Double edge resolving set and exchange property for nanosheet structure.

The exploration of edge metric dimension and its applications has been an ongoing discussion, particularly in the context of nanosheet graphs formed from the octagonal grid. Edge metric dimension is a concept that involves uniquely identifying the entire edge set of a structure with a selected subset from the vertex set, known as the edge resolving set. Let's consider two distinct edge resolving sets, denoted as Re1 and Re2, where Re1≠Re2. In such instances, it indicates that the graph G possesses a double-edge resolving set. This implies the existence of two different subsets of the vertex set, each capable of uniquely identifying the entire edge set of the graph. In this article, we delve into the edge metric dimension of nanosheet graphs derived from the octagonal grid. Additionally, we initiate a discussion on the exchange property associated with the edge resolving set. The exchange property holds significance in the study of resolving sets, playing a crucial role in comprehending the structure and properties of the underlying graph.

The Characterization and Study of Antibacterial, Free Radical Scavenging, and Anticancer Potential of Livistona chinensis-Mediated Silver Nanoparticles.

In the present research, Livistona chinensis leaf extracts were utilized as reductants to bio-fabricate silver nanoparticles (LC-AgNPs) and this was followed by the evaluation of their antioxidant, antibacterial, and anticancer potential. Multiple parameters were optimized for the formation and fidelity of LC-AgNPs. The color shift of the reaction mixture from yellow to dark brown confirmed the LC-AgNPs formation. UV/VIS spectroscopy exhibited a surface plasmon resonance (SPR) band at 436 nm. The Fourier transform infrared (FTIR) spectroscopy spectrum depicted phytochemicals in the plant extract acting as bio-reducers for LC-AgNPs synthesis. The XRD pattern confirmed the presence of LC-AgNPs by showing peaks corresponding to 2θ angle at 8.24° (111), 38.16° (200), 44.20° (220), and 64.72° (311). Zetasizer analysis exhibited size distribution by intensity of LC-AgNPs with a mean value of 255.7 d. nm. Moreover, the zeta potential indicated that the AgNPs synthesized were stable. The irregular shape of LC-AgNPs with a mean average of 38.46 ± 0.26 nm was found by scanning electron microscopy. Furthermore, the antioxidant potential of LC-AgNPs was examined using a DPPH assay and was calculated to be higher in LC-AgNPs than in leaf extracts. The calculated IC50 values of the LC-AgNPs and plant extract are 85.01 ± 0.17 and 209.44 ± 0.24, respectively. The antibacterial activity of LC-AgNPs was investigated against Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis as well as Staphylococcus aureus, and maximum potential was observed after 24 h against P. aeruginosa. Moreover, LC-AgNPs exhibited maximum anticancer potential against TPC1 cell lines compared to the plant extract. The findings suggested that LC-AgNPs could be used as antioxidant, antibacterial, and anticancer agents for the cure of free-radical-oriented bacterial and oncogenic diseases.

Immobilization of Aspergillus oryzae tyrosine hydroxylase on ZnO nanocrystals for improved stability and catalytic efficiency towards L-dopa production.

The current study focuses on the submerged fermentation of tyrosine hydroxylase (TH) from Aspergillus oryzae IIB-9 and its immobilization on zinc oxide nanocrystals (ZnO-NPs) for increased L-dopa production. The volume of Vogel's medium (75 ml), period of incubation (72 h), initial pH (5.5), and size of inoculum (1.5 ml) were optimal for maximum TH activity. The watch glass-dried (WG) and filter paper-dried (FP) ZnO-NPs were prepared and characterized using analytical techniques. The UV-Vis spectra revealed 295 and 285 nm absorption peaks for WG-ZnO-NPs and FP-ZnO-NPs dispersed in isopropanol. X-ray diffraction analysis confirmed the crystalline nature of ZnO-NPs. FTIR spectra band from 740 to 648.1/cm and 735.8/cm to 650.1/cm showed the stretching vibrations of WG-ZnO-NPs and FP-ZnO-NPs, respectively. The particle size of ZnO-NPs observed by scanning electron microscopy (SEM) images was between 130 and 170 nm. Furthermore, the stability of immobilized TH on ZnO-NPs was determined by varying the incubation period (10 min for WG-NPs and 15 min for FP-NPs) and temperature (45 °C and 30 °C for WG and FP-NPs, respectively). Incubating enzymes with various copper, iron, manganese, and zinc salts studied the catalytic efficiency of TH. Immobilization of TH on ZnO-NPs resulted in an 11.05-fold increase in TH activity, thus enhancing stability and catalytic efficiency.

In Vitro Biofilm-Mediated Biodegradation of Pesticides and Dye-Contaminated Effluents Using Bacterial Biofilms.

Overuse of pesticides in agricultural soil and dye-polluted effluents severely contaminates the environment and is toxic to animals and humans making their removal from the environment essential. The present study aimed to assess the biodegradation of pesticides (cypermethrin (CYP) and imidacloprid (IMI)), and dyes (malachite green (MG) and Congo red (CR)) using biofilms of bacteria isolated from pesticide-contaminated soil and dye effluents. Biofilms of indigenous bacteria, i.e., Bacillus thuringiensis 2A (OP554568), Enterobacter hormaechei 4A (OP723332), Bacillus sp. 5A (OP586601), and Bacillus cereus 6B (OP586602) individually and in mixed culture were tested against CYP and IMI. Biofilms of indigenous bacteria i.e., Lysinibacillus sphaericus AF1 (OP589134), Bacillus sp. CF3 (OP589135) and Bacillus sp. DF4 (OP589136) individually and in mixed culture were tested for their ability to degrade dyes. The biofilm of a mixed culture of B. thuringiensis + Bacillus sp. (P7) showed 46.2% degradation of CYP compared to the biofilm of a mixed culture of B. thuringiensis + E. hormaechei + Bacillus sp. + B. cereus (P11), which showed significantly high degradation (70.0%) of IMI. Regarding dye biodegradation, a mixed culture biofilm of Bacillus sp. + Bacillus sp. (D6) showed 86.76% degradation of MG, which was significantly high compared to a mixed culture biofilm of L. sphaericus + Bacillus sp. (D4) that degraded only 30.78% of CR. UV-VIS spectroscopy revealed major peaks at 224 nm, 263 nm, 581 nm and 436 nm for CYP, IMI, MG and CR, respectively, which completely disappeared after treatment with bacterial biofilms. Fourier transform infrared (FTIR) analysis showed the appearance of new peaks in degraded metabolites and disappearance of a peak in the control spectrum after biofilm treatment. Thin layer chromatography (TLC) analysis also confirmed the degradation of CYP, IMI, MG and CR into several metabolites compared to the control. The present study demonstrates the biodegradation potential of biofilm-forming bacteria isolated from pesticide-polluted soil and dye effluents against pesticides and dyes. This is the first report demonstrating biofilm-mediated bio-degradation of CYP, IMI, MG and CR utilizing soil and effluent bacterial flora from Multan and Sheikhupura, Punjab, Pakistan.

Diversity and Abundance of Delineated Earthworm (Annelida: Clitellata) in Pakistan: A Review.

Earthworm, a ubiquitous (but neglected) macro-invertebrate, is found in terrestrial vicinity of Pakistan. Moreover, the occurrence of earthworms is often diverse with fluctuating quantity depending upon abiotic factors and land usage patterns. The aim of this study was to summarize all the reported information related to earthworm diversity in different areas of Pakistan. Almost all the data published from year 2001 to 2021 were collected. Following data organization, total 42 earthworm's species including five families (Acanthodrilidae, Lumbricidae, Moniligastridae, Octochaetidae and Megascolecidae) were reported from various researchers. Among five families, family Acanthodrilidae was found to have only one specie (Ramiella bishambari), Lumbricidae consist of 10 species (Apporactodea rosea, Allolobophora trapezoids, Allolobophora chloroticaa, Aporrectodea longa, A. caliginosa, Bimastus parvus, Eisenia fetida, Helodrilus foetidus, Lumbricus terrestris and L. rubillus), Moniligastridae has two species (Drawida nepalensis and D. pellucida) while Octochaetidae possess only one specie (Eutyphoeus incommodus). The most abundant and diverse family Megascolecidae consist of 28 earthworm species in all habitats of different regions of Pakistan. Among geographical areas, Faisalabad was found as the richest territory with most reported earthworm species (i.e. 28). The current study suggests further in depth research to explore the unidentified and/ missing species of earthworms in Pakistan.

Bacillus licheniformis (MN900686) Mediated Synthesis, Characterization and Antimicrobial Potential of Silver Nanoparticles.

The use of bacteria in the synthesis of silver nanoparticles (AgNPs) emerges as an ecofriendly and exciting approach. In the present study, we reported the biosynthesis of AgNPs by using culture supernatant of the bacteria Bacillus licheniformis (MN900686). The biogenically synthesized AgNPs were confirmed by the change in the color of the culture filtrate from yellow to brown after the addition of AgNO3. Further characterization performed by means of UV vis-spectroscopy showed absorption peak at 414 nm which confirmed the formation of AgNPs. Fourier Transfer infrared (FTIR) confirmed the involvement of biological molecules in the formation of nanoparticles (NPs). The SEM revealed that the NPs have approximately 38 nm size. The agar well diffusion assay was used to determine antibacterial activity while tube dilution method was used to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The human pathogenic bacterial strains i.e., P. aeruginosa (MN900691) and B. subtilis (MN900684), were used as test strains. The anti-bacterial assay against test strains revealed that these NPs showed concentration dependent increased zone of inhibition (ZOI). The maximum ZOI at 25 µL of AgNPs was 20 mm against B. subtilis after 24 hours of incubation. One-way ANOVA test showed significant ZOI (p ≤ 0.05) against B. subtilis. The MIC was ranged from 4.3-6.6 µg/mL while MBC ranged from 8.3 to 6.6 µg/mL. Overall, this study suggested that the biogenically synthesized NPs are an effective alternative source of antimicrobials against pathogenic bacteria.

Purification and Characterization of Keratinase from Bacillus licheniformis dcs1 for Poultry Waste Processing.

Feather wastes-byproduct of commercial poultry processing plant is produced in large amounts. Keratinolytic enzymes produced by feather degrading bacteria can easily degrade these waste products releasing pure keratin as a residue. The aim of present study was to isolate, and characterize feather degrading bacteria as well as assess the keratinolytic potential of purified enzyme. Three feather degrading bacteria (dps3, wps1 and dcs1) were isolated from feathers of domestic chickens. Preliminary characterization of isolated bacteria revealed these isolates belonging to genus Bacillus. 16S rRNA gene sequencing identified the isolates as B. subtilis dps3 (MW255302), B. cereus wps1 (MW255303) and B. licheniformis dcs1 (MW255304). Cell free supernatant of B. licheniformis dcs1 degraded feathers completely in 14 days indicating its keratinolytic ability. Purification of keratinase enzyme from B. licheniformis dcs1 was performed using column chromatography. SDS-PAGE indicated its molecular weight as 32 KDa. Kerotinolytice activity was maximum at optimum pH of 7 and 45℃ temperature. Enzyme showed the potential to degrade keratin material such as hairs and nails of humans. Findings of current study suggested that purified enzyme possess potential to upgrade nutritional quality of poultry waste containing keratin and might play as important biotechnological tool for keratin hydrolysis.

Effective utilization of magnetic nano-coupled cloned ß-xylanase in saccharification process.

The ß-xylanase gene (DCE06_04615) with 1041 bp cloned from Thermotoga naphthophila was expressed into E. coli BL21 DE3. The cloned ß-xylanase was covalently bound to iron oxide magnetic nanoparticles coated with silica utilizing carbodiimide. The size of the immobilized MNPs (50 nm) and their binding with ß-xylanase were characterized by Fourier-transform electron microscopy (FTIR) (a change in shift particularly from C-O to C-N) and transmission electron microscopy (TEM) (spherical in shape and 50 nm in diameter). The results showed that enzyme activity (4.5 ± 0.23 U per mL), thermo-stability (90 °C after 4 hours, residual activity of enzyme calculated as 29.89% ± 0.72), pH stability (91% ± 1.91 at pH 7), metal ion stability (57% ± 1.08 increase with Ca2+), reusability (13 times) and storage stability (96 days storage at 4 °C) of the immobilized ß-xylanase was effective and superior. The immobilized ß-xylanase exhibited maximal enzyme activity at pH 7 and 90 °C. Repeated enzyme assay and saccharification of pretreated rice straw showed that the MNP-enzyme complex exhibited 56% ± 0.76 and 11% ± 0.56 residual activity after 8 times and 13 times repeated usage. The MNP-enzyme complex showed 17.32% and 15.52% saccharification percentage after 1st and 8th time usage respectively. Immobilized ß-xylanase exhibited 96% residual activity on 96 days' storage at 4 °C that showed excellent stability.

Indigenous Bacillus paramycoides spp. and Alcaligenes faecalis: sustainable solution for bioremediation of hospital wastewater.

Farmers near towns and cities are using a wide range of highly polluted wastewaters for crop irrigation in Pakistan due to severe freshwater shortage. The present study aimed to promote indigenous bacterial strains isolated from domestic, hospital, textile, pharmaceutical and mixed wastewaters to remove contaminants and colour and render these wastewaters safer for irrigation. Thirty seven bacterial strains were isolated from five wastewater samples collected from different sites in Lahore, Pakistan. Under optimal growth conditions, three isolates (D6, D7 and P1) showed >93% decolourisation potential in the treatment of hospital wastewater. 16S rDNA sequencing identified two of these isolates (D6 and D7) as showing 100% and 99.86% homology to Bacillus paramycoides spp. - novel strains from B. cereus group. Isolate P1 showed 97.47% homology to Alcaligenes faecalis. GCMS analysis of the untreated hospital wastewater revealed the presence of pharmaceutic pollutants, i.e. Phenol (876 µg/L), Salicylic acid (48 µg/L), Caffeine (7 µg/L), Naproxen (23 µg/L), Octadecene (185 µg/L) and Diazepam (14 µg/L). The analysis of treated hospital wastewaters showed percentage degradation of pharmaceutic pollutants (100%-43%) and significant reduction in the BOD5 (91%-68%), COD (89%-52%) and heavy metals concentrations. These strains therefore can represent a low-cost and low-tech alternative to bioremediate complex matrices of hospital wastewater prior to crop irrigation to support the achievement of clean re-usable water in developing countries like Pakistan.

Hospital wastewater treated with a novel bacterial consortium (Alcaligenes faecalis and Bacillus paramycoides spp.) for phytotoxicity reduction in Berseem clover and tomato crops.

Hospital wastewaters are produced in large volumes in Pakistan (∼362-745 L/bed.day) and are discharged without proper treatment. They are widely used by farmers for crop irrigation and induce a phytotoxic effect on plant growth. The study was conducted to evaluate the effect of untreated and treated hospital wastewater on seed germination of a fodder crop Trifolium alexandrinum (Berseem clover) and a food crop Solanum lycopersicum (tomato). A bacterial consortium was formed with three bacterial strains, i.e., Alcaligenes faecalis and Bacillus paramycoides spp., which were individually proven efficient in previous studies. The concentrations of untreated and treated hospital wastewater (25, 50, 75 and 100%) were used to irrigate these crop seeds. To assess the efficiency of treatment, the germination percentage, delay index, germination index, stress tolerance indices, seedling vigour index and phytotoxicity index were calculated and were statistically proven significant. The seeds grown in treated wastewater concentrations showed negative values of phytotoxicity indices (tomato: -0.36, -0.47, -0.78 and -1.11; Berseem clover: -0.23) which indicate a stimulatory or non-toxic effect on seedling growth. Our work proposes that this bacterial consortium is efficient for hospital wastewater treatment before crop irrigation.

An Artificial Intelligence-Based, Personalized Smartphone App to Improve Childhood Immunization Coverage and Timelines Among Children in Pakistan: Protocol for a Randomized Controlled Trial.

BACKGROUND: The immunization uptake rates in Pakistan are much lower than desired. Major reasons include lack of awareness, parental forgetfulness regarding schedules, and misinformation regarding vaccines. In light of the COVID-19 pandemic and distancing measures, routine childhood immunization (RCI) coverage has been adversely affected, as caregivers avoid tertiary care hospitals or primary health centers. Innovative and cost-effective measures must be taken to understand and deal with the issue of low immunization rates. However, only a few smartphone-based interventions have been carried out in low- and middle-income countries (LMICs) to improve RCI. OBJECTIVE: The primary objectives of this study are to evaluate whether a personalized mobile app can improve children's on-time visits at 10 and 14 weeks of age for RCI as compared with standard care and to determine whether an artificial intelligence model can be incorporated into the app. Secondary objectives are to determine the perceptions and attitudes of caregivers regarding childhood vaccinations and to understand the factors that might influence the effect of a mobile phone-based app on vaccination improvement. METHODS: A mixed methods randomized controlled trial was designed with intervention and control arms. The study will be conducted at the Aga Khan University Hospital vaccination center. Caregivers of newborns or infants visiting the center for their children's 6-week vaccination will be recruited. The intervention arm will have access to a smartphone app with text, voice, video, and pictorial messages regarding RCI. This app will be developed based on the findings of the pretrial qualitative component of the study, in addition to no-show study findings, which will explore caregivers' perceptions about RCI and a mobile phone-based app in improving RCI coverage. RESULTS: Pretrial qualitative in-depth interviews were conducted in February 2020. Enrollment of study participants for the randomized controlled trial is in process. Study exit interviews will be conducted at the 14-week immunization visits, provided the caregivers visit the immunization facility at that time, or over the phone when the children are 18 weeks of age. CONCLUSIONS: This study will generate useful insights into the feasibility, acceptability, and usability of an Android-based smartphone app for improving RCI in Pakistan and in LMICs. TRIAL REGISTRATION: ClinicalTrials.gov NCT04449107; https://clinicaltrials.gov/ct2/show/NCT04449107. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/22996.

Current Challenges of Digital Health Interventions in Pakistan: Mixed Methods Analysis.

BACKGROUND: Digital health is well-positioned in low and middle-income countries (LMICs) to revolutionize health care due, in part, to increasing mobile phone access and internet connectivity. This paper evaluates the underlying factors that can potentially facilitate or hinder the progress of digital health in Pakistan. OBJECTIVE: The objective of this study is to identify the current digital health projects and studies being carried out in Pakistan, as well as the key stakeholders involved in these initiatives. We aim to follow a mixed-methods strategy and to evaluate these projects and studies through a strengths, weaknesses, opportunities, and threats (SWOT) analysis to identify the internal and external factors that can potentially facilitate or hinder the progress of digital health in Pakistan. METHODS: This study aims to evaluate digital health projects carried out in the last 5 years in Pakistan with mixed methods. The qualitative and quantitative data obtained from field surveys were categorized according to the World Health Organization's (WHO) recommended building blocks for health systems research, and the data were analyzed using a SWOT analysis strategy. RESULTS: Of the digital health projects carried out in the last 5 years in Pakistan, 51 are studied. Of these projects, 46% (23/51) used technology for conducting research, 30% (15/51) used technology for implementation, and 12% (6/51) used technology for app development. The health domains targeted were general health (23/51, 46%), immunization (13/51, 26%), and diagnostics (5/51, 10%). Smartphones and devices were used in 55% (28/51) of the interventions, and 59% (30/51) of projects included plans for scaling up. Artificial intelligence (AI) or machine learning (ML) was used in 31% (16/51) of projects, and 74% (38/51) of interventions were being evaluated. The barriers faced by developers during the implementation phase included the populations' inability to use the technology or mobile phones in 21% (11/51) of projects, costs in 16% (8/51) of projects, and privacy concerns in 12% (6/51) of projects. CONCLUSIONS: We conclude that while digital health has a promising future in Pakistan, it is still in its infancy at the time of this study. However, due to the coronavirus disease 2019 (COVID-19) pandemic, there is an increase in demand for digital health and implementation of health outcomes following global social distancing protocols, especially in LMICs. Hence, there is a need for active involvement by public and private organizations to regulate, mobilize, and expand the digital health sector for the improvement of health care systems in countries.

Review: Warburg effect and renal cancer caused by errs in fumarate hydratase encoding gene.

The several types of heterogeneous kidney cancers are interrelated by their primary sites of pathology. Despite its origin in the kidney, renal cell carcinoma (RCC) is associated with its varying genetic basis. Von Hippel-Lindau (VHL) syndrome is the earliest and, thus the most highly, characterized of genetic forms of kidney cancer, which is associated with alterations in the Von Hippel-Lindau (VHL) gene. As a result of his studies and investigations, Otto Warburg reached the conclusion that cancer's fundamental cause is altered mechanism. But this theory was disdained because of the discovery of tumor suppressor genes and oncogenes. Lately, the breakthrough finding about the tumor suppressing role of gene coding for enzymes involved in Krebs cycle has revived the interest in Warburg's hypothesis. This effect has led to the uncovering of the links between metabolic alterations, mitochondrial dysfunction and cancer. One such metastatic cancer characterized by the germ-line inactivating mutation of the gene coding for fumarate hydratase (FH), a Krebs cycle's enzyme, is hereditary leiomyomatosis and renal cell carcinoma (HLRCC). In this review paper, we have discussed the background of this carcinoma, the metabolic dysfunction causing it and its therapeutic solutions.

Fungal biotransformation of synthetic levodopa to stable dopamine in l-ascorbate-mediated aerobic-thermophilic biochemical process.

In the present study, fungal biotransformation of synthetic levodopa to stable dopamine in an l-ascorbate-mediated thermophilic-aerobic biochemical reaction was investigated. A mutant strain of Aspergillus oryzae EMS-6 was used for the preparation of mycelial biomass. The mutant was previously developed through EMS-induced mutagenesis and repressed against l-cysteine HCl. Growth parameters such as rate of cultivation (48 h), initial pH (6) and incubation temperature (30 °C) supported 18.84 g/l biomass with 23 g/l glucose consumption. Thermophilic behaviour of culture was observed at 25-40 °C. Kinetic variables notably µ = 0.385 /h and Qs, exhibited consistent growth pattern. Biochemical reactions were performed aerobically using mycelial biomass as the source of enzyme 'tyrosinase' in a digital hotplate equipped with magnetic stirrers. The reaction conditions included 5 mg/ml biomass and 2.5 mg/ml levodopa as basal substrate in a thermophilic reaction of 25 min duration acidified with l-ascorbic acid. TLC and HPLC analysis of reaction mixture confirmed the presence of levodopa and dopamine using a CN-9dth (R) column. Activation enthalpy and entropy of dopa decarboxylase (DDC) and its thermal inactivation showed an improved biotransformation of levodopa to dopamine at the optimal temperature (30 °C) as compared to other temperatures being employed. Overall, 3.68 mg/ml dopamine (4.55 mg/ml proteins) synthesis from 2.38 mg/ml levodopa was accomplished. The enhancement in metabolic activity of the mutant strain is ~ 2.75-fold improved when compared to the unoptimized reaction conditions, which is highly significant (HS) indicating an eco-commercially viable (LSD ~ 0.412) bioprocess.

Isolation and Identification of Novel IndigenousBacterial Strain as a Low Cost Pectinase Source

ABSTRACT The present study was concerned with the searching of novel bacterial cultures from different samples for the lab scale production of pectinase. Keeping in view the increasing demand of pectinase specially in Faisalabad, an industrial city of Pakistan, isolation of new hyper producer bacterial strains locally is an easy and cheap way of getting the desirable products at low cost. Therefore, isolation of new strains for industrial enzyme production has been and will be remained a part of research every time. This method alone can also provide raw material for further research such as enzyme engineering or molecular directed evolution. For the identification of hyper producer strain colony PCR was done for 16S rRNA analysis. Reason to use the 16S rRNA for identification purpose is that the gene is fairly short and can be amplified quickly and easily. The bacterial isolate (sources of pectinase enzyme) was identified based on PCR amplification of 16S rRNA and for this purpose the amplified product was run in agarose gel against a known species of Bacillus licheniformis. The 16S rRNA sequencing confirmed the Bacillus status of the strain.

Optimisation of nutritional requirements for dopamine synthesis by calcium alginate-entrapped mutant strain of Aspergillus oryzae EMS-6.

The optimisation of nutritional requirements for dopamine (DA) synthesis by calcium alginate-entrapped mutant variant of Aspergillus oryzae EMS-6 using submerged fermentation technique was investigated. A total of 13 strains were isolated from soil. Isolate I-2 was selected as a better producer of DA and improved by exposing with ethyl methylsulphonate (EMS). EMS-6 was selected as it exhibited 43 µg/mL DA activity. The mutant variable was further treated with low levels of l-cysteine HCl to make it resistant against diversion and environmental stress. The conidiospores of mutant variant were entrapped in calcium alginate beads for stable product formation. EMS-6 gave maximum DA activity (124 µg/mL) when supplemented with 0.1% peptone and 0.2% sucrose, under optimised parameters viz. pH 3, temperature of 55 °C and incubation time of 70 min. The study involves the high profile of DA activity and is needed, as DA is capable to control numerous neurogenic disorders.

Biotransformation of L-tyrosine to Dopamine by a Calcium Alginate Immobilized Mutant Strain of Aspergillus oryzae.

The present research work is concerned with the biotransformation of L-tyrosine to dopamine (DA) by calcium alginate entrapped conidiospores of a mutant strain of Aspergillus oryzae. Different strains of A. oryzae were isolated from soil. Out of 13 isolated strains, isolate-2 (I-2) was found to be a better DA producer. The wild-type I-2 was chemically improved by treating it with different concentrations of ethyl methyl sulfonate (EMS). Among seven mutant variants, EMS-6 exhibiting maximal DA activity of 43 µg/ml was selected. The strain was further exposed with L-cysteine HCl to make it resistant against diversion and environmental stress. The conidiospores of selected mutant variant A. oryzae EMS-6 strain were entrapped in calcium alginate beads. Different parameters for immobilization were investigated. The activity was further improved from 44 to 62 µg/ml under optimized conditions (1.5 % sodium alginate, 2 ml inoculum, and 2 mm bead size). The best resistant mutant variable exhibited over threefold increase in DA activity (62 µg/ml) than did wild-type I-2 (21 µg/ml) in the reaction mixture. From the results presented in the study, it was observed that high titers of DA activity in vitro could effectively be achieved by the EMS-induced mutagenesis of filamentous fungus culture used.

Double mutation of Saccharomyces cerevisiae for enhanced β-D-fructofuranosidase fructohydrolase productivity and application of growth kinetics for parametric significance analysis

Abstract The kinetics of an extracellular β-D-fructofuranosidase fructohydrolase production by Saccharomyces cerevisiae in a chemically defined medium, i.e., sucrose peptone agar yeast extract at pH 6, was investigated. The wild-type was treated with a chemical mutagen, methyl methane sulfonate. Among the six mutants isolated, methyl methane sulfonate-V was found to be a better enzyme producing strain (52 ± 2.4a U/mL). The maximum production (74 ± 3.1a U/mL) was accomplished after at 48 h (68 ± 2.7a mg/mL protein). The mutants were stabilized at low levels of 5-fluoro-cytocine and the viable ones were further processed for optimization of cultural conditions and nutritional requirements. The sucrose concentration, incubation period and pH were optimized to be 30 g/L, 28 °C, and 6.5, respectively. The methyl methane sulfonate-V exhibited an improvement of over 10 folds in enzyme production when 5 g/L ammonium sulfate was used as a nitrogen source. Thin layer chromatography and high-performance liquid chromatography analysis illustrated the optimal enzyme activity supported by the higher rate of hydrolysis of sucrose into monosaccharides, particularly α-D-glucose and β-D-fructose. The values for Qp (2 ± 0.12c U/mL/h) and Yp/s (4 ± 1.24b U/g) of the mutant were considerably increased in comparison with other yeast strains (both isolates and viable mutants). The mutant could be exploited for enzyme production over a wider temperature range (26–34 °C), with significantly high enzyme activity (LSD 0.048, HS) at the optimal temperature.

Double mutation of Saccharomyces cerevisiae for enhanced ß-d-fructofuranosidase fructohydrolase productivity and application of growth kinetics for parametric significance analysis.

The kinetics of an extracellular ß-d-fructofuranosidase fructohydrolase production by Saccharomyces cerevisiae in a chemically defined medium, i.e., sucrose peptone agar yeast extract at pH 6, was investigated. The wild-type was treated with a chemical mutagen, methyl methane sulfonate. Among the six mutants isolated, methyl methane sulfonate-V was found to be a better enzyme producing strain (52±2.4(a)U/mL). The maximum production (74±3.1(a)U/mL) was accomplished after at 48h (68±2.7(a)mg/mL protein). The mutants were stabilized at low levels of 5-fluoro-cytocine and the viable ones were further processed for optimization of cultural conditions and nutritional requirements. The sucrose concentration, incubation period and pH were optimized to be 30g/L, 28°C, and 6.5, respectively. The methyl methane sulfonate-V exhibited an improvement of over 10 folds in enzyme production when 5g/L ammonium sulfate was used as a nitrogen source. Thin layer chromatography and high-performance liquid chromatography analysis illustrated the optimal enzyme activity supported by the higher rate of hydrolysis of sucrose into monosaccharides, particularly α-d-glucose and ß-d-fructose. The values for Qp (2±0.12(c)U/mL/h) and Yp/s (4±1.24(b)U/g) of the mutant were considerably increased in comparison with other yeast strains (both isolates and viable mutants). The mutant could be exploited for enzyme production over a wider temperature range (26-34°C), with significantly high enzyme activity (LSD 0.048, HS) at the optimal temperature.