Isolation and screening of l-asparaginase free of glutaminase and urease from fungal sp.

l-Asparaginase is a chemotherapeutic drug used in the treatment of acute lymphoblastic leukaemia (ALL), a malignant disorder in children. l-Asparaginase helps in removing acrylamide found in fried and baked foods that is carcinogenic in nature. l-Asparaginase is present in plants, animals and microbes. Various microorganisms such as bacteria, yeast and fungi are generally used for the production of l-asparaginase as it is difficult to obtain the same from plants and animals. l-Asparaginase from bacteria causes anaphylaxis and other abnormal sensitive reactions due to low specificity to asparagine. Toxicity and repression caused by bacterial l-asparaginase shifted focus to eukaryotic microorganisms such as fungi to improve the efficacy of l-asparaginase. Clinically available l-asparaginase has glutaminase and urease that may lead to side effects during treatment of ALL. Current work tested 45 fungal strains isolated from soil and agricultural residues. Isolated fungi were tested using conventional plate assay method with two indicator dyes, phenol red and bromothymol blue (BTB), and results were compared. l-Asparaginase activity was measured by cultivating in modified Czapek–Dox medium. Four strains have shown positive result for l-asparaginase production with no urease or glutaminase activity, among these C7 has high enzyme index of 1.57 and l-asparaginase activity of 33.59 U/mL. l-Asparaginase production by C7 was higher with glucose as carbon source and asparagine as nitrogen source. This is the first report focussing on fungi that can synthesize l-asparaginase of the desired specificity. Since the clinical toxicity of l-asparaginase is attributed to glutaminase and urease activity, available evidence indicates variants negative for glutaminase and urease would provide higher therapeutic index than variants positive for glutaminase and urease

Current trends in health-promoting potential and biomaterial applications of edible mushrooms for human wellness

Edible mushrooms are ubiquitous around the world due to their enormous health benefits. Mushrooms have been used as folk medicine and healthy food from ancient times but their health-promoting effects have not been explored. As a superfood, mushroom powder is an essential component of the human diet for improving health and immunity. Bioactive components present in them such as proteins, polysaccharides, terpenes, and lipids have recently sparked much attention to exhibit therapeutic properties such as anti-cancer, immunomodulatory, anti-hypercholesterolemia, antiviral, antidiabetic, and anti-inflammatory effects. Moreover, these isolated compounds have the potentiality to be used in dietary supplements and medicines. In addition, numerous bioactive compounds such as ergosterol, gallic acid, and cordycepin proved to be essential in preventing or reducing the severity of COVID-19. This review unveils a comprehensive understanding of the nutraceutical as well as the medicinal potential of mushrooms and their applications in food products for human wellness

Pharmacological and Clinical Importance of Integrin Antagonists in Treatment of Cancer

Integrins are heterodimeric molecules that are composed of 18 α -subunits and 8 β -subunits. They exist in 24 distinctive shapes based on combination of these sub-units and are mainly responsible for the adhesion of extracellular matrix (ECM) and immunoglobulin family molecules. Integrins mediate adhesion of epithelial cells to the basement membrane and also help in the migration, proliferation and survival of tumor cells. Studies also reveal that certain integrins act as markers for tumor cells and they also assist in both tumor progression and apoptosis. Studies reveal that unligated integrins in association with caspase 8 result in inhibition of ECM adhesion might result and integrin mediated death (IMD) on the other hand integrins in association with oncogenes or receptor tyrosine kinases can result in enhanced tumorigenesis. Among several types of integrins, αvβ 3 and α 5 β 1 have gained importance in anti-angiogenesis studie

Flexible ultra-sensitive and resistive NO2 gas sensor based on nanostructured Zn(x)Fe(1−x)2O4

Low concentration gas detection, rapid response time and low working temperature are anticipated for a varied range of toxic gas detection applications. Conversely, the existing gas sensors suffer mostly from a high working temperature along with a slow response at low concentrations of analytes. Here, we report an ultrasensitive flexible nanostructured Zn(x)Fe(1−x)2O4 (x = 0.1, 0.5 and 0.9) based chemiresistive sensor for nitrogen dioxide (NO2) detection. We evince that the prepared flexible sensor Zn(0.5)Fe(0.5)2O4 has detection potential as low as 5 ppm at a working temperature of 90 °C in a short phase. Further, the Zn(0.5)Fe(0.5)2O4 sensor exhibits excellent selectivity, stability and repeatability. The optimized sensor sensing characteristics can be helpful in tremendous development of foldable mobile devices for environmental monitoring, protection and control

Microbes Producing L-Asparaginase free of Glutaminase and Urease isolated from Extreme Locations of Antarctic Soil and Moss

L-Asparaginase (L-asparagine aminohydrolase, E.C. 3.5.1.1) has been proven to be competent in treating Acute Lymphoblastic Leukaemia (ALL), which is widely observed in paediatric and adult groups. Currently, clinical L-Asparaginase formulations are derived from bacterial sources such as Escherichia coli and Erwinia chrysanthemi. These formulations when administered to ALL patients lead to several immunological and hypersensitive reactions. Hence, additional purification steps are required to remove toxicity induced by the amalgamation of other enzymes like glutaminase and urease. Production of L-Asparaginase that is free of glutaminase and urease is a major area of research. In this paper, we report the screening and isolation of fungal species collected from the soil and mosses in the Schirmacher Hills, Dronning Maud Land, Antarctica, that produce L-Asparaginase free of glutaminase and urease. A total of 55 isolates were obtained from 33 environmental samples that were tested by conventional plate techniques using Phenol red and Bromothymol blue as indicators. Among the isolated fungi, 30 isolates showed L-Asparaginase free of glutaminase and urease. The L-Asparaginase producing strain Trichosporon asahii IBBLA1, which showed the highest zone index, was then optimized with a Taguchi design. Optimum enzyme activity of 20.57 U mL−1 was obtained at a temperature of 30 °C and pH of 7.0 after 60 hours. Our work suggests that isolation of fungi from extreme environments such as Antarctica may lead to an important advancement in therapeutic applications with fewer side effects

Optimization of solid substrate mixture and process parameters for the production of L-asparaginase and scale-up using tray bioreactor

L-asparaginase is a key enzyme that degrades asparagine and this aspect of the enzyme has found a dominant role in chemotherapeutic and food processing industry. The aim of the present study is to sequentially optimize L-asparaginase production from newly isolated Aspergillus sp. using agro-industrial residues as solid substrate. In the first stage, through simplex centroid design maximum L-asparaginase activity was observed using a ternary mixture of cotton seed cake (2/3), wheat bran (1/6), and red gram husk (1/6). In the next step, cultivation parameters such as pH, temperature and moisture content were optimized using Box-Behnken design. After 6 days of fermentation using optimized ternary mixture, maximum activity of 12.57 U/mL was obtained at temperature 35 °C, pH-8 and moisture content 70% (v/w). Optimized bounds were further translated to lab-scale tray bioreactor and L-asparaginase activity was found to be 5.41 and 6.67 U/mL in tray bioreactor with 500 g and 1000 g of substrate mixture respectively. L-asparaginase production increased 5 fold through mixture design and 1.3 times increase in enzyme activity was observed with Box-Behnken design. Present work signifies the importance of optimization in the bioprocess industry for complete understanding and evaluation of enzyme production

Studies on Production and Optimization of Glutaminase and Urease free L-Asparaginase using newly isolated Aspergillus tubingensis IBBL1

L-asparaginase is an amido-hydrolytic enzyme that degrades asparagine, an attribute that makes it as an anti-neoplastic agent and anti-acrylamide agent in therapeutic and food industry, respectively. Clinically available L-asparaginase has glutaminase and urease that may lead to side effects during treatment of Acute Lymphoblastic Leukemia (ALL). L- asparaginase from bacteria causes anaphylaxis and other abnormal sensitive reactions due to low specificity to asparagine. To overcome this, eukaryotic organisms such as fungi has been used for the production of L-asparaginase which is free of glutaminase and urease. For this study, 45 fungal isolates were subjected to screening, with a view to assess the isolates for their ability to utilize different substrates as a nitrogen source. Four isolates have shown the presence of L-asparaginase free of glutaminase and urease using semi-quntitative assay method. Among the four isolates strains C7 (Aspergillus sp.) has shown the highest zone index of 1.57. This strain was further identified as Aspergillus tubingensis IBBL1 strain at molecular level by ITS rRNA gene sequencing (GenBank: MH185806). These results suggest that this Aspergillus strain can be used successfully in developing a biological process for the degradation of L-asparagine. L-asparaginase can be produced either by solid state fermentation (SSF) or submerged fermentation (SmF). Fungal strain, C7, characterised as Aspergillus tubingensis IBBL1 which is an L-asparaginase (free of L-glutaminase and urease)- producing strain has shown highest enzyme activity of 36.1 U/mL with carbon source as glucose; asparagine as nitrogen source at inoculum volume of 5 x 107 cells/mL. Once parameters were optimized at SmF, subsequent studies were performed with SSF. SSF is preferred over submerged fermentation as it is cost effective, eco-friendly and it delivers high yield. In this work, sequential optimization of agricultural solid substrate mixture and process parameters for the production of L-asparaginase were studied using Aspergillus tubingensis IBBL1. In the first stage, through simplex centroid design maximum L-asparaginase activity was observed using a ternary mixture of cotton seed cake (2/3), wheat bran (1/6), and red gram husk (1/6). In the next step, cultivation parameters such as pH, temperature and moisture content were optimized using Box-Behnken design. After 6 days of fermentation using optimized ternary mixture, maximum activity of 34.9 U/gds was obtained at temperature of 35 °C, pH-8 and moisture content 70 %(v/w). The limited use of SSF is due to its disadvantage when considering the large scale extraction and purification of the enzyme. Scaling up of SSF bioreactor is difficult due to heat and mass transfer problem in heterogeneous system. Continuous efforts towards improving design and modeling characteristics led to the development of various bioreactors such as tray, packed bed, rotating drum and fluidized bed. By considering all the limitations of the existing bioreactors used for the SSF process, rotary bioreactor was designed as it can overcome these limitations. Additionally, L-asparaginase production was carried out using modified tray bioreactor and in-house designed and fabricated rotary bioreactor using previously optimized conditions by Aspergillus tubingensis IBBL1. Maximum L-asparaginase activity of 20.58 U/gds was observed using modified tray bioreactor with bed height of 1 cm and by applying intermittent mixing strategy at 120 h of fermentation. In case of in-house designed rotary bioreactor, L-asparaginase activity of 19.96 U/gds was obtained with 1.5 kg substrate loading, 3 min delay time of the drum and on-rotation for 30 sec. These findings demonstrate that in-house designed rotary bioreactor was easy to handle, and has shown comparable results with tray bioreactor. Correlation between growth dynamics and L-asparaginase production was explained by means of Leudeking-Piret equation. Kinetic profile of L-asparaginase production indicated that enzyme production is completely growth associated

Laboratory scale bioreactor studies on the production of l-asparaginase using Rhizopus microsporus IBBL-2 and Trichosporon asahii IBBLA1

L-asparaginase (L-asparagine aminohydrolase, E.C. 3.5.1.1, ASNase) is an enzyme that is used primarily for the production of anti-neoplastic to treat Acute Lymphoblastic Leukemia (ALL). Current formulations of the enzyme are highly bacterial in nature which have resulted in adverse reactions. The current paper focusses on production of glutaminase and urease free ASNase in a laboratory scale bioreactor using two fungal species and their efficiency in the scaling up process. The isolated species are Rhizopus microsporus IBBL-2 and Trichosporon asahii IBBLA1 isolated from wheat bran and Antarctic moss respectively. The experiments were conducted under different conditions known as initial, optimized and immobilized. Enzyme activity at these conditions for Rhizopus microsporus IBBL-2 were found to be 10.85, 13.56, 17.18 U mL−1 respectively and for Trichosporon asahii IBBLA1 were found to be 13.97, 19.53, 21.98 U mL−1 respectively in laboratory scale. The consistency of the scale-up data from the shake flask level to the laboratory scale level was found to be greater than 90% efficiency. It was also observed that aeration condition had an impact on the enzyme production with maximum enzyme activity of 13.48 U mL−1 for the Rhizopus microsporus IBBL-2 and 19.95 U mL−1 for Trichosporon asahii IBBLA1 being reported at an aeration rate of 1 Lpm. The above results proved that laboratory scale production of ASNase has shown consistency and efficiency with respect to the flask-scale studies

Fungal Lipase Production by Solid State Fermentation-An Overview

Importance of enzymes is ever-growing specifically microbial lipases which are of great industrial significance because of their applications in detergent, food, pharmaceutical, chemical and leather industry. Solid state fermentation (SSF) is an economical alternative for large scale production of enzymes that are produced by fungi. Therefore, production of lipases by solid state fermentation is a good and preferred option than submerged fermentation (SmF). The important factors in fermentation are carbon concentration, nitrogen concentration, pH, growth temperature, fermentation time and moisture content. This review mainly focuses on production of fungal lipase by solid state fermentation using various fungal strains, substrates and fermentation conditions. Enzyme characteristics, industrial application and assay methods of lipase, biomass estimation, enzyme extraction methods and engineering aspects of fermentation are also dealt with briefly. The main aim of the review is to give an overview of advancements in solid state fermentation for production of fungal lipase hithert

Solid state fermentation of mixed substrate for l -asparaginase production using tray and in-house designed rotary bioreactor

l-asparaginase cover a broad spectrum of industrial application like food, biosensor and chemotherapeutic drug. In the present work, l-asparaginase production was carried out using tray bioreactor and in-house designed, fabricated rotary bioreactor using previously optimized tri-substrate mixture using Aspergillus tubingensis IBBL1. l-asparaginase production by tray bioreactor was performed in temperature controlled chamber. Enzyme production was also performed in newly designed and fabricated rotary bioreactor at room temperature and effect of variables such as substrate loading, mixing events and particle size were evaluated. Maximum l-asparaginase activity of 20.58 U/gds was observed using tray bioreactor with bed height of 1 cm and by applying intermittent mixing strategy at 120 h of fermentation. In case of in-house designed rotary bioreactor, l-asparaginase activity of 19.96 U/gds was obtained with 1.5 kg substrate loading, 3 min delay time of the drum and on rotation for 30 s. These findings demonstrate that in-house designed rotary bioreactor was easy to handle, and has shown comparable results with tray bioreactor. At optimized conditions growth kinetics were studied in tray and rotary bioreactor using various growth model equations. Correlation between growth dynamics and l-asparaginase production was explained by means of Leudeking-Piret model. Results indicate that under the optimized conditions, l-asparaginase can be produced in bulk quantities