Burgeoning scientific literature proves that enzymes can catalyze chemical reactions in non-conventional media, are active against liquid, solid or gaseous substrates, retain their catalytic function in organic solvents, biphasic systems composed of organic solvent and water and supercritical fluids. Non-aqueous media appeared to be a promising alternative as an environment for the reactions catalyzed by enzymes. The possibility of carrying out enzymatic processes in such unusual milieus enlarged the range of applications of biocatalysis, and solved many problems witnessed by pharmaceutical, food and chemical industries. The paper describes both advantages and disadvantages of non-conventional media, and presents the examples of practical uses of selected enzymes (lipases, proteases, oxidoreductases, glycosidases) in these systems. The details on medium composition and the form of the biocatalyst are included.
Properties of lipases, which not only hydrolyze lipids in aqueous solutions, but are also active in nonaqueous systems, have attracted interest of many researchers. The presented dissertation contains a review of publications devoted to synthesis and classification of microbial lipases, both intra- and extracellular ones. Types of reactions catalyzed by these enzymes, their specificity, mechanism of reaction, molecular background of the interfacial activation, and an influence of the reaction medium on their properties are discussed.
An optimization of enzymatic synthesis of esters in nonwater environment was performedby mens of a gradient method.The studies were aimed at obtaining a model synthesis of butyl oleate catalyzed by Mucor javanicus T45 lipase.Theoptimized, changed parameters were as follows:substrated mass, lipase mass and chemical activator mass (diethanoloamine) which profitably modified the essential water layer of the lipase.For the initial concentration of the substrates equal to 57-67%, in reaction environment the obtained yield of coversion of oleic acid was in the range of 90-95%. A transfer of optimized conditions of butyl oleate synthesis onto oleates of the other aliphatic alcohols, enabled elaboration of simple in performance methods of synthesisof a broad variety of esters of oleic acid and aliphatic alcohols (C2-C18).
A mathematical model describing esters synthesis catalysed by lipase from Mucor jawanicus was designed.This model was confirmed by the syntheses of propyl stearate, butyl stearate and oleyl stearate catalysed by M.javanicus lipase.Using this model it is possible to estimate the maximum achievable yield of ester synthesis.The model can also applied for performing simulation to evaluate the effect of particular elements on the yield of ester synthesis and, consequently, it could enable the optimum selection of particular elements of the reaction system.
Enzymology of non-aqueous media, also termed a non-conventional enzymology, is a discipline focused on chemical reactions catalysed by enzymes in the media other than water. However, such environments always contain certain amount of water, either dissolved in a solvent or enzyme-bound. The latter fraction of water is called an essential one, and it is crucial for the non-aqueous enzymology, since it stabilises the conformation of protein molecule and determines its enzymatic activity. The paper presents structure, function, and methods of assays of the enzyme-bound water together with other water forms that can also be found in reaction milieu. The methods of regulation of enzymatic activity in non-aqueous media by using compounds capable of associating with polar atoms of amino acid residues involved in catalysis, and thus positively influencing activity of enzymes, have also been discussed. The factors giving rise to a decrease in catalytic activity of enzymes under non-aqueous conditions, and methods of prevention against this phenomenon have also been analysed.
Preparations of soluble and immobilized fungus lipases showing high catalityc acivity in hydrolysis of acyloglycerols in water, synthesis of esters of higher fatty acids in hydrolysis of acyloglycerols in water and synthsis of esters of higher fatty acids inorganic solvents milieu were obtained.The course of reaction andthe activity dependedon: the lipase source, the form of enzyme and the reaction milieu.The studied extacellular lipases, in a water - soluble form (especially R.nigricans lipase), exibited high activity in olive oil hydrolisis.The lipases were not active in esters synthesis in diphase system: water-organic solvent.Being drained by liophilization they demonstrated the activity in esters synthesis inorgnic solvents milieu.The endocellular lipases immobilized in situshowed the hihgest activity in the synthesis of esters in organic milieu.These enzymes can also be used for hydrolysis of acyloglycerols.The obtained liquid and immobilized mycelial preparations demonstrate activity which qualifies them for industrial scale application.
The intracellular lipases of Mucor circinelloides and Mucor racemosus immobilised in situ as well as the extracellular lipase of Rhizopus nigricans in soluble form were applied to enzymatic hydrolysis of waste fats from oil and meat industry, waste sludge of fats from municipal and industrial sewage-treatment plants and waste water containing microemulsion of fats. The reaction of hydrolysis of plant and animal waste fats was carried out by two methods. The first one was a partial hydrolysis of acylglycerols to obtain mono- and diacylglycerols. The second variant was complete hydrolysis of fats to obtain glycerol and free fatty acids. It has been demonstrated that lipase of Rhizopus nigricans partly hydrolysed waste fats. The products of hydrolysis contained 35-45% mono- and diacylglycerols. These products may be used as emulsifier in the hydrolysis of fats. The yield of hydrolysis of waste fats by immobilised Mucor lipases was from 85 up to over 95%. Waste sludge of fats from municipal and industrial sewage contain 12-62% of free fatty acids. This kind of material was hydrolysed without an emulsifier but with the addition of calcium chloride. The yield of hydrolysis of acylglycerols was from 45 to 76%. The yield of hydrolysis of microemulsion of fats in waste water by immobilised Mucor lipases was from 80 up to over 95%. As the hydrolysis products contained free fatty acids, mono- and diacylglycerols, it was easy to make emulsions which could be utilised in anaerobic-aerobic processes.
Endolipases associated with the cell structures of microorganisms are more active in comparison with purified enzymes. Due to the weak mechanical resistance lipases immobilised in situ cannot be used many times. The method of lipases encapsulation in polysacharides hydrophilic gels was elaborated. The lipases Mucor immobilised in situ were treated with oleic acid in hexane and then dispersed in an aqueous solution of sodium alginate or karagenate. Immobilisation of enzymes was achieved by intermolecular cross-linking of the polysacharide chains using the solution of calcium or potassium salts. The biocatalysts prepared under proposed conditions were active in hydrolysis of esters, as well as in esterification reaction. It was found that immobilised enzymes were active for a long time, were mechanically resistant and could be used many times in periodic and continuous processes.
A mathematical model describing sucrose esters synthesis in biphasic di-n-phentyl ether - water system by lipase from Mucor circinelloides has been elaborated. This model considers correlation between physicochemical factors, dependent on the solvent, substrates, products and temperature, catalytic factors corresponding to the relationship between lipase and the physicochemical factors of the model (Ko=F(A)), as well quantitative factors whose values may be regulated during the reaction (substrate concentration, phase volume coefficient (A) and water concentration). This model was confirmed by syntheses of caprylic and oleic sucrose esters. The maximum reaction yield for the synthesis of caprylic sucrose ester calculated on the basis of the elaborated model was 67,5% and experimental value was 67,8% for A = 44. For oleic sucrose ester, the theoretical and experimental yields of synthesis, were 71,9% and 70,5% respectively. Using this model it is possible to estimate the maximum achievable (in particular conditions) yield of the ester synthesis. It can also be applied for simulations enabling evaluation of the effect of the particular factors on the yield of ester synthesis and resulting optimisation of selected factors in the reaction system. The simulations were performed for two factors of the model in three dimensional space. It was found that the ester synthesis was mostly affected by the following invariable factors: partition coefficients of products (PE, PW) and phase volume coefficient (A). The concentration of water in the water phase and the partition coefficients of sugar (PAL) influenced the yield to a smaller extent.
Lipases Mucor circinelloides and Mucor racemosus immobilised in situ were closed in microporous polysacharides hydrophilic gel cross-linked using a solution of calcium salts. In order to increase the porosity of polysacharides matrix during its cross-linking oligomer molecules of ethylene oxide (optimal Polikol 1000) were incorporated in the structure of the matrix. Then the oligomer was removed by acetone extraction. The obtained biocatalyst preparations were tested in hydrolysis of esters and esterification of oleic acid with butanol. The hydrolysis was carried out in water saturated organic solvents medium (n-hexane and diisopropylether). It was found that the efficiency of M. racemosus lipase immobilisation in hydrolysis of n-butyl oleate, n-butyl palmitate, n-butyl stearate, n-butyl laurate amounted to 60%. The efficiency of M. circinelloides lipase immobilisation in esterification of oleic acid with 1-butanol in hexane achieved 45%.
The results of special tests which had been carried out with the following bacterail strains Bacillus sp. S2, Bacillus sp. BP, Pseudomonas sp. 141 and Micrococcus sp. R5.4 show that these strains are acive degraders of petroleum oil hydrocarbons, tolerate high concentrations of the subsrtate and have a high capability for degradation of hydrocarbons.The activity of dehydrogenases was related to the concentration of the substrate.The gas chromatography analysis of residual hydrocarbons in the medium after 9 days of cultivation proved that the main identified petroleum oil components had been metabolized with the yield 69 to 100%. Apart from the compounds originally present in crude oil no new by-products were detected in final medium.The accumulation of shorter chain length hydrocarbons and various new by-products occured in the case of another tested bacterial strain, designated 'B'.
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