ABSTRACT
The use of enzymes to generate hydrogen, instead of using rare metal catalysts, is an exciting area of study in modern biochemistry and biotechnology, as well as biocatalysis driven by sustainable hydrogen. Thus far, the oxygen sensitivity of the fastest hydrogen-producing/exploiting enzymes, [FeFe]hydrogenases, has hindered their practical application, thereby restricting innovations mainly to their [NiFe]-based, albeit slower, counterparts. Recent exploration of the biodiversity of clostridial hydrogen-producing enzymes has yielded the isolation of representatives from a relatively understudied group. These enzymes possess an inherent defense mechanism against oxygen-induced damage. This discovery unveils fresh opportunities for applications such as electrode interfacing, biofuel cells, immobilization, and entrapment for enhanced stability in practical uses. Furthermore, it suggests potential combinations with cascade reactions for CO2 conversion or cofactor regeneration, like NADPH, facilitating product separation in biotechnological processes. This work provides an overview of this new class of biocatalysts, incorporating unpublished protein engineering strategies to further investigate the dynamic mechanism of oxygen protection and to address crucial details remaining elusive such as still unidentified switching hot-spots and their effects. Variants with improved kcat as well as chimeric versions with promising features to attain gain-of-function variants and applications in various biotechnological processes are also presented.
ABSTRACT
An attractive application of hydrogenases, combined with the availability of cheap and renewable hydrogen (i.e., from solar and wind powered electrolysis or from recycled wastes), is the production of high-value electron-rich intermediates such as reduced nicotinamide adenine dinucleotides. Here, the capability of a very robust and oxygen-resilient [FeFe]-hydrogenase (CbA5H) from Clostridium beijerinckii SM10, previously identified in our group, combined with a reductase (BMR) from Bacillus megaterium (now reclassified as Priestia megaterium) was tested. The system shows a good stability and it was demonstrated to reach up to 28 ± 2 nmol NADPH regenerated s-1 mg of hydrogenase-1 (i.e., 1.68 ± 0.12 U mg-1, TOF: 126 ± 9 min-1) and 0.46 ± 0.04 nmol NADH regenerated s-1 mg of hydrogenase-1 (i.e., 0.028 ± 0.002 U mg-1, TOF: 2.1 ± 0.2 min-1), meaning up to 74 mg of NADPH and 1.23 mg of NADH produced per hour by a system involving 1 mg of CbA5H. The TOF is comparable with similar systems based on hydrogen as regenerating molecule for NADPH, but the system is first of its kind as for the [FeFe]-hydrogenase and the non-physiological partners used. As a proof of concept a cascade reaction involving CbA5H, BMR and a mutant BVMO from Acinetobacter radioresistens able to oxidize indole is presented. The data show how the cascade can be exploited for indigo production and multiple reaction cycles can be sustained using the regenerated NADPH.
Subject(s)
Hydrogenase , Hydrogenase/chemistry , NAD , Hydrogen/chemistry , NADP , OxidoreductasesABSTRACT
INTRODUCTION: Frailty used as predictive tool is still not carried out in daily practice, although many studies confirm the great clinical importance of the frailty syndrome in surgical outcomes. There is no standardized method of measuring the physiological reserves of older surgical patients. The aim of this study was to analyze a cohort of older urological patients according to various frailty indices, in order to evaluate whether they are predictors of post-operative complications after urological procedures. MATERIAL AND METHODS: This is a prospective observational study on 78 consecutive older (≥70 years) patients, subjected to major urological (both endoscopic and 'open surgical') procedures. Frailty was defined according to the Edmonton Frail Scale. Several risk models and biochemical parameters were evaluated. Post-operative outcomes were surgical and medical complications, mortality and rehospitalisation within 3 months. RESULTS: An overall prevalence of frailty of 21.8% was found. Patients with complications were frailer than those without complications (univariate analysis), considering both total patients (p = 0.002) and endoscopic (p = 0.04) and 'open surgical' patients (p = 0.013). However, in multivariate analysis, a significant correlation was not found between all frailty indices tested and the risk of major complications. Limitation of the study: the small sample size (lack of statistical power), although this is a prospective study focused on older urological patients. CONCLUSIONS: New urology-tailored pre-operative assessment tools may prove beneficial when calculating the risks/benefits of urological procedures, so that objective data can guide surgical decision- making and patient counselling. Further large clinical studies specifically focusing on elderly in urology will be needed.