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Enzyme and Microbial Technology Aug 2021Oil palm leaves (OPL) silica (SiO) can replace the energy-intensive, commercially produced SiO. Moreover, the agronomically sourced biogenic SiO is more biocompatible...
Oil palm leaves (OPL) silica (SiO) can replace the energy-intensive, commercially produced SiO. Moreover, the agronomically sourced biogenic SiO is more biocompatible and cost-effective enzyme support, which properties could be improved by the addition of magnetite (FeO) and graphene oxide (GO) to yield better ternary support to immobilize enzymes, i.e., Candida rugosa lipase (CRL). This study aimed to optimize the Candida rugosa lipase (CRL immobilization onto the ternary OPL-silica-magnetite (FeO)-GO (SiO/FeO/GO) support, for use as biocatalyst for ethyl valerate (EV) production. Notably, this is the first study detailing the CRL/SiO/Fe3O4/GO biocatalyst preparation for rapid and high yield production of ethyl valerate (EV). AFM and FESEM micrographs revealed globules of CRL covalently bound to GL-A-SiO/FeO/GO; similar to Raman and UV-spectroscopy results. FTIR spectra revealed amide bonds at 3478 cm and 1640 cm from covalent interactions between CRL and GL-A-SiO/FeO/GO. Optimum immobilization conditions were 4% (v/v) glutaraldehyde, 8 mg/mL CRL, at 16 h stirring in 150 mM NaCl at 30 °C, offering 24.78 ± 0.26 mg/g protein (specific activity = 65.24 ± 0.88 U/g). The CRL/SiO/FeO/GO yielded 77.43 ± 1.04 % of EV compared to free CRL (48.75 ± 0.70 %), verifying the suitability of SiO/FeO/GO to hyperactivate and stabilize CRL for satisfactory EV production.
Topics: Biocatalysis; Enzyme Stability; Enzymes, Immobilized; Esterification; Ferrosoferric Oxide; Graphite; Lipase; Saccharomycetales; Silicon Dioxide; Valerates
PubMed: 34116744
DOI: 10.1016/j.enzmictec.2021.109807 -
Biodegradation Oct 2021In this work, strains of Bacillus subtilis were inoculated in consortium with Rhodotorula mucilaginosa into spent soy oil as aiming to biological treatment and low-cost...
In this work, strains of Bacillus subtilis were inoculated in consortium with Rhodotorula mucilaginosa into spent soy oil as aiming to biological treatment and low-cost reuse. The microorganisms were previously isolated and selected for the lipolytic capacity of the alperujo residue generated during the processing of olive oil. For fermentation, bioassays containing Rhodotorula mucilaginosa isolated from alperujo and Candida rugosa CCMA 00371, both co-inoculated with Bacillus subtilis CCMA 0085 in medium containing (% w/v) 0.075 glucose and 0.375 (NH) PO in 75 mL of water and 75 mL of spent soy oil. Despite the low biomass productivity, it has favorable characteristics to be used in animal feed supplementation. Spent soy oil was used as a carbon source proven by Bartha respirometer. The strains of R. mucilaginosa UFLA RAS 144 and B. subtilis CCMA 0085 are promising inoculants for oil degradation and can be applied in a waste treatment system.
Topics: Biodegradation, Environmental; Hydrolysis; Microbial Consortia; Rhodotorula; Saccharomycetales; Soybean Oil; Technology
PubMed: 34046776
DOI: 10.1007/s10532-021-09951-0 -
Tropical Animal Health and Production Apr 2021Mycotic mastitis is a neglected problem type of incurable chronic mastitis in sheep flock of many countries which associated with wide economic burden. In the current...
Mycotic mastitis is a neglected problem type of incurable chronic mastitis in sheep flock of many countries which associated with wide economic burden. In the current study, a total of 600 ewes at Menofia governorate, Egypt, were subjected to clinical and molecular examination using PCR-RFLP to estimate the prevalence of chronic mycotic mastitis and identify the causative agent. A structured questionnaire is distributed to shepherds in the study area to identify the risky behavioral practices being followed and lead to increase the prevalence of mycotic mastitis cases. The results showed that out of 600 ewes examined, 150 showed clinical signs of mastitis (25%). A total of 25 ewes with clinical mastitis did not respond to antibiotic treatment for long time and suffered from mycotic mastitis (16.7%, CI 11.1-23.6%). A total of 31 fungal isolates were identified: 14 yeast spp., Candida albicans, Candida parapasilosis, Candida rugosa, and Saccharomyces spp. and 17 mold spp., Alternaria spp., and Fusarium spp. Results showed also the widespread of risky practices among shepherds which could be responsible for the increase the prevalence of mycotic mastitis among ewes in the study area including presenting of decayed food to sheep, uncontrolled usage of antibiotics for mastitis treatment, lack of usage of antiseptics, and keeping of chronic infected animals in flocks for breeding. In conclusions, using of specific ITS1 and ITS4 primer sets with PCR-RFLP technique provided a suitable method for rapid identification and genotyping of Candida spp., Scaccharomyces, Alternaria, and Fusarium vertolliodes isolated from chronic mastitis in sheep. Furthermore, this study is considered up to our knowledge one of scarce estimates available on mycotic mastitis in sheep flocks in Egypt. Mycotic mastitis existed at higher prevalence estimates in the study area and educational campaigns to shepherds are much required to increase their awareness on the threat of risky of behaviors responsible for spread of the disease among their animals.
Topics: Animals; Causality; Egypt; Female; Genotype; Mastitis; Milk; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Prevalence; Saccharomycetales; Sheep; Sheep Diseases
PubMed: 33866417
DOI: 10.1007/s11250-021-02703-5 -
Biochimica Et Biophysica Acta.... Jul 2021Bioaugmentation is a promising method of the remediation of soils polluted by persistent organic pollutants (POP). Unfortunately, it happens frequently that the...
Bioaugmentation is a promising method of the remediation of soils polluted by persistent organic pollutants (POP). Unfortunately, it happens frequently that the microorganisms inoculated into the soil die out due to the presence of enzymes secreted by autochthonous microorganisms. Especially destructive are here phospholipases C (PLC) and lipases which destruct the microorganism's cellular membrane. The composition of bacterial membranes differs between species, so it is highly possible that depending on the membrane constitution some bacteria are more resistant to PLCs and lipases than other. To shed light on these problems we applied phospholipid Langmuir monolayers as model microbial membranes and studied their interactions with α-toxin (model bacterial PLC) and the lipase isolated from soil fungus Candida rugosa. Membrane phospholipids differing in their headgroup (phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols and cardiolipins) and in their tail structure were applied. The monolayers were characterized by the Langmuir technique, visualized by Brewster angle microscopy, and the packing mode of the phospholipid molecules was verified by the application of the diffraction of synchrotron radiation. We also studied the mutual miscibility of diacylglycerols and the native phospholipids as their interaction is crucial for the understanding of the PLC and lipase activity. It turned out that all the investigated phospholipid classes can be hydrolyzed by PLC; however, they differ profoundly in the hydrolysis degree. Depending on the effects of the initial PLC action and the mutual organization of the diacylglycerol and phospholipid molecules the lipase can ruin the model membranes or can be completely neutral to them.
Topics: Clostridium perfringens; Lipase; Models, Biological; Phosphatidylcholines; Phospholipids; Saccharomycetales; Type C Phospholipases; Unilamellar Liposomes
PubMed: 33831405
DOI: 10.1016/j.bbamem.2021.183620 -
Applied Optics Apr 2021Data acquisition and processing is a critical issue for high-speed applications, especially in three-dimensional live cell imaging and analysis. This paper focuses on...
Data acquisition and processing is a critical issue for high-speed applications, especially in three-dimensional live cell imaging and analysis. This paper focuses on sparse-data sample rotation tomographic reconstruction and analysis with several noise-reduction techniques. For the sample rotation experiments, a live Candida rugosa sample is used and controlled by holographic optical tweezers, and the transmitted complex wavefronts of the sample are recorded with digital holographic microscopy. Three different cases of sample rotation tomography were reconstructed for dense angle with a step rotation at every 2°, and for sparse angles with step rotation at every 5° and 10°. The three cases of tomographic reconstruction performance are analyzed with consideration for data processing using four noise-reduction techniques. The experimental results demonstrate potential capability in retaining the tomographic image quality, even at the sparse angle reconstructions, with the help of noise-reduction techniques.
Topics: Deep Learning; Holography; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; Optical Tweezers; Rotation; Saccharomycetales; Signal-To-Noise Ratio; Tomography
PubMed: 33798139
DOI: 10.1364/AO.415284 -
Preparative Biochemistry & Biotechnology 2022Lipid-rich wastewater from the local dairy industry (cheese whey) in the Galilee, Israel was hydrolyzed by using two different sources of lipase as hydrolytic enzymes:...
Lipid-rich wastewater from the local dairy industry (cheese whey) in the Galilee, Israel was hydrolyzed by using two different sources of lipase as hydrolytic enzymes: fungal () and animal porcine pancreatic lipase(PPL). Pretreatment efficiency was verified by comparative biodegradability tests of raw and treated wastewater samples. Simultaneous hydrolysis and anaerobic digestion in the same reactors were also tested. Enzymatic pretreatment of these samples at a concentration of 0.05 w v showed organic matter removal of 90% and methane formation increases of 140% for the fungal source enzyme (i.e., AY), while for the animal source enzyme (i.e., PPL) was 86 and 130%, respectively. Enzymatic pretreatment led to significant methane formation which was obtained only for moderate substrate concentration (initial chemical oxygen demand of 15 gL); While in high concentrated lipid-rich wastewater led to methane yield inhibition. The main finding was that the combination of AY enzyme with (i.e., enzyme mixture) led to a high efficiency in methane production (+152%) and organic materials removal (more than 90%). In summary, the use of fungal hydrolytic lipase mixed with fungus is a promising method for enhancing methane production during the biodegradation of fat and grease-rich wastewaters.
Topics: Animals; Biodegradation, Environmental; Hydrolysis; Industrial Microbiology; Lipase; Lipid Metabolism; Lipids; Methane; Saccharomycetales; Swine; Wastewater
PubMed: 33787468
DOI: 10.1080/10826068.2021.1901233 -
International Journal of Biological... May 2021In this paper, an epoxy-activated cloisite (ECL) was prepared as a new heterofunctional carrier via a reaction between cloisite 30B (CL) and epichlorohydrin and utilized...
Covalent immobilization of lipase from Candida rugosa on epoxy-activated cloisite 30B as a new heterofunctional carrier and its application in the synthesis of banana flavor and production of biodiesel.
In this paper, an epoxy-activated cloisite (ECL) was prepared as a new heterofunctional carrier via a reaction between cloisite 30B (CL) and epichlorohydrin and utilized for covalent immobilization of lipase from Candida rugosa. The lipase immobilized on the ECL (LECL) was successfully used in the olive oil hydrolysis, synthesis of isoamyl acetate (banana flavor), and biodiesel production. The TGA, FT-IR, SEM, and XRD were used to characterize CL, ECL, and LECL. The influences of temperature, pH, thermal stability, and storage capacity were examined in the olive oil hydrolysis. The effects of solvent, temperature, time, water content, and substrates molar ratio on the yields of ester and biodiesel were also investigated. In the optimized conditions, the hydrolytic activity of LECL was 1.85 ± 0.05 U/ mg, and the maximum yield of ester and biodiesel was 91.6% and 95.4%, respectively. The LECL showed good thermal stability and storage capacity compared to the free lipase. Additionally, LECL was reusable for both esterification and transesterification after being used for nine cycles.
Topics: Biofuels; Enzymes, Immobilized; Esterification; Flavoring Agents; Fungal Proteins; Lipase; Saccharomycetales
PubMed: 33667558
DOI: 10.1016/j.ijbiomac.2021.02.146 -
Bioorganic Chemistry Apr 2021Candida rugosa lipase (CRL) is an enzyme commonly used in medicinal and biotechnological applications. Allosteric modulators of CRL could aid in modifying lipase-related...
Candida rugosa lipase (CRL) is an enzyme commonly used in medicinal and biotechnological applications. Allosteric modulators of CRL could aid in modifying lipase-related diseases as well as improving biotechnological processes. Thus, a combinatorial approach of computational in-silico and high-throughput in-vitro screening was used to identify allosteric modulators of CRL. The screening of natural product libraries resulted in 132 compounds of which 53 were tested in-vitro. Subsequently, four inhibitors and three enhancers were identified of which rutin and cynaroside represented the strongest inhibitors of CRL activity (IC50: 227 ± 26 µM and 446 ± 15 µM, respectively) and NP-008496 the strongest enhancer (EC50: 425 ± 18 µM). All three compounds were predicted to bind the same allosteric site suggesting a common mechanism. Therefore, the present study demonstrated a reliable work-flow, identified an allosteric site of CRL and determined inhibitors and enhancers with numerous potential medical and biotechnological applications.
Topics: Allosteric Site; Biological Products; Dose-Response Relationship, Drug; Enzyme Inhibitors; Kinetics; Lipase; Molecular Docking Simulation; Molecular Structure; Saccharomycetales; Structure-Activity Relationship
PubMed: 33639364
DOI: 10.1016/j.bioorg.2021.104732 -
Journal of Chemical Information and... Feb 2021Lipases are enzymes able to catalyze the hydrolysis or synthesis of triglycerides, depending on the reaction conditions, whereas sterol esterases show the same ability...
Lipases are enzymes able to catalyze the hydrolysis or synthesis of triglycerides, depending on the reaction conditions, whereas sterol esterases show the same ability on sterol esters. Structurally, both kinds of enzymes display an α/β-hydrolase fold, with a substrate-binding pocket formed by a hydrophobic cavity covered by a mobile lid. However, it has been reported that some lipases from the -like family display wide substrate specificity on both triglycerides and sterol esters. Among them, enzymes with different biotechnological applications, such as the lipase isoenzymes produced by and the sterol esterase from , have been exhaustively characterized and their crystal structures are available. Differences in substrate affinity among these proteins have been attributed to changes in their hydrophobicity. In this work, we analyzed the full catalytic mechanisms of these proteins using molecular dynamics tools, gaining insight into their mechanistic properties. In addition, we developed an protocol to predict the substrate specificity using and lipases as model enzymes and triglycerides and cholesterol esters with different fatty acid chain lengths as model substrates. The protocol was validated by comparing the results with those described in the literature. These results would be useful to perform virtual screening of substrates for enzymes of the -like family with unknown catalytic properties.
Topics: Candida; Lipase; Ophiostoma; Saccharomycetales; Sterol Esterase; Substrate Specificity
PubMed: 33555857
DOI: 10.1021/acs.jcim.0c01151 -
Enzyme and Microbial Technology Mar 2021The present work deals with the synthesis of lab-made carboxymethyl cellulose (CMC) and chitosan (CHI) based co-polymer cross-linked with glutaraldehyde (GLU) which is...
The present work deals with the synthesis of lab-made carboxymethyl cellulose (CMC) and chitosan (CHI) based co-polymer cross-linked with glutaraldehyde (GLU) which is used as immobilization matrix for the immobilization of Candida rugosa (CRL) lipase (CMC:CHI:GLU:CRL). This immobilized biocatalyst was subjected to characterization such as lipase-activity, kinetic-parameters, water-content, surface-texture, stability and half-life time etc. Effect of various ultrasound parameters (power, frequency, duty cycle, exposure time) on lipase activity is also tested which indicated that, developed biocatalyst has significant activity-stability and half-life-time in ultrasonicated medium. Further, this biocatalyst was applied to synthesize biomass-derived furfuryl derivative which offering excellent conversion of 99 % of bio-based furfuryl ester. The synthetic protocol is optimized in detail (with twelve reaction parameters) under ultrasonicated medium. Recyclability study offered 68 % conversion of the furfuryl ester after sixth reuse. Moreover, the developed protocol is well extended to synthesize various commercially important compounds. Besides this, we investigated thermodynamic parameters (ΔG*, ΔH*, ΔS*) which demonstrating more feasibility of biocatalytic synthesis in ultrasonicated medium than conventional medium. Finally, green metrics evaluation parameters (E-factor, carbon-efficiency and mass-intensity) are studied which indicating efficient synergetic role of immobilized CMC:CHI:GLU:CRL lipase biocatalysis and ultrasonication in green and sustainable synthesis.
Topics: Benchmarking; Biocatalysis; Biomass; Enzyme Stability; Enzymes, Immobilized; Lipase; Saccharomycetales
PubMed: 33541579
DOI: 10.1016/j.enzmictec.2020.109738