Immobilization of Rhizomucor miehei lipase onto montmorillonite K-10 and polyvinyl alcohol gel

Abstract

Immobilization of enzymes from different sources on various supports in designed systems increases enzymes’ stability by protecting the active site of it from undesired effect of reaction environment. Also, immobilization decreases the cost of separation and facilities the reuse of the enzymes. Therefore, the design of new immobilization enzyme preparations has been an inevitable area of modern biotechnology. Herein, Rhizomucor miehei lipase (RML) was immobilized on montmorillonite K-10 (MMT-RML) by adsorption and in polyvinyl alcohol (PVA-RML) by entrapment to obtain a more stable and active lipase preparation. The free and immobilized lipase preparations were characterized for p-nitrophenyl palmitate hydrolysis. The apparent Michaelis–Menten (K_mapp) constant was almost the same for the free RML and PVA-RML, whereas the corresponding value was 17.7-fold lower for MMT-RML. PVA-RML and MMT-RML have shown a 1.1 and 23.8 folds higher catalytic efficiency, respectively, than that of the free RML. The half-lives of PVA-RML and MMT-RML were found to be 7.4 and 3.4 times longer than the free RML at 35?°C, respectively. PVA-RML and MMT-RML maintained 65% and 87% of their initial activities after four reuses. These results showed that the catalytic performance of RML has improved significantly by immobilization.     

Synthesis of geranyl propionate in a solvent-free medium using Rhizomucor miehei lipase covalently immobilized on chitosan–graphene oxide beads

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.     

Application of multi-component reaction for covalent immobilization of two lipases on aldehyde-functionalized magnetic nanoparticles; production of biodiesel from waste cooking oil

Lipase from Rhizomucor miehei (RML) and Thermomyces lanuginosa lipase (TLL) were immobilized on silica core-shell magnetic nanoparticles (Fe₃O₄@SiO₂) produced by coating Fe₃O₄ core with silica shell. The nanoparticles were functionalized with aldehyde groups followed by immobilization of RML and TLL by using a multi-component reaction in an extremely mild condition. Rapid immobilization of both enzymes (1.5−12 h) with high immobilization yields (81–100%) was observed. The maximum loading capacity of the support was determined to be 81 mg for RML and 97 mg for TLL. The thermal stability of the immobilized derivatives of RML and TLL were greatly improved by retaining 54 and 97 % of their initial activities at 65 °C, respectively. The immobilized preparations were used to produce biodiesel by transesterification of waste cooking oil. In an optimization study, Response Surface Methodology (RSM) and a central composite rotatable design (CCRD) were used to study the effect of amount of biocatalyst, temperature, reaction time, water adsorbent (wt.%) and ratio of t-butanol to oil (wt.%) on the yield of biodiesel production. Biodiesel production yield by immobilized TLL reached 93.1 % under optimal conditions while the maximum yield for RML was 57.5 %. Both immobilized derivatives showed high reusability after 5 cycles of the reaction.     

Effect of moisture on lipase catalysed esterification of geraniol of palmarosa oil in non-aqueous system

Summary Optimisation of reaction conditions for the esterification of geraniol of palmarosa oil with n-butyric acid using immobilized lipase from Mucor miehei in non-aqueous system was carried out. Palmarosa oil could be easily esterified upto 95% w/w at 40°C in 24 h. Effect of moisture content was studied using Na₂SO₄ and recycling of the immobilized enzyme.     

Kinetics of solvent-free geranyl acetate synthesis by Rhizopus oligosporus NRRL 5905 lipase immobilized on to cross-linked silica

The objective of the present work was to study the kinetics of the solvent-free synthesis of geranyl acetate by a novel lipase (activity 60 U g^?1) made by immobilization of lipase from Rhizopus oligosporous NRRL 5905 on to cross-linked silica gel. Transesterification was performed with vinyl acetate as the acyl donor. Vinyl acetate was used in large excess compared to geraniol, which made the reaction pseudo first order with respect to geraniol and the reaction rate followed Michaelis–Menten kinetics for a single substrate. To obtain the highest yield for geranyl acetate, various relevant physical parameters such as shaking speed, reaction time, enzyme concentration, initial water amount and reaction temperature that influence the activity of lipase were investigated. A maximum molar conversion of 67% was achieved after 48 h of reaction at 30°C, at an enzyme concentration of 25% w/v of reaction mixture. Substrate conversion remained constant for five successive cycles; thereafter the conversion dropped by only 11%. Using a pseudo first-order kinetic model for geranyl acetate synthesis in the absence of organic solvents, apparent K_m and V_max values were evaluated as 60 mM and 141 µmol g^?1 h^?1, respectively.     

Synthesis of geranyl acetate in non-aqueous media using immobilized Pseudomonas cepacia lipase on biodegradable polymer film: Kinetic modelling and chain length effect study

Pseudomonas cepacia lipase (PCL) was immobilized on ternary blend biodegradable polymer made up of polylactic acid (PLA), chitosan (CH), and polyvinyl alcohol (PVA). Immobilized biocatalyst was characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), % water content, protein and lipase activity assay. The lipase activity assay showed enhanced activity of immobilized lipase than crude lipase. Higher half life time (t_1/2) and lower deactivation rate constant (K_d) was found for the n-hexane among various tested solvent. Influence of various reaction parameters on enzyme activity were studied in detail. When geraniol (1 mmol) and vinyl acetate (4 mmol) in toluene (3 mL) were reacted with 50 mg immobilized lipase at 55 °C; then 99% geraniol was converted to geranyl acetate after 3 h. Various kinetic parameters such as r_max, K_i(A), K_m(A), K_m(B) were determined using non-linear regression analysis for ternary-complex and Bi–Bi ping-pong mechanism. The kinetic study showed that reaction followed ternary-complex mechanism with inhibition by geraniol. Activation energy (Ea) was found to be lower for immobilized lipase (13.76 kCal/mol) than crude lipase (19.9 kCal/mol) indicating better catalytic efficiency of immobilized lipase. Immobilized biocatalyst demonstrated 4 fold increased catalytic activity than crude lipase and recycled five times.     

Geranyl acetate synthesis catalyzed by Thermomyces lanuginosus lipase immobilized on electrospun polyacrylonitrile nanofiber membrane

Isolated Thermomyces lanuginosus lipase (TLL) was immobilized by different protocols on the polyacrylonitrile nanofibers membrane. The conditions for immobilization of TLL were optimized by investigating effect of protein concentration, time and temperature on the extent of immobilization. The effect of immobilization on the catalytic activity and stability of lipase was studied thoroughly. The immobilized TLL was used as biocatalyst for geranyl acetate synthesis with geraniol and vinyl acetate as substrates and their performance was compared with free enzyme. The TLL immobilized by physical adsorption shows higher transesterification and hydrolytic activities than that of covalently linked or native TLL. There was 32 and 9 fold increase in transesterification activity of TLL immobilized by adsorption and covalent bonding, while hydrolytic activity increases only by 3.6 and 1.8 fold respectively. The optimum conditions for immobilization in both the cases were immobilization time 90–150 min, temperature 45 °C and protein concentration of 2 mg/ml. The percentage conversion of ester was more than 90% and 66% in case of physically adsorbed and covalently bonded enzyme respectively as compared to native one. However, covalently immobilized TLL shows higher operational stability than native and physically adsorbed TLL.     

Sucrose mobilization in relation to essential oil biogenesis during palmarosa (<Emphasis Type="Italic">Cymbopogon martinii</Emphasis> Roxb. Wats. var.<Emphasis Type="Italic">motia</Emphasis>) inflorescence development

Palmarosa inflorescence with partially opened spikelets is biogenetically active to incorporate [U-¹⁴C]sucrose into essential oil. The percent distribution of¹⁴C-radioactivity incorporated into geranyl acetate was relatively higher as compared to that in geraniol, the major essential oil constituent of palmarosa. At the partially opened spikelet stage, more of the geraniol synthesized was acetylated to form geranyl acetate, suggesting that majority of the newly synthesized geraniol undergoes acetylation, thus producing more geranyl acetate.In vitro development of palmarosa inflorescence, fed with [U-¹⁴C]sucrose, resulted in a substantial reduction in percent label from geranyl acetate with a corresponding increase in free geraniol, thereby suggesting the role of an esterase in the production of geraniol from geranyl acetate. At time course measurement of¹⁴CO₂ incorporation into geraniol and geranyl acetate substantiated this observation. Soluble acid invertase was the major enzyme involved in the sucrose breakdown throughout the inflorescence development. The activities of cell wall bound acid invertase, alkaline invertase and sucrose synthase were relatively lower as compared to the soluble acid invertase. Sucrose to reducing sugars ratio decreased till fully opened spikelets stage, concomitant with increased acid invertase activity and higher metabolic activity. The phenomenon of essential oil biosynthesis has been discussed in relation to changes in these physiological parameters.     

Study of lipase immobilization on zeolitic support and transesterification reaction in a solvent free-system

In order to understand the role of the acid–base, electrostatic and covalent interactions between enzyme and support, the catalytic behavior of the Rhizomucor miehei lipase (RML) immobilized on zeolite materials has been studied. The highest lipase activities were obtained when this enzyme, immobilized by adsorption, interacts through acid–base binding forces with the support surface, resulting in activation of the enzyme catalytic center. Due to the interest in biodiesel production by mild enzymatic transesterification, this heterogeneous biocatalyst has been used in transesterification of fatty acids contained in olive oil. The results show a high oleic acid conversion for several reaction cycles with a higher total biodiesel productivity compared to that using the free enzyme.     

Characterization of <Emphasis Type="Italic">Mucor miehei</Emphasis> lipase immobilized on polysiloxane-polyvinyl alcohol magnetic particles

Mucor miehei lipase was immobilized on magnetic polysiloxane-polyvinyl alcohol particles by covalent binding. The resulting immobilized biocatalyst was recycled by seven assays, with a retained activity around 10% of its initial activity. K_m and V_max were respectively 228.3 M and 36.1 U mg of protein^–1 for immobilized enzyme. Whereas the optimum temperature remained the same for both soluble and immobilized lipase (45 °C), there was a shift in pH profiles after immobilization. Optimum pH for the immobilized lipase was 8.0. Immobilized enzyme showed to be more resistant than soluble lipase when assays were performed out of the optimum temperature or pH.     

Combination of site-directed mutagenesis and yeast surface display enhances <Emphasis Type="Italic">Rhizomucor miehei</Emphasis> lipase esterification activity in organic solvent

To increase the activity of Rhizomucor miehei lipase (RML) in organic solvent, multiple sequence alignments and rational site-directed mutagenesis were used to create RML variants. The obtained proteins were surface-displayed on Pichia pastoris by fusion to Flo1p as an anchor protein. The synthetic activity of four variants showed from 1.1- to 5-fold the activity of native lipase in an esterification reaction in heptane with alcohol and caproic acid as substrates. The increase in esterification activity may be attributed to the four mutations changing the flexibility of RML or facilitating the reaction. In conclusion, this method demonstrated that multiple sequence alignments and rational site-directed mutagenesis combined with yeast display technology is a faster and more effective means of obtaining high-efficiency esterification lipase variants compared with previous similar methods.     

Enzymatic transesterification in a solvent-free system: synthesis of sn-2 docosahexaenoyl monoacylglycerol

Abstract

The enzymatic transesterification of docosahexaenoic acid (DHA) ethyl ester with glycerol was carried out by using several immobilized lipases in a solvent-free system. This reaction involves the initial formation of sn-2 docosahexaenyl monoacylglycerol. This DHA derivative is highly relevant for improving the bioavailability of DHA and it has received increasing interest in the field of nutrition. Three commercial lipases, from Rhizomucor miehei (RML), Alcaligenes sp. (AQ) and Candida antarctica-fraction B (CALB) were immobilized by interfacial adsorption on a commercial hydrophobic support (a methacrylate resin containing octadecyl groups, Sepabeads C-18) and tested for glycerolysis of DHA ethyl ester. In certain cases (e.g. immobilized CALB), the transesterification reaction continues to the formation of triacylglycerol (80%) by using a very high excess of DHA ethyl ester ((115 mmols versus 1.24 mmols of glycerol and high temperatures (50?°C). However, the same biocatalyst working at lower temperatures, 37?°C, synthetizes a 90% of sn-2 monoacylglycerol even in the presence of that a high excess of DHA ethyl ester. Interestingly, immobilized RML derivative synthesizes a 98% of sn-2 monoacylglyceride (2-MG) in 15?min at 37?°C with a 4% of immobilized biocatalyst. These high activity and regioselectivity under very mild reaction conditions are very interesting for the thermal oxidative stability of the omega-3 fatty acid as well as for the thermal stability of the biocatalyst. Using Normal Phase HPLC-ELSD and accurate commercial markers, the formation of the 2-MG was confirmed.     

Changes in volatile constituents of Zingiber officinale rhizomes during storage and cultivation

The essential oil from the fresh rhizome of Zingiber officinale was characterized by the presence of acyclic oxygenated monoterpenes mainly composed of neral, geraniol, geranial and geranyl acetate. During storage the content of neral and geranial in the rhizome increased to ca 60% of the essential oil, while the content of geraniol and geranyl acetate decreased to an undetectable amount. The change resulted from the conversion of geranyl acetate into geraniol, geranial and neral, successively. The content of geranial and neral decreased to a small extent through cultivation of the stored rhizome, whereas a large quantity of geraniol and geranyl acetate occurred in the newly propagated fresh rhizome.     

Variables that Affect Immobilization of Mucor Miehei Lipase on Nylon Membrane

Nylon membrane was used to immobilize Mucor miehei lipase. Variables that affect this immobilization procedure were studied by experimental design. A 2³ full factorial design was employed for this purpose. The protein retention and hydrolytic activity of the immobilized lipase were used as response variables. The rapid loss of enzyme activity was the main problem during repetitive use. Two strategies were used to improve the low operational stability: nylon treated with HCl and nylon coated with polyvinyl alcohol (PVA). Lipase-nylon-PVA was the best enzyme derivative, allowing performance of five consecutive assays, with a retained activity of 0.5 U mg of protein⁻¹ g of support⁻¹.     

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.     

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.     

Combination of two lipases more efficiently catalyzes methanolysis of soybean oil for biodiesel production in aqueous medium

A combination of two lipases was employed to catalyze methanolysis of soybean oil in aqueous medium for biodiesel production. The two lipase genes were cloned from fungal strains Rhizomucor miehei and Penicillium cyclopium, and each expressed successfully in Pichia pastoris. Activities of the 1,3-specific lipase from R. miehei (termed RML) and the non-specific mono- and diacylglycerol lipase from P. cyclopium (termed MDL) were 550 U and 1545 U per ml respectively, and enzymatic properties of these supernatant of fermentation broth (liquid lipase) were stable at 4 °C for >3 months. Under optimized conditions, the ratio of biodiesel conversion after 12 h at 30 °C, using RML alone, was 68.5%. When RML was assisted by addition of MDL, biodiesel conversion ratio was increased to >95% under the same reaction conditions. The results suggested that combination of lipases with different specificity, for enzymatic conversion of more complex lipid substrates, is a potentially useful strategy for biodiesel production.     

Enhancing thermostability of a Rhizomucor miehei lipase by engineering a disulfide bond and displaying on the yeast cell surface

To increase the thermostability of Rhizomucor miehei lipase, the software Disulfide by Design was used to engineer a novel disulfide bond between residues 96 and 106, and the corresponding double cysteine mutants were constructed. The R. miehei lipase mutant could be expressed by Pichia pastoris in a free secreted form or could be displayed on the cell surface. The new disulfide bond spontaneously formed in the mutant R. miehei lipase. Thermostability was examined by measuring of hydrolysis activity using 4-nitrophenyl caprylate as a substrate. The engineered disulfide bond contributed to thermostability in the free form of the R. miehei lipase variant. The variant displayed on the yeast cell surface had significantly increased residual hydrolytic activity in aqueous solution after incubation at 60°C for 5 h and increased synthetic activity in organic solvent at 60°C. These results indicated that yeast surface display might improve the stability of R. miehei lipase, as well as amplifying the thermostability through the engineered disulfide bond.     

Lipase‐Catalyzed Kinetic Resolution of (±)‐1‐(2‐Furyl) Ethanol in Nonaqueous Media

S‐1‐(2‐Furyl) ethanol serves as an important chiral building block for the preparation of various natural products, fine chemicals, and is widely used in the chemical and pharmaceutical industries. In this work, lipase‐catalyzed kinetic resolution of (R/S)‐1‐(2‐furyl) ethanol using different acyl donors was investigated. Vinyl esters are good acyl donors vis‐à‐vis alkyl esters for kinetic resolution. Among them, vinyl acetate was found to be the best acyl donor. Different immobilized lipases such as Rhizomucor miehei lipase, Thermomyces lanuginosus lipase, and Candida antarctica lipase B were evaluated for this reaction, among which C. antarctica lipase B, immobilized on acrylic resin (Novozym 435), was found to be the best catalyst in n‐heptane as solvent. The effect of various parameters was studied in a systematic manner. Maximum conversion of 47% and enantiomeric excess of the substrate (ee_s) of 89% were obtained in 2 h using 5 mg of enzyme loading with an equimolar ratio of alcohol to vinyl acetate at 60°C at a speed of 300 rpm in a batch reactor. From the analysis of progress curve and initial rate data, it was concluded that the reaction followed the ordered bi–bi mechanism with dead‐end ester inhibition. Kinetic parameters were obtained by using nonlinear regression. This process is more economical, green, and easily scalable than the chemical processes. Chirality 26:286–292, 2014. © 2014 Wiley Periodicals, Inc.     

FTIR-ATR characterization of free Rhizomucor meihei lipase (RML), Lipozyme RM IM and chitosan-immobilized RML

The synthesis and characterization of biocatalysts based on lipase from Rhizomucor miehei (RML) immobilized on chitosan-based supports were investigated. The enzyme was immobilized on chitosan following two strategies: (i) physical adsorption; and (ii) covalent bonding using glutaraldehyde. The content of enzyme bound in the supports, as precipitable protein, was analyzed using UV/visible methods. FTIR-ATR spectroscopy was employed to characterize the prepared biocatalysts, as well the native enzyme and a commercial biocatalyst Lipozyme RM IM, used as reference materials. Analysis of the amide I′ signal allowed to follow the changes in the secondary structure of the enzyme after binding to the support and its thermal stability. The hydrolysis of ethyl stearate monitored in situ by FTIR-ATR was used as a test reaction. Results showed that RML was bound to Chit and Glut–Chit with minor changes in its secondary structure, thermal stability and enzymatic activity in a selected reaction test.