Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design

Lytic polysaccharide monooxygenases (LPMOs) oxidatively break down the glycosidic bonds of crystalline polysaccharides, significantly improving the saccharification efficiency of recalcitrant biomass, and have broad application prospects in industry. To meet the needs of industrial applications, enzyme engineering is needed to improve the catalytic performance of LPMOs such as enzyme activity and stability. In this study, we engineered the chitin-active CjLPMO10A from Cellvibrio japonicus through a rational disulfide bonds design. Compared with the wild-type, the variant M1 (N78C/H116C) exhibited a 3-fold increase in half-life at 60°C, a 3.5°C higher T5015, and a 7°C rise in the apparent Tm. Furthermore, the resistance of M1 to chemical denaturation was significantly improved. Most importantly, the introduction of the disulfide bond improved the thermal and chemical stability of the enzyme without causing damage to catalytic activity, and M1 showed 1.5 times the specific activity of the wild-type. Our study shows that the stability and activity of LPMOs could be improved simultaneously by selecting suitable engineering sites reasonably, thereby improving the industrial adaptability of the enzymes, which is of great significance for applications.

CHARACTERIZATION OF PARTIALLY PURIFIED TYROSINASE ISOLATED FROM BITTER KOLA (GARCINIA KOLA)

Objective: Tyrosinase is a glycosylated, copper-containing oxidase that catalyzes the first two steps of mammalian melanogenesis as well as enzymatic browning events in damaged fruits during post-harvest handling and processing. Human skin hyperpigmentation and enzymatic browning in fruits are both undesirable. In this study, the properties and inhibitory effect of some compounds on bitter kola tyrosinase were investigated. Methods: Bitter kola tyrosinase was isolated and characterized using standard protocols. Partial purification was carried out on Sephadex G-100 loaded column chromatography. Results: Bitter kola tyrosinase was purified with a specific activity of 3.5 U/mg protein, purification fold of 2.4 and a yield of 34%. The optimum pH value was found to be 6.0 while the optimum temperature value for maximum enzyme activity was observed at 60°C. The enzyme was stable at 40oC for 20 minutes. Metals such as NaCl, KCl, MgCl2 and CaCl2 had inhibitory effect on the activity; though MgCl2 and CaCl2 had minimal effect. Also, EDTA, β-marcaptoethanol and glutathione greatly inhibited the enzyme activity at all the tested concentration. Conclusion: The properties of bitter kola tyrosinase compare very well with the tyrosinase from other sources. Also, the study was able to establish the inhibitory effect of some compounds and this could be applied in food processing industries. Peer Review History: Received: 2 November 2021; Revised: 11 December; Accepted: 25 December, Available online: 15 January 2022 Academic Editor: Dr. A.A. Mgbahurike, University of Port Harcourt, Nigeria, [email protected] UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency. Received file: Reviewer's Comments: Average Peer review marks at initial stage: 6.0/10 Average Peer review marks at publication stage: 7.5/10 Reviewers: Dr. Nazim Hussain, North East Frontier Technical University, Arunachal pradesh, India, [email protected] Ahmad Najib, Universitas Muslim Indonesia, Makassar, Indonesia, [email protected] Prof. Dr. Ali Gamal Ahmed Al-kaf, Sana'a university, Yemen, [email protected] Similar Articles: PHYTOCHEMICAL PURIFICATION OF ACTIVE CONSTITUENTS ISOLATED FROM ROOT OF THE MEDICINAL HERB, CARALLUMA QUADRANGULA

Complete wastewater discoloration by a novel peroxidase source with promising bioxidative properties

BACKGROUND Our study aimed to characterize and prospect immobilization strategies for a novel fungal peroxidase - POD (EC 1.11.1.7) and to insert it in the context of pollutant remediation, since these compounds pose risks to human and environmental health. The enzymatic extract was obtained by submerged fermentation of the fungus Trichoderma koningiopsis in an alternative substrate, consisting of fresh microalgal biomass. The immobilization efficiency was evaluated by monitoring the residual activity (RA) and the discoloration potential (DP) of a synthetic dye solution. Concomitantly, the catalytic properties of free POD were explored, and the most promising storage strategy to maintain the enzymatic activity was studied. RESULTS The novel guaiacol peroxidase expressed specific activity of up to 7801 U mg−1 in the free form, showing stability when subjected to up to 80°C in a pH range between 4.0-8.0. Furthermore, the bioproduct immobilized on magnetic nanoparticles expressed up to 689% RA and 100% DP. An increase in the RA of the enzyme, both in free and immobilized form, was also observed after storage for up to 8 months. The synthesized magnetic nanozymes showed good reusability, maintaining 13546 U mg−1 after ten cycles and removing 94% of color in a second batch. Toxicological evaluation with Allium cepa indicated that the enzymatic process of color removal with immobilized POD was essential to eliminate genotoxic effects. CONCLUSION T. koningiopsis peroxidase production and immobilization presented in our work are promising for the enzyme market and for the wastewater treatment technologies, considering its high bioxidative potential.

Motor planning brings human primary somatosensory cortex into action-specific preparatory states

Motor planning plays a critical role in producing fast and accurate movement. Yet, the neural processes that occur in human primary motor and somatosensory cortex during planning, and how they relate to those during movement execution, remain poorly understood. Here we used 7T functional magnetic resonance imaging (fMRI) and a delayed movement paradigm to study single finger movement planning and execution. The inclusion of no-go trials and variable delays allowed us to separate what are typically overlapping planning and execution brain responses. Although our univariate results show widespread deactivation during finger planning, multivariate pattern analysis revealed finger-specific activity patterns in contralateral primary somatosensory cortex (S1), which predicted the planned finger action. Surprisingly, these activity patterns were as informative as those found in contralateral primary motor cortex (M1). Control analyses ruled out the possibility that the detected information was an artifact of subthreshold movements during the preparatory delay. Furthermore, we observed that finger-specific activity patterns during planning were highly correlated to those during execution. These findings reveal that motor planning activates the specific S1 and M1 circuits that are engaged during the execution of a finger press, while activity in both regions is overall suppressed. We propose that preparatory states in S1 may improve movement control through changes in sensory processing or via direct influence of spinal motor neurons.

Dynamic targeting enables domain-general inhibitory control over action and thought by the prefrontal cortex

Over the last two decades, inhibitory control has featured prominently in accounts of how humans and other organisms regulate their behaviour and thought. Previous work on how the brain stops actions and thoughts, however, has emphasised distinct prefrontal regions supporting these functions, suggesting domain-specific mechanisms. Here we show that stopping actions and thoughts recruits common regions in the right dorsolateral and ventrolateral prefrontal cortex to suppress diverse content, via dynamic targeting. Within each region, classifiers trained to distinguish action-stopping from action-execution also identify when people are suppressing their thoughts (and vice versa). Effective connectivity analysis reveals that both prefrontal regions contribute to action and thought stopping by targeting the motor cortex or the hippocampus, depending on the goal, to suppress their task-specific activity. These findings support the existence of a domain-general system that underlies inhibitory control and establish Dynamic Targeting as a mechanism enabling this ability.

The role of methanotrophy in the microbial carbon metabolism of temperate lakes

Previous stable isotope and biomarker evidence has indicated that methanotrophy is an important pathway in the microbial loop of freshwater ecosystems, despite the low cell abundance of methane-oxidizing bacteria (MOB) and the low methane concentrations relative to the more abundant dissolved organic carbon (DOC). However, quantitative estimations of the relative contribution of methanotrophy to the microbial carbon metabolism of lakes are scarce, and the mechanism allowing methanotrophy to be of comparable importance to DOC-consuming heterotrophy remained elusive. Using incubation experiments, microscopy, and multiple water column profiles in six temperate lakes, we show that MOB play a much larger role than their abundances alone suggest because of their larger cell size and higher specific activity. MOB activity is tightly constrained by the local methane:oxygen ratio, with DOC-rich lakes with large hypolimnetic volume fraction showing a higher carbon consumption through methanotrophy than heterotrophy at the whole water column level. Our findings suggest that methanotrophy could be a critical microbial carbon consumption pathway in many temperate lakes, challenging the prevailing view of a DOC-centric microbial metabolism in these ecosystems.

RADIATION SAFETY ASSESSMENT OF CARROT AND BEET PRODUCTS CULTIVATED IN THE BLACK EARTH ZONE OF RUSSIA IN THE OREOL OF THE EASTERN CHERNOBYL TRACE

On the territory of the Plavsky radioactive hotspot of the Tula region of Russia, formed as a result of the accident at the Chernobyl nuclear power plant in 1986, an assessment of the radiation safety of growing carrots and beets was carried out in 2019. It has been established that at present the content of 137Cs in arable leached chernozems of the surveyed lands is 90–170 kBq/m2 , which is 2.5–4.5 times higher than the permissible level of density of surface radioactive contamination of soils. However, the specific activity of the radionuclide in carrot and beetroot crops does not exceed 5 Bq/kg, which is significantly less than the maximum permissible level of 137Cs accumulation in vegetables (600 Bq/kg for absolutely dry weight). The accumulation coefficients of 137Cs in the total biomass of carrots and beets are 2.0·10−2 and 7.5·10−2, and in eaten root crops – 1.1·10−2 and 2.0·10−2, respectively, which is in good agreement with the IAEA estimate for the intensity of the transition 137Cs in the production of vegetable roots from loamy and clayey soils.

Celecoxib-Induced Modulation of Colon Cancer CD133 Expression Occurs through AKT Inhibition and Is Monitored by 89Zr Immuno-PET

We developed an immuno-PET technique that monitors modulation of tumor CD133 expression, which is required for the success of CD133-targeted therapies. Methods. Anti-CD133 antibodies were subjected to sulfhydryl moiety-specific 89Zr conjugation. 89Zr-CD133 IgG was evaluated for specific activity and radiolabel stability. Colon cancer cells underwent binding assays and Western blotting. Biodistribution and PET studies were performed in mice. Results. 89Zr-CD133 IgG showed excellent target specificity with 97.2 ± 0.7 % blocking of HT29 cell binding by an excess antibody. Intravenous 89Zr-CD133 IgG followed biexponential blood clearance and showed CD133-specific uptake in HT29 tumors. 89Zr-CD133 IgG PET/CT and biodistribution studies confirmed high HT29 tumor uptake with lower activities in the blood and normal organs. In HT29 cells, celecoxib dose-dependently decreased CD133 expression and 89Zr-CD133 IgG binding that reached 19.9 ± 2.1 % ( P < 0.005 ) and 50.3 ± 10.9 % ( P < 0.001 ) of baseline levels by 50 μM, respectively. Celecoxib treatment of mice significantly suppressed tumor CD133 expression to 67.5 ± 7.8 % of controls ( P < 0.005 ) and reduced tumor 89Zr-CD133 IgG uptake from 15.5 ± 1.4 % at baseline to 12.3 ± 2.0 % ID / g ( P < 0.01 ). Celecoxib-induced CD133 reduction in HT29 cells and tumors was associated with substantial suppression of AKT activation. There were also reduced HIF-1α accumulation and IκBα/NFκB phosphorylation. Conclusion. 89Zr-CD133 IgG PET provides high-contrast tumor imaging and monitors celecoxib treatment-induced modulation of tumor CD133 expression, which was found to occur through AKT inhibition. This technique may thus be useful for screening drugs that can effectively suppress colon cancer stem cells.