D-amino Acid Oxidase as an Industrial Biocatalyst

Biocatalysis driven by D-amino acid oxidase is a significant example of the commercial production of high value-added intermediates using enzyme-based technology. The results of the most recent research on this FAD-dependent catalyst are reported here. In particular, insight is given of how in the past few years the main industrial application of this enzyme, i.e. the stereospecific bioconversion of cephalosporin C to glutaryl-7-amino cephalosporanic acid in the two-step production of 7-amino cephalosporanic acid, has been implemented by improving its production and by engineering of the biocatalyst. The set-up and the optimization of different conditions for carrying out the process under different procedures have also been updated.     

Modulation of natural killer activity in mice by prolonged administration of various doses of dietary retinoids

Groups of BALB/C mice received a diet supplement of 0 (group C), 200 (group A 200), 500 (group A 500), and 1,000 (group A 1,000) IU retinol palmitate (RP)/mouse/day in drinking water for 450 days. At progressive time intervals, mice from each group were tested for natural killer (NK) activity and for the percentage of large granular lymphocytes (LGL) in the spleen. In groups A 200, A 500 and A 1,000, a dose-dependent increase in NK activity was evident 50 days after the beginning of RP supplementation and was accompanied by a parallel increase of LGL number in the spleen. In group A 1,000, the increase of spontaneous or Poly I:C-induced cytotoxicity persisted until day 160. By contrast, inhibition of Poly I:C-induced NK cytotoxicity was found in this group at day 450.     

Overexpression of a recombinant wild-type and His-tagged Bacillus subtilis glycine oxidase in Escherichia coli.

We have cloned the gene coding for the Bacillus subtilis glycine oxidase (GO), a new flavoprotein that oxidizes glycine and sarcosine to the corresponding alpha-keto acid, ammonia and hydrogen peroxide. By inserting the DNA encoding for GO into the multiple cloning site of the expression vector pT7.7 we produced a recombinant plasmid (pT7-GO). The pT7-GO encodes a fully active fusion protein with six additional residues at the N-terminus of GO (MARIRA). In BL21(DE3)pLysS Escherichia coli cells, and under optimal isopropyl thio-beta-D-galactoside induction conditions, soluble and active chimeric GO was expressed up to 1.14 U g(-1) of cell (and a fermentation yield of 3.82 U x L(-1) of fermentation broth). An N-terminal His-tagged protein (HisGO) was also successfully expressed in E. coli as a soluble protein and a fully active holoenzyme. HisGO represents approximately 3.9% of the total soluble protein content of the cell. The His-tagged GO was purified in a single step by nickel-chelate chromatography to a specific activity of 1.06 U x mg(-1) protein at 25 degrees C and with a yield of 98%. The characterization of the purified enzyme showed that GO is a homotetramer of approximately 180 kDa with the spectral properties typical of flavoproteins. GO exhibits good thermal stability, with a Tm of 46 degrees C after 30 min incubation; its stability is maximal in the 7.0-8.5 pH range. A comparison of amino-acid sequence and substrate specificity indicates that GO has similarities to other flavoenzymes acting on primary amines and on D-amino acids.     

Defective endoplasmic reticulum-mitochondria contacts and bioenergetics in SEPN1-related myopathy

SEPN1-related myopathy (SEPN1-RM) is a muscle disorder due to mutations of the SEPN1 gene, which is characterized by muscle weakness and fatigue leading to scoliosis and life-threatening respiratory failure. Core lesions, focal areas of mitochondria depletion in skeletal muscle fibers, are the most common histopathological lesion. SEPN1-RM underlying mechanisms and the precise role of SEPN1 in muscle remained incompletely understood, hindering the development of biomarkers and therapies for this untreatable disease. To investigate the pathophysiological pathways in SEPN1-RM, we performed metabolic studies, calcium and ATP measurements, super-resolution and electron microscopy on in vivo and in vitro models of SEPN1 deficiency as well as muscle biopsies from SEPN1-RM patients. Mouse models of SEPN1 deficiency showed marked alterations in mitochondrial physiology and energy metabolism, suggesting that SEPN1 controls mitochondrial bioenergetics. Moreover, we found that SEPN1 was enriched at the mitochondria-associated membranes (MAM), and was needed for calcium transients between ER and mitochondria, as well as for the integrity of ER-mitochondria contacts. Consistently, loss of SEPN1 in patients was associated with alterations in body composition which correlated with the severity of muscle weakness, and with impaired ER-mitochondria contacts and low ATP levels. Our results indicate a role of SEPN1 as a novel MAM protein involved in mitochondrial bioenergetics. They also identify a systemic bioenergetic component in SEPN1-RM and establish mitochondria as a novel therapeutic target. This role of SEPN1 contributes to explain the fatigue and core lesions in skeletal muscle as well as the body composition abnormalities identified as part of the SEPN1-RM phenotype. Finally, these results point out to an unrecognized interplay between mitochondrial bioenergetics and ER homeostasis in skeletal muscle. They could therefore pave the way to the identification of biomarkers and therapeutic drugs for SEPN1-RM and for other disorders in which muscle ER-mitochondria cross-talk are impaired.Subject terms: Chaperones, Respiratory tract diseases     

D-amino Acid Oxidase as an Industrial Biocatalyst

Biocatalysis driven by D-amino acid oxidase is a significant example of the commercial production of high value-added intermediates using enzyme-based technology. The results of the most recent research on this FAD-dependent catalyst are reported here. In particular, insight is given of how in the past few years the main industrial application of this enzyme, i.e. the stereospecific bioconversion of cephalosporin C to glutaryl-7-amino cephalosporanic acid in the two-step production of 7-amino cephalosporanic acid, has been implemented by improving its production and by engineering of the biocatalyst. The set-up and the optimization of different conditions for carrying out the process under different procedures have also been updated.     

A process for bioconversion of cephalosporin C by Rhodotorula gracilis D-amino acid oxidase

Summary A process for the production (in a stirred tank reactor) of glutaryl-7-ACA from cephalosporin C using immobilized D-amino acid oxidase is described. Results so obtained under optimal conditions (1.2 mg coupled enzyme/L, pH 8.5, 2 mM cephalosporin C) point to a system which shows high conversion efficiency and a remarkable operational stability. No exogenous H₂O₂ is requested to shift the reaction equilibrium toward glutaryl-7-ACA production, nor any side product is detected. The immobilized system productivity was 54 g/day/mg of enzyme. This process represents the first reported case of a reactor successfully developed with a DAAO for bioconversion of cephalosporin C.