe feeling (74%) due to being in constant heart rate and rhythm control. More than half of the patients (58%) agreed or strongly agreed that they would like to use the FibriCheck® app in the future. They also found the automated reminders useful (64%). Conclusions In the TeleCheck-AF project, mHealth adherence was high, particularly in older patients ≥80 years. The app FibriCheck® is easy to use for 7 days before a scheduled teleconsultation. Patients think that remote rate and rhythm monitoring around teleconsultation by the TeleCheck-AF approach may be an alternative to traditional face-to-face consultations in the future. Abstract Figure.

Funding Acknowledgements Type of funding sources: None. OnBehalf TeleCheck-AF Investigators Aims Herein we describe the characteristics, inclusion rates and experiences from participating centres in the European TeleCheck-AF project. TeleCheck-AF is a multicentre international project initiated to maintain care delivery for patients with atrial fibrillation (AF) during COVID-19 through teleconsultations supported by an on-demand photoplethysmography-based heart rate and rhythm monitoring app (FibriCheck®). Methods Two surveys exploring centre characteristics (n = 25) and centre experiences (n = 23) were completed. Results Most centres were academic (64%) and specialized public cardiology/district hospitals (36%). Majority of centres had AF outpatient clinics (64%) and only 36% had AF ablation clinics. The time required to start patient inclusion and total number of included patients in the project was comparable for centres experienced (56%) or inexperienced in mHealth use. Within 28 weeks, 1930 AF patients were recruited, mainly for remote AF control (31% of patients) and AF ablation follow-up (42%). Average inclusion rate was highest during the lockdown restrictions and reached a steady state at a lower level after easing the restrictions (188 vs 52 weekly recruited patients). Majority (>80%) of the centres reported no problems during the implementation of the TeleCheck-AF approach. Centres agreed that the on-boarding process of their center in the TeleCheck-AF project was simple and access to the patients measurements via stand-alone cloud infrastructure was trouble-free and possible from the first day on. They also agreed that remote heart rate and rhythm assessment by the FibriCheck® app around teleconsulatation supported their medical decision making; that their patients responded positively to use FibriCheck® for seven days; and that they felt comfortable to interpret PPG recordings. Conclusions Despite different health care settings and mHealth experiences, the TeleCheck-AF approach could be set up within an extremely short time and easily used in different European centres during COVID-19. Abstract Figure.

Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Limburg Clinical Research Center OnBehalf Mobile Health Unit & Future Health Background Cryptogenic stroke (CS) and transient ischemic attack (TIA) patients have no determined aetiology at discharge. A possible cause for stroke is atrial fibrillation (AF). AF occurs in 20%-40% of the CS patients and diagnosis is highly dependent on monitoring duration. A long-term monitoring method is the insertable loop recorder (ILR), recommended by the European Society of Cardiology. However, this is not routinely used in Belgium despite reimbursement. Purpose This study aims to determine the AF detection rates of different methods used in clinical practice, ranging from short-term monitoring (monitoring in a stroke unit, 12-lead electrocardiogram (ECG), and 24-hour ECG), seven-day Holter, and long-term monitoring (smartphone application and ILR). Methods A monocentric, retrospective study was conducted in adults with CS or TIA between 1/01/17 - 1/01/20. Data were collected from the electronic medical record. The primary endpoint was the detection rate and time until first AF detection. Results A total of 368 patients suffered from a CS or TIA. Most of them were monitored in the stroke unit (96%) or with a 12-lead ECG (93%). A 24-hour ECG was used in 26%, a seven-day Holter in 38%. For long-term monitoring, a smartphone application was used in 3%. ILRs were inserted in 6%, with a median time of 102 days after stroke (IQR: 48-321). One year after ILR insertion, AF was detected in 23%. AF detection increased with monitoring duration, as shown in the figure, except for 24-hour ECG, which detected no AF. Therefore, the AF detection rate was different between short-term monitoring (5%) and seven-day Holter (10%; p=.034), and short- and long-term monitoring (16%; p=.01). The age of CS patients without AF (Mdn = 71yr) was lower than those with AF (Mdn = 79yr; p=.001). The National Institutes of Health Stroke Scale (NIHSS) and the CHA2DS2-VASc score of patients without AF (Mdn = 3) was lower than those with AF (Mdn = 6, p Conclusions Detection of AF was associated with higher age, NIHSS, and CHA2DS2-VASc scores. These variables can be used to select patients for the insertion of ILRs. The detection rate of AF was significantly higher with long-term monitoring and seven-day Holter compared to short-term monitoring. However, only 38% of the patients were monitored for a week, and only 6% had an ILR inserted. Therefore, despite guideline recommendations, long-term cardiac monitoring is underutilised in this population of CS patients. Nevertheless, AF was still detected in 14% of CS patients within one year after the stroke. These findings emphasise the need for more monitoring with a seven-day Holter, smartphone app, and ILR in this patient population. Consequently, this will result in more accurate treatment of AF as secondary prevention of CS. Abstract Figure. Time to first AF detection after stroke

Background: The yeast Komagataella phaffii, better known as Pichia pastoris, is a commonly used host for recombinant protein production. Here expression vectors are reported that address the different steps of the transcription–translation–secretion pathway of heterologous protein production.Results: Transcription and translation enhancing elements were introduced in an expression cassette for the production of recombinant Aspergillus niger feruloyl esterase A. The yield was increased by threefold as compared to the yield without these elements. Multiple copy strains were selected using a zeocin resistance marker in the expression cassette and showed another sixfold higher yield. Modification of the C-terminal amino acid sequence of the secretion signal did not significantly improve the production yield. Similar data were obtained for the production of another protein, recombinant human interleukin 8. Upscaling to fed-batch fermentation conditions resulted in a twofold increase for reference strains, while for strains with enhancing elements a tenfold improvement was observed.Conclusions: Pichia pastoris is used for recombinant protein production in industrial fermentations. By addressing the transcription and translation of mRNA coding for recombinant protein, significant yield improvement was obtained. The yield improvement obtained under microscale conditions was maintained under fed-batch fermentation conditions. These data demonstrate the potential of these expression vectors for large scale application as improved production of proteins has major implications on the economics and sustainability of biocatalyst dependent production processes e.g. for the production of pharmaceuticals and for the bioconversions of complex molecules.

A detailed analysis of the conversion of polar and apolar substrates in an heterogeneous medium is given. The enzyme is assumed to follow the mechanism: E + S⇌ES⇌E + P. The effects of various parameters, both enzymic and those of the microemulsion, are presented. In an extension to the model presented earlier, analysis of this simplified case reveals why a large difference is observed between the Km in aqueous solution and its value in microemulsions, suggesting increased affinity of the enzyme for a hydrophilic substrate and decreased affinity for an apolar substrate. It also demonstrates why, in contrast to studies in aqueous solutions, the rate constant of the reverse reaction (k-2) affects the initial rate of the reaction in microemulsions. Furthermore, it is explained that the bell-shaped relationship between the initial reaction rate and the water content of the microemulsion can depend on the value of the rate constant of the reverse reaction and on the relationship between the radius of the micelle and the molar ratio of detergent to water (w0) in the system. The model presented here predicts that bell-shaped curves will only be observed in the conversion of substrates which are, at lea partially, confined to the water pool of the microemulsion. In order to illustrate the dependence of the rate of the reaction on the composition of the microemulsion, the example of δ-chymotrypsin was chosen. Using this example, the effects of micellar parameters not accessible to systematic experimental variation can be calculated. The results of this analysis show that detailed knowledge of both microemulsion and enzymic parameters is necessary for an adequate description of the reaction rate. Based on the results of this analysis, advice to optimize the enzymatic conversion of both hydrophilic and hydrophobic substrates in heterogenic media is presented.

Phage display can be used as a protein engineering tool to select proteins with desirable binding properties from a library of randomly constructed mutants. Here, we describe the development of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has marked properties for the preparation of pharmaceutically relevant chiral compounds. The lipase gene was cloned upstream of the phage g3p encoding sequence and downstream of a modified g3p signal sequence. Consequently, the enzyme was displayed at the surface of bacteriophage fd as a fusion to its minor coat protein g3p. The phage-bound lipase was correctly folded and fully enzymatically active as determined from the hydrolysis of p-nitrophenylcaprylate with K(m)-values of 0.38 and 0.33 mM for the phage displayed and soluble lipase, respectively. Both soluble lipase and lipase expressed on bacteriophages reacted covalently with a phosphonate suicide inhibitor. The phage does not hamper lipase binding, since both soluble and phage-bound lipase have a similar half-life of inactivation of approximately 5 min. Therefore, we conclude that the Bacillus lipase can be functionally expressed on bacteriophages as a fusion to the phage coat protein g3p. The specific interaction with the suicide inhibitor offers a fast and reproducible method for the future selection of mutant enzymes with an enantioselectivity towards new substrates.

Semi-synthetic cephalosporin antibiotics belong to the top 10 of most sold drugs, and are produced from 7-aminodesacetoxycephalosporanic acid (7-ADCA). Recently new routes have been developed which allow for the production of adipyl-7-ADCA by a novel fermentation process. To complete the biosynthesis of 7-ADCA a highly active adipyl acylase is needed for deacylation of the adipyl derivative. Such an adipyl acylase can be generated from known glutaryl acylases. The glutaryl acylase of Pseudomonas SY-77 was mutated in a first round by exploration mutagenesis. For selection the mutants were grown on an adipyl substrate. The residues that are important to the adipyl acylase activity were identified, and in a second round saturation mutagenesis of this selected stretch of residues yielded variants with a threefold increased catalytic efficiency. The effect of the mutations could be rationalized on hindsight by the 3D structure of the acylase. In conclusion, the substrate specificity of a dicarboxylic acid acylase was shifted towards adipyl-7-ADCA by a two-step directed evolution strategy. Although derivatives of the substrate were used for selection, mutants retained activity on the beta-lactam substrate. The strategy herein described may be generally applicable to all beta-lactam acylases.

The large heterodimeric penicillin G acylase from Alcaligenes faecalis was displayed on the surface of phage fd. We fused the coding sequence (α subunit-internal peptide-β subunit) to the gene of a phage coat protein. A modified g3p signal sequence was used to direct the polypeptide to the periplasm. Here we show that a heterodimeric enzyme can be expressed as a fusion protein that matures to an active biocatalyst connected to the coat protein of phage fd, resulting in a phage to which the β-subunit is covalently linked and the α-subunit is non-covalently attached. The enzyme can be displayed either fused to the minor coat protein g3p or fused to the major coat protein g8p. In both cases the penicillin G acylase on the phage has the same Michaelis constant as its freely soluble counterpart, indicating a proper folding and catalytic activity of the displayed enzyme. The display of the heterodimer on phage not only allows its further use in protein engineering but also offers the possibility of applying this technology for the excretion of the enzyme into the extracellular medium, facilitating purification of the protein. With the example of penicillin acylase the upper limit for a protein to become functionally displayed by phage fd has been further explored. Polyvalent display was not observed despite the use of genetic constructs designed for this aim. These results are discussed in relation to the pore size being formed by the g4p multimer.

Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge.

In filamentous fungi, most of the strategies used for the improvement of protein yields have been based on an increase in the transcript levels of a target gene. Strategies focusing at the translational level have been also described, but are far less explored. Here the 5' untranslated sequence of the glaA mRNA, a widely used expression system for the expression of recombinant proteins, was modified by the introduction of different nucleotide elements that have positive role in the translation process. Five Aspergillus niger laccase-like multicopper oxidases (MCOs) coding genes were fused to the native glaA 5'UTR and the three synthetic versions (sUTR1, sUTR2, and sUTR3) as well, and placed under the control of the glucoamylase gene promoter. Afterwards, a total of 20 fungal transformations were done using A. niger N593 as a recipient strain and 50 transformants per transformation were isolated and analyzed. The result of the incorporation of the synthetic 5'UTRs on the overall productivity of the transformants was assessed, on one hand by monitoring the laccase activity of all the isolated transformants, and on the other hand by quantifying and comparing the activity of those secreting the highest level of each MCO. For this purpose, a high-throughput method for the screening and selection of the best producers was developed. Once the best transformants producing the highest yield of McoA, McoB, McoC, McoD, and McoJ laccases were selected, their production level was quantified in supernatants of liquid cultures. The results obtained in this work indicate that modifications in the native glaA 5'UTR can lead to improvements in protein yields. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc

Semi-synthetic cephalosporin antibiotics belong to the top 10 of most sold drugs, and are produced from 7-aminodesacetoxycephalosporanic acid (7-ADCA). Recently new routes have been developed which allow for the production of adipyl-7-ADCA by a novel fermentation process. To complete the biosynthesis of 7-ADCA a highly active adipyl acylase is needed for deacylation of the adipyl derivative. Such an adipyl acylase can be generated from known glutaryl acylases. The glutaryl acylase of Pseudomonas SY-77 was mutated in a first round by exploration mutagenesis. For selection the mutants were grown on an adipyl substrate. The residues that are important to the adipyl acylase activity were identified, and in a second round saturation mutagenesis of this selected stretch of residues yielded variants with a threefold increased catalytic efficiency. The effect of the mutations could be rationalized on hindsight by the 3D structure of the acylase. In conclusion, the substrate specificity of a dicarboxylic acid acylase was shifted towards adipyl-7-ADCA by a two-step directed evolution strategy. Although derivatives of the substrate were used for selection, mutants retained activity on the beta-lactam substrate. The strategy herein described may be generally applicable to all beta-lactam acylases.

The potential of the RNA phage MS2 to accommodate extra amino acids in its major coat protein has been examined. Accordingly, a pentapeptide was encoded in the genome as an N-terminal extension. In the MS2 crystal structure, this part of the coat protein forms a loop that extends from the outer surface of the icosahedral virion. At the RNA level, the insert forms a large loop at the top of an existing hairpin. This study shows that it is possible to maintain inserts in the coat protein of live phages. However, not all inserts were genetically stable. Some suffer deletions, while others underwent adaptation by base substitutions. Whether or not an insert is stable appears to be determined by the choice of the nucleic acid sequence used to encode the extra peptide. This effect was not caused by differential translation, because coat-protein synthesis was equal in wild-type and mutants. We conclude that the stability of the insert depends on the structure of the large RNA hairpin loop, as demonstrated by the fact that a single substitution can convert an unstable loop into a stable one.

A structurally unusual member of the adipokinetic hormone/red pigment-concentrating hormone peptide family was isolated from corpora cardiaca of the painted lady butterfly, Vanessa cardui. Its primary structure was assigned by Edman degradation and nano-electrospray-time-of-flight mass spectrometry as pQLTFTSSWGGK (Vac-AKH). Vac-AKH represents the first 11mer and the first nonamidated peptide in this family. The peptide shows significant adipokinetic activity in adult specimens of V. cardui. Injection of 10 pmol of synthetic Vac-AKH into 4-day-old decapitated males resulted in an approximately 150% increase of hemolymph lipids after 90 min. Half maximal adipokinetic activity was achieved with about 0. 1 pmol of Vac-AKH. During a 2-h incubation of corpora cardiaca/corpora allata complexes in medium containing 50 mM KCl, significant amounts of Vac-AKH were released from the glands.

Photoinduced charge separation was studied in a variety of microemulsions. The photosystem consisted of a porphyrin equipped with a hexadecyl chain, methylviologen as the electron acceptor and tributylamine as an irreversible electron donor. The cetyltrimethylammonium bromide (CTAB) based microemulsions were varied with respect to the alkyl chain of the cosurfactant (1- n -alkanol) and with respect to the concentration of the alkanol. The number of methylviologen molecules reduced per photon absorbed (yield) increased upon increasing the alkanol concentration or upon decreasing the chain lenght of the alkanol. In accordance with the literature a relationship between the yield and log P of the organic phase could be established. In addition, also a relationship between the yield and the molar concentration of the alkanol in the medium could be observed. Inspection of the conductivity of the microemulsion indicates that the increase in yield can also be caused by a change from a reversed micellar to a bicontinuous microstructure of the medium. The yield using this prophyrin was compared to that applying a similar porphyrin lacking the hexadecyl chain. No significant difference in performance could be observed despite the fact that the environment of the tailless porphyrin depends on the alkanol used as the cosurfactant whereas the hexadecyl chain modified porphyrin is insensitive to changes of the cosurfactant structure. These data suggest that the (minor) change in porphyrin location does not contribute to the overall efficiency of the reactions involved in the charge separation process.

The kinetic parameters of 20 beta-hydroxysteroid dehydrogenase were determined in aqueous solutions and in reversed micellar media composed with either an anionic, a cationic or a nonionic surfactant, at low and at high ionic strength. The velocity data were analysed in two ways: first by extrapolation to infinite concentrations of both substrates to determine 'apparent' Michaelis constants and V values, and secondly by comparison to reaction rates calculated using the model presented (see first of this series of papers in this issue of the journal). Data analysis according to the first method reveals some differences in the kinetic parameters in reversed micelles as compared to those in aqueous solution, though the kinetic parameters of the enzyme seem not to be much affected by enclosure in reversed micelles. It is shown that the changes that do occur are not caused by a shift of the intramicellar pH or by electrostatic interactions between the enzyme and the surfactant head groups. Interpretation of the data using the second method assumes that the enzyme is not affected by the enclosure in reversed micelles, and that deviations with respect to the aqueous parameters are caused by exchange phenomena between distinct aqueous droplets in the organic phase and by a high effective intramicellar substrate concentration. This model is able to predict reaction rates that agree rather well with experimentally determined rates and explains why the enzyme mechanism in reversed micelles is, at all progesterone concentrations used, the same as observed at high progesterone concentrations in aqueous solution. Furthermore it clarifies the occurrence of substrate inhibition in sodium-di(ethylhexyl)sulphosuccinate-reversed micelles and the observed low activity in Triton-reversed micelles, as arising from the high partition coefficient of progesterone and the slow rate of diffusion of progesterone into the reversed micelles. From these results, and those reported for enoate reductase (see preceding paper in this issue of the journal) it can be concluded that the theory presented before (see first of this series of papers in this issue of the journal) offers a good explanation for the observed kinetic behaviour in reversed micelles, and emphasizes the importance of exchange processes between micelles.

Enoate reductase (EC 1.3.1.31) can stereospecificially reduce a variety of α,β-unsaturated carboxylates. Its use was extended to apolar media by incorporating the enzyme into a reversed micellar medium. The kinetics of the enzyme in such a medium have been investigated using 2-methylbutenoic acid as substrate and NADH as a cofactor and compared with the reaction rates in aqueous solution. In aqueous solution the enzyme obeys a ping pong mechanism [Buhler et al. (1982) Hoppe-Seyler's Z. Physiol. Chem 363, 609–625]. In 50 mM Hepes pH = 7.0 with ionic strength of 0.05 M the Michaelis constants for NADH and 2-methylbutenoic acid are 20 μM and 6.0 mM respectively. In reversed micelles the kinetics of the reaction (Michaelis constant, maximum velocity as well as inhibitory effects) were markedly different. The rate of the enzymatic reaction of enoate reductase was studied using various concentrations of 2-methylbutenoic acid and various NADH concentrations. In reversed micelles composed of the anionic detergent sodium di(ethylhexyl)sulphosuccinate, the enzymatic reaction deviates substantially from the values in aqueous solution. Using our model (see preceding paper in this issue of the journal), all kinetics could be explained as evolving from enclosure in reversed micelles without any change in the intrinsic rate parameters of the enzyme. So the enzyme itself is unaffected by incorporation in reversed micelles, but the rate of intermicellar exchange as well as the microheterogeneity of the medium, resulting in very high local concentrations of the substrate, are the most important factors altering the reaction pattern. The effect of the composition of the reversed micellar medium was also investigated using either a nonionic or a cationic surfactant. In these solutions too, exchange and microheterogeneity of the medium proved to be the most important parameters influencing the enzymatic reaction. In all reversed micellar solutions inhibition by the enoate was observed at an overall concentration of 0.5–5 mM, implying that a concentration of substrate equal to the Km value in aqueous solution may already cause inhibition in reversed micelles. At this level no inhibition by NADH was observed. The microheterogeneity of the medium also explains this inhibition of the enzyme at relatively low 2-methylbutenoic acid concentrations.

In the literature measurements of kinetic data of enzymes in reversed micelles have been interpreted in two ways. In the first, all enzyme parameters are expressed with respect to the total volume of the reversed micellar solution. In the second, the enzymatic conversion is related only to the fraction of the volume consisting of aqueous solution (pseudophase model). In this paper equations are derived describing the rate of an enzymatic reaction for three different kinds of enzymes: enzymes obeying Michaelis-Menten kinetics, enzymes following a ping-pong bi-bi mechanism and enzymes which convert substrates according to an ordered mechanism. In deriving these equations, a distinction is made between intermicellar exchange reactions of substrate(s) and product(s) and the enzymatic reaction which takes place in the waterpool of a reversed micelle. In the description, all intrinsic rate constants of the enzyme are assumed to be independent of its environment. The rate equations show that the presence and efficiency of the intermicellar exchange reaction, which supplies the enzyme with substrate and removes product, can affect the rate of an enzymatic reaction under common experimental conditions. Whereas kinetic parameters derived from double-reciprocal plots often seem to be affected by enclosure in reversed micelles, these apparent deviations from kinetics in aqueous media can be explained by the model presented here as arising from exchange phenomena. Neither the experimentally determined maximum enzyme velocity, vmax, nor the Michaelis constants are affected by the incorporation of the enzyme in reversed micelles. The deviations of kinetic parameters from the aqueous values are shown to depend strongly on the concentration of reversed micelles, the intermicellar exchange rate and the volume fraction of water, a dependence in agreement with findings reported in the literature.

Directed evolution of secreted industrial enzymes is hampered by the lack of powerful selection techniques. We have explored surface display to select for enzyme variants with improved binding performance on complex polymeric substrates. By a combination of saturation mutagenesis and phage display we selected alpha-amylase variants, which have the ability to bind starch substrate at industrially preferred low pH conditions. First we displayed active alpha-amylase on the surface of phage fd. Secondly we developed a selection system that is based on the ability of alpha-amylase displaying phages to bind to cross-linked starch. This system was used to probe the involvement of specific beta-strands in substrate interaction. Finally, a saturated library of alpha-amylase mutants with one or more amino acid residues changed in their Cbeta4 starch-binding domain was subjected to phage display selection. Mutant molecules with good starch-binding and hydrolytic capacity could be isolated from the phage library by repeated binding and elution of phage particles at lowered pH value. Apart from the wild type alpha-amylase a specific subset of variants, with only changes in three out of the seven possible positions, was selected. All selected variants could hydrolyse starch and heptamaltose at low pH. Interestingly, variants were found with a starch hydrolysis ratio at pH 4.5/7.5 that is improved relative to the wild type a-amylase. These data demonstrate that useful a-amylase mutants can be selected via surface display on the basis of their binding properties to starch at lowered pH values. (C) 2002 Elsevier Science B.V. All rights reserved.