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Protein and Peptide Letters 2022A series of mutants of Rhizomucor miehei lipase (RML) screened through four rounds of directed evolution were studied. Mutants' triglyceride hydrolysis activity was...
BACKGROUND
A series of mutants of Rhizomucor miehei lipase (RML) screened through four rounds of directed evolution were studied. Mutants' triglyceride hydrolysis activity was assessed, and their genes were sequenced. Results showed that mutations in the propeptide can improve the activity of RML during evolution. Two parts of propeptide (wild-type and mutant) and mature region were connected by molecular simulation technology.
METHODS
The spatial structure of the most positive mutants containing the mutations in the propeptide was mainly characterized by the increase in the opening angle of the lid structure in the mature region of RML, the enhancement of the hydrophobicity of the active center, and the triad of the active center shifted outward.
RESULTS
The three indexes above explain the mechanism of propeptide mutations on the activity change of the target protein. In addition, statistical analysis of all the mutants screened in directed evolution showed that: (1) most of the mutants with increased activity contained mutations of the propeptide, (2) in the later stage of directed evolution, the number of active mutants decreased gradually, and the mutations of inactivated protein mainly occurred in the mature region, and (3) in the last round of directed evolution, the mutations distributed in the propeptide improved the mutant activity further. The results showed that the propeptide reduced RML's evolutionary pressure and delayed the emergence of the evolutionary platform.
CONCLUSION
These findings reveal the role of propeptide in the evolution of RML and provide strategies for the molecular transformation of other lipases.
Topics: Hydrolysis; Lipase; Mutation; Rhizomucor
PubMed: 35289250
DOI: 10.2174/0929866529666220314105130 -
Preparative Biochemistry & Biotechnology 2021Crude glycerol, a by-product of biodiesel production, was employed as the carbon source to produce lipase using . Under identical fermentation conditions, cell growth...
Crude glycerol, a by-product of biodiesel production, was employed as the carbon source to produce lipase using . Under identical fermentation conditions, cell growth and lipase activity were improved using crude glycerol instead of pure glycerol. The impacts of crude glycerol impurities (methyl ester, grease, glycerol, methanol, and metal ions Na, Ca, and Fe) on lipase production were investigated. Impurities accelerated entering the stationary phase. Na, Ca, and grease in waste crude glycerol were the main factors influencing higher lipase activity. Through response surface optimization of Ca, Na, and grease concentrations, lipase activity reached 1437 U/mL (15,977 U/mg), which was 2.5 times that of the control. This study highlights the economical and highly efficient valorization of crude glycerol, demonstrating its possible utilization as a carbon source to produce lipase by without pretreatment.
Topics: Culture Media; Fungal Proteins; Glycerol; Lipase; Recombinant Proteins; Rhizomucor; Saccharomycetales
PubMed: 33439089
DOI: 10.1080/10826068.2020.1870135 -
Clinical Infectious Diseases : An... May 1993Rhizomucor pusillus, a thermophilic fungus of the order Mucorales, is a rare cause of human infection. A search of the literature has produced only seven reports... (Review)
Review
Rhizomucor pusillus, a thermophilic fungus of the order Mucorales, is a rare cause of human infection. A search of the literature has produced only seven reports describing nine cases of infection caused by this organism. Recently, over a period of 17 months, four cases of R. pusillus infection in patients with leukemia were diagnosed: a cluster of three cases in a Montreal hospital and one isolated case from Quebec City. All four cases were proven both by histopathologic examination and by culture of tissues. In three cases, pulmonary involvement was confirmed following lung surgery, and in one case, disseminated infection was observed at autopsy. All patients received amphotericin B, and two underwent surgical debridement; however, none of the patients survived.
Topics: Adolescent; Adult; Anti-Bacterial Agents; Bronchoalveolar Lavage Fluid; Female; Humans; Leukemia; Leukemia, Myeloid, Acute; Male; Microbial Sensitivity Tests; Middle Aged; Mucorales; Mucormycosis; Precursor Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 8507755
DOI: 10.1093/clind/16.5.640 -
Protein Expression and Purification Apr 2021Lipase from Rhizomucor miehei (RML) is a promising biocatalyst used in food industry, fine chemicals, and biodiesel production. Yeast surface display allows direct...
Lipase from Rhizomucor miehei (RML) is a promising biocatalyst used in food industry, fine chemicals, and biodiesel production. Yeast surface display allows direct application of lipase in form of whole-cell biocatalyst, avoiding purification and immobilization process, but the protease of the host cell may affect the activity of displayed lipase. Herein, we used the protease-deficient Pichia pastoris, PichiaPink™ as host to display RML efficiently. RML gene, GCW21 gene and α-factor gene were co-cloned into plasmid pPink LC/HC and transformed into protease-deficient P. pastoris. After inducution expression for 96 h, the lipase activity of displayed RML reached 121.72 U/g in proteinase-A-deficient P. pastoris harboring high-copy plasmid, which exhibited 46.7% higher than recombinant P. pastoris without protease defect. Displayed RML occurred the maximum activity at pH 8.0 and 45 °C and the optimal substrate was p-nitrophenyl octanoate. Metal ions Li, Na, K, and Mg of 1-10 mM had activation towards displayed RML. Displayed RML was effectively improved in PichiaPink™ protease-deficient system, which may promote the further research and development for the industrial application of RML.
Topics: Cell Surface Display Techniques; Fungal Proteins; Lipase; Recombinant Proteins; Rhizomucor; Saccharomycetales
PubMed: 33276128
DOI: 10.1016/j.pep.2020.105804 -
Diagnostic Microbiology and Infectious... Oct 2020Rhizomucor miehei is a cause of bovine mycotic abortion and mastitis and has rarely been described in human disease. Here, we report the first isolation of R. miehei...
Rhizomucor miehei is a cause of bovine mycotic abortion and mastitis and has rarely been described in human disease. Here, we report the first isolation of R. miehei from native mitral valve tissue in a fatal case of endocarditis that substantiates its pathogenic potential. Apart from morphological criteria, molecular methods were a cornerstone for definite diagnosis.
Topics: Antifungal Agents; Endocarditis; Fatal Outcome; Humans; Immunocompromised Host; Male; Mitral Valve; Mucormycosis; Rhizomucor; Young Adult
PubMed: 32629298
DOI: 10.1016/j.diagmicrobio.2020.115106 -
EFSA Journal. European Food Safety... Aug 2022The food enzyme mucorpepsin (EC 3.4.23.23) is produced with the non-genetically modified strain DSM 29547 by Chr. Hansen. The food enzyme is free from viable cells of...
The food enzyme mucorpepsin (EC 3.4.23.23) is produced with the non-genetically modified strain DSM 29547 by Chr. Hansen. The food enzyme is free from viable cells of the production organism. It is intended to be used in dairy processing for cheese production. The dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.26 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 618 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 2,400. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and three matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded but is considered low except for individuals sensitised to mustard proteins, but this risk will not exceed that of mustard consumption. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.
PubMed: 35978613
DOI: 10.2903/j.efsa.2022.7457 -
Case Reports in Medicine 2011Hematopoietic stem-cell transplant recipients are at increased risk of developing invasive fungal infections. This is a major cause of morbidity and mortality. We report...
Hematopoietic stem-cell transplant recipients are at increased risk of developing invasive fungal infections. This is a major cause of morbidity and mortality. We report a case of a 17-year-old male patient diagnosed with severe idiopathic acquired aplastic anemia who developed fungal pneumonitis due to Rhizomucor sp. and rhinoencephalitis due to Scedosporium apiospermum 6 and 8 months after undergoing allogeneic hematopoietic stem-cell transplant from an HLA-matched unrelated donor. Discussion highlights risk factors for invasive fungal infections (i.e., mucormycosis and scedosporiosis), its clinical features, and the factors that must be taken into account to successfully treat them (early diagnosis, correction of predisposing factors, aggressive surgical debridement, and antifungal and adjunctive therapies).
PubMed: 21547214
DOI: 10.1155/2011/830769 -
BMJ Case Reports Oct 2017A 61-year-old man with relapsing chronic lymphocytic leukaemia, status post allogeneic stem cell transplant and multiple chemotherapy regimens presented to the emergency...
A 61-year-old man with relapsing chronic lymphocytic leukaemia, status post allogeneic stem cell transplant and multiple chemotherapy regimens presented to the emergency room after suffering a grand mal seizure. His evaluation revealed a 1.5-2 cm ring-enhancing left temporal lobe brain lesion on the CT scan. This brain lesion was resected and the histopathology revealed an invasive fungal organism resembling mucormycosis. Amplification and sequencing of the 28S ribosomal RNA gene identified the organism as The patient was treated with liposomal amphotericin B 5 mg/kg every 24 hours for 4 weeks, and then was transitioned to oral posaconazole. Serial brain imaging at 1 and 3 months, while on therapy, showed significant improvement.
Topics: Amphotericin B; Antifungal Agents; Brain Abscess; Diagnosis, Differential; Humans; Immunocompromised Host; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Mucormycosis; Rhizomucor; Seizures; Stem Cell Transplantation; Temporal Lobe; Tomography, X-Ray Computed
PubMed: 28978601
DOI: 10.1136/bcr-2017-221473 -
Preparative Biochemistry & Biotechnology Jan 2018An alternative environmentally benign support was prepared from chitosan-chitin nanowhiskers (CS/CNWs) for covalent immobilization of Rhizomucor miehei lipase (RML) to...
An alternative environmentally benign support was prepared from chitosan-chitin nanowhiskers (CS/CNWs) for covalent immobilization of Rhizomucor miehei lipase (RML) to increase the operational stability and recyclability of RML in synthesizing eugenyl benzoate. The CS/CNWs support and RML-CS/CNWs were characterized using X-ray diffraction, fluorescent microscopy, and Fourier transform infrared spectroscopy. Efficiency of the RML-CS/CNWs was compared to the free RML to synthesize eugenyl benzoate for parameters: reaction temperature, stirring rate, reusability, and thermal stability. Under optimal experimental conditions (50°C, 250 rpm, catalyst loading 3 mg/mL), a twofold increase in yield of eugenyl benzoate was observed for RML-CS/CNWs as compared to free RML, with the former achieving maximum yield of the ester at 62.1% after 5 hr. Results demonstrated that the strategy adopted to prepare RML-CS/CNWs was useful, producing an improved and prospectively greener biocatalyst that supported a sustainable process to prepare eugenyl benzoate. Moreover, RML-CS/CNWs are biodegradable and perform esterification reactions under ambient conditions as compared to the less eco-friendly conventional acid catalyst. This research provides a facile and promising approach for improving activity of RML in which the resultant RML-CS/CNWs demonstrated good operational stability for up to eight successive esterification cycles to synthesize eugenyl benzoate.
Topics: Benzoates; Chitin; Chitosan; Enzyme Stability; Enzymes, Immobilized; Esterification; Eugenol; Industrial Microbiology; Lipase; Nanostructures; Rhizomucor
PubMed: 29194017
DOI: 10.1080/10826068.2017.1405021 -
Enzyme and Microbial Technology Oct 2022The construction of methanol-resistant lipases with high catalytic activity is world-shattering for biodiesel production. A semi-rational method has been constructed to...
The construction of methanol-resistant lipases with high catalytic activity is world-shattering for biodiesel production. A semi-rational method has been constructed to enhance the properties of Rhizomucor miehei lipase with propeptide (ProRML) by introducing N-glycosylation sites in the Loop structure. The enzyme activities of the mutants N288 (1448.89 ± 68.64 U/mg) and N142 (1073.68 ± 33.87 U/mg) increased to 56.09 and 41.56 times relative to that of wild type ProRML (WT, 25.83 ± 0.73 U/mg), respectively. After incubation in 50 % methanol for 2.5 h, the residual activities of N314 and N174-1 were 95 % and 85%, which were higher than the WT (27 %). Additionally, the biodiesel yield of all mutants was increased after a one-time addition of methanol for 24 h. Among them, N288 increased the quantity of biodiesel from colza oil from 9.49 % to 88 %, and N314 increased the amount of biodiesel from waste soybean oil from 8.44% to 70%. This study provides an effective method to enhance the properties of lipase and improve its application potential in biodiesel production.
Topics: Biofuels; Glycosylation; Lipase; Methanol; Rhizomucor
PubMed: 35689964
DOI: 10.1016/j.enzmictec.2022.110072