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Jose M Guisan - One of the best experts on this subject based on the ideXlab platform.
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high stabilization of immobilized Rhizomucor Miehei lipase by additional coating with hydrophilic crosslinked polymers poly allylamine aldehyde dextran
Process Biochemistry, 2020Co-Authors: Janaina Cejudosanches, Gloria Fernandezlorente, Alejandro H Orrego, Adriana Jaimemendoza, Rohollah Ghobadi, Sonia Morenoperez, Javier Rochamartin, Jose M GuisanAbstract:Abstract Immobilized enzymes have a very large surface region which is not in contact with the support surface and, thus, have potential as a target for novel stabilization strategies. In this paper, coating the surfaces of such enzymes with a highly hydrophilic and compact cross-linked poly-aminated polymer as a strategy to increase the thermal stability of the immobilized enzymes is proposed. In particular, Rhizomucor Miehei lipase (RML) was immobilized by interfacial adsorption onto octyl-agarose and further coated with poly-allylamine (PAA), a polymer that is very rich in primary amino groups. Cross-linking of the PAA layer to coat the immobilized enzyme was carried out, in situ, by reaction with freshly oxidized dextran (aldehyde–dextran). The PAA layer only exerted moderate stabilizing effects (around 4-fold), but further cross-linking with aldehyde–dextran highly increased the stabilizing effects; the new derivative was 440-fold more stable than uncoated derivative at 55 °C and pH 7 and exhibited 6-fold more catalytic activity compared to the soluble enzyme used for immobilization. We hypothesize that the hydrophilicity of PAA reduces the exposure of internal hydrophobic pockets to the enzyme surface at high temperatures. Besides, the compactness of the polymer may reduce distortion of the enzyme surface during inactivation.
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fine modulation of the catalytic properties of Rhizomucor Miehei lipase driven by different immobilization strategies for the selective hydrolysis of fish oil
Molecules, 2020Co-Authors: Maryam Yousefi, Gloria Fernandezlorente, Jose M Guisan, Marzia Marciello, Mehdi Mohammadi, Marco FiliceAbstract:Functional properties of each enzyme strictly depend on immobilization protocol used for linking enzyme and carrier. Different strategies were applied to prepare the immobilized derivatives of Rhizomucor Miehei lipase (RML) and chemically aminated RML (NH2-RML). Both RML and NH2-RML forms were covalently immobilized on glyoxyl sepharose (Gx-RML and Gx-NH2-RML), glyoxyl sepharose dithiothreitol (Gx-DTT-RML and Gx-DTT-NH2-RML), activated sepharose with cyanogen bromide (CNBr-RML and CNBr-NH2-RML) and heterofunctional epoxy support partially modified with iminodiacetic acid (epoxy-IDA-RML and epoxy-IDA-NH2-RML). Immobilization varied from 11% up to 88% yields producing specific activities ranging from 0.5 up to 1.9 UI/mg. Great improvement in thermal stability for Gx-DTT-NH2-RML and epoxy-IDA-NH2-RML derivatives was obtained by retaining 49% and 37% of their initial activities at 70 °C, respectively. The regioselectivity of each derivative was also examined in hydrolysis of fish oil at three different conditions. All the derivatives were selective between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA. The highest selectivity (32.9 folds) was observed for epoxy-IDA-NH2-RML derivative in the hydrolysis reaction performed at pH 5 and 4 °C. Recyclability study showed good capability of the immobilized biocatalysts to be used repeatedly, retaining 50–91% of their initial activities after five cycles of the reaction.
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hydrolysis of fish oil by hyperactivated Rhizomucor Miehei lipase immobilized by multipoint anion exchange
Biotechnology Progress, 2011Co-Authors: Marco Filice, Jose M Guisan, Marzia Marciello, Lorena Betancor, Alfonso V Carrascosa, Gloria FernandezlorenteAbstract:Rhizomucor Miehei lipase (RML) is greatly hyperactivated (around 20- to 25-fold toward small substrates) in the presence of sucrose laurate. Hyperactivation appears to be an intramolecular process because it is very similar for soluble enzymes and covalently immobilized derivatives. The hyperactivated enzyme was immobilized (in the presence of sucrose laurate) on cyanogen bromide-activated Sepharose (very mild covalent immobilization through the amino terminal residue), on glyoxyl Sepharose (intense multipoint covalent immobilization through the region with the highest amount of Lys residues), and on different anion exchangers (by multipoint anionic exchange through the region with the highest density of negative charges). Covalent immobilization does not promote the fixation of the hyperactivated enzyme, but immobilization on Sepharose Q retains the hyperactivated enzyme even in the absence of a detergent. The hydrolysis of fish oils by these hyperactivated enzyme derivatives was sevenfold faster than by covalently immobilized derivatives and three and a half times faster than by the enzyme hyperactivated on octyl-Sepharose. The open structure of the hyperactivated lipase is fairly exposed to the medium, and no steric hindrance should interfere with the hydrolysis of large substrates. These new hyperactivated derivatives seem to be more suitable for hydrolysis of oils by RML immobilized inside porous supports. In addition, the hyperactivated derivatives are fairly stable against heat and organic cosolvents. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
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kinetically controlled synthesis of monoglyceryl esters from chiral and prochiral acids methyl esters catalyzed by immobilized Rhizomucor Miehei lipase
Bioresource Technology, 2011Co-Authors: Andreina Acosta, Marco Filice, Gloria Fernandezlorente, Jose M Palomo, Jose M GuisanAbstract:Abstract Partial acylation of only one primary hydroxyl group of glycerol generates a chiral center at position 2 . Rhizomucor Miehei lipase (RML) catalyzes the kinetically controlled transesterification of different aromatic carboxylic acids methyl esters with glycerol. High synthetic yields of glyceryl esters (around 70–80%) were obtained even in the presence of significant concentrations of water (from 5% to 20%). After a long incubation of the reaction mixture in the presence of the biocatalyst only pure free acid was obtained. Other lipases (from Geobacillus thermocatenulatus and from Thermomyces lanuginose ) also catalyzed similar kinetically controlled transesterifications although less efficiently. RML immobilized on Sepharose-Q showed a high activity and specificity, compared to the immobilization by other techniques, only producing monoglyceryl esters with all substrates. In particular, monoglyceryl-phenylmalonate product was synthesized in 82% overall yield and >99% diastereomeric excess at pH 7.0 and 37 °C and 90% glycerol.
Zhengqiang Jiang - One of the best experts on this subject based on the ideXlab platform.
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a novel aspartic protease from Rhizomucor Miehei expressed in pichia pastoris and its application on meat tenderization and preparation of turtle peptides
Food Chemistry, 2018Co-Authors: Qian Sun, Fusheng Chen, Fang Geng, Yongkang Luo, Siyi Gong, Zhengqiang JiangAbstract:A novel aspartic protease gene (RmproA) was cloned from the thermophilic fungus Rhizomucor Miehei CAU432 and expressed in Pichia pastoris. The RmproA was successfully expressed in P. pastoris as an active extracellular protease. High protease activity of 3480.4 U/mL was obtained by high cell-density fermentation. The protease was purified by the two step protocols to homogeneity. The molecular mass of the RmproA was estimated to be 52.4 kDa by SDS-PAGE and 50.6 kDa by gel filtration. The purified enzyme was optimally active at pH 5.5 and 55 °C, respectively. The enzyme exhibited a broad range of substrate specificity. RmproA-treated pork muscle showed lower shear force than papain-treated sample at a relative low concentration, suggesting its effectiveness on meat tenderization. Moreover, turtle hydrolysis by RmproA resulted in a large amount of small peptides, which exhibited high ACE-inhibitory activity. Thus, RmproA may be a potential candidate for several industrial applications.
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Jiang Z: Biochemical characterization of a first fungal esterase from Rhizomucor Miehei showing high efficiency of ester synthesis
2016Co-Authors: Yu Liu, Qiaojuan Yan, Shaoqing Yang, Xiaojie Duan, Zhengqiang JiangAbstract:Background: Esterases with excellent merits suitable for commercial use in ester production field are still insufficient. The aim of this research is to advance our understanding by seeking for more unusual esterases and revealing their characterizations for ester synthesis. Methodology/Principal Findings: A novel esterase-encoding gene from Rhizomucor Miehei (RmEstA) was cloned and expressed in Escherichia coli. Sequence analysis revealed a 975-bp ORF encoding a 324-amino-acid polypeptide belonging to the hormone-sensitive lipase (HSL) family IV and showing highest similarity (44%) to the Paenibacillus mucilaginosus esterase/lipase. Recombinant RmEstA was purified to homogeneity: it was 34 kDa by SDS-PAGE and showed optimal pH and temperature of 6.5 and 45uC, respectively. The enzyme was stable to 50uC, under a broad pH range (5.0–10.6). RmEstA exhibited broad substrate specificity toward p-nitrophenol esters and short-acyl-chain triglycerols, with highest activities (1,480 U mg21 and 228 U mg21) for p-nitrophenyl hexanoate and tributyrin, respectively. RmEstA efficiently synthesized butyl butyrate (92 % conversion yield) when immobilized on AOT-based organogel. Conclusion: RmEstA has great potential for industrial applications. RmEstA is the first reported esterase from Rhizomucor Miehei
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crystal structure and characterization of a novel l serine ammonia lyase from Rhizomucor Miehei
Biochemical and Biophysical Research Communications, 2015Co-Authors: Zhen Qin, Qiaojuan Yan, Zhengqiang JiangAbstract:L-serine ammonia-lyase, as a member of the beta-family of pyridoxal-5'-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (a-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor Miehei (RmSDH) was solved at 1.76 A resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic beta-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted alpha/beta structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-Iyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 degrees C, respectively. It was stable in the pH range of 7.0-9.0 and at temperatures below 40 degrees C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of beta-elimination enzymes and will provide a basis for further enzyme engineering. (C) 2015 Elsevier Inc. All rights reserved.
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structural insights into the substrate specificity of two esterases from the thermophilic Rhizomucor Miehei
Journal of Lipid Research, 2015Co-Authors: Shaoqing Yang, Qiaojuan Yan, Xiaojie Duan, Zhen Qin, Zhengqiang JiangAbstract:Two hormone-sensitive lipase (HSL) family esterases (RmEstA and RmEstB) from the thermophilic fungus Rhizomucor Miehei, exhibiting distinct substrate specificity, have been recently reported to show great potential in industrial applications. In this study, the crystal structures of RmEstA and RmEstB were determined at 2.15 A and 2.43 A resolutions, respectively. The structures of RmEstA and RmEstB showed two distinctive domains, a catalytic domain and a cap domain, with the classical α/β-hydrolase fold. Catalytic triads consisting of residues Ser161, Asp262, and His292 in RmEstA, and Ser164, Asp261, and His291 in RmEstB were found in the respective canonical positions. Structural comparison of RmEstA and RmEstB revealed that their distinct substrate specificity might be attributed to their different substrate-binding pockets. The aromatic amino acids Phe222 and Trp92, located in the center of the substrate-binding pocket of RmEstB, blocked this pocket, thus narrowing its catalytic range for substrates (C2–C8). Two mutants (F222A and W92F in RmEstB) showing higher catalytic activity toward long-chain substrates further confirmed the hypothesized interference. This is the first report of HSL family esterase structures from filamentous fungi.jlr The information on structure-function relationships could open important avenues of exploration for further industrial applications of esterases.
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high level expression of a novel α galactosidase gene from Rhizomucor Miehei in pichia pastoris and characterization of the recombinant enyzme
Protein Expression and Purification, 2015Co-Authors: Zhou Chen, Zhengqiang Jiang, Qiaojuan Yan, Yu LiuAbstract:Abstract The second α-galactosidase gene (designated as RmgalB ) was cloned from the thermophilic fungus Rhizomucor Miehei and expressed in Pichia pastoris . The gene belonging to glycoside hydrolase (GH) family 36 has an open reading frame (ORF) of 2241 bp encoding 746 amino acids with two introns. The recombinant α-galactosidase (RmgalB) was secreted at high levels of 1953.9 U ml −1 in high cell density fermentor, which is the highest yield obtained for a α-galactosidase. The purified enzyme as a tetramer gave a single band corresponding to a molecular mass of 83.1 kDa in SDS–PAGE. The enzyme exhibited a very high specific activity of 505.5 U mg −1 . The optimum temperature and pH of RmgalB were determined to be 55 °C and pH 5.5, respectively. It was stable within pH 5.5–9.5 and up to 55 °C. RmgalB displayed specificity toward raffinose and stachyose, and completely hydrolyzed the anti-nutritive raffinose family oligosaccharides (RFOs). These properties make RmgalB useful in the food and feed industries.
Roswanira Abdul Wahab - One of the best experts on this subject based on the ideXlab platform.
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Rhizomucor Miehei lipase immobilized on reinforced chitosan chitin nanowhiskers support for synthesis of eugenyl benzoate
Preparative Biochemistry & Biotechnology, 2018Co-Authors: Fatin Myra Abdul Manan, Nursyafreena Attan, Nashi Widodo, Hassan Y Aboulenein, Roswanira Abdul WahabAbstract:ABSTRACTAn 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 sustai...
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enzymatic esterification of eugenol and benzoic acid by a novel chitosan chitin nanowhiskers supported Rhizomucor Miehei lipase process optimization and kinetic assessments
Enzyme and Microbial Technology, 2018Co-Authors: Fatin Myra Abd Manan, Nursyafreena Attan, Zainoha Zakaria, Aemi Syazwani Abdul Keyon, Roswanira Abdul WahabAbstract:A biotechnological route via enzymatic esterification was proposed as an alternative way to synthesize the problematic anti-oxidant eugenyl benzoate. The new method overcomes the well-known drawbacks of the chemical route in favor of a more sustainable reaction process. The present work reports a Box-Behnken design (BBD) optimization process to synthesize eugenyl benzoate by esterification of eugenol and benzoic acid catalyzed by the chitosan-chitin nanowhiskers supported Rhizomucor Miehei lipase (RML-CS/CNWs). Effects of four reaction parameters: reaction time, temperature, substrate molar ratio of eugenol: benzoic acid and enzyme loading were assessed. Under optimum conditions, a maximum conversion yield as high as 66% at 50°C in 5h using 3mg/mL of RML-CS/CNWs, and a substrate molar ratio (eugenol: benzoic acid) of 3:1. Kinetic assessments revealed the RML-CS/CNWs catalyzed the reaction via a ping-pong bi-bi mechanism with eugenol inhibition, characterized by a Vmax of 3.83mMmin-1. The Michaelis-Menten constants for benzoic acid (Km,A) and eugenol (Km,B) were 34.04 and 138.28mM, respectively. The inhibition constant for eugenol (Ki,B) was 438.6mM while the turnover number (kcat) for the RML-CS/CNWs-catalyzed esterification reaction was 40.39min-1. RML-CS/CNWs were reusable up to 8 esterification cycles and showed higher thermal stability than free RML.
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synthesis of geranyl propionate in a solvent free medium using Rhizomucor Miehei lipase covalently immobilized on chitosan graphene oxide beads
Preparative Biochemistry & Biotechnology, 2017Co-Authors: Abdurrahman Adamu Isah, Nursyafreena Attan, Naji Arafat Mahat, Fahrul Huyop, Joazaizulfazli Jamalis, Iffah Izzati Zakaria, Roswanira Abdul WahabAbstract:The chemical route of producing geranyl propionate involves the use of toxic chemicals, liberation of unwanted by-products as well as problematic separation process. In view of such problems, the use of Rhizomucor Miehei lipase (RML) covalently bound onto activated chitosan-graphene oxide (RML-CS/GO) support is suggested. Following analyses using Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and thermogravimetry, properties of the RML-CS/GO were characterized. A response surface methodological approach using a 3-level-four-factor (incubation time, temperature, substrate molar ratio, and stirring rate) Box-Behnken design was used to optimize the experimental conditions to maximize the yield of geranyl propionate. Results revealed that 76 ± 0.02% of recovered protein had yielded 7.2 ± 0.04 mg g-1 and 211 ± 0.3% U g-1 of the maximum protein loading and esterification activity, respectively. The actual yield of geranyl propionate (49.46%) closely agreed with the predicted value (49.97%) under optimum reaction conditions (temperature: 37.67°C, incubation time: 10.20 hr, molar ratio (propionic acid:geraniol): 1:3.28, and stirring rate: 100.70 rpm) and hence, verifying the suitability of this approach. Since the method is performed under mild conditions, the RML-CS/GO biocatalyst may prove to be an environmentally benign alternative for producing satisfactory yield of geranyl propionate.
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statistical modelling of eugenol benzoate synthesis using Rhizomucor Miehei lipase reinforced nanobioconjugates
Process Biochemistry, 2016Co-Authors: Fatin Myra Abd Manan, Ida Nurhazwani Abd Rahman, Nur Haziqah Che Marzuki, Naji Arafat Mahat, Fahrul Huyop, Roswanira Abdul WahabAbstract:The chemical route for synthesizing eugenol benzoate is concomitant with a myriad of environmentally unfavorable practices viz. the use toxic chemicals, tedious separation process and emancipation of harmful unwanted by-products. In this perspective, an alternative technique utilizing Rhizomucor Miehei lipase (RML) immobilized onto activated chitosan-multiwalled carbon nanotubes (RML/CS/MWCNTs) is proposed. The properties and morphology of the RML/CS/MWCNTs were characterized using field emission scanning electron microscopy (FESEM), Fourier-transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). The approach of response surface methodology employing the central composite design (CCD) based on four parameters (incubation time, temperature, substrate molar ratio, and enzyme loading) were used to optimize the experimental conditions for the RML/CS/MWCNTs catalyzed synthesis of eugenol benzoate. The study found that the high yield production of eugenol benzoate is greatly affected by factors such as temperature and incubation time. Under optimized conditions, the highest yield of eugenol benzoate was obtained (56.13%) at 60 °C, 6 h of incubation time, enzyme loading (15 mg) and molar ratio of eugenol/benzoic acid of 4:1. Therefore, the RML/CS/MWCNTs developed here appear to be a promising alternative yet environmentally friendly biocatalyst for a sustainable production of eugenol benzoate.
Mehdi Mohammadi - One of the best experts on this subject based on the ideXlab platform.
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fine modulation of the catalytic properties of Rhizomucor Miehei lipase driven by different immobilization strategies for the selective hydrolysis of fish oil
Molecules, 2020Co-Authors: Maryam Yousefi, Gloria Fernandezlorente, Jose M Guisan, Marzia Marciello, Mehdi Mohammadi, Marco FiliceAbstract:Functional properties of each enzyme strictly depend on immobilization protocol used for linking enzyme and carrier. Different strategies were applied to prepare the immobilized derivatives of Rhizomucor Miehei lipase (RML) and chemically aminated RML (NH2-RML). Both RML and NH2-RML forms were covalently immobilized on glyoxyl sepharose (Gx-RML and Gx-NH2-RML), glyoxyl sepharose dithiothreitol (Gx-DTT-RML and Gx-DTT-NH2-RML), activated sepharose with cyanogen bromide (CNBr-RML and CNBr-NH2-RML) and heterofunctional epoxy support partially modified with iminodiacetic acid (epoxy-IDA-RML and epoxy-IDA-NH2-RML). Immobilization varied from 11% up to 88% yields producing specific activities ranging from 0.5 up to 1.9 UI/mg. Great improvement in thermal stability for Gx-DTT-NH2-RML and epoxy-IDA-NH2-RML derivatives was obtained by retaining 49% and 37% of their initial activities at 70 °C, respectively. The regioselectivity of each derivative was also examined in hydrolysis of fish oil at three different conditions. All the derivatives were selective between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA. The highest selectivity (32.9 folds) was observed for epoxy-IDA-NH2-RML derivative in the hydrolysis reaction performed at pH 5 and 4 °C. Recyclability study showed good capability of the immobilized biocatalysts to be used repeatedly, retaining 50–91% of their initial activities after five cycles of the reaction.
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co immobilization of Rhizomucor Miehei lipase and candida antarctica lipase b and optimization of biocatalytic biodiesel production from palm oil using response surface methodology
Renewable Energy, 2019Co-Authors: Mansour Shahedi, Zohreh Habibi, Mehdi Mohammadi, Maryam Yousefi, Mohammad Ali AshabiAbstract:Abstract Lipases from Candida antarctica B (nonspecific lipase) and Rhizomucor Miehei (1,3-specific lipase) were simultaneously immobilized on epoxy functionalized silica gel under mild conditions. The results showed rapid and simple immobilization of 4–15 mg of CALB:RML (different ratios 4:1, 2:1, 1.5:1, 1:1) on 1 g of support after 6 h. The thermal stability of derivatives and also their stability in methanol were greatly improved compared to the single immobilized enzyme. All the derivatives were also used to catalyze the transesterification of palm oil with methanol to produce fatty acid methyl esters (FAMEs). Response surface methodology (RSM) and a central composite rotatable design (CCRD) was used to study the effects of five factors, reaction temperature, methanol/oil ratio, reaction time, t-butanol concentration and CALB:RML ratio on the fatty acid methyl esters (FAME) yield. A quadratic polynomial equation was obtained for methanolysis reaction by multiple regression analysis. The optimum combinations for the reaction were CALB:RML ratio (2.5:1), t-butanol to oil (39.9 wt%), temperature (35.6 °C), methanol:oil ratio (5.9), reaction time 33.5 h. FAME yield of 78.3.5%, which was very close to the predicted value of 75.2%, was obtained. Verification experiment confirmed the validity of the predicted model.
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a novel approach for bioconjugation of Rhizomucor Miehei lipase rml onto amine functionalized supports application for enantioselective resolution of rac ibuprofen
International Journal of Biological Macromolecules, 2018Co-Authors: Mehdi Mohammadi, Zohreh Habibi, Somayyeh Gandomkar, Maryam YousefiAbstract:Abstract Since the amine groups are highly reactive toward various functional moieties, the formation of covalent bonds between a biomolecule and an insoluble amine-functionalized support is the most frequently used technique in immobilization protocols. A new approach based on the Ugi four-component reaction was used for immobilization of Rhizomucor Miehei lipase (RML) as a model enzyme on amine-functionalized silica and silica nanoparticles (SBA-15). For this, the amine-modified supports were prepared and the structural properties of the functionalized supports, prior to and after functionalization were characterized by using IR, SEM, TGA, DTA, TEM. Immobilization of RML on the aminated carriers was performed under extremely mild conditions (25 °C, pH 7). The results revealed very rapid immobilization of 150 and 200 mg of RML on 1 g of silica-NH2 and SBA-NH2, respectively, producing 95–100% of immobilization yield. The specific activity and optimum pH of the immobilized preparations and the effect of temperature and co-solvents on their stabilities as well as the reusability of the derivatives were tested. The immobilized preparations were also used as enantioselective catalyst in kinetic resolution of racemic ibuprofen. Among them, Silica-RML showed the best selectivity with 92.2% enantiomeric excess (ee) and E-value of 33.9.
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the use of isocyanide based multicomponent reaction for covalent immobilization of Rhizomucor Miehei lipase on multiwall carbon nanotubes and graphene nanosheets
RSC Advances, 2016Co-Authors: Mehdi Mohammadi, Maryam Yousefi, Maryam Ashjari, Maryam Garmroodi, Ali Asghar KarkhaneAbstract:We describe here a novel and simple method for making bioconjugation and immobilization of Rhizomucor Miehei lipase (RML) on carboxylated multiwall carbon nanotubes (MWCNT-COOH) and carboxylated graphene nanosheets (Gr-COOH) by using an isocyanide-based four-component reaction. In this approach, the enzyme supplies amino groups, the support supplies carboxylic acid groups, and the missing components (isocyanide and aldehyde) are added to the reaction medium. This coupling reaction was carried out in water at 25 °C, in which rapid and high enzyme loading were achieved. The maximum loading capacity of 530 mg and 680 mg was obtained for Gr-COOH and MWCNT-COOH, respectively. A variety of techniques including FTIR, Raman spectroscopy, XRD, SEM, and TGA were employed to characterize the immobilized derivatives of RML. The immobilized preparations showed significantly increased thermal stability and co-solvent stability as compared to the soluble enzyme. Kinetic parameters and optimum pH activity of RML and its immobilized preparations were also determined. The Km values of 0.44, 0.23, and 0.18 mM and the maximum reaction rates (Vmax) of 0.09, 0.1, and 0.08 mM min−1 were obtained for MWCNTs-RML, Gr-RML, and free RML, respectively. This approach may provide a general and efficient method to attach biomolecules on a variety of carboxylated solid surfaces at ambient conditions.
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rapid and high density covalent immobilization of Rhizomucor Miehei lipase using a multi component reaction application in biodiesel production
RSC Advances, 2015Co-Authors: Mehdi Mohammadi, Maryam Yousefi, Shaghayegh Dezvarei, Maryam Ashjari, Mohadese Babaki, Javad MohammadiAbstract:Aldehyde-functionalized silica and silica nanoparticles (SBA-15) were prepared as a matrix system for enzyme immobilization. Immobilization of Rhizomucor Miehei lipase (RML) on these supports was performed via a multicomponent reaction under extremely mild conditions (25 °C, pH 7). Investigation on the mechanism of this reaction confirmed the Ugi four-component immobilization approach. The loading capacity of the supports and specific activity of the immobilized derivatives were interestingly improved. The results revealed very rapid immobilization of 10 and 60 mg of RML on 1 g of aldehyde-functionalized silica and SBA-15 after 10 and 30 minutes, respectively. Leaching experiments were performed by incubation of the immobilized derivatives in 1 M NaCl solution. The lack of the free lipase in the solution confirmed the covalent nature of the linkage. The thermal stability and co-solvent stability of the derivatives in the presence of three polar organic solvents (1-propanol, 2-propanol and dioxane) were greatly improved compared to the soluble enzyme. Both the derivatives were also used to catalyze the transesterification of colza oil with methanol to produce fatty acid methyl esters (FAMEs). In the case of RML immobilized on SBA-15 (SBA-RML), the presence of 40% of tert-butanol (v/v) as solvent in the reaction medium largely improved the conversion yield.
Maryam Yousefi - One of the best experts on this subject based on the ideXlab platform.
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fine modulation of the catalytic properties of Rhizomucor Miehei lipase driven by different immobilization strategies for the selective hydrolysis of fish oil
Molecules, 2020Co-Authors: Maryam Yousefi, Gloria Fernandezlorente, Jose M Guisan, Marzia Marciello, Mehdi Mohammadi, Marco FiliceAbstract:Functional properties of each enzyme strictly depend on immobilization protocol used for linking enzyme and carrier. Different strategies were applied to prepare the immobilized derivatives of Rhizomucor Miehei lipase (RML) and chemically aminated RML (NH2-RML). Both RML and NH2-RML forms were covalently immobilized on glyoxyl sepharose (Gx-RML and Gx-NH2-RML), glyoxyl sepharose dithiothreitol (Gx-DTT-RML and Gx-DTT-NH2-RML), activated sepharose with cyanogen bromide (CNBr-RML and CNBr-NH2-RML) and heterofunctional epoxy support partially modified with iminodiacetic acid (epoxy-IDA-RML and epoxy-IDA-NH2-RML). Immobilization varied from 11% up to 88% yields producing specific activities ranging from 0.5 up to 1.9 UI/mg. Great improvement in thermal stability for Gx-DTT-NH2-RML and epoxy-IDA-NH2-RML derivatives was obtained by retaining 49% and 37% of their initial activities at 70 °C, respectively. The regioselectivity of each derivative was also examined in hydrolysis of fish oil at three different conditions. All the derivatives were selective between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA. The highest selectivity (32.9 folds) was observed for epoxy-IDA-NH2-RML derivative in the hydrolysis reaction performed at pH 5 and 4 °C. Recyclability study showed good capability of the immobilized biocatalysts to be used repeatedly, retaining 50–91% of their initial activities after five cycles of the reaction.
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co immobilization of Rhizomucor Miehei lipase and candida antarctica lipase b and optimization of biocatalytic biodiesel production from palm oil using response surface methodology
Renewable Energy, 2019Co-Authors: Mansour Shahedi, Zohreh Habibi, Mehdi Mohammadi, Maryam Yousefi, Mohammad Ali AshabiAbstract:Abstract Lipases from Candida antarctica B (nonspecific lipase) and Rhizomucor Miehei (1,3-specific lipase) were simultaneously immobilized on epoxy functionalized silica gel under mild conditions. The results showed rapid and simple immobilization of 4–15 mg of CALB:RML (different ratios 4:1, 2:1, 1.5:1, 1:1) on 1 g of support after 6 h. The thermal stability of derivatives and also their stability in methanol were greatly improved compared to the single immobilized enzyme. All the derivatives were also used to catalyze the transesterification of palm oil with methanol to produce fatty acid methyl esters (FAMEs). Response surface methodology (RSM) and a central composite rotatable design (CCRD) was used to study the effects of five factors, reaction temperature, methanol/oil ratio, reaction time, t-butanol concentration and CALB:RML ratio on the fatty acid methyl esters (FAME) yield. A quadratic polynomial equation was obtained for methanolysis reaction by multiple regression analysis. The optimum combinations for the reaction were CALB:RML ratio (2.5:1), t-butanol to oil (39.9 wt%), temperature (35.6 °C), methanol:oil ratio (5.9), reaction time 33.5 h. FAME yield of 78.3.5%, which was very close to the predicted value of 75.2%, was obtained. Verification experiment confirmed the validity of the predicted model.
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a novel approach for bioconjugation of Rhizomucor Miehei lipase rml onto amine functionalized supports application for enantioselective resolution of rac ibuprofen
International Journal of Biological Macromolecules, 2018Co-Authors: Mehdi Mohammadi, Zohreh Habibi, Somayyeh Gandomkar, Maryam YousefiAbstract:Abstract Since the amine groups are highly reactive toward various functional moieties, the formation of covalent bonds between a biomolecule and an insoluble amine-functionalized support is the most frequently used technique in immobilization protocols. A new approach based on the Ugi four-component reaction was used for immobilization of Rhizomucor Miehei lipase (RML) as a model enzyme on amine-functionalized silica and silica nanoparticles (SBA-15). For this, the amine-modified supports were prepared and the structural properties of the functionalized supports, prior to and after functionalization were characterized by using IR, SEM, TGA, DTA, TEM. Immobilization of RML on the aminated carriers was performed under extremely mild conditions (25 °C, pH 7). The results revealed very rapid immobilization of 150 and 200 mg of RML on 1 g of silica-NH2 and SBA-NH2, respectively, producing 95–100% of immobilization yield. The specific activity and optimum pH of the immobilized preparations and the effect of temperature and co-solvents on their stabilities as well as the reusability of the derivatives were tested. The immobilized preparations were also used as enantioselective catalyst in kinetic resolution of racemic ibuprofen. Among them, Silica-RML showed the best selectivity with 92.2% enantiomeric excess (ee) and E-value of 33.9.
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the use of isocyanide based multicomponent reaction for covalent immobilization of Rhizomucor Miehei lipase on multiwall carbon nanotubes and graphene nanosheets
RSC Advances, 2016Co-Authors: Mehdi Mohammadi, Maryam Yousefi, Maryam Ashjari, Maryam Garmroodi, Ali Asghar KarkhaneAbstract:We describe here a novel and simple method for making bioconjugation and immobilization of Rhizomucor Miehei lipase (RML) on carboxylated multiwall carbon nanotubes (MWCNT-COOH) and carboxylated graphene nanosheets (Gr-COOH) by using an isocyanide-based four-component reaction. In this approach, the enzyme supplies amino groups, the support supplies carboxylic acid groups, and the missing components (isocyanide and aldehyde) are added to the reaction medium. This coupling reaction was carried out in water at 25 °C, in which rapid and high enzyme loading were achieved. The maximum loading capacity of 530 mg and 680 mg was obtained for Gr-COOH and MWCNT-COOH, respectively. A variety of techniques including FTIR, Raman spectroscopy, XRD, SEM, and TGA were employed to characterize the immobilized derivatives of RML. The immobilized preparations showed significantly increased thermal stability and co-solvent stability as compared to the soluble enzyme. Kinetic parameters and optimum pH activity of RML and its immobilized preparations were also determined. The Km values of 0.44, 0.23, and 0.18 mM and the maximum reaction rates (Vmax) of 0.09, 0.1, and 0.08 mM min−1 were obtained for MWCNTs-RML, Gr-RML, and free RML, respectively. This approach may provide a general and efficient method to attach biomolecules on a variety of carboxylated solid surfaces at ambient conditions.
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rapid and high density covalent immobilization of Rhizomucor Miehei lipase using a multi component reaction application in biodiesel production
RSC Advances, 2015Co-Authors: Mehdi Mohammadi, Maryam Yousefi, Shaghayegh Dezvarei, Maryam Ashjari, Mohadese Babaki, Javad MohammadiAbstract:Aldehyde-functionalized silica and silica nanoparticles (SBA-15) were prepared as a matrix system for enzyme immobilization. Immobilization of Rhizomucor Miehei lipase (RML) on these supports was performed via a multicomponent reaction under extremely mild conditions (25 °C, pH 7). Investigation on the mechanism of this reaction confirmed the Ugi four-component immobilization approach. The loading capacity of the supports and specific activity of the immobilized derivatives were interestingly improved. The results revealed very rapid immobilization of 10 and 60 mg of RML on 1 g of aldehyde-functionalized silica and SBA-15 after 10 and 30 minutes, respectively. Leaching experiments were performed by incubation of the immobilized derivatives in 1 M NaCl solution. The lack of the free lipase in the solution confirmed the covalent nature of the linkage. The thermal stability and co-solvent stability of the derivatives in the presence of three polar organic solvents (1-propanol, 2-propanol and dioxane) were greatly improved compared to the soluble enzyme. Both the derivatives were also used to catalyze the transesterification of colza oil with methanol to produce fatty acid methyl esters (FAMEs). In the case of RML immobilized on SBA-15 (SBA-RML), the presence of 40% of tert-butanol (v/v) as solvent in the reaction medium largely improved the conversion yield.
