The study of lipase immobilization and its application in transesterification reaction
Organic synthesis plays an important role in chemistry, biochemistry, medicine and other ﬁelds. One of the major challenges in organic synthesis ﬁeld is to design green synthetic routes. With the growing environmental concern new trends have been introduced in synthesis. Green catalysis is one of the exciting trend. Specially enzyme catalyzed reactions attract researchers. Enzyme as catalyst offers many advantages like low energy consumption, less environmental pollution, clean reaction with fewer by products. Considering all the above facts, we decided to work with enzyme catalyst. For that purpose we go through many papers, sites and collect all the supportive data. There are six classes of enzymes. We take hydrolase class for our study. From that, lipase class was chosen and study of lipase immobilization was done. The main advantage of immobilized enzyme is that, easy separation of the enzyme from product occur which also improves the economy of the reaction. It was also observed that the stability of immobilized enzyme increases through which a better control of reaction can be achieved. Some drawback was also observed. They include mass transfer limitations, change in properties like selectivity, cost of carriers etc. which have to be improved for good result. There are different methods of immobilization stated in literature such as adsorption, entrapment, cross-linking and covalent bonding. We use entrapment method for immobilization of lipase. We prepared a ﬁlm of polymer which entraps our lipase. Advantages of this method are no chemical modiﬁcation, relatively stable form, easy handling. The lipase enzyme has natural function is to hydrolase ester bonds of tryglyceroids. So its better application was found in transesterification reaction which involve reaction between an ester and alcohol to produce anthor pair of ester and alcohol. Now we use immobilized enzyme in transesteriﬁcation reaction.
Keywords: entrapment, hydolase enzyme
|GC-MS||Gas Chromatography Mass Spectroscope|
|FID||Flame Ionization Detectro|
1) Homogeneous catalyst
2) Heterogeneous catalyst
3) Biocatalyst or enzyme catalyst
From the above 3 types biocatalyst is proved to be very useful. It has great advantages like low environmental impact, extremely selective nature, they can achieve transformation not possible using conventional chemical reagent etc. Mostly immobilized enzyme was use for this purpose. The immobilization of an enzyme is an enzyme attached to an inert insoluble material. This can provide increased resistance to changes in conditions such as pH or temperature. It also lets enzyme be held in place throughout the reaction, following which they are easily separated from the products and may be used again. Immobilized enzymes are very important for commercial uses as they possess many beneﬁts to their expenses and processes of the reaction which include:
1. Convenience - Minuscule amounts of proteins dissolve in the reaction, so workup can be much easier. Upon completion, reaction mixtures typically contain only solvent and reaction products.
2. Economy – the immobilized enzyme is easily removed from the reaction making it easy to recycle the biocatalyst
3. Stability – immobilized enzyme typically have greater thermal and operational stability than the soluble form of the enzyme.
There are various ways by which one can immobilize an enzyme. They are as follows:
1. Adsorption – Enzyme is attached to the outside of an inert material. Though this method is slowest method for immobilization of an enzyme it is simple and economical.
2. Entrapment – In this method, the enzyme is trapped in insoluble beads or microspheres. However this insoluble substances hinders the arrival of the substrate and the exit of the product. To overcome this one can use another method which include enzyme trapping on a biodegradable polymer which is very useful. Also it provides easy handling and no chemical modiﬁcation.
3. Cross Linkage – Enzyme molecules are covalently bonded to each other to create a matrix consisting of almost only enzyme. This method produces a three dimensional cross linked enzyme aggregate. The reaction ensure that the binding site does not cover the enzymes active site.
4. Covalent Bonding – The enzyme is bound covalently to an insoluble support such as silica gel. This approach provides the strongest enzyme/ support interaction. The functional group that take part in this binding are amino group, carbonyl group, thiol group, phenolic group, hydroxyl group, etc.
Thus considering all the methods of immobilization we can put the advantage of this method as follows:
1. Enzyme can be recovered at the end of reaction thereby can be reused
2. Economy of the reaction is improved
3. Easy separation of enzyme from the product
4. Stability of immobilized enzyme increases
5. Better control of reaction can be achieved
Now we see some application of immobilized enzyme.
1] Industrial production of antibiotics, beverages, amino acids etc. uses immobilized enzyme.
2] It is used in treatment of sewage and industrial eﬄuents
3] Immobilization of lipase enzyme for effective dirt removal from cloth
4] It is useful in scouring, bio-polishing and desizing of fabrics
5] Enzymes like pectinases and cellulases immobilized on suitable carriers are successfully used in the production jams, jellies and syrups from fruits and vegetables.
6] It has wide application in production of biodesel from vegetable oil.
7] A research activity extensively uses many enzymes. The use of immobilized enzyme allows researchers to increase the eﬃciency of the different enzyme.
Now after looking all prospective of immobilizer enzymes we decided to apply immobilized enzyme and perform transesteriﬁcation reaction. The transesteriﬁcation reaction involves reaction between an ester and alcohol in the presence of lipase enzyme to give another pair of ester and alcohol. These reactions are often catalyzed by the addition of acid or base catalyst. The reaction can also be accomplished with the help of enzymes (biocatalyst) particularly lipase. Lipase has natural function to hydrolase ester bonds of tryglyceroids. Since both the reactants and products are an ester and alcohol, the reaction is reversible and the equilibrium constant is equal to one.
The transesteriﬁcation reaction has also a great application, they are listed below:
1. Polyester production - The largest scale application of transesteriﬁcation is in the synthesis of polyester. In this application diesters undergo transesteriﬁcation with diols to form macromolecules.
2. Methanolysis – The reverse reaction methanolysis, is also an example of transesteriﬁcation. This process has been used to recycle polyester into individual monomers.
3. Biodiesel production – It is also used to convert fats (triglycerides) into biodiesel. Transesteriﬁed vegetable oil (biodiesel) was used to generate power for heavy duty vehicle in South Africa before WW 2.
4. Synthesis – Transesteriﬁcation is used to synthesis enol derivatives, which are diﬃcult to produce by other means. Vinyl acetate, which is cheaply available, undergoes transesterification, giving access to vinyl ethers.
MATERIALS AND METHODS
Chemicals and Enzymes
Terahydrofuran (THF), Toluene, 1, 4-Dioxane and other chemicals used as solvent were bought from Hi-Media Pvt. Ltd., India. The lipase enzyme was bought from sigma Aldrich Pvt. Ltd., India.
Immobilization of Lipase
The immobilization of lipase was done by using following steps - ﬁrst we take 10 ml of distill water in clean beaker along with a small magnetic bid and put it on a magnetic stirrer. Then we add slowly 200 mg (w/ w) GG to it and leave it for 3 hr. the rotation speed was 350 rpm. In between we add 3 ml of water to it to avoid clots in it. After 3 hr GG properly mix with water and a uniform mixture was formed. In that mixture 50 mg (w/ w) of enzyme was added. For that we add 50 mg of enzyme to 3 ml of distill water separately in test tube and this mixture was added to GG mix. Note that the rotation speed was reduced to 130 rpm after the addition of enzyme. Finally after the 1 hr the mixture was poured into a Teﬂon petridish and uniformly spread and allow to dry at 45-46◦c for 24 hr. The prepared ﬁlm was cut into small pieces of 2-3. This biocatalyst ﬁlms were used in furtherstudy.
The reaction was carried out in round bottom ﬂask. The reaction mixture consists of alcohol (1 mmol) and VE (3 mmol). 3 ml of hexane was added to this mixture as a solvent. The mixture was stir for 2-3 min; then reaction was initiated by addition of 50 mg immobilized biocatalyst & kept .at 45◦c in a rotary orbital shaker with speed of 130 rpm.
Analysis was done by withdrawing 50 µl of sample at speciﬁc time interval, diluted it and analyzed by GC (Thermoﬁsher scientiﬁc- trace 1110) having FID and capillary column (TG-5MS, 30m × 0.25mm × 0.25µm). The temperature of the GC oven was set at 100◦C for 2 min with a rise of 25◦C min−1 up to 280◦C for 30 min while the temperature of both the detector and injector was kept 300◦C. Conversion was determined based on the area under the curve of limiting reactant; for this purpose standard sample was used.
Recyclability of Immobilised Enzyme
After completion of reaction, the mass was ﬁltered through Whatman ﬁlter paper and rinsed 2-3 times with solvent to remove the traces of reactant/ product. Then it was dried at 40-42◦C for 5-6 hr. and stored at 4-6◦C until used for next cycle.
RESULT AND DISCUSSION
Influence of Solvent
Organic solvent should be chosen in such way that it does not dissolve the substrate but also maintain the suitable conditions for catalytic activity of lipase. It was well reported that lipases work suitably with non-polar solvents rather than polar solvents. This is because the non-polar solvent shows molecular lubrication effect which is absent in case of polar solvents. In the present study the polar solvents like acetone and 1, 4-dioxane gave 58 and 64% conversion respectively. (ﬁg.4). On the other hand the non-polar solvents such as toluene, hexane and cyclohexane gave 85, 89 and 93% conversion respectively. Finally hexane was selected as a best solvent for further study.
Influence of Enzyme Loading
Concerned with the application view of enzyme, the amount of enzyme is an important factor. The effect of catalyst amount ranging from 20 to 60 mg was tested. Higher biocatalyst loading leads to increase in exposure of substrate to active catalytic sites of immobilized enzymes. Thus, more bio-catalyst loading enzyme has more immobilized to adsorb more substrate. Thus it was observed that % conversion increases with increase in catalyst loading from 20 to 60 mg. Further increase in amount of enzyme did not have signiﬁcant changes on ﬁnal conversion. The reason behind that is the excess amount of catalyst does not take part in reaction; indicating that 50 mg of immobilized lipase was suﬃcient to carry out biotransformation. Thus, remaining all experiments was carried out with loading of 50 mg of biocatalyst.
Influence of Temperature
Enzymes are very sensitive to temperature. At higher temperature enzyme loss its activity. Therefore temperature is crucial factor while studying enzyme catalytic reactions. It also increases the molecular collision–interaction and improves the solubility of the substrates in reaction media. So, it is obligatory to ﬁnd out optimum reaction temperature for an enzymatic reaction. The effect of temperature was studied in the range of 25 to 55 ◦c . It was observed that reaction rate grows up as we go from 25 to 45 ◦c but as we increase the temperature from 45 to 55◦c there is slight change in conversion. So, 45◦c temperature was applied for further experiments.
Influence of Molar Ratio
In any reaction the molar ratio is also an important factor along with other parameters. The molar ratios have great inﬂuence on reaction. We studied molar ratios ranging from 1:1 to 1:4. We observed that as the molar ratio varies the time required for reaction changes and also it affects % conversion. We got better results for 1:3 molar ratio. That means 1 mmol of alcohol reacts with 3 mmol of ester. This ratio was applied for next experiments.
The reusability of the immobilized lipase for four consecutive recycles was studied by transesteriﬁcation reaction following the above mentioned procedure in section 2.4. In addition, at the end of each recycle the ﬁltered immobilized lipase was collected carefully, dried at 40-42◦C for 5-6 h and was then used for next recycle.
The immobilization of enzyme was carried out on a environmentally friendly and biodegradable GG polymer. The immobilization of lipase on polymer matrix was shown better lipase activity. Thus making it more eligible biocatalyst for an transesteriﬁcation reaction. The various vinyl esters have been synthesis by using immobilized lipase, which has several applications in food and ﬂavor industry. Various reaction parameters was optimized, which include temperature, amount of enzyme, solvent, ratio of alcohol to ester etc. Thus the immobilization of lipase was successfully studied.
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4. Budujar K.C. and Bhanage B.M. , Enhanced catalytic avtivity of lipase immobilized on biodegradable copolymer of chitosan and polyvinyl alcohol support for synthesis of propionate ester: kinetik approch .(12th june 2014)
5. Dhake K.P. , Tambade P.J. , Qureshi Z.S. , Singhal R.S. and Bhanage B. M. HPMC-PVA Film immobilized Lipase as a biocatalyst for transesteriﬁcation reaction (1 march 2011)Rhizopus Oryzae
6. Budgujar K.C. and Bhanage B.M. , Application of Lipase immoblized on biocompatible ternary blande polymer matrix for synthesis of citronellyl acetate in non-aqueous media : Kinetik modelling study
7. Badgujar K.C. and Banage B.M. Immoblisation of lipase on biocompatible co-polymer of polyvinyl alcohl chitosan for synthesis of laurate compounds in supercritical carbondioxide using response surface methodology, (9th may 2015)
8. Budgujar K.C., Dhake K.P. and Bhanage B.M. immobilization of candida cylandracea lipase on poly lactic acid , polyvinyl alcohol and chitosan based polymer ﬁlm : characterization, activity, stabilty and its application for N-acylation reaction. Process biochem 2013;48 :1335-47.
9. Badgujar K.C. and Banage B.M. solvent stability study with thermodynamic analysis and superior biocatalytic activity Lipase immobilized on biocompatible hybrid matrix of polyvinyl alcohol and hypromellose. J Phys Chem B 2014 ;118 :1480
With great pleasure, I express my gratitude to my project guide Dr. B.M.Bhanage, professor, Department of Chemistry, Institute of Chemical Techonology,Mumbai for his guidance. I am also thankful to colleague Mrs. Priyanka Jawale and Miss. Manasi Patil for their guidance and helpful disscussion during project.Also I express my gratitude to my labmates for co-operation during project.I express my thanks to my profesor Dr. Mayur Desai, Department of Chemistry, Gogate-Joglekar College, Ratnagiri for his recommendation letter. I am also thankful to my professor Dr. Aparna Kulkarni, Department of Chemistry, Gogate-Joglekar College, Ratnagiri for counseling and motivating me. I am extremely grateful to the Indian Academy of Sciences for providing me with the fellowship and such a great platform and a life changing experience. The Author Cafe platform was extremely user-friendly and helped me in compiling my report. Last but not the least I would like to thank my parents and friends who have always been very supportive, without the help of whom this project would not have been possible.