Miehei Lipase Research Articles

Shifting-Nitroxides to Investigate Enzymatic Hydrolysis of Fatty Acids by Lipases Using Electron Paramagnetic Resonance in Turbid Media.

While optical methods are not efficient enough for the easy, fast, and efficient detection of enzymatic activity in turbid media, the properties of the electron paramagnetic resonance (EPR) technique make it suitable for use in such media. Nitroxides which exhibit a change in their EPR hyperfine coupling constants upon enzymatic activity and are selective to lipases were developed under the name of shifting-nitroxides. Several fatty acids, exhibiting saturated and unsaturated chains of various lengths, were coupled with the shifting-nitroxide via an enol ester link and tested against several lipases. As the solubility of fatty acids is low in HEPES buffer, experiments were performed in turbid aqueous solution. Almost all labeled fatty acids were hydrolyzed by Candida rugosa lipase, and more selectivity is observed with Porcine Pancreas lipase type II. No activity was observed for lipase AK Amano 20, Candida antartica lipase B, and Mucor miehei lipase.

Co-immobilization of Rhizomucor miehei lipase and Candida antarctica lipase B and optimization of biocatalytic biodiesel production from palm oil using response surface methodology

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.

Rhizomucor miehei Lipase Supported on Inorganic Solids, as Biocatalyst for the Synthesis of Biofuels: Improving the Experimental Conditions by Response Surface Methodology

Two inorganic solids have been evaluated as supports of Lipozyme RM IM, a Rhizomucor miehei lipase immobilized on a macroporous anion exchange resin, in order to improve its application as a biocatalyst in the synthesis of biofuels. The experimental conditions have been optimized to get the selective transesterification of sunflower oil, by using a multi-factorial design based on the response surface methodology (RSM). In this way, the effects of several reaction parameters on the selective ethanolysis of triglycerides to produce Ecodiesel, a biodiesel-like biofuel constitute by one mole of monoglyceride (MG) and two moles of fatty acid ethyl ester (FAEE), have been evaluated. Thus, it was obtained that a 6:1 oil/ethanol molar ratio, 0.215 g of biocatalyst supported in silica-gel (0.015 g Lipase/0.2 g silica-gel), 50 µL of 10 N NaOH, together with previous optimized reaction parameters, 35 °C reaction temperature and 120 min of reaction time, gave the best results (conversions around 70%; selectivity around 65%; kinematic viscosities about 9.3 mm2/s) in the reaction studied. Besides, Lipozyme RM IM, supported on silica-gel, biocatalyst exhibited a very good stability, remaining its activity even after 15 cycles.

Ternary Blended Chitosan/Chitin/ $$\hbox {FE}_{3}\hbox {O}_{4}$$ FE 3 O 4 Nanosupport for Lipase Activation and Stabilization

To overcome the drawbacks and high costs of the synthetic route to produce pentyl valerate (PeVa), i.e., a fuel additive, the biotechnological route of utilizing Rhizomucor miehei lipase conjugated to magnetite carrier support (RML-CS/CH/MNPs) is proposed. The magnetized RML-CS/CH/MNPs were developed for enabling high yield production of PeVa at relatively short reaction time $$(<$$ 3 h), while facilitating easy removal of excess RML-CS/CH/MNPs. Efficacy of the developed RML-CS/CH/MNPs biocatalyst was assessed for relevant esterification factors, viz. time, enzyme loading, temperature, substrate molar ratio and stirring speed. The highest yield of PeVa (96%) was reached under an optimized condition ( $$50\,^{\circ }\hbox {C}$$ , using valeric acid/pentanol molar ratio of 1:2, with a lipase loading of 1.5 mg/mL, incubation time of 3 h), thus indicating the RML was catalyzing at its fullest potential due to improved activity and stability after immobilization onto CS/CH/MNPs. RML-CS/CH/MNPs retained a 90% activity after 40 days of storage and leached an initial 3.5% of the lipase likely due to insufficient rinsing during preparation. Since the RML-CS/CH/MNPs was prepared without the use of acids, it appears to be a greener catalyst for the esterification production of PeVa and presumably for other types of commercially important esters, too.

THE EFFECT OF ACUTE CONSUMPTION OF HYDROLYZED OILS ON SWIMMING CAPACITY ENDURANCE OF MICE (MUS MUSCULUS)

Objective: The aim of this study was to investigate the effect of hydrolyzed oils of different fatty acids composition on swimming capacity of mice.Methods:The oils used were Virgin Coconut oil (VCO) and Palm Kernel oil (PKO) as medium chain triglyceride (MCT) oils, Palm oil and Corn oil as long chain triglyceride (LCT) oils. Hydrolysis of the oil was performed with Rhizomucor miehei lipase enzyme. The mice were acclimatized and tested motoric to select the mice and divided into twenty groups, each group consisted of 5 mice. Each oil in the experiment were 0.1 ml, 0.2 ml, 0.4 ml/ 20 g BW, positive control was caffeine 13 mg/kg bw. Then, the mice were placed in a glass aquarium. Then swimming capacty endurance was measured.Results:.Non-hydrolyzed oils with dose of 0.1ml significantly induced swimming capacity of mice compared with negative control (distilled water) from 184.8 increased to 248.0 seconds by MCT oil, to 239.8 seconds by unsaturated LCT oil and to 214.8 seconds by saturated LCT oil. The hydrolyzed oils increased swimming capacity ± 65% by hydrolyzed MCT oils, ±54% by hydrolyzed unsaturated LCT oil and 21% by hydrolyzed saturated LCT oil. The hydrolyzed corn oil at high dose (0.4ml) was more effective to increase swimming capacity than saturated MCT (VCO and PKO) and LCT oil (palm oil).Conclusion:The results suggest that hydrolyzed MCT and LCT oils are more effective to increase swimming capacity than non-hydrolyzed.

THE EFFECT OF ACUTE CONSUMPTION OF HYDROLYZED OILS ON SWIMMING CAPACITY ENDURANCE OF MICE (MUS MUSCULUS)

Objective: The aim of this study was to investigate the effect of hydrolyzed oils of different fatty acids composition on swimming capacity of mice.Methods:The oils used were Virgin Coconut oil (VCO) and Palm Kernel oil (PKO) as medium chain triglyceride (MCT) oils, Palm oil and Corn oil as long chain triglyceride (LCT) oils. Hydrolysis of the oil was performed with Rhizomucor miehei lipase enzyme. The mice were acclimatized and tested motoric to select the mice and divided into twenty groups, each group consisted of 5 mice. Each oil in the experiment were 0.1 ml, 0.2 ml, 0.4 ml/ 20 g BW, positive control was caffeine 13 mg/kg bw. Then, the mice were placed in a glass aquarium. Then swimming capacty endurance was measured.Results:.Non-hydrolyzed oils with dose of 0.1ml significantly induced swimming capacity of mice compared with negative control (distilled water) from 184.8 increased to 248.0 seconds by MCT oil, to 239.8 seconds by unsaturated LCT oil and to 214.8 seconds by saturated LCT oil. The hydrolyzed oils increased swimming capacity ± 65% by hydrolyzed MCT oils, ±54% by hydrolyzed unsaturated LCT oil and 21% by hydrolyzed saturated LCT oil. The hydrolyzed corn oil at high dose (0.4ml) was more effective to increase swimming capacity than saturated MCT (VCO and PKO) and LCT oil (palm oil).Conclusion:The results suggest that hydrolyzed MCT and LCT oils are more effective to increase swimming capacity than non-hydrolyzed.

Lipase esterification in the Centrifugal Partition Reactor: Modelling and determination of the specific interfacial area

Recently the Centrifugal Partition Chromatography (CPC) device, called Centrifugal Partition Reactor (CPR), was proposed to operate biocatalytic reactions in two phase systems in continuous mode. However, information about hydrodynamic behavior in this device for operation of enzymatic reactions is still missing. In this study, in order to propose a modelling of this type of reactor, we firstly determined in a batch reactor the kinetics constants of a model reaction, the esterification of oleic acid with n-butanol, catalyzed by Rhizomucor miehei lipase. This reaction was described by a Ping Pong bi-bi mechanism with alcohol inhibition and specific experiments were done to determine the kinetic constants. Specific interfacial area in the batch reactor has to be known and its determination was done by measuring droplets size of organic phase by using a microscope. Experimental conversion values of oleic acid obtained in the continuous operation of the CPR, enabled the modelling to give an estimation of the value of the specific interfacial area in the CPR, which was found around 15,000 m2. m−3. The value of the specific interfacial area was shown here to be independent of the mobile phase flow rate, an important feature to characterize such an intensified enzymatic reactor.

Lipase-catalysed synthesis of palm oil-omega-3 structured lipids.

In this work, Candida antarctica lipase A was applied to selectively remove saturated fatty acids from palm oil to prepare palm oil acylglycerol concentrate (POAC), where palmitic acid decreased from 40.0 to 28.7% and oleic acid increased from 40.0 to 50.5% after 3 h of hydrolysis. Lipozyme RMIM from Rhizomucor miehei was then used to incorporate either alpha linolenic acid (ALA) or eicosapentaenoic acid (EPA) into the resulting POAC. Optimum omega-3 incorporation was achieved when POAC to omega-3 ratio was 6 : 3, reaction temperature was 40 °C and reaction time was 18 h. Under these conditions, the ALA content in the separated ALA incorporated structured lipid (POAC-ALA) was 27.1%, and the EPA content in the EPA incorporated structured lipids (POAC-EPA) was 30.9%. The formed structured lipids had lower levels of saturated fatty acids, and significantly lower melting points, in both cases below 8 °C. The enzymatic process developed produces new structured lipids, with lower saturated fat and higher omega-3, with potential as a healthy palm oil derived lipid ingredient.

Different construction strategies affected on the physiology of Pichia pastoris strains highly expressed lipase by transcriptional analysis of key genes

ABSTRACT We demonstrated previously that expression of Rhizomucor miehei lipase (RML) in Pichia pastoris could be significantly increased by addition of gene propeptide, optimized signal peptide codons and manipulation of gene dosage. In this study, effects of various strategies on the protein synthesis and secretion pathways were analyzed. Using nine strains previously constructed, we evaluated cell culture properties, enzymatic activities, and analyzed transcriptional levels of nine genes involved in protein synthesis and secretion pathways by qPCR. We observed that (i) Addition of propeptide decreased lipase folding stress by down-regulated four UPR-related genes. (ii) Signal peptide codons optimization had no effect on host with no change in the nine detected genes. (iii) Folding stress and limited transport capacity produced when rml gene dosage exceed 2. Different limiting factors on lipase expression in strains with different construction strategies were identified. This study provides a theoretical basis for further improving RML by transforming host.

Multipurpose fixed-bed bioreactor to simplify lipase production by solid-state fermentation and application in biocatalysis

A multipurpose fixed-bed bioreactor was evaluated, to simplify the process of producing lipase and its application in biocatalysis. Rhizomucor miehei lipase was produced by solid-state fermentation (SSF) in the fixed-bed bioreactor and generated a fermented solid with 30 U/g lipase activity and a synthesis capacity of 80% (conversion at 6 h of reaction in a standard stirred reactor). The fermented solid was then dried by forced aeration in the same column (1.0 L air/min, 35 °C) and the dried fermented solid (5 wt.% water) showed the same synthesis capacity as the freeze-dried standard, with high storage stability at 4 °C and 25 °C (14 months with no decrease in synthesis capacity). The reaction performed in the multipurpose bioreactor (one-pot system) showed relatively low conversion (30% of the conversion obtained in the stirred reactor at 6 h) when only babassu cake was employed as the fermentation substrate, due to the preferred pathways formed as a consequence of bed retraction during drying. Therefore, SSF was carried out with a mixture of 60:40 wt.% babassu cake and palm fiber (used to give structure to the fermented solid and prevent retraction during drying), and the reaction in the one-pot system reached a maximum conversion of 71%.

Efficient biotechnological synthesis of flavor esters using a low-cost biocatalyst with immobilized Rhizomucor miehei lipase.

In this work, the synthesis of two fruit flavor esters, namely methyl and ethyl butyrate, by lipase from Rhizomucor miehei immobilized onto chitosan in the presence of the surfactant sodium dodecyl sulfate SDS was investigated. In the optimized conditions, maximum esterification yield for ethyl butyrate and methyl butyrate was (92 ± 1%) and (89 ± 1%), respectively. Esterification yields for both reactions were comparable or even superior to the ones achieved when the synthesis was catalyzed by a commercial enzyme, Lipozyme®, at the same reaction conditions. For ethyl butyrate, the developed biocatalyst was used for seven consecutive cycles of reaction with retention of its catalytic activity. For methyl butyrate synthesis the biocatalyst was used for four consecutive cycles without loss of its catalytic activity. The results show that chitosan may be employed in obtaining biocatalysts with high catalytic efficiency and can successfully replace the currently commercial available biocatalysts.

Improvement of activity and stability of Rhizomucor miehei lipase by immobilization on nanoporous aluminium oxide and potassium sulfate microcrystals and their applications in the synthesis of aroma esters

In this study, Rhizomucor miehei lipase (RML) was immobilized on the hexagonally-ordered nanoporous aluminium oxide membranes (RML-Al2O3-NP) by adsorption and as protein-coated microcrystals (RML-PCMCs) by simultaneously precipitating RML on micron-sized potassium sulfate crystals (K2SO4) in pre-chilled acetone. The hydrolytic activities of immobilized lipase preparations were investigated in terms of p-nitrophenyl palmitate hydrolysis and their esterification activities were examined for the synthesis of some aroma esters such as butyl acetate, isoamyl acetate, hexyl acetate, heptyl acetate, and geranyl acetate. The immobilization yields were 33.8 and 25.1%, respectively for RML immobilized on Al2O3-NP membranes and potassium sulfate crystals. The catalytic efficiency ratios of RML-Al2O3-NP and RML-PCMCs were 2.3- and 3.9-fold higher than that of the free lipase, respectively in terms of hydrolytic activity. The free lipase was stabilized as 4.1- and 10.5-fold, respectively at 40 and 50 °C when immobilized on Al2O3-NP. The corresponding stabilization factors were 4.6- and 12.8-fold higher for RML-PCMCs. RML-Al2O3-NP and RML-PCMCs maintained 84 and 86% of their initial hydrolytic activities, respectively after 10 reuses. Of the synthesized aroma esters, the highest yield was obtained for the geranyl acetate. After 4 h reaction time, no geraniol was detected in the preparative-scale (196 g/L) synthesis of geranyl acetate for both the immobilized lipases when the initial geraniol amount, vinyl acetate amount, RML-PCMCs amount, and reaction temperature values were 1 mmol, 3 mmol, 100 mg (or 300 mg RML-Al2O3-NP), and 50 °C, respectively. These results show that the immobilization of R. miehei lipase by adsorption on nanoporous aluminium oxide and as protein-coated microcrystals leads to the obtention of highly stable, catalytically more active, and reusable lipase preparations.

Enzymatic Synthesis and Polymerization of Isosorbide-Based Monomethacrylates for High-Tg Plastics

Isosorbide is a stiff bicyclic diol derived from glycose-based polysaccharides, and is thus an attractive building block for novel rigid bioplastics. In the present work, a highly regioselective biocatalytic approach for the synthesis of isosorbide 5-methacrylate was developed. The Lipozyme RM IM (Rhizomucor miehei lipase)-catalyzed process is straightforward, easily scalable, and chromatography-free; a simple extractive workup afforded the monomer at >99% purity and in 87% yield. The developed strategy was applied for the synthesis of a series of monomethacrylated isosorbide derivatives. Radical polymerization of the monomers produced rigid polymethacrylates with a certain side group in either endo or exo configuration, exclusively, which generated materials with great diversity of properties. For example, the two regioisomeric polymers carrying hydroxyl groups reached a glass transition temperature at Tg = 167 °C. The polymer tethered with dodecanoate chains in exo position showed crystallinity with an ...

Identification and engineering of the key residues at the crevice-like binding site of lipases responsible for activity and substrate specificity.

Rational engineering of the crevice-like binding site of lipases for improvement of lipases' catalytic properties. The residues located at the crevice-like binding site of four representative lipases including Thermomyces lanuginosus lipases (TLL and Lip), Rhizopus oryzae lipase (ROL), and Rhizomucor miehei lipase (RML) were identified through structural analysis. The residues at the bottom of the crevice-like binding site recognizing the substrates with short/medium carbon chain length and those located at the right-hand wall of the surface crevice region affecting the product release were changed by site-directed mutagenesis. The corresponding double mutants exhibited ~ 5 to 14-fold higher activity towards p-nitrophenyl esters than their wild types, and their substrate preference shifted to acyl moiety with shorter carbon chain length. In addition, the mutations led to an increase of B-factor, resulting in decrease of their optimum temperature by 10-20°C. The key residues located at the crevice-like binding site play important roles in determining lipase activity, substrate preference and optimum temperature, which offers a useful new paradigm for facilitating rational design of lipases.

Immobilization of Rhizomucor miehei lipase onto the organic functionalized SBA-15: Their enzymatic properties and glycerolysis efficiencies for diacylglycerols production

In this study, mesoporous silica SBA-15 was modified by organic functional groups through silanization. Series of organosilane compounds were grafted onto the SBA-15, and the obtained functionalized carriers were then used to immobilize the lipase from Rhizomucor miehei (RML). The enzymatic properties of the obtained immobilized RML samples were evaluated, and the catalytic efficiencies in glycerolysis of triacylglycerols (TAG) reaction were studied. Compared with the parent SBA-15 immobilized RML, the organic modification gave a maximum improvement of enzymatic activity from 200.00 to 13211.11 U/g; in addition, TAG conversion and diacylglycerols (DAG) content increased from 21.28 to 84.24% and 15.45 to 59.03% respectively. The organic modification also decreased the sensitivity of immobilized RML in extreme pH values and increased their thermostability.

Production of Structured Phosphatidylcholine with High Content of Myristic Acid by Lipase-Catalyzed Acidolysis and Interesterification

Synthesis of structured phosphatidylcholine (PC) enriched with myristic acid (MA) was conducted by acidolysis and interesterification reactions using immobilized lipases as catalysts and two acyl donors: trimyristin (TMA) isolated from ground nutmeg, and myristic acid obtained by saponification of TMA. Screening experiments indicated that the most effective biocatalyst for interesterification was Rhizomucor miehei lipase (RML), whereas for acidolysis, the most active were Thermomyces lanuginosus lipase (TLL) and RML. The effect of the molar ratio of substrates (egg-yolk PC/acyl donor), enzyme loading, and different solvent on the incorporation of MA into PC and on PC recovery was studied. The maximal incorporation of MA (44 wt%) was achieved after 48 h of RML-catalyzed interesterification in hexane using substrates molar ratio (PC/trimyristin) 1/5 and 30% enzyme load. Comparable results were obtained in toluene with 1/3 substrates molar ratio. Interesterification of PC with trimyristin resulted in significantly higher MA incorporation than acidolysis with myristic acid, particularly in the reactions catalyzed by RML.

Enzymatic Synthesis of Structured Monogalactosyldiacylglycerols Enriched in Pinolenic Acid.

We enzymatically prepared structured monogalactosydiacylglycerols (MGDGs) enriched in pinolenic acid (PLA). PLA-enriched free fatty acids (FFAs) containing ∼86 mol % PLA were produced from an FFA fraction obtained from pine nut oil (PLA content, ∼13 mol %) by urea crystallization. Commercial MGDGs (5 mg) were acidolyzed with PLA-enriched FFAs using four commercial immobilized lipases as biocatalysts. The reaction was performed in acetone (4 mL) in a stirred-batch reactor. Lipozyme RM IM (immobilized Rhizomucor miehei lipase) was the most effective biocatalyst for the reaction. Structured MGDGs containing 42.1 mol % PLA were obtained under optimal reaction conditions: temperature, 25 °C; substrate molar ratio, 1:30 (MGDGs/PLA-enriched FFAs); enzyme loading, 20 wt % of total substrates; and reaction time, 36 h. The structured MGDGs were separated from the reaction products at a purity of 96.6 wt % using silica column chromatography. The structured MGDGs could be possibly used as emulsifiers with appetite-suppression effects.

Production of lipases in cottonseed meal and application of the fermented solid as biocatalyst in esterification and transesterification reactions

Abstract Cottonseed meal is a co-product from the cotton industry that is produced in large quantities worldwide. The aim of this work was to add value to this by-product through the production of Rhizomucor miehei lipases and application of the dry fermented solid in esterification reactions between oleic acid and ethanol or methanol and transesterification of a vegetable acid oil. Reactions were carried out in a solvent-free system and conversions higher than 85% were obtained in 4 h in the esterification reactions. The reusability of the biocatalyst was evaluated and the washing of the biocatalyst with solvent (ethanol) was necessary to recover the activity of the lipase, being obtained conversion of 74% in relation to the initial conversion after 4 sucessive reactions. The dried fermented solid was also able to catalyze the production of ethyl and methyl esters by simultaneous transesterification/esterification reactions of macauba acid oil, with production of an ester content of about 76% after 24 h. This is the first time that fermented solids produced in cottonseed meal without any supplementation are used as biocatalyst of esterification and transesterification reactions.

N-Butyl levulinate synthesis using lipase catalysis: comparison of batch reactor versus continuous flow packed bed tubular microreactor

Levulinic acid and its derivatives have applications in the synthesis of various organic chemicals because of the presence of carboxylic acid and ketone functional groups in the same moiety. Levulinate esters have potential applications in biodiesel as blending components, and fragrance and flavor industries. In the present work, n-butyl levulinate was synthesized by different commercial immobilized lipases such as Rhizomucor miehei lipase (RM IM), Thermomyces lanuginosus lipase (TL IM) and Novozym 435 using a continuous flow packed bed tubular microreactor and also a stirred tank batch reactor. Novozym 435 was the best catalyst. The continuous flow tubular microreactor gave 85% conversion of levulinic acid in 2 min at 60 °C when mole ratio 1: 3 of levulinic acid to n-butanol was used in methyl tert-butyl ether (MTBE) as solvent. The batch reactor needed 2 h to achieve the same conversion thereby proving superiority of the continuous flow reactors.

A novel approach for bioconjugation of Rhizomucor miehei lipase (RML) onto amine-functionalized supports; Application for enantioselective resolution of rac-ibuprofen

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.