Two-substrate enzyme engineering using small libraries that combine the substrate preferences from two different variant lineages.

Improving the range of substrates accepted by enzymes with high catalytic activity remains an important goal for the industrialisation of biocatalysis. Many enzymes catalyse two-substrate reactions which increases the complexity in engineering them for the synthesis of alternative products. Often mutations are found independently that can improve the acceptance of alternatives to each of the two substrates. Ideally, we would be able to combine mutations identified for each of the two alternative substrates, and so reprogramme new enzyme variants that synthesise specific products from their respective two-substrate combinations. However, as we have previously observed for E. coli transketolase, the mutations that improved activity towards aromatic acceptor aldehydes, did not successfully recombine with mutations that switched the donor substrate to pyruvate. This likely results from several active site residues having multiple roles that can affect both of the substrates, as well as structural interactions between the mutations themselves. Here, we have designed small libraries, including both natural and non-natural amino acids, based on the previous mutational sites that impact on acceptance of the two substrates, to achieve up to 630× increases in kcat for the reaction with 3-formylbenzoic acid (3-FBA) and pyruvate. Computational docking was able to determine how the mutations shaped the active site to improve the proximity of the 3-FBA substrate relative to the enamine-TPP intermediate, formed after the initial reaction with pyruvate. This work opens the way for small libraries to rapidly reprogramme enzyme active sites in a plug and play approach to catalyse new combinations of two-substrate reactions.

Comparative immunohistochemical analysis of WT-1, Syndecan and Snail in Ameloblastoma and odontogenic keratocyst: A retrospective study.

Background: The purpose of this experimental study was to evaluate and compare the degree of expression of Wilm's Tumor Gene-1 (WT-1), Syndecan (CD 138) and Snail in Ameloblastoma and odontogenic keratocyst (OKC) and to analyse their potential role in pathogenesis. Methods and Material: Immunohistochemical analysis was performed to evaluate WT-1, Syndecan and Snail expression in Ameloblastoma (n = 20) and OKC (n = 20). Topographical immunoexpression pattern of Ameloblast-like cells, Stellate Reticulum-like cells in Ameloblastoma and basal layer as well as suprabasal layer of cells of OKC were also compared. The results obtained were subjected to ANOVA test and Tukey HSD test through SPSS software 20.0 for Microsoft Windows. Results: WT-1 and Snail overexpression was seen in both Ameloblastoma and OKCs. Syndecan, responsible for maintaining normal cellular morphology, cell-cell adhesion and differentiation was significantly downregulated in both the lesions. The Ameloblasts-like cells and the basal cells showed significantly higher immunopositivity for WT-1 and Syndecan as compared to that of basal cells. An inverse relation was noted for Snail protein. The ANOVA test predicted a statistically significant difference of expression across the lesions with a P value <0.0001 for Syndecan and Snail. Conclusions: The under-expression of epithelial membrane protein Syndecan-1 and upregulation of EMT transcription factor Snail can promote local invasion and is indicative of poor prognosis of these lesions. The overexpression of WT-1 results in tumorigenesis, proliferation and localized aggressiveness of Ameloblastoma and intrabony growth of OKC. Further investigation on the biologic behaviour of OKC is still recommended to arrive at more specific conclusions regarding its nature.

Implications of ß-thalassemia on oral health status in patients: A cross-sectional study.

Background: ß-Thalassemia is a chronic disease of autosomal recessive origin that is identified by the presence of a severe form of anemia. This hematological disease has been shown to directly influence a person's physical as well as psychological well-being along with their families. Aim: This study aimed to find an association between dental health status and oral health-related quality of life (QoL) among children who have been diagnosed with ß-thalassemia. Materials and Methods: This prospective cross-sectional study was carried out in the dental outpatient department; blood bank and pediatric outpatient departments that were associated with the primary institute. All study participants were age-ranged from 3 to 15 years. Informed written consent was obtained from caregivers or parents of all the study participants. This study was conducted for a total duration of 1 year (from June 2020 to June 2021). All study participants were categorized into two groups: (a) Group I (n = 150) comprised children who were diagnosed with ß-thalassemia and (b) Group II (n = 150) comprised normal controls. Exclusion criteria in the study included children suffering from any systemic disease that predisposes them to dental caries or periodontal diseases. The intra-oral examination was performed using Decayed-Missing-Filled Teeth Index (DMFT/dmft Index) and Oral Hygiene Index-Simplified (OHI-S). Assessment of QoL was done by using the "Child Perceptions Questionnaire for children." Collected data were recorded in Microsoft Excel workbook, 2007. Statistical comparison between both the groups was performed by using statistical tools such as the Chi-square test, Fisher's exact test, independent t-test, and Mann-Whitney U test. The probability values lesser than 0.05 were considered to be statistically significant. Results: Maxillofacial findings-rodent facies, saddle nose, lip incompetence, pale oral mucosa, anterior open-bite, lower anterior teeth crowding, and maxillary anterior teeth spacing or crowding-were seen. Class II malocclusion was present in significant numbers of subjects. On comparing dmft/DMFT scores, no significance was observed while on comparing OHI-S index, statistical significance was seen. A statistically significant difference in the QoL was noted between thalassemic children and the control group. Conclusion: Thalassemic children showed a significant association between dental health and QoL.

Inner-View of Nanomaterial Incited Protein Conformational Changes: Insights into Designable Interaction.

Nanoparticle bioreactivity critically depends upon interaction between proteins and nanomaterials (NM). The formation of the "protein corona" (PC) is the effect of such nanoprotein interactions. PC has a wide usage in pharmaceuticals, drug delivery, medicine, and industrial biotechnology. Therefore, a detailed in-vitro, in-vivo, and in-silico understanding of nanoprotein interaction is fundamental and has a genuine contemporary appeal. NM surfaces can modify the protein conformation during interaction, or NMs themselves can lead to self-aggregations. Both phenomena can change the whole downstream bioreactivity of the concerned nanosystem. The main aim of this review is to understand the mechanistic view of NM-protein interaction and recapitulate the underlying physical chemistry behind the formation of such complicated macromolecular assemblies, to provide a critical overview of the different models describing NM induced structural and functional modification of proteins. The review also attempts to point out the current limitation in understanding the field and highlights the future scopes, involving a plausible proposition of how artificial intelligence could be aided to explore such systems for the prediction and directed design of the desired NM-protein interactions.

Nanomaterials in the Pharmaceuticals: Occurrence, Behaviour and Applications.

Nanoparticles are an emerging class of multi-functional materials defined by size-dependent properties. The ever-growing interest in nanotechnology research has been attributed to the significant properties and parameters of the nanoparticles that make them more versatile than their bulk counter parts that are expected to have large spectrum impact on existing technologies such as drug delivery, biomedical, therapeutics, healthcare and pharmaceutical. The main objectives of this review are to study the impact, occurrence and behaviour of nanomaterials and their applications in pharmaceuticals. Nano-approaches are being explored globally to enhance bioavailability of drugs and diagnostics. This is due to the extremely small size and large specific surface areas of the nanoparticles that may interact directly with cellular system. Biomacromolecule surface recognition by nanoparticles acts as artificial receptors, which provide a potential tool for controlling cellular and extracellular processes for numerous biological applications such as diagnostics, therapy, drug delivery and biosensing. This review article is expected to broaden our understanding towards the development of nanoparticles based diagnostics, combination therapies in treating different diseases and other pharmaceutical applications.

Enhanced functionality and stabilization of a cold active laccase using nanotechnology based activation-immobilization.

A simple nanotechnology based immobilization technique for imparting psychrostability and enhanced activity to a psychrophilic laccase has been described here. Laccase from a psychrophile was supplemented with Copper oxide nanoparticles (NP) corresponding to copper (NP-laccase), the cationic activator of this enzyme and entrapped in single walled nanotube (SWNT). The activity and stability of laccase was enhanced both at temperatures as low as 4°C and as high as 80°C in presence of NP and SWNT. The enzyme could be released and re-trapped (in SWNT) multiple times while retaining significant activity. Laccase, immobilized in SWNT, retained its activity after repeated freezing and thawing. This unique capability of SWNT to activate and stabilize cold active enzymes at temperatures much lower or higher than their optimal range may be utilized for processes that require bio-conversion at low temperatures while allowing for shifts to higher temperature if so required.

Improved production of reducing sugars from rice husk and rice straw using bacterial cellulase and xylanase activated with hydroxyapatite nanoparticles.

Purified bacterial cellulase and xylanase were activated in the presence of calcium hydroxyapatite nanoparticles (NP) with concomitant increase in thermostability about 35% increment in production of d-xylose and reducing sugars from rice husk and rice straw was obtained at 80°C by the sequential treatment of xylanase and cellulase enzymes in the presence of NP compared to the untreated enzyme sets. Our findings suggested that if the rice husk and the rice straw samples were pre-treated with xylanase prior to treatment with cellulase, the percentage increase of reducing sugar per 100g of substrate (starting material) was enhanced by about 29% and 41%, respectively. These findings can be utilized for the extraction of reducing sugars from cellulose and xylan containing waste material. The purely enzymatic extraction procedure can be substituted for the harsh and bio-adverse chemical methods.

Nanotechnology enabled enhancement of enzyme activity and thermostability: study on impaired pectate lyase from attenuated Macrophomina phaseolina in presence of hydroxyapatite nanoparticle.

In this paper we show that hydroxyapatite nanoparticles (NP) can not only act as a chaperon (by imparting thermostability) but can serve as a synthetic enhancer of activity of an isolated extracellular pectate lyase (APL) with low native state activity. The purified enzyme (an attenuated strain of Macrophomina phaseolina) showed feeble activity at 50°C and pH 5.6. However, on addition of 10.5 µg/ml of hydroxyapatite nanoparticles (NP), APL activity increased 27.7 fold with a 51 fold increase in half-life at a temperature of 90°C as compared to untreated APL. The chaperon like activity of NP was evident from entropy-enthalpy compensation profile of APL. The upper critical temperature for such compensation was elevated from 50°C to 90°C in presence of NP. This dual role of NP in enhancing activity and conferring thermostability to a functionally impaired enzyme is reported for the first time.

Degumming of ramie fiber and the production of reducing sugars from waste peels using nanoparticle supplemented pectate lyase.

Banana, citrus and potato peels were subjected to treatment with hydroxyapatite nanoparticle (NP) supplemented purified pectate lyase (NP-PL), isolated from Bacillus megaterium AK2 to produce reducing sugar (RS). At both 50 and 90°C production of RS by NP-PL was almost twofold greater than that by untreated pectate lyase (PL) from each of the three peels. The optimal production of RS from banana and citrus peels were after 24 and 6h of incubation while it was 24 and 4h for potato peels at 50 and 90°C, respectively, on NP-PL treatment. NP-PL could degum raw, decorticated ramie fibers as well as enhance fiber tenacity and fineness. The weight loss of the fibers were 24% and 31% better (compared to PL treatment) after 24 and 48 h of processing. These findings have potential implications for the bio-ethanol, bio-fuel and textile industries.

Thermostability, pH stability and dye degrading activity of a bacterial laccase are enhanced in the presence of Cu2O nanoparticles.

The present study relates to a nanotechnology enabled method in which purified laccase from Escherichia coli AKL2 was supplemented with 100 µM copper oxide nanoparticles (Cu(2)O) (NP-laccase). The activity, half life and stability of NP-laccase were enhanced by 4, 42 and 36-fold respectively at high temperature (80 °C) and also over a wide range of pH (4-12) than laccase (in the presence of 0.18 mM CuSO(4)). Thermodynamic analysis of the nanoparticle-induced enzyme stability revealed an enhanced entropy-enthalpy compensation at 80 °C, which reflected the maintenance of its native structure. This was further supported by CD studies. The enhanced activity and thermostability of NP-laccase can be utilized for efficient decolorisation of dyes (both phenolic and azo).

Improvement of thermostability and activity of pectate lyase in the presence of hydroxyapatite nanoparticles.

The activity and half-life of pectate lyase (PL) from Bacillus megaterium were nine- and 60-fold, respectively, higher at 90 °C in the presence of hydroxyapatite nanoparticles (NP-PLs) than in the presence of 1mM CaCl(2). Thermodynamic analysis of the nanoparticle-induced stability revealed an enhanced entropy-enthalpy compensation by the NP-PLs since a reciprocal linearity of the enthalpy-entropy change to 90 °C was observed. Without nanoparticles, the linearity range was 70 °C. Such compensation reflected the maintenance of the native structure of proteins. The remarkable enhancement of activity and stability of the NP-PL system at high temperatures may be utilized commercially e.g. in the food industry or the processing of natural fibers that may require a thermotolerant enzyme.