Displaying publications 1 - 20 of 41 in total

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  1. El Enshasy HA, Hatti-Kaul R
    Trends Biotechnol, 2013 Dec;31(12):668-77.
    PMID: 24125745 DOI: 10.1016/j.tibtech.2013.09.003
    For centuries, mushrooms have been used as food and medicine in different cultures. More recently, many bioactive compounds have been isolated from different types of mushrooms. Among these, immunomodulators have gained much interest based on the increasing growth of the immunotherapy sector. Mushroom immunomodulators are classified under four categories based on their chemical nature as: lectins, terpenoids, proteins, and polysaccharides. These compounds are produced naturally in mushrooms cultivated in greenhouses. For effective industrial production, cultivation is carried out in submerged culture to increase the bioactive compound yield, decrease the production time, and reduce the cost of downstream processing. This review provides a comprehensive overview on mushroom immunomodulators in terms of chemistry, industrial production, and applications in medical and nonmedical sectors.
  2. Eid AM, El-Enshasy HA, Aziz R, Elmarzugi NA
    Int J Nanomedicine, 2014;9:4685-95.
    PMID: 25336948 DOI: 10.2147/IJN.S66180
    There is an increasing trend among pharmaceutical industries to use natural bioactive materials as medicinal agents and to use new technologies such as self-nanoemulsifying systems. The solubility and bioavailability of poorly soluble drugs can be enhanced by self-nanoemulsifying systems. Swietenia oil is frequently used because of its antimicrobial, antimutagenic, and anticancer bioactive medical properties. This study was conducted to develop self-nanoemulsifying systems for Swietenia oil that will enhance the anti-inflammatory activity of the oil. The self-emulsifying systems developed for Swietenia oil in this study were constructed using ternary phase diagrams and contained the nonionic surfactants Labrasol(®), Tween 20, Capmul(®), and Labrafil(®). The effect of these surfactants on the formulation was examined. The mean droplet size of Swietenia oil as well as their distribution, appearance, viscosity, and spreading times were studied to find the optimum formula, which contained droplets that were less than 200 nm. The next step was to test the anti-inflammatory properties of the optimum formula using a carrageenan-induced rat paw edema test. The results from this test were compared to the oil solution. Different oil/surfactants mixtures had various emulsification properties that were related to the size of their droplets. Tween 20 is a good surfactant to use in self-emulsifying systems because it produces droplets of nano-size. Mixtures of Capmul/Labrasol at a ratio of 2:1 and Labrafil/Tween 20 at a ratio of 1:2 were able to produce self-nanoemulsifying formulations containing Swietenia oil concentrations that ranged from 20%-50%. Nanoemulsion occurred when the size of the droplets fell below 200 nm with low size distribution (<0.3) after being gently mixed with water. It was found that the hydrophilic/lipophilic balance value affected the ternary phase diagram behavior of Swietenia oil and surfactants. In addition, the anti-inflammatory properties of Swietenia oil were greater in the self-nanoemulsifying systems than in the oil solution.
  3. Dkhil MA, Delic D, El Enshasy HA, Abdel Moneim AE
    Oxid Med Cell Longev, 2016;2016:7468524.
    PMID: 27148432 DOI: 10.1155/2016/7468524
  4. Elsayed EA, Farid MA, El-Enshasy HA
    BMC Biotechnol, 2019 07 16;19(1):46.
    PMID: 31311527 DOI: 10.1186/s12896-019-0546-2
    BACKGROUND: Natamycin is an antifungal polyene macrolide antibiotic with wide applications in health and food industries. Currently, it is the only antifungal food additive with the GRAS status (Generally Regarded as Safe).

    RESULTS: Natamycin production was investigated under the effect of different initial glucose concentrations. Maximal antibiotic production (1.58 ± 0.032 g/L) was achieved at 20 g/L glucose. Under glucose limitation, natamycin production was retarded and the produced antibiotic was degraded. Higher glucose concentrations resulted in carbon catabolite repression. Secondly, intermittent feeding of glucose improved natamycin production due to overcoming glucose catabolite regulation, and moreover it was superior to glucose-beef mixture feeding, which overcomes catabolite regulation, but increased cell growth on the expense of natamycin production. Finally, the process was optimized in 7.5 L stirred tank bioreactor under batch and fed-batch conditions. Continuous glucose feeding for 30 h increased volumetric natamycin production by about 1.6- and 1.72-folds in than the batch cultivation in bioreactor and shake-flasks, respectively.

    CONCLUSIONS: Glucose is a crucial substrate that significantly affects the production of natamycin, and its slow feeding is recommended to alleviate the effects of carbon catabolite regulation as well as to prevent product degradation under carbon source limitation. Cultivation in bioreactor under glucose feeding increased maximal volumetric enzyme production by about 72% from the initial starting conditions.

  5. Mustafa SM, Chua LS, El-Enshasy HA
    Molecules, 2019 Jun 26;24(13).
    PMID: 31247970 DOI: 10.3390/molecules24132357
    The issues of lactose intolerance and vegetarianism have encouraged the introduction of non-dairy fermented food into the market. Therefore, this study aims to evaluate the effect of agitation speed on the bioactive compounds and functional characteristics of probioticated pomegranate juice. Pomegranate juice was fermented with Lactobacillus casei at different agitation speeds ranging from 0 (microaerophilic) to 150 rpm at 37 °C. The functional properties of probioticated pomegranate juice were evaluated in terms of growth (biomass), lactic acid production, antioxidant activity, total phenolic content, and key metabolites using LC-MS/MS. The growth kinetics of fermentation was monitored at the optimal condition using one factor at a time method. High cell growth (3.58 × 1010 cfu/mL or 7.9 gL-1) was observed for L. casei probioticated pomegranate juice agitated at 0 rpm. The findings of this study reveal the potential of pomegranate juice as a medium for L. casei cultivation without nutrient supplementation. The improvement of antioxidant activity in the probioticated juice could be due to the increment of quercetin-3-glucoside. Therefore, L. casei grew well in pomegranate juice with a high cell viability and antioxidant activity at a non-agitated condition. Probioticated pomegranate juice is a potentially functional drink.
  6. Awad HM, El-Enshasy HA, Hanapi SZ, Hamed ER, Rosidi B
    Nat Prod Res, 2014;28(24):2273-7.
    PMID: 25078877 DOI: 10.1080/14786419.2014.939083
    This study discusses the isolation and identification of a new Streptomycetes highly active chitinase producer. Fifteen strains were isolated from Malaysian soil samples. The isolate WICC-A03 was found to be the most active chitinase producer. Its antifungal activity was evaluated against many phytopathogens. The identification of WICC-A03 using phenotypic and genotypic methods strongly indicated that strain WICC-A03 belonged to the genus Streptomyces and displayed similarity (91%) with Streptomyces glauciniger. Thus, it was given the suggested name S. glauciniger WICC-A03 with accession number: JX139754. WICC-A03 produces extracellular chitinase in a medium containing 1.5% colloidal chitin in submerged culture on 144 h. The produced enzyme was partially characterised and its molecular weight of 50 kDa was determined by using SDS-PAGE. This study indicates that WICC-A03 is a potential chitinase producer for biocontrol of plant pathogens. Further experiments are being carried out to optimise medium composition and cultivation conditions under lab and bioreactor scale.
  7. Kalam S, Basu A, Ahmad I, Sayyed RZ, El-Enshasy HA, Dailin DJ, et al.
    Front Microbiol, 2020;11:580024.
    PMID: 33193209 DOI: 10.3389/fmicb.2020.580024
    Acidobacteria represents an underrepresented soil bacterial phylum whose members are pervasive and copiously distributed across nearly all ecosystems. Acidobacterial sequences are abundant in soils and represent a significant fraction of soil microbial community. Being recalcitrant and difficult-to-cultivate under laboratory conditions, holistic, polyphasic approaches are required to study these refractive bacteria extensively. Acidobacteria possesses an inventory of genes involved in diverse metabolic pathways, as evidenced by their pan-genomic profiles. Because of their preponderance and ubiquity in the soil, speculations have been made regarding their dynamic roles in vital ecological processes viz., regulation of biogeochemical cycles, decomposition of biopolymers, exopolysaccharide secretion, and plant growth promotion. These bacteria are expected to have genes that might help in survival and competitive colonization in the rhizosphere, leading to the establishment of beneficial relationships with plants. Exploration of these genetic attributes and more in-depth insights into the belowground mechanics and dynamics would lead to a better understanding of the functions and ecological significance of this enigmatic phylum in the soil-plant environment. This review is an effort to provide a recent update into the diversity of genes in Acidobacteria useful for characterization, understanding ecological roles, and future biotechnological perspectives.
  8. Kato T, Azegami J, Yokomori A, Dohra H, El Enshasy HA, Park EY
    BMC Genomics, 2020 Apr 23;21(1):319.
    PMID: 32326906 DOI: 10.1186/s12864-020-6709-7
    BACKGROUND: Ashbya gossypii naturally overproduces riboflavin and has been utilized for industrial riboflavin production. To improve riboflavin production, various approaches have been developed. In this study, to investigate the change in metabolism of a riboflavin-overproducing mutant, namely, the W122032 strain (MT strain) that was isolated by disparity mutagenesis, genomic analysis was carried out.

    RESULTS: In the genomic analysis, 33 homozygous and 1377 heterozygous mutations in the coding sequences of the genome of MT strain were detected. Among these heterozygous mutations, the proportion of mutated reads in each gene was different, ranging from 21 to 75%. These results suggest that the MT strain may contain multiple nuclei containing different mutations. We tried to isolate haploid spores from the MT strain to prove its ploidy, but this strain did not sporulate under the conditions tested. Heterozygous mutations detected in genes which are important for sporulation likely contribute to the sporulation deficiency of the MT strain. Homozygous and heterozygous mutations were found in genes encoding enzymes involved in amino acid metabolism, the TCA cycle, purine and pyrimidine nucleotide metabolism and the DNA mismatch repair system. One homozygous mutation in AgILV2 gene encoding acetohydroxyacid synthase, which is also a flavoprotein in mitochondria, was found. Gene ontology (GO) enrichment analysis showed heterozygous mutations in all 22 DNA helicase genes and genes involved in oxidation-reduction process.

    CONCLUSION: This study suggests that oxidative stress and the aging of cells were involved in the riboflavin over-production in A. gossypii riboflavin over-producing mutant and provides new insights into riboflavin production in A. gossypii and the usefulness of disparity mutagenesis for the creation of new types of mutants for metabolic engineering.

  9. Mustafa SM, Chua LS, El-Enshasy HA, Abd Majid FA, Hanapi SZ, Abdul Malik R
    J Food Biochem, 2019 04;43(4):e12805.
    PMID: 31353583 DOI: 10.1111/jfbc.12805
    This study was focused on the effects of fermentation temperature and pH on the quality of Punica granatum juice probioticated with Lactobacillus species: Lactobacillus plantarum, Lactobacillus casei, Lactobacillus bulgaricus, and Lactobacillus salivarius. The whole fruit juice of P. granatum which is rich with phytonutrients appeared to be a good probiotic carrier. The probiotication was carried out for 24 hr at 30, 35, and 37°C and pH 2.5, 4.0, and 5.5 under microaerophilic conditions. The results found that P. granatum juice cultivated with L. casei had a better growth profile with a higher biomass density at 37°C around pH 3.5-4.0. Probiotication could maintain the scavenging activity of P. granatum juice cultivated with L. casei. The scavenging activity achieved up to 90% inhibition at the concentration of 5 mg/ml. The whole fruit-squeezed P. granatum juice was suitable for the growth of Lactobacillus species even without supplementation during cultivation. PRACTICAL APPLICATIONS: The findings of this study presented the potential of P. granatum juice (whole fruit) to be used as a good probiotic carrier, particularly for Lactobacillus species without supplementation. High nutritious P. granatum juice catered the need of probiotic bacteria during fermentation. Probiotication could maintain the antioxidant capacity of the juice in term of its radical scavenging activity. The antioxidant capacity was mainly attributed to the metabolites such as phenolic acids (romarinic acid and caftaric acid) and flavonoids (quercetin, quercetin 3-glucoside, rutin and kaempferol rutinoside). With the optimized temperature (37°C) and pH (4.00), probiotic bacteria could growth well up to a cell viability of 2.46 × 1010  cfu/ml. This offers P. granatum to be developed into functional food to cater to the needs of the consumers who are lactose intolerant to dairy products.
  10. Singh V, Haque S, Kumari V, El-Enshasy HA, Mishra BN, Somvanshi P, et al.
    Sci Rep, 2019 04 24;9(1):6482.
    PMID: 31019210 DOI: 10.1038/s41598-019-42740-7
    Arterial/venous thrombosis is the major cardiovascular disorder accountable for substantial mortality; and the current demand for antithrombotic agents is extensive. Heparinases depolymerize unfractionated heparin (UFH) for the production of low molecular-weight heparins (LMWHs; used as anticoagulants against thrombosis). A microbial strain of Streptomyces sp. showing antithrombotic activity was isolated from the soil sample collected from north India. The strain was characterized by using 16S rRNA homology technique and identified as Streptomyces variabilis MTCC 12266 capable of producing heparinase enzyme. This is the very first communication reporting Streptomyces genus as the producer of heparinase. It was observed that the production of intracellular heparinase was [63.8 U/mg protein (specific activity)] 1.58 folds higher compared to extracellular heparinase [40.28 U/mg protein]. DEAE-Sephadex A-50 column followed by Sepharose-6B column purification of the crude protein resulted 19.18 folds purified heparinase. SDS-PAGE analysis of heparinase resulted an estimated molecular-weight of 42 kDa. It was also found that intracellular heparinase has the ability to depolymerize heparin to generate LMWHs. Further studies related to the mechanistic action, structural details, and genomics involved in heparinase production from Streptomyces variabilis are warranted for large scale production/purification optimization of heparinase for antithrombotic applications.
  11. Akhtar N, Ilyas N, Yasmin H, Sayyed RZ, Hasnain Z, A Elsayed E, et al.
    Molecules, 2021 Mar 12;26(6).
    PMID: 33809305 DOI: 10.3390/molecules26061569
    Plant growth-promoting rhizobacteria (PGPR) mediate heavy metal tolerance and improve phytoextraction potential in plants. The present research was conducted to find the potential of bacterial strains in improving the growth and phytoextraction abilities of Brassica nigra (L.) K. Koch. in chromium contaminated soil. In this study, a total of 15 bacterial strains were isolated from heavy metal polluted soil and were screened for their heavy metal tolerance and plant growth promotion potential. The most efficient strain was identified by 16S rRNA gene sequencing and was identified as Bacillus cereus. The isolate also showed the potential to solubilize phosphate and synthesize siderophore, phytohormones (indole acetic acid, cytokinin, and abscisic acid), and osmolyte (proline and sugar) in chromium (Cr+3) supplemented medium. The results of the present study showed that chromium stress has negative effects on seed germination and plant growth in B. nigra while inoculation of B. cereus improved plant growth and reduced chromium toxicity. The increase in seed germination percentage, shoot length, and root length was 28.07%, 35.86%, 19.11% while the fresh and dry biomass of the plant increased by 48.00% and 62.16%, respectively, as compared to the uninoculated/control plants. The photosynthetic pigments were also improved by bacterial inoculation as compared to untreated stress-exposed plants, i.e., increase in chlorophyll a, chlorophyll b, chlorophyll a + b, and carotenoid was d 25.94%, 10.65%, 20.35%, and 44.30%, respectively. Bacterial inoculation also resulted in osmotic adjustment (proline 8.76% and sugar 28.71%) and maintained the membrane stability (51.39%) which was also indicated by reduced malondialdehyde content (59.53% decrease). The antioxidant enzyme activities were also improved to 35.90% (superoxide dismutase), 59.61% (peroxide), and 33.33% (catalase) in inoculated stress-exposed plants as compared to the control plants. B. cereus inoculation also improved the uptake, bioaccumulation, and translocation of Cr in the plant. Data showed that B. cereus also increased Cr content in the root (2.71-fold) and shoot (4.01-fold), its bioaccumulation (2.71-fold in root and 4.03-fold in the shoot) and translocation (40%) was also high in B. nigra. The data revealed that B. cereus is a multifarious PGPR that efficiently tolerates heavy metal ions (Cr+3) and it can be used to enhance the growth and phytoextraction potential of B. nigra in heavy metal contaminated soil.
  12. El Enshasy HA, Elsayed EA, Suhaimi N, Malek RA, Esawy M
    BMC Biotechnol, 2018 11 09;18(1):71.
    PMID: 30413198 DOI: 10.1186/s12896-018-0481-7
    BACKGROUND: Pectinase enzymes present a high priced category of microbial enzymes with many potential applications in various food and oil industries and an estimated market share of $ 41.4 billion by 2020.

    RESULTS: The production medium was first optimized using a statistical optimization approach to increase pectinase production. A maximal enzyme concentration of 76.35 U/mL (a 2.8-fold increase compared with the initial medium) was produced in a medium composed of (g/L): pectin, 32.22; (NH4)2SO4, 4.33; K2HPO4, 1.36; MgSO4.5H2O, 0.05; KCl, 0.05; and FeSO4.5H2O, 0.10. The cultivations were then carried out in a 16-L stirred tank bioreactor in both batch and fed-batch modes to improve enzyme production, which is an important step for bioprocess industrialization. Controlling the pH at 5.5 during cultivation yielded a pectinase production of 109.63 U/mL, which was about 10% higher than the uncontrolled pH culture. Furthermore, fed-batch cultivation using sucrose as a feeding substrate with a rate of 2 g/L/h increased the enzyme production up to 450 U/mL after 126 h.

    CONCLUSIONS: Statistical medium optimization improved volumetric pectinase productivity by about 2.8 folds. Scaling-up the production process in 16-L semi-industrial stirred tank bioreactor under controlled pH further enhanced pectinase production by about 4-folds. Finally, bioreactor fed-batch cultivation using constant carbon source feeding increased maximal volumetric enzyme production by about 16.5-folds from the initial starting conditions.

  13. El-Baz AF, El-Enshasy HA, Shetaia YM, Mahrous H, Othman NZ, Yousef AE
    Probiotics Antimicrob Proteins, 2018 Mar;10(1):77-88.
    PMID: 28634812 DOI: 10.1007/s12602-017-9291-9
    A new yeast strain with promising probiotic traits was isolated from the Red Sea water samples. The isolate (YMHS) was subjected to genetic characterization and identified as Cryptococcus sp. Nucleotide sequence analysis of the rRNA gene internal transcribed spacer regions showed 95% sequence similarity between the isolate and Cryptococcus albidus. Cryptococcus sp. YMHS exhibited desirable characteristics of probiotic microorganisms; it has tolerance to low pH in simulated gastric juice, resistance to bile salts, hydrophobic characteristics, broad antimicrobial activity, and in vitro ability to degrade cholesterol. The isolate grew well in a semi-defined medium composed of yeast extract, glucose, KH2PO4, (NH4)2SO4, and MgSO4, yielding cell mass of 2.32 and 5.82 g/l in shake flask and in bioreactor cultures, respectively. Fed-batch cultivation, with controlled pH, increased the biomass gradually in culture, reaching 28.5 g/l after 32 h cultivation. Beside the feasible use as a probiotic, the new strain also could be beneficial in the development of functional foods or novel food preservatives. To our knowledge, this is the first report of yeast with probiotic properties isolated from the Red Sea.
  14. Prabha R, Singh DP, Gupta S, Gupta VK, El-Enshasy HA, Verma MK
    Microorganisms, 2019 Nov 23;7(12).
    PMID: 31771141 DOI: 10.3390/microorganisms7120608
    Multifunctionalities linked with the microbial communities associated with the millet crop rhizosphere has remained unexplored. In this study, we are analyzing microbial communities inhabiting rhizosphere of kodo millet and their associated functions and its impact over plant growth and survival. Metagenomics of Paspalum scrobiculatum L.(kodo millet) rhizopshere revealed taxonomic communities with functional capabilities linked to support growth and development of the plants under nutrient-deprived, semi-arid and dry biotic conditions. Among 65 taxonomically diverse phyla identified in the rhizobiome, Actinobacteria were the most abundant followed by the Proteobacteria. Functions identified for different genes/proteins led to revelations that multifunctional rhizobiome performs several metabolic functions including carbon fixation, nitrogen, phosphorus, sulfur, iron and aromatic compound metabolism, stress response, secondary metabolite synthesis and virulence, disease, and defense. Abundance of genes linked with N, P, S, Fe and aromatic compound metabolism and phytohormone synthesis-along with other prominent functions-clearly justifies growth, development, and survival of the plants under nutrient deprived dry environment conditions. The dominance of actinobacteria, the known antibiotic producing communities shows that the kodo rhizobiome possesses metabolic capabilities to defend themselves against biotic stresses. The study opens avenues to revisit multi-functionalities of the crop rhizosphere for establishing link between taxonomic abundance and targeted functions that help plant growth and development in stressed and nutrient deprived soil conditions. It further helps in understanding the role of rhizosphere microbiome in adaptation and survival of plants in harsh abiotic conditions.
  15. Fazeli-Nasab B, Sayyed RZ, Farsi M, Ansari S, El-Enshasy HA
    Physiol Mol Biol Plants, 2020 Jan;26(1):107-117.
    PMID: 32158124 DOI: 10.1007/s12298-019-00732-x
    Mango (Mangifera indica) is one of the most important tropical fruits in the world. Twenty-two genotypes of native mangoes from different regions of southern Iran (Hormozgan and Kerman) were collected and analyzed for the ribosomal genes. GC content was found to be 55.5%. Fu and Li's D* test statistic (0.437), Fu and Li's F* test statistic (0.500) and Tajima's D (1.801) were positive and nonsignificant. A total of 769 positions were identified (319 with insertion or deletion including 250 polymorphic and 69 monomorphic loci; 450 loci without any insertion or deletion including 35 Singletons and 22 haplotypes). Nucleotide diversity of 0.309 and a high genetic differentiation including Chi square of 79.8; P value of 0.3605 and df value of 76 was observed among mango genotypes studied. The numerical value of the ratio dN/dS (0.45) indicated a pure selection in the examined gene and the absence of any key changes. Cluster analysis differentiated the mango used in this research (M. indica L.) into two genotypes but could not differentiate their geographical locations. The results of this study indicated that a high genetic distance exists between HajiGholam (Manojan) and Arbabi (Rodan) genotypes and showed higher genetic diversity in mango of Rodan region. Results of present study suggested that for successful breeding, the genotypes of Rodan region mango especially Arbabi mango can be used as a gene donor and ITS can be a suitable tool for genetic evaluations of inter and intra species.
  16. Sayyed RZ, Wani SJ, Alyousef AA, Alqasim A, Syed A, El-Enshasy HA
    PLoS One, 2019;14(6):e0212324.
    PMID: 31211775 DOI: 10.1371/journal.pone.0212324
    Poly-β-hydroxybutyrate (PHB) depolymerase is known to decompose PHB, biodegradable polymers and therefore has great commercial significance in the bioplastic sector. However, reports on PHB depolymerases from isolates obtained from plastic-contaminated sites that reflect the potential of the source organism is scarce. In this study, we evaluated the production of extracellular PHB depolymerase from Microbacterium paraoxydans RZS6 isolated from the plastic-contaminated site in the municipal area of Shahada, Maharashtra, India, for the first time. The isolate was identified using 16S rRNA gene sequencing, gas chromatographic analysis of fatty acid methyl esters (GC-FAME), and BIOLOG method. Ithydrolyzed PHB on minimal salt medium (MSM) containing PHB as the only source of carbon. The isolate produced PHB depolymerase at 45°C during 48 h of incubation. The enzyme was purified most efficiently using octyl-sepharose CL-4B column, with the highest purification yield of 6.675 Umg-1mL-1. The activity of the enzyme was enhanced in the presence of Ca2+ and Mg2+ ions but inhibited by Fe2+ (1 mM) ions and mercaptoethanol (1000 rpm). the nzyme kinetic analysis revealed that the enzyme was a metalloenzyme; requiring Mg2+ ions, that showed optimum enzyme activity at 30°C (mesophilic) and under neutrophilic (pH 7) conditions. Scale-up from the shake-flask level to a laboratory-scale bioreactor further enhanced the enzyme yield by 0.809 UmL-1. The molecular weight of the enzyme (40 kDa), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, closely resembled the PHB depolymerase of Aureobacterium saperdae. Our findings highlighted the applicability of M. paraoxydans as a producer of extracellular PHB depolymerase having potential of degrading PHB under diverse conditions.
  17. Sayyed RZ, Bhamare HM, Sapna, Marraiki N, Elgorban AM, Syed A, et al.
    PLoS One, 2020;15(6):e0229968.
    PMID: 32497077 DOI: 10.1371/journal.pone.0229968
    Although laccase has been recognized as a wonder molecule and green enzyme, the use of low yielding fungal strains, poor production, purification, and low enzyme kinetics have hampered its large-scale application. Thus,this study aims to select high yielding fungal strains and optimize the production, purification, and kinetics of laccase of Aspergillus sp. HB_RZ4. The results obtained indicated that Aspergillus sp. HB_RZ4 produced a significantly large amount of laccase under meso-acidophilic shaking conditions in a medium containing glucose and yeast extract. A 25 μM CuSO4 was observed to enhance the enzyme yield. The enzyme was best purified on a Sephadex G-100 column. The purified enzyme resembled laccase of A. flavus. The kinetics of the purified enzyme revealed high substrate specificity and good velocity of reaction,using ABTS as a substrate. The enzyme was observed to be stable over various pH values and temperatures. The peptide structure of the purified enzyme was found to resemble laccase of A. kawachii IFO 4308. The fungus was observed to decolorize various dyes independent of the requirement of a laccase mediator system.Aspergillus sp. HB_RZ4 was observed to be a potent natural producer of laccase, and it decolorized the dyes even in the absence of a laccase mediator system. Thus, it can be used for bioremediation of effluent that contains non-textile dyes.
  18. Kato T, Azegami J, Kano M, El Enshasy HA, Park EY
    Appl Microbiol Biotechnol, 2021 Oct;105(20):7813-7823.
    PMID: 34559286 DOI: 10.1007/s00253-021-11595-2
    This study focuses on sirtuins, which catalyze the reaction of NAD+-dependent protein deacetylase, for riboflavin production in A. gossypii. Nicotinamide, a known inhibitor of sirtuin, made the color of A. gossypii colonies appear a deeper yellow at 5 mM. A. gossypii has 4 sirtuin genes (AgHST1, AgHST2, AgHST3, AgHST4) and these were disrupted to investigate the role of sirtuins in riboflavin production in A. gossypii. AgHST1∆, AgHST3∆, and AgHST4∆ strains were obtained, but AgHST2∆ was not. The AgHST1∆ and AgHST3∆ strains produced approximately 4.3- and 2.9-fold higher amounts of riboflavin than the WT strain. The AgHST3∆ strain showed a lower human sirtuin 6 (SIRT6)-like activity than the WT strain and only in the AgHST3∆ strain was a higher amount of acetylation of histone H3 K9 and K56 (H3K9ac and H3K56ac) observed compared to the WT strain. These results indicate that AgHst3 is SIRT6-like sirtuin in A. gossypii and the activity has an influence on the riboflavin production in A. gossypii. In the presence of 5 mM hydroxyurea and 50 µM camptothecin, which causes DNA damage, especially double-strand DNA breaks, the color of the WT strain colonies turned a deeper yellow. Additionally, hydroxyurea significantly led to the production of approximately 1.5 higher amounts of riboflavin and camptothecin also enhanced the riboflavin production even through the significant difference was not detected. Camptothecin tended to increase the amount of H3K56ac, but the amount of H3K56ac was not increased by hydroxyurea treatment. This study revealed that AgHst1 and AgHst3 are involved in the riboflavin production in A. gossypii through NAD metabolism and the acetylation of H3, respectively. This new finding is a step toward clarifying the role of sirtuins in riboflavin over-production by A. gossypii.Key points• Nicotinamide enhanced the riboflavin production in Ashbya gossypii.• Disruption of AgHST1 or AgHST3 gene also enhanced the riboflavin production in Ashbya gossypii.• Acetylation of H3K56 led to the enhancement of the riboflavin production in Ashbya gossypii.
  19. El-Sohaimy SA, Androsova NV, Toshev AD, El Enshasy HA
    Plants (Basel), 2022 Oct 24;11(21).
    PMID: 36365277 DOI: 10.3390/plants11212825
    (1) Background: Hemp seeds are a source of plant-based protein, making them an appropriate supplement to a plant-based diet. The current work was focused on the preparation of the protein isolate from the hemp seeds with eco-friendly and cheap technology. Moreover, it evaluated the physicochemical and functional properties of hemp protein isolate for its potential application in food manufacturing. (2) Methods: The protein content of hemp seeds has been isolated through two main steps: (1) extraction of the protein content of an alkaline pH (10-12); (2) precipitation of the extracted protein on an acidic pH as an isoelectric point (pH = 4.5). (3) Results: The edastin protein is the most predominant protein in the protein profile with a molecular weight of 58.1 KDa beside albumin with a molecular weight of 31.5 KDa. The FTIR spectrum detected the absorption peaks of the amide I at 1750 and 1600 cm-1, which pointed to C=O stretching while N-H stretching at 1650-1580 cm-1. The peak at 3250 is found to be related to N-H stretching of the aliphatic primary amine (3400-3300 cm-1) and the N-H stretching for the secondary (II) amine appeared at 3350-3310 cm-1. The Hemp protein isolate (HPI) showed a high content of arginine (15.52 g/100 g), phenylalanine + tyrosine (9.63 g/100 g), methionine + cysteine (5.49 g/100 g), leucine + isoleucine (5.21 g/100 g), and valine (4.53 g/100 g). It contains a moderate level of threonine (3.29 g/100 g) and lysine (2.50 g/100 g) with tryptophan as the limiting amino acid (0.22 g/100 g). The HPI showed an appropriate water-and-oil holding capacity (4.5 ± 2.95 and 2.33 ± 1.88 mL/g, respectively). The foaming capacity of the HPI was increased with increasing the pH values to reach the maximum value at pH 11 (67.23 ± 3.20%). The highest emulsion ability index of the HPI was noted at pH 9 (91.3 ± 2.57 m2/g) with low stability (19.15 ± 2.03). (4) Conclusions: A strong positive correlation (r = 0.623) was shown between protein concentration and solubility. The current easy-to-use, cheap, and eco-friendly technology provides the industrial sector with a cheap protein isolate for manufacturing protein-rich diet and beverages. The HPI showed a good nutritional quality and functional properties that might be helpful in utilizing it in different food products such as beverages and bakery products.
  20. Kato T, Yokomori A, Suzuki R, Azegami J, El Enshasy HA, Park EY
    J Appl Microbiol, 2022 Feb;132(2):1176-1184.
    PMID: 34496097 DOI: 10.1111/jam.15296
    AIMS: Effects of a proteasome inhibitor, MG-132, on the riboflavin production in Ashbya gossypii were investigated to elucidate the relationship of the riboflavin production with flavoprotein homeostasis.

    METHODS AND RESULTS: The addition of MG-132 to the liquid medium reduced the specific riboflavin production by 79% in A. gossypii at 25 μM after 24 h. The addition of the inhibitor also caused the accumulation of reactive oxygen species and ubiquitinated proteins. These results indicated that MG-132 works in A. gossypii without any genetic engineering and reduces riboflavin production. In the presence of 25 μM MG-132, specific NADH dehydrogenase activity was increased by 1.4-fold compared to DMSO, but specific succinate dehydrogenase (SDH) activity was decreased to 52% compared to DMSO. Additionally, the amount of AgSdh1p (ACR052Wp) was also reduced. Specific riboflavin production was reduced to 22% when 20 mM malonate, a SDH inhibitor, was added to the culture medium. The riboflavin production in heterozygous AgSDH1 gene-disrupted mutant (AgSDH1-/+ ) was reduced to 63% compared to that in wild type.

    CONCLUSIONS: MG-132 suppresses the riboflavin production and SDH activity in A. gossypii. SDH is one of the flavoproteins involved in the riboflavin production in A. gossypii.

    SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that MG-132 has a negative influence on the riboflavin production and SDH activity in A. gossypii and leads to the elucidation of the connection of the riboflavin production with flavoproteins.

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