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Journal of Microbiology and... Feb 2017NRRL 5282 and NRRL 1526 can produce lipases with high synthetic activities in wheat bran-based solid-state culture. In this study, the purification and biochemical...
NRRL 5282 and NRRL 1526 can produce lipases with high synthetic activities in wheat bran-based solid-state culture. In this study, the purification and biochemical characterization of the lipolytic activities of these lipases are presented. SDS-PAGE indicated a molecular mass of about 55 and 35 kDa for the purified and enzymes, respectively. -Nitrophenyl palmitate (NPP) hydrolysis was maximal at 40°C and pH 7.0 for the lipase, and at 30°C and pH 5.2 for the enzyme. The enzymes showed almost equal affinity to NPP, but the of the lipase was about 1.13 times higher than that determined for using the same substrate. For both enzymes, a dramatic loss of activity was observed in the presence of 5 mM Hg, Zn, or Mn, 10 mM -bromosuccinimide or sodium dodecyl sulfate, and 5-10% (v/v) of hexanol or butanol. At the same time, they proved to be extraordinarily stable in the presence of -hexane, cyclohexane, -heptane, and isooctane. Moreover, isopentanol up to 10% (v/v) and propionic acid in 1 mM concentrations increased the NPP hydrolyzing activity of lipase. Both enzymes had 1,3-regioselectivity, and efficiently hydrolyzed -nitrophenyl (NP) esters with C8-C16 acids, exhibiting maximum activity towards NP-caprylate () and pNP-dodecanoate (). The purified lipases are promising candidates for various biotechnological applications.
Topics: Bromosuccinimide; Butanols; Caprylates; Electrophoresis, Polyacrylamide Gel; Esterification; Heptanes; Hexanes; Hexanols; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Laurates; Lipase; Manganese; Mercury; Nitrobenzenes; Palmitates; Propionates; Rhizomucor; Rhizopus; Sodium Dodecyl Sulfate; Zinc
PubMed: 27780957
DOI: 10.4014/jmb.1608.08005 -
Cryobiology Aug 2021Thermophilic fungi have several biotechnological and industrial applications such as thermostable enzyme production, biodegradation, and tobacco processing, etc....
Thermophilic fungi have several biotechnological and industrial applications such as thermostable enzyme production, biodegradation, and tobacco processing, etc. Thermophilic fungi cannot survive at temperatures below 20 °C. Owing to their inability to grow at low temperatures, they are not stable, so stocking is very difficult. Although a large number of different storage methods are available and described, no method can be universally applied to all fungi. Thermophilic fungi present "heat-loving" characteristics, and therefore a new challenge for its preservation and there is no universal protocol for the preservation of thermophilic fungi. The aim of this study was to evaluate the viability, contamination and stability of thermophilic fungi stored under different preservation methods. In this work, 25 thermophilic fungal isolates of species Thermomyces thermophilus, Rhizomucor pusillus, Trichocladium griseum, Melanocarpus albomyces, Malbranchea cinnamomea, Thermothelomyces thermophilus, Thermothelomyces hinnuleus,Thermothielavioidesterrestris, Mycothermus thermophilus, Humicola insolens maintained constant sub-culturing at room temperature, +4 °C and +20 °C, lyophilization at +4 °C, freezing at -20 °C, freezing block at -20 °C and a new technique liquid preservation at room temperature for the periods ranging 5 years. We evaluated the effect of preservation methods by sub-culturing onto either sabouraud dextrose agar (SDA) or yeast extract soluble starch agar (YpSs) on growth, production and viability of spores and macro- and micromorphology. In this study, preservation methods for thermophilic fungi were investigated extensively for the first time and it is clearly shown that freezing block at -20 °C method and lyophilization were better methods for long-term preservation up to 5 years.
Topics: Ascomycota; Cryopreservation; Eurotiales; Fungal Genus Humicola; Fungi; Onygenales; Rhizomucor; Sordariales
PubMed: 34153346
DOI: 10.1016/j.cryobiol.2021.06.005 -
Extremophiles : Life Under Extreme... May 2020In contrast to mesophiles, in which levels of trehalose and phosphatidic acids (PA) increased only under heat shock (HS), in thermophiles trehalose and PA were...
In contrast to mesophiles, in which levels of trehalose and phosphatidic acids (PA) increased only under heat shock (HS), in thermophiles trehalose and PA were predominant under optimal growth conditions. To study the role of trehalose protection in the adaptation of thermophiles to various stressors, the composition of osmolytes and membrane lipids in the thermophilic fungus Rhizomucor miehei was studied under cold (CS), osmotic (OS) and oxidative (OxS) shocks. CS resulted in no accumulation of glycerol in the mycelium, while the amount of trehalose decreased. The main lipid changes were the increase in the PA proportion with simultaneous decrease of sterols (St), the increase of the unsaturation degree of polar lipids and the decrease of the ergosterol proportion in total St. OS did not cause changes in the lipid composition, but led to the decrease of ergosterol proportion too. Despite the low ability of Mucorales to produce polyols, increase in the level of arabitol and glycerol was observed under OS. OxS led to the decrease of trehalose level and had no effect on the lipid composition. Thus, our results show the similarity (OS) and the difference (CS and OxS) between adaptation mechanisms of thermophiles and mesophiles.
Topics: Membrane Lipids; Osmosis; Oxidative Stress; Rhizomucor; Trehalose
PubMed: 32144516
DOI: 10.1007/s00792-020-01163-3 -
Scientific Reports Apr 2022The use of enzymes immobilized on nanomagnetic supports has produced surprising results in catalysis, mainly due to the increase in surface area and the potential for...
The use of enzymes immobilized on nanomagnetic supports has produced surprising results in catalysis, mainly due to the increase in surface area and the potential for recovery and reuse. However, the meticulous control of the process and difficulties in reproducibility have made industrial-scale applications unfeasible. Furthermore, the role of conjugation strategies in the catalytic activity and recycling of catalysts is unclear. Therefore, the objective of this study was to compare the conjugation of enzymes on nanomagnetic supports through physical adsorption (naked) or covalent bonding with mercaptopropyltrimethoxysilane (MPTS) and aminopropyltriethoxysilane (APTS) ligands. The free lipase obtained from Rhizomucor miehei was used as a model enzyme. Total protein and enzyme activity were determined using spectrophotometry (UV-Vis) and the p-nitrophenyl palmitate (p-NPP) hydrolysis method. The results indicated that a more significant enzyme surface loading does not always mean better immobilization success. The physical adsorption binding strategy had higher surface loading and low catalytic activity. On the other hand, covalent coupling with free NH2 had an excellent catalytic activity with very low surface loading. Finally, we show that recyclability can be improved with conjugation mediated by disulfide bonds. The findings presented here are essential for developing nanoconjugates with high enzymatic activity, which can guarantee the success of several industrial applications.
Topics: Adsorption; Enzymes, Immobilized; Hydrolysis; Lipase; Reproducibility of Results
PubMed: 35474328
DOI: 10.1038/s41598-022-10721-y -
Frontiers in Microbiology 2021Daqu is an important saccharifying and fermenting agent. It provides various microorganisms and enzymes for the fermentation of Baijiu and plays a vital role in the...
Daqu is an important saccharifying and fermenting agent. It provides various microorganisms and enzymes for the fermentation of Baijiu and plays a vital role in the formation of Baijiu flavor. However, it is difficult to obtain information on microbial growth and metabolism in time for Daqu production. Therefore, the "Qu Xiang" obtained by smelling is an important index in the traditional production process to evaluate the microbial fermentation in the process of Daqu-making, "Qu Xiang" mainly represents the volatile flavor compounds in Daqu. The microbial diversity and volatile metabolites on 0, 6, 16, and 29 days of the fermentation process were measured using high-throughput sequencing and gas chromatography-mass spectrometry. Significant differences were found in the composition of the microbial community. , , , and were the main bacterial genera. , , and are the main fungal genera. A total of 32 differential volatile metabolites were detected in samples at four time points using differential metabolic analysis. The correspondence of prevailing microorganisms with differential metabolites distinguished by Spearman correlation and two-way orthogonal partial least square analysis show that Saccharopolyspora exhibited a significant connection for the 12 differential metabolites. A significant positive correlation was observed between and 13 different metabolites. These findings further understanding of the metabolism of microorganisms in Daqu fermentation and also help to control the microorganisms in the Daqu-making process, to obtain more stable Baijiu products.
PubMed: 34630343
DOI: 10.3389/fmicb.2021.688981 -
A Rapid and Specific Real-Time PCR Assay for the Detection of Clinically Relevant Mucorales Species.International Journal of Molecular... Dec 2022Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of...
Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of a standardized PCR (polymerase chain reaction) assay for early diagnosis of this fungal infection, this work was aimed to develop a new PCR assay able to detect the presence of Mucorales genera in clinical specimens. Here, we describe a novel diagnostic TaqMan MGB probe assay for precise and rapid detection of the most common clinical species of Mucorales. Zygomycete-specific oligonucleotides were designed to specifically amplify and bind highly conserved sequences of fungal 28S rRNA gene. Additionally, we succeeded in differentiating Mucorales species (i.e., , , , and ) in artificially infected serum samples, suggesting that the quantitative capability of this real-time PCR assay could potentially optimize the diagnosis of mucormycosis.
Topics: Humans; Mucorales; Real-Time Polymerase Chain Reaction; Mucormycosis; RNA, Ribosomal, 28S; Immunocompromised Host
PubMed: 36499395
DOI: 10.3390/ijms232315066 -
Frontiers in Microbiology 2023Mucormycosis, an invasive fungal disease with severe consequences, poses a significant threat to immunocompromised individuals. However, the timely and accurate...
Mucormycosis, an invasive fungal disease with severe consequences, poses a significant threat to immunocompromised individuals. However, the timely and accurate identification of Mucorales infection continues to present difficulties. In this study, novel detection techniques utilizing recombinase polymerase amplification (RPA) and quantitative real-time polymerase chain reaction (qPCR) were developed, specifically targeting the mitochondrial gene, in order to address this challenge. The specificity of the RPA and qPCR assay was assessed by adding genomic DNAs extracted from 14 non-targeted strains, as well as human and mouse blood. No false-positive results were observed. Additionally, genomic DNAs from 13 species in five genera of order Mucorales were tested and yielded positive results in both methods. To further evaluate the sensitivity of the assays, DNAs from and were utilized, with concentrations ranging from 1 ng/μL to 1 fg/μL. The limit of detection (LoD) for the RPA assay was determined to be 1 pg., with the exception of which had a LoD of 1 ng. The LoD for the qPCR assay varied between 10 fg and 1 pg., depending on the specific species being tested. Sensitivity analysis conducted on simulated clinical samples revealed that the LoD for RPA and qPCR assays were capable of detecting DNA extracted from 10 and 10 colony forming units (CFU) conidia in 200 μL of blood and serum, respectively. Consequently, the real-time RPA and qPCR assays developed in this study exhibited favorable sensitivity and specificity for the diagnosis of mucormycosis.
PubMed: 37954252
DOI: 10.3389/fmicb.2023.1273073 -
Histopathology Mar 2024Mucormycosis is a fast-progressing disease with a high mortality rate. The most important factor determining survival of patients is early and accurate diagnosis....
AIMS
Mucormycosis is a fast-progressing disease with a high mortality rate. The most important factor determining survival of patients is early and accurate diagnosis. Although histopathology often recognises invasive mould infections at first, histomorphology alone is insufficient in providing an accurate diagnosis. Unbiased molecular methods to detect and identify fungi are promising, yet their role in complementing routine histopathological workflows has not been studied sufficiently.
METHODS AND RESULTS
We performed a retrospective single-centre study examining the clinical value of complementing histopathology with internal transcribed spacer (ITS) sequencing of fungal DNA in the routine diagnosis of mucormycosis. At our academic centre, we identified 14 consecutive mucormycosis cases diagnosed by histopathology and subsequent ITS sequencing. Using histomorphological examination, fungal hyphae could be detected in all cases; however, morphological features were unreliable regarding specifying the taxa. Subsequent ITS sequencing identified a remarkable phylogenetic diversity among Mucorales: the most common species was Rhizopus microsporus (six of 14; 42.9%), followed by Lichtheimia corymbifera (three of 14, 21.4%) and single detections of Rhizopus oryzae, Actinomucor elegans, Mucor circinelloides, Rhizomucor pusillus and Rhizomucor miehei (one of 14; 7.1%, respectively). In one case, we additionally detected Pneumocystis jirovecii in the same lung tissue specimen, suggesting a clinically relevant co-infection. Fungal culture was performed in 10 cases but yielded positive results in only two of 10 (20%), revealing its limited value in the diagnosis of mucormycosis.
CONCLUSIONS
Our study demonstrates that a combination of histopathology and ITS sequencing is a practically feasible approach that outperforms fungal culture in detecting Mucorales in tissue-associated infections. Therefore, pathologists might adapt diagnostic workflows accordingly when mucormycosis is suspected.
Topics: Humans; Mucormycosis; Retrospective Studies; Phylogeny
PubMed: 38192085
DOI: 10.1111/his.15131 -
Frontiers in Microbiology 2021The fungi present during pile-fermentation of Sichuan dark tea play a pivotal role in the development of its aroma and physical characteristics. Samples of tea leaves...
The fungi present during pile-fermentation of Sichuan dark tea play a pivotal role in the development of its aroma and physical characteristics. Samples of tea leaves were collected on days 0 (YC-raw material), 8 (W1-first turn), 16 (W2-second turn), 24 (W3-third turn), and 32 (W4-out of pile) during pile-fermentation. High-throughput sequencing revealed seven phyla, 22 classes, 41 orders, 85 families, 128 genera, and 184 species of fungi. During fermentation, the fungal diversity index declined from the W1 to W3 stages and then increased exponentially at the W4 stage. A bar plot and heatmap revealed that , , , , , , and were abundant during piling, of which was the most abundant. Cluster analysis revealed that the W4 stage of fermentation is critical for fungal growth, diversity, and the community structure in Sichuan dark tea. This study revealed the role of fungi during pile-fermentation in the development of the essence and physical characteristics of Sichuan dark tea. This study comes under one of the Sustainable Development Goals of United Nations Organization (UNO) to "Establish Good Health and Well-Being."
PubMed: 34421866
DOI: 10.3389/fmicb.2021.706714 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Sep 2021L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has...
L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.
Topics: Asparaginase; Bacillus subtilis; Industrial Microbiology; Protein Engineering; Rhizomucor; Sequence Alignment
PubMed: 34622632
DOI: 10.13345/j.cjb.200759