Taking advantage of the catalytic promiscuity of L-carbamoylase from Geobacillus stearothermophilus CECT43 (BsLcar) and N-succinyl-amino acid racemase from Geobacillus kaustophilus CECT4264 (GkNSAAR), we have evaluated the production of different optically pure L-α-amino acids starting from different racemic N-formyl- and N-carbamoyl-amino acids using a dynamic kinetic resolution approach. The enzymes were immobilized on two different solid supports, resulting in improved stability of the enzymes in terms of thermostability and storage when compared to the enzymes in solution. The bienzymatic system retained up to 80% conversion efficiency after 20 weeks at 4 °C and up to 90% after 1 week at 45 °C. The immobilization process also resulted in a great enhancement of the activity of BsLcar toward N-formyl-tryptophan, showing for the first time that substrate specificity of L-carbamoylases can be influenced by this approach. The system was effective for the biosynthesis of natural and unnatural L-amino acids (enantiomeric excess (e.e.) >99.5%), such as L-methionine, L-alanine, L-tryptophan, L-homophenylalanine, L-aminobutyric acid, and L-norleucine, with a higher performance toward N-formyl-α-amino acid substrates. Biocatalyst reuse was studied, and after 10 reaction cycles, over 75% activity remained.

amino acid racemase; racemase; amino acid; enzymatic cascade Abbreviations: N-succinylamino acid racemase (NSAR); o-succinylbenzoate synthase (OSBS); N-succinylamino acid racemase/o-succinylbenzoate synthase subfamily (NSAR/OSBS); N-acetyl-amino or N-acyl-amino acid racemase (NAAR); Nsubstituted-amino acid (NxA); N-substituted-amino acid racemase (NxAR); Kinetic resolution (KR); Dynamic kinetic resolution (DKR); N-acetyl-(NA); N-acetyl-amino acid (NAA); N-succinyl-(NS); N-succinyl-amino acid (NSA); N-carbamoyl-(NC); Ncarbamoyl-amino acid (NCAA); N-chloroacetyl-(NCh); N-chloroacetyl-amino acid (NChAA); N-formyl-(NF); N-formyl-amino acid (NFAA); N-butyril (NBt); N-butyril amino acid (NBtA); N-propionyl (NPr); N-propionyl amino acid (NPrA); N-benzoyl (NBzA); N-benzoyl amino acid (NBzA); (1R,6R)-2-succinyl-6-hydroxy-2,4cyclohexadiene-1-carboxylate (SHCHC); Amycolatopsis sp. TS-1-60 NSAR (AmyNSAR); Geobacillus kaustophilus NSAR (GkaNSAR); Geobacillus stearothermophilus CECT 49 NSAR (GstNSAR); Streptomyces atratus Y-53 NSAR (SatNSAR); Sebekia benihana NSAR (SebeNSAR); Chloroflexus aurantiacus NSAR (CauNSAR); Thermus thermophilus NSAR (TteNSAR); Deinococcus radiodurans NSAR (DraNSAR); Exiguobacterium sp. AT1b OSBS (ExiOSBS); Alicyclobacillus acidocaldarius OSBS (AacOSBS); Enterococcus faecalis V583 NSAR (EfaNSAR); Listeria innocua Clip11262 NSAR (LinNSAR); Lysinibacillus varians GY32 NSAR (LvaNSAR); Roseiflexus castenholzii HLO8 NSAR (RcaNSAR); Amycolatopsis mediterranei S699 NSAR (AmedNSAR).

No abstract

Formamidases (EC 3.5.1.49) are poorly characterized proteins. In spite of this scarce knowledge, ammonia has been described as playing a central role in the pathogenesis of human pathogens such as Helicobacter pylori, for which formamidase has been shown to participate in the nitrogen metabolic pathway. Sequence analysis has revealed that at least two different groups of formamidases are classified as EC 3.5.1.49: on the one hand, the derivatives of the FmdA-AmdA superfamily, which are the best studied to date, and on the other hand, the derivatives of Helicobacter pylori AmiF. Here we present the cloning, purification, and characterization of a recombinant formamidase from Bacillus cereus CECT 5050T (BceAmiF), the second member of the AmiF subfamily to be characterized, showing new features of the enzyme further supporting its relationship with aliphatic amidases. We also present homology modeling-based mutational studies confirming the importance of the Glu140 and Tyr191 residues in the enzymatic activities of the AmiF family. Moreover, we can conclude that a second glutamate residue is critical in several members of the nitrilase superfamily, meaning that what has consistently been identified as a C-E-K triad is in fact a C-E-E-K tetrad.

Background: Severe and morbid obesity are increasing globally, particularly in women. As BMI increases, the likelihood of anovulation is higher. The primary aim of the EMOVAR clinical trial is to examine, over the short (16 weeks) and medium (12 months) term, the effects of a supervised physical exercise program (focused primarily on aerobic and resistance training) on ovarian function in women with severe/morbid obesity who have undergone bariatric surgery. Secondary objectives are to examine the effects of the intervention on chronic inflammation, insulin resistance, arterial stiffness, physical fitness, and health-related quality of life. Methods: This is a randomized controlled trial in which ∼40 female bariatric surgery patients, aged between 18 and 45 years old, will be included. Participants assigned to the experimental group will perform a total of 48 sessions of supervised concurrent (strength and aerobic) training (3 sessions/week, 60 min/session) spread over 16 weeks. Patients assigned to the control group will receive lifestyle recommendations. Outcomes will be assessed at baseline, week 16 (i.e., after the exercise intervention) and 12 months after surgery. The primary outcome is ovarian function using the Sex-Hormone Binding Globuline, measured in serum. Secondary outcomes are serum levels of anti-mullerian hormone, TSH, T4, FSH, LH, estradiol, prolactine, and free androgen index, as well as oocyte count, the diameters of both ovaries, endometrial thickness, and uterine arterial pulsatility index (obtained from a transvaginal ultrasound), the duration of menstrual bleeding and menstrual cycle duration (obtained by personal interview) and hirsutism (Ferriman Gallwey Scale). Other secondary outcomes include serum markers of chronic inflammation and insulin resistance (i.e., C-reactive protein, interleukin 6, tumor necrosis factor-alpha, leptin, glomerular sedimentation rate, glucose, insulin and the HOMA-IR), arterial stiffness, systolic, diastolic and mean blood pressure, body composition, and total weight loss. Physical fitness (including cardiorespiratory fitness, muscular strength, and flexibility), health-related quality of life (SF-36 v2) and sexual function (Female Sexual Function Index) will also be measured. Discussion: This study will provide, for the first time, relevant information on the effects of exercise training on ovarian function and underlying mechanisms in severe/morbid obese women following bariatric surgery. Trial registration number: ISRCTN registry (ISRCTN27697878).