Abstract

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.

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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.3.1.1.3) 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.

(Fruity flavor)

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)

Reactions:

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).

Transesterification Reactions

Acidolysis

Alcoholysis

Ester Exchange

Aminolysis

Hydrolysis

Ether synthesis

Figure 3: Figure depicting various Candida rugosa Lipase catalysed reactions.

Bacterial Lipase
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)

Fungal Lipase
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)
Sr. No.
Organism
Specificity
Molecular

Weight

(kDa)
PH

Optima
Temp.

Optima
Specific

Activity

(U/mg)
1
Chromobacterium viscosum
Unspecific
30
6.5-7.0
70
22.75
2
Pseudomonas sp.
Regio l, 3
32
7.8
47
7.80
3
P. fluorescens
Regio 1,3
32
7.2
50-55
3.05
4
C curvata
18:1>16.0=14.0
195
5.0-8.0
60
4
5
C. deformans
Regio 1,3
207
7.0
80
19
6
Aspergillus niger
Regio 1,3
38
5.6
25
9.02
7
Candida

cylindracea
Unspecific
120
7.2
45
53.22
8
Humicola

lanuginosa
Unspecific
27.5
8.0
6.0
5.16
9
Mucor miehei
Regio 1,3

8.0
40
3.25
10
Lipase A
Unspecific
27
7.5
35
0.09(A+B)
11
Lipase B
Unspecific
36
5.8
40

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.

Sources

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.

Applications

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)

Industry
Effect
Product
Bakery
Flavour improvement and shelf-life prolongation
Backer products
Beverages
Improved aroma
Beverages
Chemical
Enantioselectivity
Chiral building blocks and chemicals.
Cleaning
Synthesis

Hydrolysis
Chemicals

Removal of cleaning agents like surfactants.
Dairy
Hydrolysis of milk fat Cheese ripening fat

Modification of butter
Flavour  agents

Cheese

Butter
Cosmetics
Synthesis
Emulsifiers, moisturizing agent
Fats and Oils
Tans-esterification

Hydrolysis
Coco butter, margarine

Fatty acids, glycerol, mono- and diglycerides
Food dressing
Quality improvement
Mayormaise, dressings and whippings.
Health food
Trans-esterification
Health food
Leather
Hydrolysis
Leather products
Meat and Fish
Flavour development

And fat removal
Meat and fish
Paper
Hydrolysis
Paper products
Pharmaceuticals
Trans-esterification

Hydrolysis
Specially lipids

Digestive aids

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.

http://www.ebi.ac.uk/cgi-bin/dbfetch?db=emblcds&id=CAA45957

P20261|LIP1_CANRU Lipase 1 – Candida rugosa (Yeast) (Candida cylindracea).

MELALALSLIASVAAAPTATLANGDTITGLNAIINEAFLGIPFAEPPVGNLRFKDPVPYS

GSLDGQKFTSYGPSCMQQNPEGTYEENLPKAALDLVMQSKVFEAVSPSSEDCLTINVVRP

PGTKAGANLPVMLWIFGGGFEVGGTSTFPPAQMITKSIAMGKPIIHVSVNYRVSSWGFLA

GDEIKAEGSANAGLKDQRLGMQWVADNIAAFGGDPTKVTIFGESAGSMSVMCHILWNDGD

NTYKGKPLFRAGIMQSGAMVPSDAVDGIYGNEIFDLLASNAGCGSASDKLACLRGVSSDT

LEDATNNTPGFLAYSSLRLSYLPRPDGVNITDDMYALVREGKYANIPVIIGDQNDEGTFF

GTSSLNVTTDAQAREYFKQSFVHASDAEIDTLMTAYPGDITQGSPFDTGILNALTPQFKR

ISAVLGDLGFTLARRYFLNHYTGGTKYSFLSKQLSGLPVLGTFHSNDIVFQDYLLGSGSL

IYNNAFIAFATDLDPNTAGLLVKWPEYTSSSQSGNNLMMINALGLYTGKDNFRTAGYDAL

FSNPPSFFV

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.

References:

1.         Lehninger A.L., N. D. L. a. C. M. M. (1993) Principles of Biochemistry, 2nd Ed., CBS publishers

2.         M.T, R. (2002) Current Opinion in Chemical Biology 6, 145-150

3.         Saxena R.K., G. P. K., Gupta R.,Davidson W.S., Bradoo S., Gulati R. (1999) Current Science 77, 101-111

4.         M.T., J. K. E. a. R. (1998) Trends Biotechnology 16, 396-403

5.         R., V. (1989) Methods in Enzymology 64, 340-392

6.         D., S. K. a. M. (2000) Process Biochemistry 36, 607-611

7.         R. Gupta, N. G. a. P. R. (2004) Applied Microbiology and Biotechnology 64, 763-781

8.         145-150., R. M. T. (2002) Current Opinion in Chemical Biology 6, 145-150.

9.         M, R. M. a. C. (1993) Journal of Biological Chemistry 268(12843-12847)

10.       C. Torres, C. O. (1995) Journal of Molecular Catalysis A: Chemical 97, 119-134

 

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