**3. Lipase-active heterogeneous biocatalysts for vegetable oil and fatty acid bioconversion**

Heterogeneous lipase-active biocatalysts are of great importance due to remarkable properties of their enzymatic active component, namely lipase (triacylglycerol acyl hydrolase, EC 3.1.1.3.), that catalyzes a variety of reactions involving triglycerides—hydrolysis, esterification (synthesis of esters), inter-(or trans-)esterification (intra- and intermolecular exchange between fatty acids' residues), alcoholysis, and aminolysis [2, 9]. Nowadays, the main areas of industrial application of lipases and lipase-active biocatalysts are as follows: (1) hydrolysis of oily and fat stains on clothes by smart washing powders containing lipases as additives; (2) a large scale process of interesterification of the oil-fat blend to produce valuable ingredients, in particular for various spreads and margarine; (3) alcoholysis (methanolysis/ ethanolysis) of triglycerides of vegetable rapeseed and soybean oils, as well as waste cooking and algal oils, into methyl/ethyl esters of fatty acids for production of biodiesel as an additive to fuel for engines; and (4) esterification of organic acids and synthesis of marketable valuable products including various esters of fatty acids for food and cosmetic industries, as well as enantiomers for pharmaceutical industry.

The most interesting and unique property of lipases is their ability to catalyze reaction in anhydrous media of organic solvents with water content less than 1%. Despite nonconventional conditions, these processes are characterized by high efficiency such as 100% selectivity, high conversion, and yield of final product.

### *Heterogeneous Biocatalysts for the Final Stages of Deep Processing of Renewable Resources… DOI: http://dx.doi.org/10.5772/intechopen.89411*

One of the best results of oils' methanolysis by commercial biocatalyst Novozym® is that conversion of triglycerides into methyl esters of fatty acids (biodiesel) was 99% for 50 h at 45°C [10]. In 2007, the first biocatalytic process of the biodiesel production was implemented by methanolysis of edible oils' waste with the productivity of 10,000 tons; the biocatalyst was prepared by immobilization of lipase from *Candida* sp. [11].

NOVO (NOVOZYMES) Company is a leader in the production and sale of heterogeneous biocatalysts prepared by immobilizing the recombinant lipases on various supports. The Lipozyme® TL IM biocatalyst is prepared by immobilization of recombinant thermostable 1,3-specific *T. lanuginosus* lipase on silica. This biocatalyst is widely used in the industrial interesterification of fat-oil blends in order to produce valuable products, such as specialized fats and spreads without undesirable *trans*-isomers of fatty acids, as well as substitutes of cocoa butter and dairy fat. The Novozym®435 biocatalyst is prepared by immobilization of nonspecific *Candida antarctica* lipase on a macroporous polyacrylic polymer. The commercial Novozym® biocatalysts type are used in biodiesel production by methanolysis of vegetable oil (rapeseed and soya been) and waste oils of cooking. Nowadays, the NOVO biocatalysts are intensively studied for application in various processes, including organic synthesis.

It is well-known that methanol and ethanol inactivate enzymes rapidly. Therefore, in order to reduce the biocatalysts' inactivation, methanol was added stepwise in small portions during the reaction cycle of the biodiesel production. Another acylating reagent—methyl or ethyl acetate—was examined to be used. It was found that this reagent did not reduce the activity of the commercial biocatalyst Novozym® even at a molar ratio of oil to methyl acetate equal to 1:12; and under optimal operating conditions, the yield of methyl esters was equal to 96% and the biocatalyst's half-life time (t½) increased 20-fold in comparison with t½ in reaction with methanol [12]. Acyl derivatives of glycerol produced in interesterification of vegetable oils with methyl or ethyl acetate are valuable commercial products also. For example, mono- and triacyl glycerol are employed as fuel additives. Triacylglycerol (triacetin) is widely used in the food industry due to its good moisture-retaining properties. If the linseed oil is used in biocatalytic interesterification with ethyl acetate, the produced mixture of ethyl esters of ω3-, ω6-unsaturated fatty acids (vitamin F) is a valuable product for cosmetics industry and fodder additives production.

Nowadays, enzymatic esterification is considered as a competitive alternative to the chemical organic synthesis of various esters that are valuable commercial products commonly used in manufacturing flavors, fragrances, emollients, lubricants, antimicrobial agents, and nontoxic surfactants. The requirement of consumers for such natural products is constantly increasing. Compared with organic synthesis using strong liquid and solid acids as catalysts and temperature above 100°C (usually, 120–150°C), the enzymatic esterification is currently of great commercial interest since this method of esters' production proceeds efficiently at a low temperature (usually, at 20–40°C) without the formation of any by-products and with high specificity toward substrates. The heterogeneous lipase-active biocatalysts for the low-temperature esters'synthesis are prepared, as mentioned above, by immobilizing lipases on solid supports by various chemical origins and texture. These heterogeneous biocatalytic processes realized in periodic or continuous modes in anhydrous reaction media fully satisfy the requirements of "green" chemistry and they are promising for implementation into the organic synthesis industry [2].

The authors and their collaborators have developed and researched systematically the heterogeneous biocatalysts in which the enzymatic active component was a

Comparing these data with the Corning's results in [4], the t½ of GlucoSib

10 months upon storage at ambient temperature (18–22°C).

commercially attractive.

*Molecular Biotechnology*

per 1 kg of biocatalysts.

**acid bioconversion**

industry.

**8**

**2.1 Conclusion for the part 2**

biocatalysts was estimated to be higher, 350 h vs. 150 h at 60°C, respectively. Longterm stability was sufficient also; the biocatalysts retained initial activity for

A technological scheme has been proposed and tested on a laboratory scale using the GlucoSib biocatalyst and immersed vortex reactor IVR for production of starch treacle and glucose syrups by heterogeneous dextrin hydrolysis. The advantages of this technological scheme are as follows: (1) significant acceleration of dextrin hydrolysis; (2) energy and resource saving in comparison with traditional starch processing; (3) a high quality of the final products due to the lack of protein impurities; and also, very importantly, (4) easily regulated carbohydrate composition of the treacle by simply stopping rotation of reactor body. It should be noted that when comparing the efficiency of the process of dextrin hydrolysis in vortex reactor with parameters of traditional packed-bed reactor the productivity of the IVR was higher by 1.2–1.5 times. The productivity in a novel proposed technology was calculated to be 5.3 tons of glucose per 1 kg of biocatalysts GlucoSib that is quite

The highly active and stable heterogeneous biocatalysts for dextrin hydrolysis were prepared by adsorption of glucoamylase on mesoporous carbon support Sibunit™. Under technological conditions (32 w/v% dextrin, 60°C, pH 5), the maximal activity was observed to be equal to 750 U/g, and inactivation half-life time (t½) was 350 h. The immersed vortex reactor designed specially for the biocatalytic diffusion-controlled heterogeneous processes was used to carry out starch saccharification with enhanced productivity roughly estimated as 5 tons of glucose

**3. Lipase-active heterogeneous biocatalysts for vegetable oil and fatty**

Heterogeneous lipase-active biocatalysts are of great importance due to remarkable properties of their enzymatic active component, namely lipase (triacylglycerol acyl hydrolase, EC 3.1.1.3.), that catalyzes a variety of reactions involving triglycerides—hydrolysis, esterification (synthesis of esters), inter-(or trans-)esterification (intra- and intermolecular exchange between fatty acids' residues), alcoholysis, and aminolysis [2, 9]. Nowadays, the main areas of industrial application of lipases and lipase-active biocatalysts are as follows: (1) hydrolysis of oily and fat stains on clothes by smart washing powders containing lipases as additives; (2) a large scale process of interesterification of the oil-fat blend to produce valuable ingredients, in particular for various spreads and margarine; (3) alcoholysis (methanolysis/ ethanolysis) of triglycerides of vegetable rapeseed and soybean oils, as well as waste cooking and algal oils, into methyl/ethyl esters of fatty acids for production of biodiesel as an additive to fuel for engines; and (4) esterification of organic acids and synthesis of marketable valuable products including various esters of fatty acids for food and cosmetic industries, as well as enantiomers for pharmaceutical

The most interesting and unique property of lipases is their ability to catalyze reaction in anhydrous media of organic solvents with water content less than 1%. Despite nonconventional conditions, these processes are characterized by high efficiency such as 100% selectivity, high conversion, and yield of final product.

recombinant *T. lanuginosus* lipase. These lipase-active biocatalysts were prepared both by entrapment of fully disrupted cells (lysates) of recombinant strainproducer r*E*.*coli*/lip inside silica xerogel and its nanocarbon-containing composites [13, 14, 17] and by adsorption of *T. lanuginosus* lipase produced by recombinant lipase on silica [15–18] and carbon aerogel [19]. These biocatalysts were studied in the reactions of tributyrin hydrolysis [13, 19], interesterification of oil-fat blends and vegetable oil triglycerides with ethyl acetate [13, 14], and esterification of fatty acids [15–19]. The two biocatalysts prepared by adsorptive immobilization of recombinant *T. lanuginosus* lipase are described briefly below.

activity but also by simplicity of its implementation and economical enzyme consumption. For example, a minimal volume of lipase solution used for spontaneous adsorption on 1 g of silica was 3.0 mL, whereas for forcible adsorption, it was 0.8 mL equal to total pore volume (VΣ) of SiO2. All results described here referred to the lipase-active heterogeneous biocatalysts (designated as LipoSil) prepared by forcible adsorption on silica of recombinant lipase, which were used predominantly

*Heterogeneous Biocatalysts for the Final Stages of Deep Processing of Renewable Resources…*

Biocatalytic processes of enzymatic esterification were performed at ambient conditions (20 2°C, 1 bar) in unconventional anhydrous media of organic solvents such as hexane and diethyl ether. The saturated fatty acids differing in the number of carbon atoms (C2–C10, C18), as well as aliphatic alcohols differing in the structure of the molecules, namely, the number of carbon atoms (C2–12, C16), the isomerism of the carbon skeleton (*n*- and *iso*-) and OH-group position (*prim-, sec-,* and *tert-*) were studied as substrates in esterification by LipoSil. There were some peculiarities of operation of the preliminary dried lipase-active biocatalysts in nonaqueous organic solvents. A considerable increase of the activity was observed during the 1st–3rd reaction cycles. This phenomena, named preconditioning stage, was due to the ongoing accumulation of formed product—water—in the vicinity of the adsorbed lipase inside the silica-based biocatalyst, and this stage proceeded faster and the higher than the activity of the biocatalyst. For example, if the biocatalytic activities were about 5 and 500 U/g, then the activation of the biocatalysts (preconditioning) proceeded within 24 and 0.5 h, respectively. Calculation showed that under studied conditions upon full conversion of fatty acid, maximal 0.1 mL of water was formed inside for one reaction cycle. Since the total pore volume of silica (0.8 mL/g) was multifold greater than the volume of the water formed, this amount of H2O was firmly held inside KSK™ silica commonly applied as a dehumidifier for industrial gases. Therefore, during esterification, the favorable aqua microenvironment was created for adsorbed lipase, and the activities of the dried biocatalysts increased by 2–4 times. After preconditioning stage, the biocatalytic activity, named stationary, was measured in batch reactor during several tens of reaction cycles. Each reaction cycle was completed to the full conversion of acid, close to 85– 90%. Then, the reaction medium was removed by decantation and the biocatalysts were washed by solvent for 20 h. The next reaction cycle was started by adding fresh reaction medium containing substrates of lipase—acid S1 and double molar excess of alcohol S2. Stationary activity of the biocatalysts was fluctuated in magnitude during the consecutive reaction cycles of the periodic esterification process, perhaps due to the presence of ester (product) residues inside the operating biocatalyst. As it can be seen in **Figure 3**, the operational stability of the prepared biocatalyst was sufficiently high; its stationary activity was retained completely after 38 cycles (900 h) of esterification of various fatty acid. Also, the biocatalysts possessed a high long-term stability; the activity was determined to be 80% of initial one after storage for 9 months in the solvent (hexane and diethyl ether) at

in esterification processes [15, 16, 18].

*DOI: http://dx.doi.org/10.5772/intechopen.89411*

ambient temperature and in dried state in refrigerator.

uct per 1 kg of LipoSil biocatalyst.

**11**

Obviously, a very important property of heterogeneous biocatalysts is the high

The study of the functional properties of enzymes after their immobilization, such as activity, stability, and, importantly, specificity, is of great interest. Of particular scientific and practical interest is the research of the possibility of modulating these properties, by engineering heterogeneous biocatalysts, in particular, by

operational stability, since the productivity calculated by multiplying average activity by 2t½ increases significantly. The esterifying activity of the prepared lipase-active biocatalysts did not practically change during 500–1000 h of operation. Under studded conditions, the productivity was evaluated as 2 tons of prod-
