Cofactor switch: Development of A Nad+-dependent cascade for the production of ursodeoxycholic acid (UDCA)

The employment of alcohol dehydrogenases in cascade reactions is often limited by the different cofactor specificity of the enzymes involved: the employment of additional cofactor regeneration systems and the excess amount of sacrificial substrates frequently increase the environmental impact and the costs of biocatalytic processes. Additionally, a NADP+-dependent process is generally less desirable, inasmuch this cofactor is more expensive, unstable and less naturally available than NAD+, leading to an increase of the process costs. Nowadays, protein engineering offers the possibility to switch the cofactor dependency of enzymes introducing few targeted mutation1. Please click Additional Files below to see the full abstract

Next-generation sequencing approach to hyperCKemia: A 2-year cohort study

Next-generation sequencing (NGS) was applied in molecularly undiagnosed asymptomatic or paucisymptomatic hyperCKemia to investigate whether this technique might allow detection of the genetic basis of the condition

Distribution of Exonic Variants in Glycogen Synthesis and Catabolism Genes in Late Onset Pompe Disease (LOPD)

Pompe disease (PD) is a monogenic autosomal recessive disorder caused by biallelic pathogenic variants of the GAA gene encoding lysosomal alpha-glucosidase; its loss causes glycogen storage in lysosomes, mainly in the muscular tissue. The genotype-phenotype correlation has been extensively discussed, and caution is recommended when interpreting the clinical significance of any mutation in a single patient. As there is no evidence that environmental factors can modulate the phenotype, the observed clinical variability in PD suggests that genetic variants other than pathogenic GAA mutations influence the mechanisms of muscle damage/repair and the overall clinical picture. Genes encoding proteins involved in glycogen synthesis and catabolism may represent excellent candidates as phenotypic modifiers of PD. The genes analyzed for glycogen synthesis included UGP2, glycogenin (GYG1-muscle, GYG2, and other tissues), glycogen synthase (GYS1-muscle and GYS2-liver), GBE1, EPM2A, NHLRC1, GSK3A, and GSK3B. The only enzyme involved in glycogen catabolism in lysosomes is alpha-glucosidase, which is encoded by GAA, while two cytoplasmic enzymes, phosphorylase (PYGB-brain, PGL-liver, and PYGM-muscle) and glycogen debranching (AGL) are needed to obtain glucose 1-phosphate or free glucose. Here, we report the potentially relevant variants in genes related to glycogen synthesis and catabolism, identified by whole exome sequencing in a group of 30 patients with late-onset Pompe disease (LOPD). In our exploratory analysis, we observed a reduced number of variants in the genes expressed in muscles versus the genes expressed in other tissues, but we did not find a single variant that strongly affected the phenotype. From our work, it also appears that the current clinical scores used in LOPD do not describe muscle impairment with enough qualitative/quantitative details to correlate it with genes that, even with a slightly reduced function due to genetic variants, impact the phenotype

Allogeneic haematopoietic stem cell transplantation for mitochondrial neurogastrointestinal encephalomyopathy

Haematopoietic stem cell transplantation has been proposed as treatment for mitochondrial neurogastrointestinal encephalomyopathy, a rare fatal autosomal recessive disease due to TYMP mutations that result in thymidine phosphorylase deficiency. We conducted a retrospective analysis of all known patients suffering from mitochondrial neurogastrointestinal encephalomyopathy who underwent allogeneic haematopoietic stem cell transplantation between 2005 and 2011. Twenty-four patients, 11 males and 13 females, median age 25 years (range 10-41 years) treated with haematopoietic stem cell transplantation from related (n = 9) or unrelated donors (n = 15) in 15 institutions worldwide were analysed for outcome and its associated factors. Overall, 9 of 24 patients (37.5%) were alive at last follow-up with a median follow-up of these surviving patients of 1430 days. Deaths were attributed to transplant in nine (including two after a second transplant due to graft failure), and to mitochondrial neurogastrointestinal encephalomyopathy in six patients. Thymidine phosphorylase activity rose from undetectable to normal levels (median 697 nmol/h/mg protein, range 262-1285) in all survivors. Seven patients (29%) who were engrafted and living more than 2 years after transplantation, showed improvement of body mass index, gastrointestinal manifestations, and peripheral neuropathy. Univariate statistical analysis demonstrated that survival was associated with two defined pre-transplant characteristics: human leukocyte antigen match (10/10 versus <10/10) and disease characteristics (liver disease, history of gastrointestinal pseudo-obstruction or both). Allogeneic haematopoietic stem cell transplantation can restore thymidine phosphorylase enzyme function in patients with mitochondrial neurogastrointestinal encephalomyopathy and improve clinical manifestations of mitochondrial neurogastrointestinal encephalomyopathy in the long term. Allogeneic haematopoietic stem cell transplantation should be considered for selected patients with an optimal donor

Development of an enzymatic tool-box for lignin oxidation/degradation. Sviluppo di un tool-box enzimatico per l\u2019ossidazione e la degradazione della lignina.

Lignin is an amorphous polymer characterized by a wide range of molecular mass components, a disordered and branched three-dimensional structure, insoluble in water and in most common solvents. In order to perform lignin degradation, enzymatic treatment could represent an environmentally friendly alternative to chemical methods. The main purpose of this PhD project was to develop an "enzymatic tool-box" for an efficient oxidation and degradation of lignin into aromatic monomers. Biochemical properties of available commercial and recombinant ligninolytic enzymes (laccase, Mn peroxidase and lignin peroxidase) were evaluated under identical experimental conditions, with the final goal to identify interesting biocatalysts for lignin degradation. The effect of pH, temperature, NaCl, DMSO and Tween-80 on the enzymatic activity has been investigated. The activity of novel enzymes, such as the membrane-bound polyphenol oxidase from the marine bacterium Marinomonas mediterranea (MmPPOA) and a peroxidase produced by Nonomuraea gerenzanensis, was also evaluated. A new high-throughput colorimetric screening to assay the oxidation/degradation of lignin by different enzymes was developed: this method facilitates the identification of optimal conditions for a lignin treatment based on the combined use of various laccases and peroxidases. On this side, coupling the colorimetric assay with a size-exclusion chromatography analysis allows to identify changes in lignin molecular mass distribution due to enzymatic treatment. Finally, based on recent literature, a chemo-enzymatic process to depolymerise lignin was carried out on two lignin linkage model compounds and on four technical lignins. This could represent an innovative and feasible way for valorisation of lignin under mild conditions

Atividade de enzimas antioxidativas e absorção de silício em plantas de pimentão submetidas a estresse salino

No Brasil, o cultivo protegido aliado a fertirrigação tem se estabelecido como alternativa econômica para o cultivo de diversas espécies hortícolas, assim cultivou-se pimentão (Capsicum annuum L.) híbrido Elisa, com o objetivo de estudar a absorção de silício e atividade de enzimas antioxidativas em plantas submetidas a estresse salino. Para a condução do experimento, foram adotados dois manejos para a aplicação de fertilizantes. O primeiro sistema considerou as recomendações para a cultura, visando manter a condutividade elétrica (C.E.) do solo em torno de 1,5 dS m-1. O segundo manejo foi estabelecido com alta concentração salina objetivando elevar a C.E. a valores três vezes superior ao tratamento controle (4,5 dS m-1). Esta concentração salina, baseada na aplicação de KCl, foi proposta para induzir estresse às plantas. Aplicou-se 1,82 t ha-1 de silicato de cálcio (23% SiO2) como fonte de silício. Observou-se que o aumento da salinidade do solo interferiu em parâmetros como produção de frutos, área foliar e composição química das plantas. Os frutos produzidos sob estas condições apresentaram-se reduzidos e deformados, independentemente da presença de silício. As plantas que receberam adição de silício no solo apresentaram maior área foliar, em ambas as condições de C.E. do solo. Plantas sob estresse apresentaram maior concentração de L-prolina nas folhas, maior atividade da enzima SOD e CAT. O silicato de cálcio contribuiu de alguma forma na eficiência das respostas antioxidativas monitoradas.The protect cultivation allied to a ferti-irrigation system has been established in Brazil as an economic alternative to a variety of horticultural species, so sweet pepper (Capsicum annuum L.) Elisa hybrid was conducted in order to study the silicon absorption and antioxidant enzymes activity in plants under salt stress. Two managements of fertilizer application were used to carry out this experiment. The first system maintained the soil electric conductivity (E.C.) near 1,5 dS m-1 as recommended for the culture. The second one, was established with high salt concentration, to increase the E.C. to triple score of the control (4,5 dS m-1). This salt concentration was proposed to induce the stress in plants. 1,82 t ha-1 of calcium silicate (23% SiO2) was applied as source of silicon. It was observed that the high salinity changed some characteristics like fruit production, leaf area and chemical composition. The fruits under this condition showed small size and deformity, with or without silicon. The plants supplemented with silicon had higher leaf area for both soil E.C. condition. Plants under stress condition showed higher concentration of prolina in leafs and higher activity of SOD and CAT. The calcium silicate has contributed at antioxidant efficiency results.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Latest development in the synthesis of ursodeoxycholic acid (UDCA): a critical review

Ursodeoxycholic acid (UDCA) is a pharmaceutical ingredient widely used in clinics. As bile acid it solubilizes cholesterol gallstones and improves the liver function in case of cholestatic diseases. UDCA can be obtained from cholic acid (CA), which is the most abundant and least expensive bile acid available. The now available chemical routes for the obtainment of UDCA yield about 30% of final product. For these syntheses several protection and deprotection steps requiring toxic and dangerous reagents have to be performed, leading to the production of a series of waste products. In many cases the cholic acid itself first needs to be prepared from its taurinated and glycilated derivatives in the bile, thus adding to the complexity and multitude of steps involved of the synthetic process. For these reasons, several studies have been performed towards the development of microbial transformations or chemoenzymatic procedures for the synthesis of UDCA starting from CA or chenodeoxycholic acid (CDCA). This promising approach led several research groups to focus their attention on the development of biotransformations with non-pathogenic, easy-to-manage microorganisms, and their enzymes. In particular, the enzymatic reactions involved are selective hydrolysis, epimerization of the hydroxy functions (by oxidation and subsequent reduction) and the specific hydroxylation and dehydroxylation of suitable positions in the steroid rings. In this minireview, we critically analyze the state of the art of the production of UDCA by several chemical, chemoenzymatic and enzymatic routes reported, highlighting the bottlenecks of each production step. Particular attention is placed on the precursors availability as well as the substrate loading in the process. Potential new routes and recent developments are discussed, in particular on the employment of flow-reactors. The latter technology allows to develop processes with shorter reaction times and lower costs for the chemical and enzymatic reactions involved

Combination of Asymmetric Organo- and Biocatalysis in Flow Processes and Comparison with their Analogous Batch Syntheses

Schober L, Tonin F, Hanefeld U, Gröger H. Combination of Asymmetric Organo- and Biocatalysis in Flow Processes and Comparison with their Analogous Batch Syntheses. European Journal of Organic Chemistry . 2022;(7): e202101035.A sequential-type as well as a tandem-type chemoenzymatic flow cascade combining an organocatalytic aldol reaction and a biocatalytic reduction to form stereoselectively a 1,3-diol with two stereogenic centers were developed. Initially, a comprehensive screening of 24 alcohol dehydrogenases was carried out and the identified candidates were applied in different multistep flow cascades. All four stereoisomers of the desired 1,3-diol product are accessible via a sequential flow approach with product formation-related conversions of up to 76% over two steps, isolated yields of up to 64% and enantiomeric excess of >99% in all cases. In addition, a tandem-type flow process, performing both reaction steps simultaneously, was established leading to 51 % conversion with >99% ee and 8:1 d.r. and representing a combination of the fields of asymmetric chemo-catalysis, biocatalysis and flow chemistry