24 Biotechnological Applications of lipases

Prof. M. N. Gupta

1. Objectives

To learn:

1.) Why and how enzymes are used in biotechnology

2.) Why lipase is among the most frequently used enzyme in biotechnology

3.) How change in reaction medium leads to use of lipases in synthesis work

4.) The use of glycerol which is obtained as a by-product in the production of biodiesel

2. Concept Map

2. Description

Chemical Industry is often blamed for our industrial problems. Chemists have long ago started developing strategies for synthesis of compounds and materials which hurt the environment lot less.

An early outcome of that was the development of a new branch of chemistry which is called Green Chemistry. One of the important notion there is that catalysts should be used. Enzymes with their high catalytic efficiencies obviously have a place in the development of greener strategies in the production of chemicals. Lipids, as a renewable resource, and lipases which catalyse these reactions are the subject of these next few modules.

A 2002 estimate records that number of industrial scale processes in which biocatalysts are used have doubled since 1992!

Hydrolases (44%) dominated these applications. Hydrolases are the enzymes which hydrolyze carbohydrates, proteins, nucleic acids, lipids etc.

Along with proteases and carbohydrases, lipases constitute the most important class among hydrolases.

The shift in favour of biocatalysts is due to the fact that biocatalysis follow all the principles of green chemistry.

•  The feedstock for growing microorganisms (sugars and amino acids) is renewable
•  Atom economy is maximized. Selectivity makes it possible to not having to use protection and deprotection chemistry.
•  Milder reaction conditions and high catalytic efficiency ensure low energy consumption.

Why lipases became the most frequently used in biotechnology?

Lipases in detergents

Detergents are used all over the world to clean clothes, utensils and many surfaces. Enzymes, as additives, allow “catalytic cleaning”. The function of the enzyme is to create fragments of “dirt” or “stains” by hydrolysis so that surfactants present in the detergent can take over and do “chemical cleaning”.

Hence, it is obvious that hydrolases is the class of enzymes which are added to the detergents. Proteases were incorporated in detergents about 80-90 years ago. Currently, household detergents also contain amylases, cellulases and lipases.

Addition of lipases is necessitated as any oil based food etc are major causes of stains on the clothes. It is estimated that enzymes in detergents constitute >50% of the market for industrial enzymes. Hence, for lipases too, use in detergents is one of the major applications of lipases.

“cleaning” as an activity is associated since ancient times. Around 5000 BC, scouring in river was the way to clean. Around 2100 BC, historical accounts mention that humans living on the banks of Tigris river used olive oil and wood ash to make soap. It was around 1928 that sulfation of fatty acids was carried out to obtain synthetic detergent.

It as Otto Rohm who in 1931 patented the use of pancreatic enzymes in detergents. Over the next couple of decades the challenges were to create enzymes which could withstand alkaline pH and other harsh conditions of washing, create granular detergents so that the enzyme dust did not cause allergy.

It was in 1988 that Novo introduced Lipolase, a lipase for adding to the detergents. Around 1994, gist-Brocades introduced Lipomax and Genecor International introduced Lumfast; both were lipases tailored for incorporating in detergents.

Table 1: Commercial enzymes used in detergent formulations and other applications

Hence, while proteases, amylases and cellulases were blended in detergents much earlier, use of lipases happened much later. To screen the suitable lipase, SLM-test has been developed.

After the ash, products are analysed by HPLC for checking the efficiency of triolein hydrolysis. This test quickly allows screening of many lipase candidates.

As the market is huge, lipase engineering by site directed mutagenesis to improve ther wash performance was carried out right from the early years of this application. Hence, screening test was helpful to assess engineered mutants.

Lipases in Oil and Fat Processing Industry

Oil and fat processing industry is very old. Many lipases were commercially available for fat splitting and for incorporating in detergents.

As these are large scale applications, these enzymes were produced in bulk. For such applications and given the advantages of bulk productions, these industrial level preparations were not as costly as research grade enzymes.

As these preparations did not require high purity, the downstream processing steps were not much required. That cut down on the overall production costs.

So as new applications emerged, this readily available class of enzymes were tried more extensively. For the same reason, proteases and amylases were also readily used extensively in biotechnology even in its early years.

Lipases, however, had two distinctive features because of which they score over these other classes of industrial enzymes. Both features have to do with the nature of the emerging applications.

Klibanov’s work at MIT in late 1980’s showed convincingly that hydrolases can be used for synthesis if the reaction medium is changed from conventional water rich to low water conditions.

Principle of microscopic reversibility implies that every reaction in principle is reversible. For hydrolases, water as also a reactant for hydrolysing bonds. If no water is there, hydrolysis cannot take place.

In such media, let us say anhydrous octane, a protease can form peptide bond instead of hydrolysing it. While Klibanov in those early papers used proteases to form esters of amino acids, his group soon thereafter showed that proteases can indeed synthesize peptides.

More laboratories joined in. Notable early contributions came from two other groups. The Swedish group showed how to optimize the process for peptide synthesis in organic media using proteases.

Lipases also turned out to be very stable in organic solvents. A classical work, again by Klibanov’s group showed that a lipase could be heated at 100 ᵒC in an anhydrous organic solvent and still retained almost all its activity.

The second important feature of lipases are these enzymes show broad specificity. Lipases not only catalyse reactions involving fats/oils and esters, these also carry out very different reactions such as aldol condensation. This promiscuous nature has already been discussed in an earlier module.

Fats and oils constitute very important feedstock for chemical industries. These are also what are known as renewable chemicals. Both fatty acids and glycerol can be the starting point for obtaining many chemicals and materials.

Oils and fats of non-edible variety are especially interesting since their use does not deplete our edible oil stocks. Such crops include castor bean, Jatropha curcas, Crambe abyssimica and Macaw palm (Acrocomia aculeate) and Licania rigida (oiticia).

In Brazil, many of these have been exploited for biodiesel production. A particular atytractive feature of these crops are tolerance to harsh conditions and ability to grow on low-fertility soils.

In an application like biodiesel production, feedstock cost constitutes about 70-88% of the final price. This, ofcourse, is based upon the use of chemical catalysts. In case of use of lipases, the cost of catalysts is substantially higher.

In addition to triglycerides, the unrefined oils/fats also contain mono- and di- glycerides, free fatty acids, phospholipids, unsaponifiable lipids such as carotenoids, steroids, chlorophyll, terpenes and even hydrocarbons.

In addition to that, the biorefinery concept involves not just the use of fats/oils in production of chemical/materials. Biofuels but also use rest of the plant material for obtaining value added products. Extensive enzymatic/ non-enzymatic routes for valorization of the waste for oleochemical industries have been developed.

The fatty acids present in the triglycerides from oil crops affect the properties of the biodiesel ultimately produced. The fatty acids with higher C-chain leads to the biofuel with higher cloud point and cold filter plugging point.

Biodiesel produced from such fatty acids is not suitable for use in places where ambient temperature is low. Similarly a biofuel with better combustion quality (cetane number) is produced from saturated fatty acids or MUFA.

The presence of many double bonds in the fatty acid chain will also make biofuel more susceptible to oxidation, degradation and polymerization. In general, biodiesel from fatty acid such as oleic acid or ricinoleic acid is reported to produce high quality biodiesel.

Waste fat from animal sources has also been used for various biotechnological applications which include biodiesel production. Rendering consists of heating carcasses with water at high temperature or even steam to release fat.

One key parameter in the biodiesel production is the presence of free fatty acids. The alkali catalyst most frequently used in biodiesel production produces soap with the free fatty acids. Use of refining to remove FFA increases the production cost.

Biodiesel is alkyl ester of long chain fatty acids. The alkyl group is generally from short chain alcohols such as methyl alcohol and ethyl alcohol. The alkyl esters of higher chain alcohols are also important as these can be used as lubricants.

The esterification of fatty acids by short chain alcohols, as mentioned earlier, is m ost frequently carried out by NaOH or KOH at moderately high temperatures. This consumes energy, corrodes production vessel and produces soap from free fatty acids.

Apart from biodiesel and biolubricants, esters of different acids and alcohols are also used as flavours and fragrances. We will discuss this aspect more in the next module when we discuss organic synthesis with the help of lipases.

As we mentioned in the earlier module, lipases show broad specificity. Part of that is that these enzymes can also act as esterases. So, the reverse synthesis can produce esters.

Esters like ascorbyl palmitate have been produced by lipases. Ascorbyl palmitate is the fat soluble anti oxidant which we mentioned earlier. Acylated sugars like palmitoyl glucose etc are amphiphilic and hence constitute important food grade biosurfactants.

Use of lipases in producing these esters: biodiesel, biolubricants, fragrances and flavors and biosurfactants allows the use of milder conditions in terms of temperature and pressure. For example, in china, a plant producing biodiesel from soybean with the use of a lipase has been operating for many years and some more similar plants are being commissioned elsewhere.

Glycerol and its products

One advantage of using lipase for producing biodiesel is that the by-product glycerol is of cleaner quality and does not require extensive downstream processing. This makes glycerol obtained through this lipase based technology useful for some sectors like pharmaceuticals and cosmetics.

Glycerol is a colourless, odourless viscous liquid which is also called glycerine. Crude glycerol is 70-80% pure and is generally purified to a level of 99-99.5% purity before being made commercially available.

It is useful as an ingredient in food products, cosmetics, toiletries and drugs. It is generally used as a humectant, sweetner, solvent or a preservative.

Traditional uses of glycerol included its use in synthesis of trinitroglycerine, alkyl resins and polyurethans. Its use in alkyl resins and regenerated celluloses is as a softner. Surface coatings and paints use this as an ingredient as it imparts toughness. Special quality of paper use glycerol.

As a food additive, it is used as an additive called E422. Its use prevents baked goods from drying. It is also used in cough syrups and expectorants.

During World War , glycerol was a national defense priority in Europe as soap industry was not producing enough of it to meet the demand as a precursor for dynamite.

To meet this challenge, production route for making glycerol from epichlorohydrin was established. Epichlorohydrin is made from propylene and thus glycerol production became dependant upon petroleum industry.

Advent of biodiesel changed this picture drastically. In 1992, about 600,000 tons of glycerol was available as by product of biodiesel synthesis. In 2008, this figure rose to 1.5 million tons. At present, this is the only source of glycerol!

About 100 kg of oil produces 100 kg of methyl esters (biodiesel) and 10.5 kg of glycerol in KOH catalysed transesterification of a vegetable oil as an equilibrium controlled reaction.

This large amount of glycerol availability has led to extensive research and development efforts in searching for alternative and new applications of glycerol. These applications are independent of the route by which biodiesel production is carried out. However, lipase based route produces better quality glycerol.

Glycerol has been found to be a useful solvent for carrying out many organic reactions such as aza-Michael reactions of amines.

Some other solvents derived from glycerol have been developed. A family of 1,3-dialkyl-2-propanols and 1,2,3-trialkoxypropanes have been found to be useful solvents for epoxidation reactions. Even for enzymatic reactions, glycerol has been explored as a greener solvent.

Glycerol as a platform molecule

Syngas or synthetic gas is a mixture of H2 + CO and is used for synthesis of methanol. Syngas can be obtained from glycerol by using a Pt-Re catalyst at 225-300 ᵒC without using any organic solvent.

Similarly, another aqueous phase reforming process has been developed which rapidly converts glycerol to H2 with very low consumption of energy. Small amount of CO is also produced in this process based upon favourable water-gas shift thermodynamics.

Both H2 and methanol are considered cleaner fuels. H2 is considered as a gold standard among alternative fuels. Methanol, on the other hand, is easy to store and transport.

Syngas can be used as a source of fuels and chemicals and extensive experience with syngas and its applications is available to the chemical industry.

1,2-propanediol is obtained from petroleum sources. Obtaining this important commodity chemical at commercial level by reduction of glycerol has been a great achievement.

The route is inexpensive and utilizes copper chromite as catalyst at 200 ᵒC. Davy process technology has developed as alternative process for achieving the same.

1,3-propanediol (PDO) can be now obtained using glycerol as a feedstock in the fermentation processes using bacterial strains from citrobacter, enterobacter, klrbsiella and lactobacillus.

The dehydratase action followed by NAD+-linked oxidoreductase catalysis leads to accumulation of PDO. A new antifreeze mixture of 70% PDO + 30% glycerol has been produced using biodiesel production facilities.

1,3-dichloro-2-propanol can be obtained by halogenation of glycerol and can be converted to epichlorohydrin by dehydrochlorination. Epoxy resins are used in marine, automative, housing and canning industries and have over billion UDS market.

Epichlorohydrin is also used as a precursor for 1-chloro-2,3-propanediol. This compound is used by pharma industries in cough mixtures.

In 2007, Solvay has started a 10,000 ton plant facility for producing epichlorohydrin from glycerol. The glycerol was sourced from a French biodiesel (from rapeseed oil) producing company. Dow chemical, similarly has 150,000 ton plant in China for producing epichlorohydrin from glycerol obtained from small local producers. These producers obtain their glycerol from biodiesel production from mostly rapeseed oil and palm oil.

Biorefineries are an important part of global efforts to reduce dependence upon depleting petroleum resources. Simultaneously, these represent a shift from bioremediation to green processes which produce less waste. In fact, the strategy is to abolish the concept of waste and establish valorization processes. Whole lot of products have come out based upon glycerol. Lot of new chemistry and biological transformations were developed. Lipase based process gives better quality glycerol.

Summary

•  Use of enzymes in biotechnology
•  Lipases as most frequently used enzyme in biotechnology
•  Production of esters including biodiesel by medium engineering of lipase catalysed reactions
•  Applications of glycerol produced as a by-product of biodiesel