Search

Enzymatic cycling system (coupled dehydrogenase-catalyzed biosystem being composed of two elementary enzymatic reactions mediated by NAD(P)H + NAD(P)+) is industrially attractive for reducing prochiral carbonyl compounds to the corresponding chiral hydroxyl compounds. The reaction rate equation of the batch-wise biosystem was generally derived by ordered Bi Bi mechanism of two-substrate enzyme reaction on several reasonable assumptions. The rate equations of the batch-wise biosystem was generalized by transforming them into the dimensionless forms. The dimensionless forms were solved numerically. It was revealed that the batch-wise biosystem was generally made up of unique 3 phases, i.e., phases I, II and III. Phase I was very short transient so that the biosystem entered rapidly phase II. In phase II the consumption rate dynamically balanced with its formation rate so that the concentration of NAD(P)H was invariable with time (and hence NAD(P)+ concentration was, too). Phase III was substrate-exhausting phase, and the coenzyme concentration became finally only [NAD(P)+] or only [NAD(P)H] depending on the initial molar ratio of the prochiral carbonyl compound to the substrate of the coenzyme regeneration reaction ( [ S ] 0 / [ S ′ ] 0 ) > or [ S ] 0 / [ S ′ ] 0 1.0 , were proposed. As the main assumption irreversibility of the two elemental enzymatic reactions was discussed. Validity of the proposed rate equations was mentioned.

There is considerable interest in the development of simple methods for quantifying production of the biodegradable polyester poly(3-hydroxybutyrate) [P(3HB)] by bacteria. Cells of Cupriavidus necator were grown on agar medium containing different concentrations of glucose (10–25 g/L) as a sole carbon source. Trace amounts (100±5 μg) of dried C. necator cells were directly subjected to thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) in the presence of tetramethylammonium hydroxide (TMAH). On the resulting chromatograms, a series of characteristic peaks, attributed to the THM products from poly(3-hydroxybutylate) accumulated in the bacterial cells, were clearly observed without any interfering component. Based on the peak intensities, the contents of P(3HB) in C. necator were determined precisely and rapidly without using any cumbersome sample pretreatment. Furthermore, the values of the P(3HB) contents coincided overall with those obtained by the conventional method involving solvent extraction followed by gravimetric determination.

Kinetics of batch-wise enzymatic cycling system (oxidoreductase-catalyzed reaction system involving enzyme-coupled cofactor regeneration) has been studied covering a broad range of the conserved total cofactor concentration, [C]0 (=NAD(P)+ + NAD(P)H), based on reasonable several assumptions. It is composed of two elementary reactions, i.e. product synthesis reaction and cofactor regeneration reaction, both of which have been expressed by Michaelis–Menten type rate equations. A novel dimensionless variable, r, has been introduced, which is defined as the concentration of one of the two cofactor components, [X] (NADH+ or NADPH+), divided by [C]0, i.e. r .e[X]/[C]0. The following results have been obtained. (1) The fundamental equation of the batch-wise enzymatic cycling system has been transformed to a differential equation whose formula is: dr/dT = N(r)/D(r) (N(r) and D(r) are quadratic equations of r having different coefficients). (2) It has been elucidated that the batch-wise enzymatic cycling s...

A novel general principle was proposed to transform enzyme kinetic equations into unified dimensionless forms. The principle consists of six steps. The principle was applied to ten complex biochemical reaction systems and their characteristics were mathematically analyzed. The approach has six merits.: (i) the number of parameters is reduced, (ii) the rate expressions and their progress curves become much simpler, easily understood and are analyzed globally by the newly defined dimensionless time, T, (iii) the relative magnitude of kinetic constants and of the maximum velocity involved in the rate equations are straightforwardly estimated by three type m values, (iv) mass balance and stoichiometric relationship of the dimensionless rate expressions are exactly the same format as of the actual rate expressions, (v) 2D or 3D plots of the unified dimensionless equations can be depicted without inputs of the actual experimental data of the variables and kinetic constants, and (vi) the newly defined dimensionless inactivation constant, K i a c t , is useful to estimate the effect of the enzyme inactivation on the batch reactions. The principle is beneficial for the mathematical analyses of complex biochemical reactions involving two or more kinds of enzymes, for those involving two or more substrates or for those involving both.

Amyloid β-protein (Aβ) is the major component of extracellular plaques in the brains of patients with Alzheimer's disease. It has been suggested that the interaction of Aβ with membrane cholesterol is essential for Aβ to exert neurotoxicity; however, the effect of phospholipids, another major membrane lipid component, on Aβ-induced neurotoxicity remains unclarified. Here we report the protective effect of phosphatidylcholine (PC) on primary cultured neurons against Aβ1-42-induced damage. Aβ1-42 caused neuronal death as demonstrated by lactose dehydrogenase (LDH) release, which was completely prevented by a pretreatment with PC in a dose-dependent manner. PC containing unsaturated long-chain acyl groups, 1,2-dioleoyl-PC (DOPC), also prevented neuronal death caused by Aβ1-42. The oleic acid ethyl-ester (OAEE) partially prevented Aβ1-42-induced neurotoxicity. Neurons that were pretreated with DOPC or OAEE for 24h, washed out, and exposed to Aβ1-42 in the absence of either of these reagents, were still resistant to Aβ1-42-induced neurotoxicity. In contrast, treatment with phosphotidylserine (PS) or docosahexaenoic acid etyl-ester (DHAEE) had no protective effect on neurons against Aβ1-42-induced damage. These results suggest that the control of cellular PC content, not PS content, may prove useful in the prevention or treatment of Alzheimer's disease.

In our previous paper, thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) measurements of poly(butylene succinate- co -butylene adipate) (PBSA) in the presence of tetramethylammonium hydroxide had revealed that the butylene adipate (BA) content in the PBSA films gradually decreased with soil burial degradation time. In order to clarify the cause of this change in copolymer composition, biodegradation behavior of PBSA with lowered crystallinity was evaluated by THM-GC. PBSA film samples with lower degree of crystallinity, prepared by heating and cooling quickly the original commercially available films, were subjected to a soil burial biodegradation test at 30 °C for up to 4 weeks. The copolymer compositions between BA and butylene succinate (BS) units in various stages of the degraded film samples were estimated on the basis of chromatograms obtained by THM-GC with less than 5% of the relative standard deviations. As a result, the change in copolymer composition for the heated PBSA films during soil burial was relatively small compared to the original films, suggesting that biodegradation for the heated films proceeded with the comparable rate for both BA and BS-rich moieties due to lowered crystallinity. Based on these results, the reason for the change in copolymer composition observed for the original PBSA films was clarified as follows: (1) the BA-rich moieties in the copolymer chains could show relatively lower crystallinity than the BS-rich moieties and (2) the BA-rich moieties were preferentially biodegraded during soil burial test, leading to the decrease in the BA content as the biodegradation proceeded.

Summary A novel, simple, compact, efficient system based on a unit operation of bubble stripping was developed for intensive methanol (MeOH) removal from crude biodiesel (BDF) which was synthesized by potassium hydroxide-catalyzed transesterification reaction of triacylglycerol (TAG) with MeOH. As TAG both rapeseed oil and used cooking oil were tested. In the system, gas was supplied by a gas pump through a gas bubbler into the crude biodiesel, then the exit gas containing MeOH vapor passed through a condenser (0°C) and the condensed MeOH was collected in a vessel. MeOH-free gas from the condenser returned into the inlet of the gas pump, thus gas was circulated in a closed system. After the intensive MeOH removal by the bubble stripping, insoluble sediment always appeared in the biodiesel so that it was removed by centrifugation. As a final step, trace amounts of potassium (K) and free glycerol were removed by adsorbent treatment. MeOH, K, free glycerol, fatty acid methyl ester, and water contents, acid, and iodine values and oxidation stability of the purified biodiesel cleared the specifications of EN 14214 Standard. The proposed dry process consisting of the three steps will be applicable to an industrial production of high-quality biodiesel. Practical applications: Bubble stripping that has been developed in this study is simple but very effective to remove almost completely residual methanol from the crude biodiesel. Compared with the conventional wet process, the dry process that authors' have proposed has no necessity of washing water, hence no waste water treatment. Elimination of sediment after the intensive residual MeOH removal by the bubble stripping before the final adsorption step will make it possible to decrease significantly the amount of the adsorbent (kg) required per kg of the crude biodiesel or to prolong the life span of its packed bed column, resulting in the cost reduction of the biodiesel production. The proposed dry process consisting of the three steps will be applicable to an industrial production of high-quality biodiesel.

In order to produce microbiologically large amount of l (+)-lactic acid (LA) from glucose, batch and fed-batch (intermittent addition of sterilized glucose powder aseptically) cultures of Rhizopus oryzae NBRC 5384 (identical to NRRL 395 and ATCC 9363) whose mycelia were immobilized in situ within sponge-like cubic particles (3.5 mm edge long) were carried out at 37°C in a three baffled shake flask. Appropriately calculated amount of fine powdery calcium carbonate (CaCO 3 ) was added initially or intermittently to control pH of the culture liquids. High accumulations of LA (145 g/L and 231 g/L, in reality 176 g/L and 280 g/L as anhydrous calcium lactate) were achieved by a batch (glucose concentration = 150 g/L) and a fed-batch cultures (the initial glucose concentration = 150 g/L and the intermittent addition of glucose equivalent to 100 g/L). In these cultures the yields and productivities of LA were, 95.0%, 1.42 g/L·h and 92.5%, 1.83 g/L·h, respectively. Existence of considerable amounts of calcium lactate (Ca(LA) 2 ) as crystals in the culture broth was experimentally proved by two evidences: (i) heating up (70°C) followed by quick low centrifugal force to remove remaining CaCO 3 solids from culture broth and then cooling down (37°C) followed by incubation of the culture supernatant at 37°C to observe recrystallization of Ca(LA) 2 , and (ii) the measurement of solubility of Ca(LA) 2 in the culture media. It was conceptually discussed to be able to avoid the product inhibition by means of crystallization fermentation for the high accumulation of LA by R. oryzae .

An extracellular lipase was isolated from a culture filtrate of Fusarium oxysporum f. sp. lini SUF 402 by hydrophobic chromatography. Purity of the preparation was 38-fold and recovery yield was 32%. The molecular mass of the isolated enzyme was 30 kDa. The lipase had a sugar chain, and the N-terminal amino acid was modified. The optimun pH at 37°C was 7.0. The enzyme had higher activity toward p-nitrophenyl esters with long and middle chain fatty acids (C8-C18) than with a short chain fatty acid (C4). The enzyme was not inhibited by phenylmethylsulfonyl fluoride or 2-mercaptoethanol. In the case of the lipase, both the hydrolysis rate of tristearin and final concentration of fatty acid content were higher than those of triolein. The lipase was examined with respect to its ability of concentrating the n-3 polyunsaturated fatty acid (n-3 PUFA) content of partially hydrolyzed glycerides (TG and DG) obtained from two kinds of fish oil (cod liver oil and refined sardine oil). The lipase gave increases in n-3 PUFA contents as the hydrolysis progressed. The lipase concentrated only docosahexaenoic acid with little increase in icosapentaenoic acid. Maximal total n-3 PUFA contents were about 30% in both fish oils.

Yeast's extracellular expression provides a cost-efficient means of producing industrially useful recombinant proteins. However, depending on the protein to be expressed, the production results in a poor yield, which is occasionally accompanied with loss of the expression plasmid and hence hampered growth of the host in the inducing medium. Here we propose an alternative approach, high cell-density expression, to improve the yield of a certain range of so-called difficult-to-express proteins. In this expression system, recombinant yeast cells resting in stationary phase (OD(660)=3-4) are suspended in a small aliquot of inducing medium to form a high cell-density culture (e.g., OD(660)=15). When applied to the yeast strains harboring Lentinula edodes laccase (Lcc1 or Lcc4) expressing plasmids, the high cell-density system allowed the host cells to synthesize elevated amounts of the laccase which resulted in >1000- to 6000-fold higher yield than those synthesized in a classical growth-associated manner. The resting cells required aerobic agitation for the maximum production. The production system also worked for other foreign enzymes but not for beta-galactosidase from Aspergillus oryzae or Escherichia coli, likely suggesting an involvement of chaperons that act on a certain range of secretory proteins.

Biodegradability of poly(butylene succinate-co-butylene adipate) (PBSA) was studied based on its copolymer composition determined by thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) using trace amounts (20 µg) of the film samples. The copolymer compositions of butylene adipate (BA) units gradually decreased with soil burial degradation time, reflecting preferential biodegradation of BA moieties during soil burial due to their lower crystallinity. Furthermore, local differences in biodegradability of a given PBSA film after soil burial were also successfully evaluated.

Stereoselective ethanolysis of monoacid TAG by immobilized Rhizomucor miehei lipase (RML) was studied for preparation of optically pure sn-2,3-DAG. Trioctanoylglycerol (TO) was used as a model substrate. The enantiomeric purity of the product, sn-2,3-dioctanoylglycerol (sn-2,3-DO), was very high (percent enantiomeric excess >99%) when an excess of ethanol was used. The result indicated that RML was highly stereoselective toward the sn-1 position of TO under conditions of excess ethanol. The stereoselectivity of RML depended on the amount of ethanol. The larger the amount of ethanol was, the higher the stereoselectivity became. After optimizing the parameters such as reactant molar ratio, water content, and temperature, (ethanol/TO molar ratio =31∶1 and water content =7.5 wt% of the reactants at 25°C), optically pure sn-2,3-DO was obtained at 61.1 mol% in the glyceride fraction in 20 min. The above conditions were further applied for ethanolysis of monoacid TAG with different acyl groups such as tridecanoylglycerol (C10∶0), tridodecanoylglycerol (C12∶0), tritetradecanoylglycerol (C14∶0) and trioctadecenoylglycerol [triolein, (C18∶1)]. The yields and enantiomeric purities of 1,2(2,3)-DAG were dramatically reduced when TAG with FA longer than decanoic acid were used.

To engineer proteins with desirable characteristics from a naturally occurring protein, high-throughput screening (HTS) combined with directed evolutional approach is the essential technology. However, most HTS techniques are simple positive screenings. The information obtained from the positive candidates is used only as results but rarely as clues for understanding the structural rules, which may explain the protein activity. In here, we have attempted to establish a novel strategy for exploring functional proteins associated with computational analysis. As a model case, we explored lipases with inverted enantioselectivity for a substrate p-nitrophenyl 3-phenylbutyrate from the wild-type lipase of Burkhorderia cepacia KWI-56, which is originally selective for (S)-configuration of the substrate. Data from our previous work on (R)-enantioselective lipase screening were applied to fuzzy neural network (FNN), bioinformatic algorithm, to extract guidelines for screening and engineering processes to be followed. FNN has an advantageous feature of extracting hidden rules that lie between sequences of variants and their enzyme activity to gain high prediction accuracy. Without any prior knowledge, FNN predicted a rule indicating that "size at position L167," among four positions (L17, F119, L167, and L266) in the substrate binding core region, is the most influential factor for obtaining lipase with inverted (R)-enantioselectivity. Based on the guidelines obtained, newly engineered novel variants, which were not found in the actual screening, were experimentally proven to gain high (R)-enantioselectivity by engineering the size at position L167. We also designed and assayed two novel variants, namely FIGV (L17F, F119I, L167G, and L266V) and FFGI (L17F, L167G, and L266I), which were compatible with the guideline obtained from FNN analysis, and confirmed that these designed lipases could acquire high inverted enantioselectivity. The results have shown that with the aid of bioinformatic analysis, high-throughput screening can expand its potential for exploring vast combinatorial sequence spaces of proteins.

A novel HPLC-based method for direct separation of the three isomers of mono-acid diacylglycerols (DAGs), i.e., 1,2-DAG, 2,3-DAG and 1,3-DAG, has been established. The method employs a tandem column system, in which two different columns (a conventional silica gel column and a chiral stationary phase column) are connected in series. Two isomeric mixtures of DAGs (i.e., dicapryloylglycerol and dioleoylglycerol) and lipase-catalyzed reaction mixtures were successfully resolved on the tandem column HPLC system without any derivatization prior to the analysis. According to the established analytical method, stereoselectivity of two lipases toward mono-acid triacylglycerols in ethanolysis reaction was investigated. The tested enzymes were immobilized Candida antarctica lipase B (CALB) and Rhizomucor miehei lipase (RML). Analyses of the enantiomeric purity of 1,2-DAG and 2,3-DAG, generated as intermediates during the reaction, revealed that CALB and RML have sn-3 and sn-1 stereopreference, respectively.

Expression of a functional antibody fragment (Fab) using an Escherichia coli cell-free expression system has been reported previously [Jiang et al., FEBS Lett., 514, 290-294 (2002)]. The low yield of the synthesized antibody, however, limits the usefulness of the cell-free expression system, partly due to the degradation of product by endogenous proteases from the E. coli extract. To determine which proteases are responsible for the degradation, we compared the expression of a 6D9 Fab fragment under conditions whereby several protease inhibitors were added into the cell-free system. The addition of serine protease inhibitor increased the amount of the Fab fragment, indicating that serine proteases caused the antibody degradation. Therefore, several serine protease-deficient mutants of E. coli BW25113 were constructed by targeted homologous recombination. The use of extract from a double protease-deficient mutant (DeltadegP-ompT) significantly increased the amount and antigen-binding activities of an anti-HSA scFv and a 6D9 Fab fragment. These results suggest that the DegP- and OmpT-deleted mutant is a useful source of S30 extract for the production or screening of antibodies using the cell-free expression system.

The completion of human genome sequencing has shifted the focus of research from genes to proteins. In this regard, a protein library chip has become a useful tool for cell-free protein synthesis. In this study, we attempted to make a highly-integrated protein chip from a DNA library using in vitro protein synthesis on a microchamber array fabricated by using PDMS (polydimethyl siloxane), a hydrophobic surface, and glass, a hydrophilic bottom substrate. These structural properties prevented cross-contamination among the chambers. The minimum volume capacity of the smallest chamber was about 1 pl. The total number of chambers per chip was 10,000 on one chip (capacity 150 pl) and 250,000 on two others (1 and 5 pl). Next, we attempted in vitro protein synthesis using this microchamber array. The fluorescence of Green Fluorescent Protein (GFP) expressed on the chamber was rapidly detected (within just 1 h). GFP expression was also successful using immobilized DNA molecules on polymer beads. DNA immobilized beads were added as the source to each microchamber. Protein was successfully synthesized from DNA immobilized beads, which allowed easy handling of the DNA molecules.

Microbial poly(3-hydroxyalkanoates) (PHAs) with fluorinated phenoxy side groups were produced by Pseudomonas putida when fluorophenoxyalkanoic acids were used as carbon sources. 11-(2-Fluorophenoxy)undecanoic acid (2FPUDA), 11-(3-fluorophenoxy)undecanoic acid (3FPUDA), 11-(4-fluorophenoxy)undecanoic acid (4FPUDA), 11-(2,4-difluorophenoxy)undecanoic acid (2,4DFPUDA), 11-(2,4,6-trifluorophenoxy)undecanoic acid (2,4,6TFPUDA), and 11-(2,3,4,5,6-pentaflurophenoxy)undecanoic acid (2,3,4,5,6PFPUDA) were used as carbon sources in the present study. When cells were grown with 2,4DFPUDA, the production of homo poly(3-hydroxy-5-(2,4-difluorophenoxy)pentanoate) was confirmed by NMR and GC/MS analyses. Fluorine atoms inserted into the side chain of the PHA dramatically affected its physical properties. In marked contrast to medium chain length (MCL) PHA, this fluorinated PHA was opaque, cream colored, and possessed greater crystallinity and a higher melting point (∼100 °C) than did the other MCL PHAs. Surface contact angle evaluation revealed that the PHA with two fluorine atoms possessed water-shedding properties. The number of substituted fluorine atoms in the carbon source affected cell growth and difluorine-substituted phenoxyalkanoic acids reduced cell growth, and polymer production compared to non-substituted phenoxyalkanoic acids. No polymeric materials were obtained using either 2,4,6TFPUDA or 2,3,4,5,6PFPUDA.

We have applied a combination of in vitro saturation mutagenesis and the single-molecule-PCR-linked in vitro expression (SIMPLEX) for rapid generation and screening of antibody mutants with improved affinity. A pool of anti-human serum albumin scFv mutant genes was created by in vitro saturation mutagenesis of a parent’s CDR-H3. Individual mutant genes were amplified by single-molecule PCR, and the PCR products were used directly as templates for in vitro coupled transcription/translation. Thereafter, all expressed scFv mutants were screened by competition ELISA. Two mutants showing improved affinities were selected and kinetically characterized. Because the single-molecule PCR, in vitro expression, and selection of distinct mutants were carried out parallelly in an array format, the process was very speedy and perfectly suited automation.

Blends of bacterial poly(3-hydoroxyalkanoate) (PHA) with a medium-length side chain and polylactide (PLA), and blends of the chemically modified PHA (ePHA) with PLA, were prepared. The morphologies, some physical properties, and thermal behavior of the blends based on PLA were investigated by electron microscopies, testing machines, and a differential scanning calorimeter, respectively. A blend of uncrystallized rubbery second components, PHA and ePHA, produced an increase in the impact toughness of the PLA blends in contrast to a decrease in the tensile strength value. PHA, especially, with an inserted epoxy group side chain was more effective in improving the morphology and the physical properties than PHA. The ePHA particles existed in the PLA domain as a dispersed phase having a size of 0.1–1 μm. The results of the biodegradation test demonstrated that the PLA blends still maintained their biodegradability and were more accessible to hydrolysis and microbial attack, resulting in a greater weight loss than the pure PLA. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 2363–2369, 2004

The Dam1 complex, also known as DASH complex, is the outer kinetochore protein complex of yeast that plays a crucial role in attachment of kinetochore to microtubule. The Dam1 complex is formed by at least nine proteins including Dam1p, Duo1p, Dad1p, Spc19p and Spc34p. In this study, domains of Spc34p that physically interact with other subunits of the complex were mapped using a high-throughput methodology. The method is a combination of two-hybrid screening of a random truncation library of the Spc34 gene and a unique PCR-based amplification that converge the selected DNA fragments to a few short fragments. Duo1p, Dam1p, Dad1p and Spc19p binding domains of Spc34p were mapped on M1-E59, M1-D47, M1-D47 or T207-E295 and S154-Q294, respectively. Most of the boundaries were located at less conserved regions among fungal Spc34p homologs, which is consistent with the boundaries of the putative secondary structures. The accuracy of the mapped domain boundaries was verified using truncated Spc34p polypeptides. The results and methodology we demonstrated herein not only shed light on the molecular architecture of the protein complex but also pave the road to the high-throughput identification of specific interaction domains of proteins whose possible interaction partners have been identified in genome-scale analyses.

A novel entirely in vitro strategy for generation and screening of a combinatorial protein library in an array format has been developed and is experimentally demonstrated. The strategy exploits virtues of PCR and in vitro coupled transcription/translation. Our new approach provides high-throughput construction and screening of the in vitro protein library, and compatibility with various selection methods.

We found that the affinity tag fused to the carboxyl (C-) terminal of a single-chain Fv (scFv) antibody was proteolytically degraded in a wheat germ cell-free protein synthesis system. The addition of two extra residues of glycine to the tail of the cMyc tag significantly increased the stability of the tag, suggesting that wheat endogenous carboxypeptidase(s) play a primary role in the C-terminal tag-specific degradation. In addition to the modification of the tag sequence, addition of diisopropyl fluorophosphate, which is known as an inhibitor of carboxypeptidases, prevented the cMyc tag sequence degradation. The effects of other protease inhibitors on the translation reaction and stability of the synthesized protein are also reported.

The cDNAs encoding the zeta class of glutathione S-transferases (GSTs) (GSTZs), which are multifunctional enzymes, were cloned from Arabidopsis thaliana L. and three lines of Brassica napus L. by RT PCR, and named AtGSTZ for A. thaliana ecotype Columbia gll, and BnGSTZ-A, BnGSTZ-B and BnGSTZ-C for B. napus cv. Shan 2A, Shan 213 and Ken C1, respectively. Sequence analysis revealed that the sequences of Shan 2A and Shan 2B were identical, and Ken C1 was different only in 3.8% of the sequence. Comparison of these sequences with published sequences in GeneBank showed that the sequences of the Brassica species were unpublished. The cDNAs were then inserted into two vectors for in vivo expression in Escherichia coli and in vitro expression in a wheat-germ cell-free protein synthesis system. All GSTZs were well expressed both in vivo and in vitro as an enzymatically active form, showing that all of them had both dichloroacetic acid dechlorinating and maleylacetone isomerase activities.

By thein vitro combinatorial mutagenesis, which is a sequential reaction of PCR mutagenesis andin vitro coupled transcription/translation withEscherichia coli S30 extract, S65 and E222 of green fluorescent protein ofAequarea victoria were comprehensively changed to all possible combinations of amino acids, thus totally 400 mutant (including a wild type) proteins were simultaneously produced and their fluorescent properties were analyzed. Although a few mutations had been reported so far at the 222nd position, replacement E222 to all other19 amino acids gave fluorescent signal to the mutants by changing Ser 65 to Ala together. Among the mutants, replacement to G, A, S, Q, H and C gave relatively high fluorescence. Thein vitro combinatorial mutagenesis, therefore, has been proved valuable for comprehensive structure-function studies of proteins.

Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans , codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR , suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[( R )-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.

The mature lipase of Burkholderia cepacia KWI-56 was synthesized in an enzymatically active form using an in vitro Escherichia coli S30 coupled transcription/translation system by expressing the mature lipase gene (rlip) in the presence of its specific activator. To investigate the substrate specificity of the lipase comprehensively, a large number of mutant lipases were constructed and analyzed in a high throughput manner by combining overlapping PCR and in vitro protein synthesis. In this paper, Phe119 and Leu167, which are located in the acyl portion of the substrate-binding pocket of the lipase of B.cepacia KWI-56, were substituted with six hydrophobic amino acid residues by the in vitro combinatorial mutagenesis. The wild-type and 35 mutant genes amplified by PCR were directly used as templates for the in vitro transcription/translation. The acyl chain-length selectivity of the in vitro expressed lipases against p-nitrophenyl butyrate, p-nitrophenyl caprylate and p-nitrophenyl palmitate, was compared by their relative hydrolysis rates. Two mutant lipases, L167V and F119A/L167M, which showed a significant shift in substrate selectivity were further expressed in vivo and refolded in vitro. It was found that L167V raised its preference for the short-chain ester, whereas F119A/L167M improved its selectivity for the long-chain ester.

The comonomer compositional distribution and thermal and morphological characteristics were investigated for five bacterially synthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] samples with 3HV content of 45, 49, 70, 80, and 96 mol %. All these samples were fractionated into many fractions with widely different 3HV content by changing solvent/nonsolvent volume ratio of chloroform/n-heptane mixtures. Bacterial P(3HB-co-3HV) samples investigated in this study were found to have broad comonomer compositional distribution. The tendencies of the fractional precipitation of the P((3HB-co-3HV)s with 3HV content lower than 60 mol % and those with 3HV higher than 80 mol % were found to be contrary. The 3HV content dependences of the thermal properties and crystalline structures were investigated for bacterial poly(3-hydroxybutyrate) [P(3HB)] and a series of compositionally well-fractionated P(3HB-co-3HV) samples with 3HV content ranged from 14 to 98 mol % by DSC, WAXD, and solid-state (13)C NMR. It was found that P(3HB-co-3HV) samples with 3HV content lower than about 47 mol % form the crystalline lattice having the P(3HB) homopolymer type lattice including the 3HV unit as the crystal constituent, and those with a 3HV content higher than about 52 mol % form the crystalline lattice having the P(3HV) homopolymer type lattice including the 3HB units. Thus, P(3HB-co-3HV)s show the crystalline structural change in a very narrow range of 3HV content.

The specific intermolecular hydrogen-bonding interaction between some poly(hydroxyalkanoic acid)s (PHAs) and 4,4’-thiodiphenol (TDP) were studied by DSC and FT-IR. Chemosynthetic poly(3-hydroxypropionate), bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and bacterial poly(3-hydroxybutyrate- co-4-hydroxybutyrate) were used as the PHA samples. A curve-fitting program for line-shape analysis of FT-IR spectra was also applied to investigate the fractions of associated carbonyl groups. The measurements show there are strong intermolecular hydrogen bonds between the carbonyl groups of PHAs and the hydroxyl groups of TDP. The content of intermolecular hydrogen bonds is influenced by both the content of TDP in the PHA/TDP blends and the crystallizability of PHAs. The crystallizability is related to the flexibility of the molecular chains. With the addition of TDP to PHAs, the decrease of melting point Tm and the increase of glass transition temperature Tg of the component of PHAs in the blends were found. When the content of TDP was more than 40%, phase separation took place, which was so serious that Tg dropped obviously as soon as the content of TDP was up to 80%. At this time, it is difficult for the molecules of PHAs and TDP to form intermolecular hydrogen bonds. As indicated by the study of FT-IR, TDP has more ability to form the self-hydrogen-bond of TDP-HO···HO-TDP type than the inter-hydrogen-bond of PHA-C=O···HO-TDP type. Based on the analysis on the eutectic behavior in different PHA/TDP pairs, it was found that the bulkiness of the pendant alkyl groups in the chains of PHAs affects the eutectic behavior.

Symmetrically structured triacylglycerols (TG) rich in docosahexaenoic acid (DHA) with caprylic acid (CA) at the outer positions were synthesized enzymatically form bonito oil in a two-step process: (i) ethanolysis of bonito oil TG to 2-monoacylglycerols (2-MG) and fatty acid ethyl esters, and (ii) reesterification of 2-MG with ethyl caprylate. Ethanolysis catalyzed by immobilized Candida antarctica lipase (Novozym 435) yielded 92.5% 2-MG with 43.5% DHA content in 2 h. The 2-MG formed were reesterified with ethyl caprylate by immobilized Rhizomucor miehei lipase (Lipozyme IM) to give structured TG with 44.9% DHA content [based on fatty acid composition with caprylic acid (CA) excluded] in 1 h. The final structured lipids comprised 85.3% TG with two CA residues and one original fatty acid residue, 13% TG with one CA residue and two original fatty acid residues, and 1.7% tricaprylolglycerol (weight percent). The amount of TG with two CA residues and one C22 residue (22∶6=DHA, 22∶5, and 22∶4) was 51 wt%. The 1,3-dicapryloyl-2-docosahexaenoylglycerol to 1,2(2,3)-dicapryloyl-3 (1)-docosahexaenoylglycerol ratio (based on high-performance liquid chromatography peak area percentages) was greater than 50∶1. The recovery of TG as structured lipids after silica gel column purification was approximately 71%. Ethyl esters and 2-MG formed at 2 h of ethanolysis could be used to determine the positional distribution of fatty acids in the intial TG owing to the high 1,3-regiospecificity of Novozym 435 and the reduced acyl migration in the system.

Paracoccus denitrificans degraded poly(3-hydroxybutyrate) (PHB) in the cells under carbon source starvation. Intracellular poly(3-hydroxyalkanoate) (PHA) depolymerase gene ( phaZ ) was identified near the PHA synthase gene ( phaC ) of P. denitrificans . Cell extract of Escherichia coli carrying lacZ–phaZ fusion gene degraded protease-treated PHB granules. Reaction products were thought to be mainly D (−)-3-hydroxybutyrate (3HB) dimer and 3HB oligomer. Diisopropylfluorophosphonate and Triton X-100 exhibited an inhibitory effect on the degradation of PHB granules. When cell extract of the recombinant E. coli was used, Mg 2+ ion inhibited PHB degradation. However, the inhibitory effect by Mg 2+ ion was not observed using the cell extract of P. denitrificans .

A highly efficient enzymatic method for the synthesis of regioisomerically pure 1,3-dicapryloyl-2-docosahexaenoyl glycerol (CDC) in two steps was established. 2-Monoglyceride (2-MG) formation by ethanolysis of tridocosahexaenoylglycerol (DDD) with immobilized Candida antarctica lipase (Novozym 435) as catalyst was the key step of the synthesis. CDC was finally obtained by reesterification of 2-MG with ethylcaprylate (EtC) catalyzed by Rhizomucor miehei lipase (Lipozyme IM). The regiospecificity of Novozym 435 depended on the type of reaction and the initial composition of the reaction medium. It displayed strict 1,3-regiospecificity for ethanolysis at a high excess of ethanol in the reaction mixture although it displayed no regiospecificity in transesterification and esterification reactions. The highest yield of CDC (85.4%) was obtained by ethanolysis at a 4∶1 weight ratio of ethanol/DDD for 6 h followed by reesterification at a 20∶1 molar ratio of EtC/initial DDD for 1.5 h. The regioisomeric purity of CDC was 100%. Good results were obtained also for the synthesis of 1,3-dicapryloyl-eicosapentaenoylglycerol (CEC) by the same method: 84.2% yield and 99.8% regioisomeric purity at the same reactant ratios as above. The yield of the reesterification step and the regioisomeric purity of the product were influenced by the molar ratio of the reactants for both CDC and CEC syntheses: higher excess of EtC favored higher yields and regioisomeric purity of the products.

A novel multivariable control strategy is developed for alcohol (ethanol and n-pentanol) concentrations in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(HB-co-HV), a biodegradable polymer by Paracoccus denitrificans ATCC 1774. This controller, which is developed to control the mole fraction of P(HB-co-HV), consists of two parts: one is for ethanol concentration control and the other is for mole fraction control, based on the concept of metabolic flux distribution control. A simple metabolic reaction (MR) model is constructed for flux distribution analysis. The relationship between mole ratio of specific consumption rate of the two alcohols (ethanol and n-pentanol) and the mole fraction of 3HV units in the polymer is linear. This result suggests that the split ratio at a branched point of 3-ketovaleryl-CoA in the P(HB-co-HV) synthetic pathway is constant for several fermentation conditions. When the mole fraction of 3HV units has a target value, the feed rate of n-pentanol becomes a function of the feed rate of ethanol and the set value of 3HV, based on the MR model. The mole fraction of 3HV units successfully reached the target value using this strategy. The mole fraction control strategy is combined with an optimal production strategy based on the optimal profile of the specific growth rate. The combined strategy is realized using multivariable controllers and P(3HB-co-3HV) production is maximized with a given value of mole fraction of 3HV units at the final step of fermentation.

A simple method for direct optical resolution of some asymmetric triacylglycerols (TGs) has been established. The method employs chiral-phase high-performance liquid chromatography (HPLC). An enantiomeric pair of TGs comprising 1-eicosapentaenoyl-2,3-dicapryroyl-sn-glycerol (ECC) and 1,2-dicapryroyl-3-eicosapentaenoyl-sn-glycerol (CCE) was resolved on a CHIRALCEL OF™ or on a CHIRALCEL OD™ column. The separation of another pair of asymmetric TGs, 1-docosahexaenoyl-2,3-dicapryroyl-sn-glycerol (DCC) and 1,2-dicapryroyl-3-docosahexaenoyl-sn-glycerol (CCD), was achieved with the CHIRALCEL OD column. The chiral-phase HPLC method in combination with silver-ion HPLC and high-temperature gas chromatography was used for monitoring two interesterification reactions, whose products were chiral TGs. Interesterification of tricapryloylglycerol with ethyleicosapentaenoate or with ethyldocosahexaenoate was performed using Rhizomucor miehei lipase as the catalyst. The products targeted were the asymmetric pair of TGs, ECC and CCE or DCC and CCD. The amounts of sn-1-substituted products (ECC or DCC) were greater than their sn-3-substituted counterparts (CCE or CCD) throughout the reaction period, suggesting that R. miehei lipase had a stereopreference towards the sn-1 position over the sn-3 position.

Synthesis of 1,3-dicapryloyl-2-eicosapentaenoylglycerol (CEC) catalyzed by Lipozyme IM (immobilized Rhizomucor miehei lipase) was performed by interesterification of trieicosapentaenoylglycerol (EEE) with caprylic acid (CA) (acidolysis) and EEE with ethyl caprylate (EtC) (interesterification). Both methods involved two steps: (i) transesterification at an optimized water content and temperature for the high yield conversion of the substrate to CEC, 1-capryloyl-2-eicosapentaenoylglycerol (CEOH) and 2-eicosapentaenoylglycerol (OHEOH), and (ii) reesterification of CEOH and OHEOH to CEC by water removal under reduced pressure. Interesterification had clear advantages over acidolysis. The reaction rates for interesterification were higher and the reaction times shorter. The final yield of CEC by interesterification was higher, and the extent of acyl migration, indicated by the tricapryloylglycerol content, was lower. The disadvantage of the higher price of EtC used for interesterification (approximately 10 times higher than the price of CA) was overcome by synthesizing it directly in the same reaction vessel prior to the interesterification step. EtC was rapidly synthesized by esterification of CA with ethanol in high yield (92% obtained in 2.5 h). The amount of water added to the reaction mixture and the reaction temperature influenced the yields of CEC, CEOH, and OHEOH in the transesterification step for both interesterification and acidolysis methods. The regioisomeric purity of CEC was 100% for both methods at temperatures of 40°C or less. The highest yield of CEC (81%) was obtained for the interesterification of EEE with EtC, formed directly in the same reaction vessel, at a CA/EEE molar ratio of 20∶1 and 30°C.

Dicapryloyl-2-eicosapentaenoylglycerol (CEC) was synthesized by interesterification of trieicosapen- taenoylglycerol (EEE) with ethyl caprylate (EtC) catalyzed by Lipozyme TM . After some of the reaction conditions were opti- mized, the maximal molar content of CEC in the glycerides of the reaction mixture was 91%. Among the parameters studied in the optimization, the critical ones were: (i) the water content, which influenced the conversion of EEE to CEC and 1-capryl- oyl-2-eicosapentaenoylglycerol (CEOH), and (ii) the timing of water removal under reduced pressure for the reesterification of CEOH to form CEC. The complete synthesis of CEC from ethyl eicosapentaenoate (EtE) was performed in three steps: (i) hydrol- ysis of EtE to free eicosapentaenoic acid (EPA), (ii) esterification of glycerol with EPA to form EEE, and (iii) interesterification of EEE with EtC under the optimized conditions. The first two steps were catalyzed by Novozym TM and the third by Lipozyme TM . The total yield over all the steps was 88%, and no purification of the intermediates was necessary. The regioisomeric purity of the product was 100% by silver-ion high-pressure liquid chro- matography. Paper no. J9400 in JAOCS 77, 501-506 (May 2000). Triglycerides containing a polyunsaturated fatty acid (PUFA), such as eicosapentaenoic or docosahexaenoic acid (EPA or DHA, respectively), at the second position of the glycerol backbone and two medium-chain acids (MCA) at the outer positions are a group of structured glycerides with proven therapeutic and nutritional properties. These lipids provide the essential fatty acids and retain the assimilation advantages of the medium-chain triglycerides (1,2). The purpose of this paper is the establishment of a feasible method for the synthesis of 1,3-dicapryloyl-2-eicosapen- taenoylglycerol (CEC) for pharmaceutical use. Chemically pure starting materials and intermediates as well as high re- gioisomeric purity of the product are basic requirements for the medical use of the product. There are a few possible strategies for the synthesis of such triglycerides. Classical chemical esterification methods start either from the 1,3-diglyceride of MCA or the 2-monoglyc- eride of PUFA. These compounds are obtained chemically by multistep methods that use toxic reactants and catalysts. Te- dious purification after each step employs large amounts of organic solvents. These partial glycerides are quite unstable and undergo acyl migration even at ambient temperature. A straightforward method that combines an enzymatic with a chemical step was established in our laboratory (3). The symmetrical 1,3-diglyceride of caprylic acid (CA) was obtained enzymatically by direct esterification of glycerol with CA in a solvent-free system followed by chemical con- densation with EPA. The product was a mixture of approxi- mately 90% CEC and 10% 1,2-dicapryloyl-3-eicosapen- taenoylglycerol, and the total yield was less than 40%.

To reduce the freezing point of sesame oil, the lipase-catalyzed interesterification of sesame oil in a solvent free system was studied. The lipase was immobilized on Celite and refined sesame oil was used as the only substrate for the reaction. After interesterification, the oil did not solidify at 0 degrees C even after 24 h, and even longer storage at 2-4 degrees C did not result in solidification. The change of physical behavior was investigated with a differential scanning calorimeter and X-ray diffraction, and reduction in the thermodynamic and crystallographic stability of the interesterified oil was demonstrated. The change in triacylglycerol species composition after the reaction was analyzed, showing that content of trisaturated acylglycerol was decreased.

Poly(3-hydroxyalkanoate) (PHA) with a thiophenoxy group at the end of the side chain was obtained from Pseudomonas putida 27N01, using 11-thiophenoxy undecanoic acid as the sole carbon source. NMR and GC/MS analyses indicated that the polymer consisted of 3-hydroxy-5-thiophenoxypentanoate as the primary monomer unit and 3-hydroxy-7-thiophenoxyheptanoate as a minor unit. The polymer had a white cream color and was elastometric. X-ray analysis suggested that the PHA had an amorphous-like diffraction pattern. The final dry cell concentration was 693 mg/L, and the PHA content of the dry cells was 19.5%. GPC measurement with polystyrene as a standard showed that the number-average molecular weight was 81 000 with a polydispersity of 1.8. The glass transition temperature was approximately 4 °C according to DSC. Thus, we were able to successfully characterize this PHA and to show that a sulfur atom can be incorporated into the bacterial polymer produced from P. putida 27N01.

Production of structured triacylglycerols (sTAG) containing eicosapentaenoic acid (EPA) at the sn-1 (or 3) position using Lipozyme in a solvent-free system was studied. Optimal water activity (a(w)) for the synthesis of the sTAG was investigated. Vacuum was applied to shift reaction equilibrium toward the synthesis reaction by removing by-products. During vacuum application, the water level of the reaction system was controlled at the optimal level by addition of a suitable amount of water at a predetermined interval. Intermittent periodic addition of a suitable amount of water into the reaction mixture made the reaction rate faster than that without adding water. A molar yield of 89.7% of the targeted sTAG was obtained after 16 h reaction with a(w) control during the vacuum application as compared with the yield of 87.0% after 24 h of reaction without a(w) control during the vacuum application.

A novel bioreactor using a hollow-fiber membrane was developed for the coupled transcription/ translation system using T7 RNA polymerase and Escherichia coli S30 extract. The large surface area per the reaction volume of the reactor assured rapid mass transfers of substrates into the reaction mixture and of wastes out from it across the membrane by their molecular diffusion. The flux was large enough to maintain nucleotide concentrations for more than 3 h, which increased the protein synthesis greatly. In addition, the T7 terminator sequence downstream from the reporter genes was found to increase the synthesized protein significantly, especially when the product of polymerase chain reaction (PCR) was used as a template. Implementation of this finding and use of the bioreactor developed multiplied the productivity of protein by the in vitro direct expression from PCR template.

A high resolution approach to silver ion HPLC was studied for the separation of positional isomers of triacylglycerols (TAGs) containing long chain polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA) in enzymatically synthesized structured TAGs. Isopropanol was used as a novel modifier in a hexane-acetonitrile based mobile phase for silver ion HPLC. Peak identification was based on HPLC-mass spectroscopy and selectivities of lipases. Positional isomers of TAGs containing one molecule of EPA, DHA, or DPA with saturated fatty acids (FAs) such as caprylic acid and palmitic acid were separated within 13 min using a gradient of hexane-isopropanol-acetonitrile as mobile phase. TAGs containing two or more EPA, DHA, or DPA were also separated from each other within 25 min, but their positional isomers were unresolved. The retention characteristics of the TAG were found to be related to the number of carbon atoms in the FAs present in addition to the number of double bonds and their isomeric configuration. One isomer with an unsaturated FA in the sn-2 position eluted faster than the other with the unsaturated FA in the sn-1 or 3 position. Species with longer chain FAs attached to TAGs with the same degree of unsaturation eluted faster than those that have shorter chain FAs. For example, docosapentaenoylhexadecanoyloctanoin (DPA/C16/C8) was eluted faster than dioctanoyldocosapentaenoin (DPA/C8/C8).

The lipase-catalyzed acidolysis of a single-cell oil (SCO) containing docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) with caprylic acid (CA) was investigated. The targeted products were structured lipids containing CA residues at the sn-1 and -3 positions and a DHA or DPA residue at the sn-2 position of glycerol. Rhizomucor miehei lipase (RML) and Pseudomonas sp. KWI-56 lipase (PSL) were used as the biocatalysts. When PSL was used > 60 mol% of total SCO fatty acids (FA) were exchanged with CA, with DHA and DPA as well as the other saturated FA being exchanged. The content of the triacylglycerols (TG) containing two CA and one DHA or DPA (number of carbon atoms = 41, i.e., C41) residue was high (36%), and the isomer with the desired configuration (unsaturated FA residue at the sn-2 position) represented 77–78% of C41. In the case of RML, CA content reached only 23 mol% in the TG. A large amount of DHA and DPA residues remained unexchanged with RML, so that the resulting oil was rich in TG species containing two or three DHA or DPA residues (46%). TG C41 amounted to 22%, almost all of which had the desired configuration. This result suggested that the difference in the degree of acidolysis by the two enzymes was due to their different selectivity toward DHA and DPA, as well as the difference in their positional specificities.

6-Hydroxynicotinate 3-monooxygenase, a membrane-bound, 42-kDa monomeric enzyme from Pseudomonas fluorescens TN5 was purified and characterized. The enzyme catalyzes the oxidative decarboxylation of 6-hydroxynicotinate and depends on O2, NADH and FAD with the holoenzyme containing 1 M of FAD per 1 M of enzyme. The isolated enzyme was used for the synthesis of 2,5-dihydroxypyridine, a precursor for the chemical synthesis of 5-aminolevulinic acid, which is applied as a plant growth hormone, a herbicide and in cancer therapy. A 1.8-kbp DNA fragment, which contains the ORF encoding 6-hydroxynicotinic acid 3-monooxygenase, was cloned, sequenced and expressed in Escherichia coli. The deduced 385 amino acid sequence of the cloned ORF is in agreement with the enzyme molecular mass, amino acid sequence of an internal peptide, contains a putative FAD-binding site and is homologous to similar flavoproteins such as salicylate 1-monoxygenase.

Phospholipase D (PLD), secreted into the culture medium of an actinomycete, Streptoverticillium cinnamoneum, has been purified to homogeneity and characterized. The Stv. cinnamoneum PLD efficiently catalyzes both the hydrolysis and transphosphatidylation of various phospholipids, including phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylserine (PS). However, the substrate specificity differs between the two reactions; PE serves as the most preferred substrate for the hydrolysis, but PC and PS are better substrates than PE for the transphosphatidylation. In addition, the transphosphatidylation but not the hydrolysis of PE and PC is markedly activated on the addition of metal ions, especially Al3+. Nucleotide and amino acid sequence determination of the Stv. cinnamoneum PLD revealed the presence of common structural motifs identified in all PLD sequences from various species.

A high-resolution method based on silver ion high-performance liquid chromatography (HPLC) and high-temperature gas chromatography (HTGC) with on-column injection was established for the separation of triacylglycerols (TAG) which contained eicosapentaenoic acid (EPA). A solvent system based on n-hexane, 2-propanol, and acetonitrile in HPLC for the separation of EPA-containing TAG was developed. It was dependent upon the number of EPA molecules and their isomeric distribution on the glycerol backbone. This technique was used to monitor the incorporation of EPA onto specific positions on the glycerol backbone. For quantitative monitoring of the synthesis reaction of the TAG containing EPA, an HTGC analysis was developed. For monitoring of the synthesis of structured TAG containing EPA, transesterification of tricaprylin with EPA ethyl ester in a solvent-free system was performed. The transesterification reaction using various lipases was studied at a different molar ratios of substrates, several initial water activities (aw), and various reaction temperatures.