RESUMO
Arthrobacter sp. CGMCC 3584 is used for the industrial production of cyclic adenosine monophosphate (cAMP). However, because of the paucity of genetic engineering tools for genetic manipulation on Arthrobacter species, only a few metabolically engineered Arthrobacter have been constructed and investigated. In this study, for the first time, we constructed an arpde knockout mutant of Arthrobacter without any antibiotic resistance marker by a PCR-targeting-based homologous recombination method. Our results revealed that the deletion of arpde had little effect on biomass production and improved cAMP production by 31.1%. Furthermore, we compared the transcriptomes of the arpde knockout strain and the wild strain, aiming to understand the capacities of cAMP production due to arpde inactivation at the molecular level. Comparative transcriptomic analysis revealed that arpde inactivation had two major effects on metabolism: inhibition of glycolysis, PP pathway, and amino acid metabolism (phenylalanine, tryptophan, branched-chain amino acids, and glutamate metabolism); promotion of the purine metabolism and carbon flux from the precursor 5'-phosphoribosyl 1-pyrophosphate, which benefited cAMP production.
Assuntos
Arthrobacter , AMP Cíclico/biossíntese , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Genes Bacterianos , Arthrobacter/genética , Arthrobacter/metabolismo , AMP Cíclico/genéticaRESUMO
We aimed to identify risk factors for early hypocalcemia after parathyroidectomy in patients with secondary hyperparathyroidism. We retrospectively enrolled 106 of 120 consecutive patients with secondary hyperparathyroidism who underwent parathyroidectomy between January 2019 and July 2021. Perioperative laboratory parameters, preoperative computerized tomography (CT) images, and postoperative histology were evaluated. Parathyroid calcification was defined as hyperdense regions with a density of > 130 Hounsfield Units on CT images of the parathyroid. Subtotal parathyroidectomy, total parathyroidectomy without auto-transplantation, or total parathyroidectomy with auto-transplantation were performed in the present study. Postoperative hypocalcemia was defined as a serum calcium concentration < 2.1 mmol/L within 4 days of surgery. The participants were categorized according to the presence (n = 33) or absence (n = 73) of postoperative hypocalcemia. The demographics, comorbidities, and surgical details were similar in the two groups. Multivariate analysis showed that the preoperative alkaline phosphatase activity, serum intact parathyroid hormone and calcium concentrations, and parathyroid calcification were independent risk factors for postoperative hypocalcemia (all P < 0.05). Receiver operating characteristic analysis generated areas under the curves for preoperative alkaline phosphatase, intact parathyroid hormone, and parathyroid calcification of 0.82, 0.80, and 0.70, respectively (all P < 0.05). Cut-off values for preoperative alkaline phosphatase (> 242.9 IU/L) and intact parathyroid hormone (> 2,104 pg/mL) were found to be predictive of postoperative hypocalcemia. High preoperative alkaline phosphatase activity and serum intact parathyroid hormone concentration and low serum calcium are associated with higher risks of postoperative hypocalcemia. Calcification of the parathyroid may represent a novel radiologic means of predicting postoperative hypocalcemia.
Assuntos
Hiperparatireoidismo Secundário , Hipocalcemia , Humanos , Paratireoidectomia/métodos , Hipocalcemia/etiologia , Cálcio , Fosfatase Alcalina , Estudos Retrospectivos , Diálise Renal/efeitos adversos , Hiperparatireoidismo Secundário/etiologia , Hiperparatireoidismo Secundário/cirurgia , Hormônio Paratireóideo , Fatores de RiscoRESUMO
In this research article, we reported a self-assembly approach to prepare a copper polyphosphate kinase 2 hybrid nanoflower and established a cofactor ADP regeneration system from AMP using the nanoflower. First, the structure of the hybrid nanoflower was confirmed by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy, which indicated the successful loading of the enzyme in the hybrid nanoflower. Moreover, compared to the free enzyme, the hybrid nanoflower exhibited a better performance in ADP production and possessed wider catalytic pH and temperature ranges as well as improved storage stability. The hybrid nanoflower also exhibited well reusability, preserving 71.7% of initial activity after being used for ten cycles. In addition, the phosphorylation of glucose was conducted by utilizing ADP-dependent glucokinase coupled with the ADP regeneration system, in which the hybrid nanoflower was used for regenerating ADP from AMP. It was observed that the ADP regeneration system operated effectively at a very small amount of AMP. Thus, the hybrid nanoflower had great application potential in industrial catalytic processes that were coupled with ADP-dependent enzymes.
RESUMO
Self-assembly of the functional units onto the surface of nanoparticles is a powerful approach to generate functional nanosystems. In this work, we first expressed a recombinant class III polyphosphate kinase 2 (ArPPK2) with his-tag. It is able to synthesize ATP from AMP by a single enzyme, simplifying two-step reaction of ATP regeneration from AMP. Then we chose the Fe-based metal-organic frameworks (MOF)s as carriers to produce the enzyme-MOF biocomposite, based on the interaction between the his-tags and coordinatively unsaturated metal sites present on the external surface of MOFs by a self-assembly process. It was found that ArPPK2@MIL-101-NH2@Fe3O4-COOH exhibited better reusability than other candidates during cycle analysis, preserving 70.1% of initial activity after reusing thirteen times, and also retained high storage stability. The optimum pH of the enzyme-MOF biocomposite was increased from 8.0 to 9.0 and the optimum temperature was increased from 30â to 45â. Compared to free ArPPK2, the enzyme-MOF biocomposite showed increased thermal and pH stability. In addition, we successfully constructed an ATP regeneration system from AMP using the enzyme-MOF biocomposite, coupled with amide bond formation catalyzed by the adenylation domain of tyrocidine synthetase A (TycA-A). The immobilized ArPPK2 will provide a promising route for ATP regeneration from AMP in industrial processes. And the generation of the enzyme-MOF biocomposite by the self-assembly approach can be extended to efficiently immobilize other recombinant his-tagged enzymes.