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The use of lipase immobilized on an octyl-agarose support to obtain the optically pure enantiomers of chiral drugs in reactions carried out in organic solvents is a great challenge for chemical and pharmaceutical sciences. Therefore, it is extremely important to develop optimal procedures to achieve a high enantioselectivity of the biocatalysts in the organic medium. Our paper describes a new approach to biocatalysis performed in an organic solvent with the use of CALB-octyl-agarose support including the application of a polypropylene reactor, an appropriate buffer for immobilization (Tris base—pH 9, 100 mM), a drying step, and then the storage of immobilized lipases in a climatic chamber or a refrigerator. An immobilized lipase B from Candida antarctica (CALB) was used in the kinetic resolution of (R,S)-flurbiprofen by enantioselective esterification with methanol, reaching a high enantiomeric excess (eep = 89.6 ± 2.0%). As part of the immobilization optimization, the influence of different buffers was investigated. The effect of the reactor material and the reaction medium on the lipase activity was also studied. Moreover, the stability of the immobilized lipases: lipase from Candida rugosa (CRL) and CALB during storage in various temperature and humidity conditions (climatic chamber and refrigerator) was tested. The application of the immobilized CALB in a polypropylene reactor allowed for receiving over 9-fold higher conversion values compared to the results achieved when conducting the reaction in a glass reactor, as well as approximately 30-fold higher conversion values in comparison with free lipase. The good stability of the CALB-octyl-agarose support was demonstrated. After 7 days of storage in a climatic chamber or refrigerator (with protection from humidity) approximately 60% higher conversion values were obtained compared to the results observed for the immobilized form that had not been stored. The new approach involving the application of the CALB-octyl-agarose support for reactions performed in organic solvents indicates a significant role of the polymer reactor material being used in achieving high catalytic activity. [ABSTRACT FROM AUTHOR]

Thermal stability is one of the essential parameters characterizing biocatalysts with potential applications in the chemical and pharmaceutical industries. Therefore, it is extremely important to develop standardized procedures for enzyme stability studies. The paper attempts to assess the thermal stability of immobilized lipases in aqueous buffers: lipase B from Candida antarctica (CALB) and lipase from Candida rugosa (CRL-OF) immobilized on the Octyl-Sepharose CL-4B carrier. As part of the optimization conditions of the immobilization, the influence of time on the catalytic activity and lipase loading, as well as the effect of temperature on lipase activity (optimal incubation—14 h at 4 °C), was determined. The thermal stability test procedure was carried out for 7 days using a climatic chamber (65 °C) and a refrigerator (4 °C). The studies of immobilized lipases included the assessment of the impact of various solvents (water, citrate buffer, 1,2-dichloropropane—DCP), temperature, light in the visible spectral range (400–800 nm), and additions of calcium ions. The highest value of residual activity (564.5 ± 21.6%) was received by storing the immobilized CALB in citrate buffer (pH 4.0, 500 mM) with the addition of calcium ions (Ca2+). On the other hand, residual activity values for immobilized CRL-OF after storage in the climatic chamber were lower than 5%. A combining of techniques: immobilization onto the support in high ionic strength and low pH, with a technique of extremally high-temperature applied in a climatic chamber, with the addition of Ca2+ allowed to achieve of excellent thermal stability of the immobilized CALB, with increasing of catalytic activity more than five-fold. Additionally, performing studies on the thermal stability of the tested lipases using a climatic chamber seems to be particularly promising in the context of unifying and standardizing storage guidelines, enabling the comparison of results between different laboratories, as well as enhancing catalytic activity. [ABSTRACT FROM AUTHOR]

The application of the climatic chamber presented in this paper to assess the storage stability of immobilized lipases is a new approach characterized by the potential of unifying the study conditions of biocatalysts created in various laboratories. The data achieved from storing lipases in the climatic chambers may be crucial for the chemical and pharmaceutical industry. Our paper describes the developed protocols for immobilization via interfacial activation of lipase B from Candida antarctica (CALB) and lipase OF from Candida rugosa (CRL-OF) on the Octyl-Sepharose CL-4B support. Optimization included buffers with different pH values of 4–9 and a wide range of ionic strength from 5 mM to 700 mM. It has been shown that the optimal medium for the CALB immobilization process on the tested support is a citrate buffer at pH 4 and high ionic strength of 500 mM. Implementing new optimal procedures enabled the hyperactivation of immobilized CALB (recovery activity 116.10 ± 1.70%) under the applicable reaction conditions using olive oil as a substrate. Importantly, CALB storage stability tests performed in a climatic chamber under drastic temperature and humidity conditions proved good stability of the developed biocatalyst (residual activity 218 ± 7.3% of dry form, after 7 days). At the same time, the low storage stability of CRL OF in a climatic chamber was demonstrated. It should be emphasized that the use of a climatic chamber to test the storage stability of a dry form of the studied lipases immobilized on Octyl-Sepharose CL-4B is, to our knowledge, described for the first time in the literature. [ABSTRACT FROM AUTHOR]

Abstract: A novel approach has been developed for activating the highly bioactive long-chain n-3 polyunsaturated fatty acids EPA and DHA as oxime esters and incorporating them exclusively to the end-positions of glycerol and enantiopure 1-O-alkylglycerols. The Candida antarctica lipase B was observed to display a superb regioselectivity when using the acetoxime esters of EPA and DHA as acyldonors under mild condition to keep acyl-migration side-reaction under complete control. Regiopure 1,3-diacylglycerols, 1-O-alkyl-3-acyl-sn-glycerols and their antipodes possessing EPA and DHA were afforded in very high purity and yields. [Copyright &y& Elsevier]

Abstract: Synthesis of symmetrically structured triacylglycerols possessing bioactive n−3 polyunsaturated fatty acids (eicosapentaenoic acid or docosahexaenoic acid) at the 2-position and a short-chain fatty acid (C2, C4, C6) located at the end-positions by a highly efficient two-step chemoenzymatic process is described. Full regiocontrol devoid of any acyl-migration side reactions was obtained in both a lipase promoted step to introduce the short-chain fatty acids exclusively into the primary alcohol positions of glycerol using activated vinyl esters at low temperature and a subsequent coupling reaction involving free EPA and DHA using EDAC as a coupling agent. [Copyright &y& Elsevier]

This paper focuses on a detailed evaluation of commercially available immobilized lipases and simple monohydric alcohols for the production of alkyl esters from sunflower oil by enzymatic alcoholysis. Six lipases were tested with seven alcohols, including straight and branched-chain primary and secondary alcohols. The reactions were conducted in a batch stirred reaction vessel using stoichiometric amounts of substrafes under solvent-free conditions. Dramatic differences in alcoholysis performance were observed among the different lipases. For most of the alcohols, Novozym 435 produced the highest yield of FA alkyl esters, with yields well over 90% for methanol, absolute ethanol, and 1-propanol. Overall, 96% ethanol was the preferred alcohol for all lipases except Novozym 435, and ethanolysis reactions reached the maximal conversion efficiency. Increasing the water content in the system resulted in an increased degree of conversion for all lipases except Novozym 435. The secondary alcohol 2-propanol significantly reduced the alcoholysis reaction with all lipases; however, the branch-chain isobutanol was more advantageous than linear 1-butanol for Novozym 435, Lipozyme RM IM, and Lipase PS-C. Many commercial immobilized lipases are highly efficient and promising for the production of alkyl esters, offering high reaction yields and a simple operation process. [ABSTRACT FROM AUTHOR]

The objective of the work described in this paper was to study a possible FA selectivity of digestive lipolytic enzymes isolated from salmon and trout intestines toward astaxanthin diesters of various FA composition and compare it with the FA selectivity of microbial lipase. Astaxanthin diesters of varying FA composition were prepared in excellent yields (>90%) by chemical esterification using a carbodiimide coupling agent. The astaxanthin diesters were screened in a hydrolysis reaction by various commercially available lipases. The highest conversion rates were observed with the Candida rugosa lipase, which discriminated against n-3 PUFA. The rate of hydrolysis was determined by HPLC. Digestive lipolytic enzymes isolated from salmon and rainbow trout intestines displayed reversed FA selectivity. Thus, astaxanthin diesters highly enriched with n-3 PUFA including EPA and DHA were observed to be hydrolyzed at a considerably higher rate than the more saturated esters. Similar trends in FA selectivity were observed in the hydrolysis of fish oil TAG by the digestive lipolytic enzyme mixtures. [ABSTRACT FROM AUTHOR]

Abstract: A highly efficient two-step chemoenzymatic synthesis of enantiopure structured ether lipids of the 1-O-alkyl-2,3-diacyl-sn-glycerol type has been developed. Chimyl, batyl and selachyl alcohols possessing pure saturated fatty acid (SFA) attached to the sn-3 position and pure EPA and DHA attached to the sn-2 position were obtained under full regiocontrol. This was offered by mild conditions and a highly efficient lipase that operated at room temperature. High-resolution 1H NMR spectroscopy was used to monitor the progress of the reactions and to evaluate the full regiocontrol of the reactions involved by keeping track of all prospective adducts involved in these reactions. This was extended to preparation of a focused library of eight monoacyl intermediate adducts for all even-numbered SFA ranging from C2–C16 and the corresponding EPA and DHA structured diacyl glycerol ethers (DAGE) products for chimyl, batyl and selachyl alcohols, the total of 72 compounds. [Copyright &y& Elsevier]

Abstract: The synthesis of nine enantiopure structured triacylglycerols (TAGs) of the AAB type is described, all possessing the (S)-absolute configuration. Six of them possess one saturated and two identical unsaturated fatty acyl chains, whereas the remaining three possess two identical saturated fatty acids along with one unsaturated fatty acid. The former group was synthesized by a five-step chemoenzymatic route involving a highly regioselective immobilized Candida antarctica lipase, starting from enantiopure (R)-solketal. The second group was prepared by a fully chemical five-step approach based on the same chiral precursor. Such enantiopure TAGs are strongly required as standards for the enantiospecific analysis of intact TAGs in fats and oils. [Copyright &y& Elsevier]

The synthesis of twelve enantiopure structured triacylglycerols (TAGs) of the ABC type possessing three different fatty acids is described by a six-step chemoenzymatic approach starting from (S)-solketal. Eight of the TAGs possess two different saturated fatty acyl groups located in the sn -1 and sn -2 positions with an unsaturated fatty acyl group in the remaining sn -3 position of the glycerol skeleton, whereas the remaining four possess two different saturated acyl groups in the terminal sn -1 and sn -3 positions with an unsaturated acyl group in the sn -2 position. The former group was synthesised by a six-step chemoenzymatic route involving a highly regioselective immobilised Candida antarctica lipase. The second group was prepared by a similar six-step approach, that required two separate lipase steps. Such enantiopure TAGs are strongly demanded as standards for enantiospecific analysis of intact TAGs in fats and oils. Image 1 • Twelve enantiostructured ABC type triacylglycerols (TAGs) were synthesised. • Unique TAGs as standards for chiral separation. • Facile methodology to synthesise both enantiomers of ABC type TAGs. • Highly regioselective lipase. [ABSTRACT FROM AUTHOR]