Production of Glucose Isomerase from Streptomyces roseiscleroticus

This study was aimed at the isolation and characterization of a microbial strain capable of producing glucose isomerase. Microbial strain was isolated from soil using starch casein agar as a differential media. The isolated microbial strain was capable of producing glucose isomerase which was tested using 2, 3, 5 - triphenyltetrazolium solution as a chromogenic substrate. The microbial strain was identified as Streptomyces species based on its morphological and microscopic characteristics. It was further subjected to molecular characterization using 16S rRNA sequencing and was subsequently confirmed as Streptomyces roseiscleroticus. Glucose isomerase was produced from Streptomyces roseiscleroticus after 120 hr of submerged fermentation at pH 6.8 and at 37°C utilizing xylose as the sole carbon source and a compendium of peptone, beef and yeast extracts as nitrogen sources. These findings suggest that the microbial strain, Streptomyces roseiscleroticus can be a useful bacterial source for the production of glucose isomerase needed for commercial and industrial utilization.

Robust chitinolytic activity of crab-eating monkey (Macaca fascicularis) acidic chitinase under a broad pH and temperature range

Diet of the crab-eating monkey (Macaca fascicularis) consists of both plants and animals, including chitin-containing organisms such as crabs and insects. This omnivorous monkey has a high expression of acidic chitinase (CHIA) in the stomach and here, we report on its enzymatic properties under different conditions. When we compared with Mus musculus CHIA (Mm-CHIA), Macaca fascicularis CHIA (Mf-CHIA) exhibits higher chitinolytic activity at broad pH (1.0–7.0) and temperature (30–70 ℃) range. Interestingly, at its optimum pH (5.0), Mf-CHIA showed the highest activity at 65 °C while maintaining it at robust levels between 50 and 70 °C. The degradation efficiency of Mf-CHIA was superior to Mm-CHIA toward both polymeric chitin as well as an artificial chromogenic substrate. Our results show that unique features of Mf-CHIA including its thermostability warrant the nomination of this enzyme for potential agricultural and biomedical applications.

Ce-MOF with Intrinsic Haloperoxidase-Like Activity for Ratiometric Colorimetric Detection of Hydrogen Peroxide

Metal–organic framework (MOF) nanozymes, as emerging members of the nanozymes, have received more and more attention due to their composition and structural characteristics. In this work, we report that mixed-valence state Ce-MOF (MVCM) has intrinsic haloperoxidase-mimicking activity. MVCM was synthesized by partial oxidation method using Ce-MOF as a precursor. In the presence of H2O2 and Br−, MVCM can catalyze oxidative bromination of chromogenic substrate phenol red (PR) to produce the blue product bromophenol blue (Br4PR), showing good haloperoxidase-like activity. Because of the special chromogenic substrate, we constructed a ratiometric colorimetric-sensing platform by detecting the absorbance of the MVCM-(PR, Br−) system at wavelengths of 590 and 430, for quantifying H2O2, where the detection limit of the H2O2 is 3.25 μM. In addition, the haloperoxidase-mimicking mechanism of the MVCM is proposed. Moreover, through enzyme kinetics monitoring, the Km (H2O2 and NH4Br) of the MVCM is lower than that of cerium oxide nanomaterials, indicating that the MVCM has a stronger binding affinity for H2O2 and NH4Br than other materials. This work provides more application prospects for the development of nanozymes in the field of biosensors in the future.

Thein Vitroprocoagulant Effects of Standard and Extended Half-Life Recombinant Factor IX Concentrates in Patients on Prophylaxis with Emicizumab

Introduction: Emicizumab is a humanized, monoclonal, bispecific antibody that binds factor (F) FX and FIXa allowing thrombin generation in the absence of FVIII, which is used for routine treatment of patients with Hemophilia A (HA) with and without inhibitors. Plasma level of FIX will be an important limiting factor for the formation of the FX-FIXa-emicizumab ternary complex in the absence of FVIII, suggesting the potential use of FIX concentrates to regulate the procoagulant function of emicizumab and therefore, to use it as an alternative treatment in certain circumstances to stop or prevent bleedings in patients on prophylaxis with emicizumab. At the present time there are several recombinant FIX concentrates with differences in their content of activated FIX (FIXa) and in their half-life (standard or extended) that may differ in their procoagulant effects when combined with emicizumab. Aim: The aim of this study was to evaluate if there were differences in thein vitroprocoagulant effects of two recombinant FIX concentrates, one with standard half-life (rFIX Nonacog Alfa, BeneFIX®, Pfizer) and the other with extended half-life (rFIX fused to rAlbumin, Albutrepenonacog Alfa, Idelvion®, CLS Behring) in samples from patients on prophylaxis with Emicizumab. Methods: This is a prospective and transversal pilot study that was approved by the Ethics Committee from La Paz University Hospital. Two patients with haemophilia A (HA) with inhibitors in prophylaxis with emicizumab were recruited and one haemophilia B (HB) patient was included as a control for the effects of FIX. Blood samples were collected in tubes with corn trypsin inhibitor (CTI, Haematologic Technologies, USA), to block thecontact phase and to only evaluate coagulation mediated by the extrinsic pathway. Levels of FIXa in concentrates of FIX were quantified using the Spectrozyme® FIXa chromogenic substrate (LOXO) and measuring the increase in OD at 405 nm. Rotational thromboelastometry (ROTEM) was performed using whole blood activated by a low concentration of tissue factor solution (final dilution 1:50,000 of EXTEM reagent) plus recalcification. Parameters evaluated in ROTEM were CT (cloting time), defined as time until detection of a clot firmness of 2 mm; and CFT (clot formation time), defined as time between detection of a clot firmness from 2 to 20 mm. Calibrated automated thrombogram (CAT)was performed using platelet free plasma (PFP) activated by low concentration of tissue factor plus phospholipids (PPP-Reagent LOW®, Stago). Parameters evaluated in CAT were: Peak, defined as maximum thrombin concentration reached, in nM; and ETP, defined as the total amount of thrombin generated over time, in nMxmin. Results: The presence of FIXa activity assayed by a chromogenic substrate was not detectable with 20 IU of Idelvion while BeneFIX® showed a specific concentration-dependent FIX activity that was blocked with the serine protease inhibitor EGR-chloromethylketone (Figure 1). ROTEM (Figure 2) and CAT (Figure 3) results showed that the addition of increased concentrations of both concentrates of rFIX produces an enhanced procoagulant effect of Emicizumab similar to the effect produced by the addition of the bypassing agent rFVIIa (NovoSeven®, NovoNordisk). These results also showed that it is necessary three-four times higher concentration (U/dl) of Idelvion® to get similar procoagulant effects that those obtained with BeneFIX®. The higher procoagulant effects of BeneFIX® were also observed in samples of a patient with severe HB. Conclusion: Global coagulation assays suggest that increasing endogenous FIX levels with two rFIX concentrates that have different FIXa content and half-life, produce an enhanced procoagulant effect of Emicizumab opening the idea of the use of these concentrates as an alternative treatment for bleedings in patients with inhibitors on prophylaxis with Emicizumab. Further studies need to be performed to evaluate the procoagulant activity of the concomitant use of different rFIX concentrates and Emicizumab, and to assess security of this therapeutic approach. This work was supported by grants from FIS-FONDOS FEDER (PI19/00631 and P19/00772). EMM holds a predoctoral fellowship from Fundación Española de Trombosis y Hemostasia (FETH-SETH). Disclosures Fernandez-Bello: Novartis:Speakers Bureau;Stago:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;NovoNordisk:Current Employment, Research Funding, Speakers Bureau;Roche:Speakers Bureau;SOBI,:Research Funding;Pfizer:Speakers Bureau.García Barcenilla:Pfizer,:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;Roche:Speakers Bureau;Bayer:Speakers Bureau;Novartis:Speakers Bureau;NovoNordisk:Research Funding, Speakers Bureau.Alvarez Román:Roche:Speakers Bureau;Novartis:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;Pfizer,:Research Funding, Speakers Bureau;Bayer:Consultancy;SOBI,:Consultancy, Research Funding, Speakers Bureau;Grifols:Research Funding;NovoNordisk,:Research Funding, Speakers Bureau.Martín:NovoNordisk:Speakers Bureau;SOBI:Research Funding;Pfizer:Research Funding, Speakers Bureau;Roche:Speakers Bureau;Novartis:Speakers Bureau.Rivas Pollmar:Novartis:Speakers Bureau;Roche:Speakers Bureau;Pfizer:Speakers Bureau.Canales:Roche:Honoraria;Celgene:Honoraria;Roche:Honoraria;Janssen:Honoraria;Novartis:Honoraria;Sandoz:Speakers Bureau;Sandoz:Honoraria;Janssen:Speakers Bureau;iQone:Honoraria;Sandoz:Honoraria;Gilead:Honoraria;Takeda:Speakers Bureau;Novartis:Honoraria;Karyopharm:Honoraria;Janssen:Honoraria;Takeda:Speakers Bureau;Janssen:Speakers Bureau;Roche:Speakers Bureau;Sandoz:Speakers Bureau;Roche:Speakers Bureau;Karyopharm:Honoraria.Butta:Grifols:Research Funding;Novartis:Speakers Bureau;ROCHE:Research Funding, Speakers Bureau;Pfizer:Speakers Bureau;SOBI:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;NovoNordisk:Speakers Bureau.Jimenez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer:Honoraria;F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer:Consultancy;Grifols, Novo Nordisk, Takeda, Sobi, Pfizer:Research Funding.

The Mimic Enzyme Properties of Au@PtNRs and the Detection for Ascorbic Acid Based on Their Catalytic Properties

Being superior to natural enzymes, nanoenzymes are drawing a great deal of attention in the field of biosensing. Herein, we developed an ultrasensitive, stable and selective colorimetric assay having dual functionalities of Au-tipped Pt nanorods (NRs). The optical and catalytic properties of Au-tipped Pt NRs were monitored using a spectrophotometer and the chromogenic substrate 3, 3′, 5, 5′-tetramethylbenzidine (TMB) in the presence of H2O2, respectively. We found that Au-tipped Pt NRs exhibited excellent peroxidase-like activity, which decomposed hydrogen peroxide (H2O2) into oxygen (O2). The produced O2 oxidized the chromogenic substrate into a blue color product. The oxidation rate of the chromogenic substrate could be monitored using a spectrophotometer at 652 nm. Notably, the peroxidase-like activity of Au-tipped Pt NRs decreased in the presence of ascorbic acid (AA). The produced O2 preferentially reacted with AA, generating ascorbyl radicals (AA·) instead of oxidizing TMB, and thereby decreased the oxidation rate of TMB. Based on this inhibitory property, a selective colorimetric assay was developed using Au-tipped Pt NRs for the detection of AA. This work offers a novel detection method for AA.