scholarly journals Development of a Hybrid Bioinorganic Nanobiocatalyst: Remarkable Impact of the Immobilization Conditions on Activity and Stability of β-galactosidase

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4152
Author(s):  
Luigi Tavernini ◽  
Oscar Romero ◽  
Carla Aburto ◽  
Fernando López-Gallego ◽  
Andrés Illanes ◽  
...  

Hybrid bioinorganic biocatalysts have received much attention due to their simple synthesis, high efficiency, and structural features that favor enzyme activity and stability. The present work introduces a biomineralization strategy for the formation of hybrid nanocrystals from β-galactosidase. The effects of the immobilization conditions were studied, identifying the important effect of metal ions and pH on the immobilization yield and the recovered activity. For a deeper understanding of the biomineralization process, an in silico study was carried out to identify the ion binding sites at the different conditions. The selected β-galactosidase nanocrystals showed high specific activity (35,000 IU/g biocatalyst) and remarkable thermal stability with a half-life 11 times higher than the soluble enzyme. The nanobiocatalyst was successfully tested for the synthesis of galacto-oligosaccharides, achieving an outstanding performance, showing no signs of diffusional limitations. Thus, a new, simple, biocompatible and inexpensive nanobiocatalyst was produced with high enzyme recovery (82%), exhibiting high specific activity and high stability, with promising industrial applications.

2021 ◽  
Author(s):  
Moataz Dowaidar

Gastric cancer is the world's second-largest death cause. Developing suitable medical therapies can help individuals live longer. So far, GC treatment has depended on several pharmaceutical techniques. Chemotherapy and surgery are GC patients' most frequent treatment choices. The most major hurdles to effective GC therapy are chemotherapeutic resistance and non-selective targeting. Recent GC-targeted therapeutic research has focused on building more selective and effective anti-GC pharmacological approaches. Because molecular focused therapy can greatly exacerbate the current inefficacy of normal GC therapy procedures, peptide base synthesis can be used as a carrier to deliver radiation or other fatal chemicals to tumor locations with precise protein overexpression. Different types of peptides with special binding affinity to GC overexpressed receptors have been identified for targeted therapy and imaging. Although some of these peptides have excellent GC targeting ability, they also need great GC penetration capacity and no systemic in vivo toxicity before they can be employed in clinical studies. One of these peptides' most notable limitations is their short plasma half-life, limiting their efficient delivery to tumor locations. Sluggish binding pharmacokinetics, along with in vivo instability, can produce targeted treatment failure. Using an appropriate modification strategy to boost blood circulation time may be advantageous.The key to producing successful, innovative anti-cancer targeting drugs with specific targeting capabilities is to mark the peptide with distinct diagnostic and therapeutic radioisotopes. Although a peptide's radiolabeling or enzymatic degradation may not affect its targeting capabilities, the radiation dose delivery impact on it is obvious. Selecting an appropriate type of radionuclide to achieve high-specific activity, using a simple and high-efficiency radiolabeling process, and selecting an adequate spacer and chelator to manage peptide biodistribution are all important considerations when designing a peptide-based radiopharmaceutical. High internalization and significant systemic circulation washout are other essential tumor targeting needs. Many of the peptides described in this work lack these critical features. The radiolabeled peptide should also remain intact and have a short blood washout period, allowing targeted imaging and therapy. SPECT and PET are the most extensively used technologies in nuclear medicine. Although PET has a greater resolution, SPECT technology gives a comparable sensitivity at a lesser cost. Combining fast binding pharmacokinetics with suitable stability in vivo can result in efficient tumor contrast. Non-target liver and kidney accumulation is required when employing radiolabeled peptides to target GC. When a radiolabeled peptide accumulates more in the liver and intestine than in the GC tumor, the image quality degrades. However, using the proper chelator and spacer can assist decrease non-target accumulation in the kidneys. Finally, considering all these conditions and being positive, it is conceivable to produce a unique peptide with avid binding to GC cells.


2021 ◽  
Vol 8 ◽  
Author(s):  
Michiel Van de Voorde ◽  
Charlotte Duchemin ◽  
Reinhard Heinke ◽  
Laura Lambert ◽  
Eric Chevallay ◽  
...  

Samarium-153 (153Sm) is a highly interesting radionuclide within the field of targeted radionuclide therapy because of its favorable decay characteristics. 153Sm has a half-life of 1.93 d and decays into a stable daughter nuclide (153Eu) whereupon β− particles [E = 705 keV (30%), 635 keV (50%)] are emitted which are suitable for therapy. 153Sm also emits γ photons [103 keV (28%)] allowing for SPECT imaging, which is of value in theranostics. However, the full potential of 153Sm in nuclear medicine is currently not being exploited because of the radionuclide's limited specific activity due to its carrier added production route. In this work a new production method was developed to produce 153Sm with higher specific activity, allowing for its potential use in targeted radionuclide therapy. 153Sm was efficiently produced via neutron irradiation of a highly enriched 152Sm target (98.7% enriched, σth = 206 b) in the BR2 reactor at SCK CEN. Irradiated target materials were shipped to CERN-MEDICIS, where 153Sm was isolated from the 152Sm target via mass separation (MS) in combination with laser resonance enhanced ionization to drastically increase the specific activity. The specific activity obtained was 1.87 TBq/mg (≈ 265 times higher after the end of irradiation in BR2 + cooling). An overall mass separation efficiency of 4.5% was reached on average for all mass separations. Further radiochemical purification steps were developed at SCK CEN to recover the 153Sm from the MS target to yield a solution ready for radiolabeling. Each step of the radiochemical process was fully analyzed and characterized for further optimization resulting in a high efficiency (overall recovery: 84%). The obtained high specific activity (HSA) 153Sm was then used in radiolabeling experiments with different concentrations of 4-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA). Even at low concentrations of p-SCN-Bn-DOTA, radiolabeling of 0.5 MBq of HSA 153Sm was found to be efficient. In this proof-of-concept study, we demonstrated the potential to combine neutron irradiation with mass separation to supply high specific activity 153Sm. Using this process, 153SmCl3 suitable for radiolabeling, was produced with a very high specific activity allowing application of 153Sm in targeted radionuclide therapy. Further studies to incorporate 153Sm in radiopharmaceuticals for targeted radionuclide therapy are ongoing.


Catalysts ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 885
Author(s):  
Ren Kuan Li ◽  
Xi Juan Ying ◽  
Zhi Lin Chen ◽  
Tzi Bun Ng ◽  
Zhi Min Zhou ◽  
...  

Agarases catalyze the hydrolysis of agarose to oligosaccharides which display an array of biological and physiological functions with important industrial applications in health-related fields. In this study, the gene encoding agarase (Aga-ms-R) was cloned from Microbulbifer sp. BN3 strain. Sequence alignment indicated that Aga-ms-R belongs to the GH16 family and contains one active domain and two carbohydrate binding module (CBM) domains. The mature Aga-ms-R was expressed successfully by employing the Brevibacillus system. Purified rAga-ms-R was obtained with a specific activity of 100.75 U/mg. rAga-ms-R showed optimal activity at 50 °C and pH 7.0, and the enzyme activity was stable at 50 °C and also over the pH range of 5.0–9.0. After exposure of rAga-ms-R to 70 °C for 30 min, only partial enzyme activity remained. Thin layer chromatographic analysis of the enzymatic hydrolysate of agar obtained using rAga-ms-R disclosed that the hydrolysate comprised, in a long intermediate-stage of the hydrolysis reaction, mainly neoagarotetraose (NA4) and neoagarohexaose (NA6) but ultimately, predominantly neoagarotetraose and trace amounts of neoagarobiose (NA2). Hydrolysates of the raw red seaweeds Gracilaria sjoestedtii and Gelidium amansii, produced by incubation with rAga-ms-R, were mainly composed of neoagarotetraose. The results demonstrate the high efficiency of rAga-ms-R in producing neoagaraoligosaccharide under low-cost conditions.


2000 ◽  
Vol 66 (9) ◽  
pp. 3727-3734 ◽  
Author(s):  
Ho-Geun Yoon ◽  
Hee-Yun Kim ◽  
Young-Hee Lim ◽  
Hye-Kyung Kim ◽  
Dong-Hoon Shin ◽  
...  

ABSTRACT A thermostable chitosanase gene from the environmental isolateBacillus sp. strain CK4, which was identified on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kDa enzyme. The deduced amino acid sequence of the chitosanase from Bacillus sp. strain CK4 exhibits 76.6, 15.3, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows thatBacillus sp. strain CK4 belongs to cluster III withB. subtilis. The gene was similar in size to that of the mesophile B. subtilis but showed a higher preference for codons ending in G or C. The enzyme contains 2 additional cysteine residues at positions 49 and 211. The recombinant chitosanase has been purified to homogeneity by using only two steps with column chromatography. The half-life of the enzyme was 90 min at 80�C, which indicates its usefulness for industrial applications. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, with trimers through hexamers as the major products.


2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Liya Wang ◽  
Hanan Sharif ◽  
Sara Saellström ◽  
Henrik Rönnberg ◽  
Staffan Eriksson

Abstract Background Thymidine kinase 1 (TK1) catalyzes the initial phosphorylation of thymidine in the salvage pathway synthesis of dTTP, an essential building block of DNA. TK1 is a cytosolic enzyme with its highest level during the S-phase of the cell cycle. In cancer cells TK1 is upregulated and excess TK1 is leaked into the blood. Therefore, serum TK1 has been used as biomarker for cancer diagnosis and prognosis in human medicine. Feline TK1 shows high sequence similarity to TK1 from other species. The aim of this study was to characterize feline TK1 and evaluate if serum TK1 can be used as a diagnostic biomarker. Results Feline TK1 was cloned, expressed and affinity purified. The purified feline TK1 phosphorylated not only pyrimidine deoxyribonucleosides but also pyrimidine ribonucleosides and to some extent purine deoxynucleosides. A number of anticancer and antiviral nucleoside analogs also served as substrates with fairly high efficiency. ATP and dATP were the preferred phosphate donor. Serum TK1 activity in felines with malignant diseases was significantly higher than that in healthy individuals. ROC analysis revealed an area under the curve (AUC) of 0.98 with a sensitivity of 0.83 and a specificity of 0.95 for felines with lymphoma. Serum TK1 activity in felines with IBD or inflammatory disease was within the same range as healthy ones. Furthermore, in felines with lymphoma serum TK1 activity returned to normal levels in response to treatment. Conclusion Feline TK1 has high specific activity and a broader substrate specificity in comparison with TK1 from other species. Serum TK1 activity in felines with malignant diseases is significantly higher than that in normal felines and in felines with inflammatory diseases. These results suggest that serum TK1 may be a promising biomarker for the diagnosis and monitoring of malignant diseases and for the differential diagnosis of certain inflammatory disease.


2021 ◽  
Vol 28 ◽  
Author(s):  
Fatima Akram ◽  
Rabia Akram ◽  
Ikram ul Haq ◽  
Ali Nawaz ◽  
Zuriat Jabbar ◽  
...  

Background: Chitin, the second most abundant polysaccharide in nature, is a constantly valuable and renewable raw material after cellulose. Due to advancement in technology, industrial interest has grown to take advantage of the chitin. Objective: Now, biomass is being treated with diverse microbial enzymes or cells for the production of desired products under best industrial conditions. Glycosidic bonds in chitin structure are degraded by chitinase enzymes, which are characterized into number of glycoside hydrolase (GHs) families. Methods: Thermophilic microorganisms are remarkable sources of industrially important thermostable enzymes, having ability to survive harsh industrial processing conditions. Thermostable chitinases have an edge over mesophilic chitinases as they can hydrolyse the substrate at relatively high temperatures and exhibit decreased viscosity, significantly reduced contamination risk, thermal and chemical stability and increased solubility. Various methods are employed to purify the enzyme and increase its yield by optimizing various parameters such as temperature, pH, agitation, and by investigating the effect of different chemicals and metal ions etc. Results: Thermostable chitinase enzymes show high specific activity at elevated temperature which distinguish them from mesophiles. Genetic engineering can be used for further improvement of natural chitinases, and unlimited potential for the production of thermophilic chitinases has been highlighted due to advancement in synthetic biological techniques. Thermostable chitinases are then used in different fields such as bioremediation, medicine, agriculture and pharmaceuticals. Conclusion: This review will provide information about chitinases, biotechnological potential of thermostable enzyme and the methods by which they are being produced and optimized for several industrial applications. Some of the applications of thermostable chitinases have also been briefly described.


2020 ◽  
Vol 51 (3) ◽  
pp. 767-776
Author(s):  
Al-easawi & et al.

This study was aimed, extraction and purification of beta-Galactosidase from local almond(Amygdalus communis)  for lactose intolerance treatment. The best one among 10 methods method of the extraction was using sodium phosphate buffer at 0.2 molar. Which was  achieves the highest specific activity amounted to 3.66unit/mg protein. Then, partial purification of enzyme was done using five methods. The highest specific activity was obtained using the method of precipitation with ammonium sulphate at 30-70% since the specific activity was 15.85units/mg protein. Which represented the best way to precipitation the enzyme. Three iso enzymes were obtained. One of them was taken for its high specific activity(20.10units/mg protein) and ion exchange chromatography was used and followed by gel filtration technique using sephadex-100 column to increase purification. The specific activity was increased to 21.95units/mg protein. Lactose hydrolysis efficiency test was performed and the purified enzyme showed high efficiency in standard lactose hydrolysis test.          


1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.


1962 ◽  
Vol 08 (03) ◽  
pp. 425-433 ◽  
Author(s):  
Ewa Marciniak ◽  
Edmond R Cole ◽  
Walter H Seegers

SummarySuitable conditions were found for the generation of autoprothrombin C from purified prothrombin with the use of Russell’s viper venom or trypsin. DEAE chromatographed prothrombin is structurally altered and has never been found to yield autoprothrombin C and also did not yield it when Russell’s viper venom or trypsin were used. Autoprothrombin C is derived from prothrombin with tissue extract thromboplastin, but not in large amounts with the intrinsic clotting factors. With the latter thrombin and autoprothrombin III are the chief activation products. Autoprothrombin III concentrates were prepared from serum and upon activation with 25% sodium citrate solution or with Russell’s viper venom large amounts of autoprothrombin C were obtained, and this was of high specific activity. Theoretically trypsin is not a thrombolytic agent, but on the contrary should lead to intravascular clotting.


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