Lipase is an ideal tool for organic chemistry due to their important characteristics they display. They exhibit exquisite chemo selectivity, regio selectivity and stereo selectivity and they are available in large quantity produced in high yield from microbial organisms. Crystal structure of many lipases has been solved facilitating the design of rational engineering strategies. They do not usually require cofactors nor do they catalyze side reactions. The present contribution discussed the factors affecting the lipase reaction. The mode of action and the different reactions are summarized. The open and closed forms of the protein are also discussed. The factors limiting the study of the reaction of Candida rugosa lipase in organic medium and a possible experiment design is also suggested. The DNA sequence and the deduced amino acid sequence of Candida rugosa lipase is also deduced.
1. Introduction: The word enzyme is derived from the Greek meaning ‘in yeast’. Enzymes are highly specialized proteins. They are reaction catalysts (i.e. they speed up the rates of reactions without themselves undergoing any permanent change) of biological system and have extraordinary catalytic power, often far greater than that of synthetic catalysts. They have a high degree of specificity for their substrates which is the outstanding characteristics of these biocatalysts, they accelerate specific chemical reactions, and they function in aqueous solutions under very mild conditions of temperature and pH.(1) The study of enzymes also has immense practical importance. In some diseases, especially inheritable genetic disorders, there may be a deficiency or even a total absence of one or more enzymes in the tissues.
In the absence of enzymes most of the reactions of cellular metabolism would not occur even over a time period of years and life could not exist. The application of recombinant DNA techniques to the study of enzymes has produced some remarkable new insights. It has proved possible to alter catalytic activity and specificity in a rational manner by introducing mutation at defined positions using site directed mutagenesis. This has helped in understanding the mechanism of enzyme action and has also opened the prospect of designing enzymes with specific required properties.
Figure.1. Pie-chart showing the frequency of use of particular biocatalysts in biotransformations.
Proteases, esterases and lipase account for more than half all biotransformations (Faber et al 1993)
The demand for industrial enzyme, particularly of microbial origin, is ever increasing owing to their application in a wide variety of processes. Enzyme mediated reactions are attractive alternatives to tedious and expensive methods.
2. Main Body: Lipases stand amongst the most important biocatalysts carrying out novel reactions in both aqueous and non-aqueous media. Among lipases of plant, animal and microbial origin it is the microbial lipase, which finds immense application, because microbes can be easily cultivated and their lipases can catalyze a wide variety of hydrolytic and synthetic reactions.
Lipase comes under the category of hydrolases. Lipase (E.C.18.104.22.168) catalyzes the hydrolysis of triacylglycerol in aqueous medium to release free fatty acid and glycerol.
Triacylglycerol Lipase Free fatty acid + Glycerol.
These reactions usually proceed with high regio and/or enantioselectivity, making lipase an important group of biocatalyst in organic chemistry.(2). This hydrolytic reaction is reversible. In the presence of decreased amounts of H2O, often in the presence of organic solvents, the enzymes are effective catalysts for various intersterification and transesterification reactions, synthesis of esters in organic solvents formed from glycerol and long chain fatty acids.
(e.g.) Ethyl hexanoate Lipase Ethanol + Hexanoic acid.
There is no strict definition available for the term “long chain” but glycerol esters with an acyl chain length of greater than or equal to 10 ‘C’ atoms can be regarded lipase substrate with trioleoylglycerol being the standard substrate. The reasons for the enormous biotechnological potential of microbial lipases include the facts that they are
1) Stable in organic solvents;
2) Do not require cofactors
3) Possess a broad substrate specificity;
4) Exhibit a high enantio-selectivity
Mode of action
Lipase acts at oil water face substrate is in equilibrium between oil phase and emulsion and is constantly changing. Actual reaction rate depends on the amount of substrate in the emulsion rather than amount in the oil. But the amount in the emulsion depends on the concentration of substrate in the oil (3).
The interfacial area can be increased substantially to its saturation limit by the use of emulsifier as well as agitation. The saturation limit also depends on the ingredient used as well as physical condition deployed. Lipase required activation and several types of activation are known such as making more substrate available by better emulsification using surface-active agents, mild detergents such as Tween or bile salts (3).
Figure 2. Reaction Mechanism of Lipases(4)
In nature lipases available from various sources have considerable variation in their reaction specificities, this property is generally referred to as enzyme specificity. Thus, from fatty acid side enzyme lipase has affinity for short chain fatty acid (Acetic, butyric, caproic, capriylic, capric etc.), some have preferences for unsaturated fatty acids (oleic, linoleic, linolenic etc.), and many are non-specific and randomly split the fatty acids from the triglycerides. From the glycerol side of the triglyceride (TG) the lipase often shown positional specificity and attack the fatty acids at 1 or 3 position of carbon of glycerol or at both the positions but not the fatty acid at position 2 of the glycerol molecule.
Lipases function at the oil water interface. The amount of oil available at the interface determines the activity of the lipases. This interface area can be increased substantially to its saturation limit by the use of emulsifiers as well as by agitation. (5).
Lipases are not involved in any anabolic processes, since this enzyme acts at the oil water interface it can be used as catalyst for preparation of industrially important compounds. As lipases act on ester bonds they have been used in fat splitting, inter esterification, development of different flavors in cheese, improving palatability of beef fat for making dog food etc. currently application of lipase involves using lipase in water deficient organic solvents for synthesizing different value added esters from organic acids and alcohols. Lipases which are stable and work at alkaline pH of 8 to 11 which are suitable wash conditions for enzymatic detergent powders and liquids, have also been found, these hold good potential for use in detergent industry.(6)
Under nature conditions lipase catalyze hydrolysis of the ester bonds at the interphase between an insoluble substrate phase and aqueous phase in which enzyme is dissolved. In experimental condition, such as in the absence of water they are capable of reversing the reaction. The reverse reaction leads to esterification and formation of glycerides from fatty acid and glycerol (3).
Figure 3: Figure depicting various Candida rugosa Lipase catalysed reactions.
Bacterial lipases are glycoproteins but some extracellular lipases are lipoproteins Winkler et.al. reported that enzyme production in most of the bacteria is affected by polysaccharides. Most of the bacterial lipases are constitutive and non-specific in their substrate specificity and a few bacterial lipases are thermostable.
Among bacteria Achromobacter sp., Alcaligenes sp., Arthrobacter sp., and chromobacterium sp., have been exploited for the production of lipases. Staphylococcal lipases are lipoprotein in nature. Lipases purified from S. aureus and S. hyicus show molecular weight ranging between 34-46 kDa. They are stimulated by Ca2+ and inhibited by EDTA. The optimum pH varies between 7.5 and 9.0.(7)
Lawrence, Brockerhoff and Jensen have studied and presented their reviews on fungal lipases.These lipases are being exploited due to their low cost of extraction, thermal and pH stability, substrate specificity and activity in organic solvents. The chief produces of commercial lipases are Aspergillus niger, Candida cylindracea, Humicola lanuginosa, Mucor miehei, Rhizopus oryzae, R. delemar, R. miveus and R. arrhizus.Lipase purification
Lipases have been purified from animal, plant, fungal and bacterial sources by different methods. Ammonium sulphate precipitation, gel filtration and ion exchange chromatography affinity chromatography techniques are used to decrease the number of steps necessary for lipase purification and increase specify. Currently reverse micellar two phase systems, membrane process and immuno purification are being used for the purification of lipases.(3)
Table:1 Properties of Lipase of some microorganisms:(8)
Regio l, 3
Mechanism of lipase action is broken down into following steps :
1 Adsorption of lipase to interface;
2 Binding of substrate to enzyme;
3 Chemical reaction;
4 Release of product.
Adsorption of lipase to an interface is an interactive process. In aqueous media the non-polar residues of the enzymes lid interact with the non polar residues around the catalytic triad, placing the residues in the interior of the enzyme. The polar residues on the lid are on the exterior of the enzyme interacting with the aqueous medium. Near the lid is a cavity made up of polar residues. In an aqueous medium, the cavity is filled with water molecules.
Figure 4. Open and Closed forms of Candida rugosa lipase .(9)
Lipases undergo conformational changes to lower the energy of the system. The water molecules in the polar cavity are pushed out. At the same time the polar residues of the cavity begin pulling on the residues at lids exterior. This action is favourable because there non polar residues buried beneath the lid. As the enzyme is pulled closer to the interface, the conformational changes become greater until parts of the enzyme are enveloped by the hydrophobic medium at the interface. The catalytic triad reacts with the carbonyl group. Since the carbonyl group must be very near the active site the acyl chain must also be near the surface of the enzyme. It is the interaction of the carbonyl group and acyl chain with the enzyme that allows the substrate to bind. When the carbonyl group is in position near the active site, the chemical reaction can occur. The chemical reaction occurs due to the action of the catalytic triad (Fig.4). Here the first step is to make serine alcohol forming an oxyanion. The oxyanion is stabilized by amino acids which hydrogen bond to it, the electrons are pushed back to the carbonyl carbon; the portion on the histidine is transferred to diacylglycerol which is subsequently released.
The main sources of lipases are microorganisms, plant and animals of which most widely used are microbial sources. Plant sources are obtained from seeds and animal sources from pancreas. Microbial sources are stable, cheap and abundantly compared to others. Under microbial classification most bacteria are glycol-proteins with rest extracellular called lipoproteins(3). Fungal lipases are exploited are due to low cost of extraction, pH and thermal stability, substrate specificity and enzyme activity inorganic solvents. Based on the occurrence they may be classified as plant lipases, animal lipases and microbial lipases.
Lipase from Candida rugosa have varied applications in various pharmaceutical and other industries. The applications are compiled in the table below.
Table: 2 Effect of lipases at industrial level and their products.(5)
Flavour improvement and shelf-life prolongation
Chiral building blocks and chemicals.
Removal of cleaning agents like surfactants.
Hydrolysis of milk fat Cheese ripening fat
Modification of butter
Emulsifiers, moisturizing agent
Fats and Oils
Coco butter, margarine
Fatty acids, glycerol, mono- and diglycerides
Mayormaise, dressings and whippings.
Meat and Fish
And fat removal
Meat and fish
Gene and amino acid sequence of Candida rugosa lipase.
SQ Sequence 1650 BP; 299 A; 541 C; 490 G; 320 T; 0 other; 2613492056 CRC32;
atggagctcg ctcttgcgct cctgctcatt gcctcggtgg ctgctgcccc caccgccacg 60
ctcgccaacg gcgacaccat caccggtctc aacgccatca tcaacgaggc gttcctcggc 120
attccctttg ccgagccgcc ggtgggcaac ctccgcttca aggaccccgt gccgtactcc 180
ggctcgctcg atggccagaa gttcacgctg tacggcccgc tgtgcatgca gcagaacccc 240
gagggcacct acgaggagaa cctccccaag gcagcgctcg acttggtgat gcagtccaag 300
gtgtttgagg cggtgctgcc gctgagcgag gactgtctca ccatcaacgt ggtgcggccg 360
ccgggcacca aggcgggtgc caacctcccg gtgatgctct ggatctttgg cggcgggttt 420
gaggtgggtg gcaccagcac cttccctccc gcccagatga tcaccaagag cattgccatg 480
ggcaagccca tcatccacgt gagcgtcaac taccgcgtgt cgtcgtgggg gttcttggct 540
ggcgacgaga tcaaggccga gggcagtgcc aacgccggtt tgaaggacca gcgcttgggc 600
atgcagtggg tggcggacaa cattgcggcg tttggcggcg acccgaccaa ggtgaccatc 660
tttggcgagc tggcgggcag catgtcggtc atgtgccaca ttctctggaa cgacggcgac 720
aacacgtaca agggcaagcc gctcttccgc gcgggcatca tgcagctggg ggccatggtg 780
ccgctggacg ccgtggacgg catctacggc aacgagatct ttgacctctt ggcgtcgaac 840
gcgggctgcg gcagcgccag cgacaagctt gcgtgcttgc gcggtgtgct gagcgacacg 900
ttggaggacg ccaccaacaa cacccctggg ttcttggcgt actcctcgtt gcggttgctg 960
tacctccccc ggcccgacgg cgtgaacatc accgacgaca tgtacgcctt ggtgcgcgag 1020
ggcaagtatg ccaacatccc tgtgatcatc ggcgaccaga acgacgaggg caccttcttt 1080
ggcaccctgc tgttgaacgt gaccacggat gcccaggccc gcgagtactt caagcagctg 1140
tttgtccacg ccagcgacgc ggagatcgac acgttgatga cggcgtaccc cggcgacatc 1200
acccagggcc tgccgttcga cacgggtatt ctcaacgccc tcaccccgca gttcaagaga 1260
atcctggcgg tgctcggcga ccttggcttt acgcttgctc gtcgctactt cctcaaccac 1320
tacaccggcg gcaccaagta ctcattcctc ctgaagcagc tcctgggctt gccggtgctc 1380
ggaacgttcc actccaacga cattgtcttc caggactact tgttgggcag cggctcgctc 1440
atctacaaca acgcgttcat tgcgtttgcc acggacttgg accccaacac cgcggggttg 1500
ttggtgaagt ggcccgagta caccagcagc ctgcagctgg gcaacaactt gatgatgatc 1560
aacgccttgg gcttgtacac cggcaaggac aacttccgca ccgccggcta cgacgcgttg 1620
ttctccaacc cgccgctgtt ctttgtgtaa
DNA Sequence of Candida rugosa lipase (NCBI TAX ID:44332), Gene LIP1.
P20261|LIP1_CANRU Lipase 1 – Candida rugosa (Yeast) (Candida cylindracea).
Protein sequence of Candida rugosa lipase, LIP1_CANRU,
Primary accession number: P20261, http://www.expasy.org/uniprot/P20261
Enough information is known about the functionality and specificity of Candida rugosa lipase in both the reaction medium (aqueous and non-aqueous). The importance of its activity in organic medium is being explored a considerable. But, till date the rate of reaction in aqueous medium is high compared to the reaction in organic medium. Factors like the water content and the solvent nature have a very important affect in the stability of enzymes in organic media. It is also considered that the enzymes in the solvents having the partition coefficient (degree of hydrophobicity) >2 higher stability (10). The regio-and stereo specificity of the protein is also very high, but there are some fundamental questions still to be answered. There are issues of higher rate of reactions, high stability and high activity.
Therefore, some experiments that mimic the aqueous system have to be performed. The solvent nature of the reaction medium has also to be optimized. Immobilized enzyme preparations that mimic the aqueous medium and provide stability and integrity of the enzyme can help in accelerating the reactions in organic media.
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